WO2011142545A2 - Method for inhibiting the induction of cell death by inhibiting the synthesis or secretion of age-albumin in cells of the mononuclear phagocyte system - Google Patents

Method for inhibiting the induction of cell death by inhibiting the synthesis or secretion of age-albumin in cells of the mononuclear phagocyte system Download PDF

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WO2011142545A2
WO2011142545A2 PCT/KR2011/003147 KR2011003147W WO2011142545A2 WO 2011142545 A2 WO2011142545 A2 WO 2011142545A2 KR 2011003147 W KR2011003147 W KR 2011003147W WO 2011142545 A2 WO2011142545 A2 WO 2011142545A2
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albumin
age
cells
synthesis
disease
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Korean (ko)
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WO2011142545A3 (en
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이봉희
변경희
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가천의과학대학교 산학협력단
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Priority to US13/697,332 priority Critical patent/US9662347B2/en
Priority claimed from KR1020110039984A external-priority patent/KR101351181B1/en
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/13—Amines
    • A61K31/14—Quaternary ammonium compounds, e.g. edrophonium, choline
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/195—Carboxylic acids, e.g. valproic acid having an amino group
    • A61K31/197—Carboxylic acids, e.g. valproic acid having an amino group the amino and the carboxyl groups being attached to the same acyclic carbon chain, e.g. gamma-aminobutyric acid [GABA], beta-alanine, epsilon-aminocaproic acid or pantothenic acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/20—Carboxylic acids, e.g. valproic acid having a carboxyl group bound to a chain of seven or more carbon atoms, e.g. stearic, palmitic, arachidic acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/21—Esters, e.g. nitroglycerine, selenocyanates
    • A61K31/215—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids
    • A61K31/22—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids of acyclic acids, e.g. pravastatin
    • A61K31/221—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids of acyclic acids, e.g. pravastatin with compounds having an amino group, e.g. acetylcholine, acetylcarnitine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/4164—1,3-Diazoles
    • A61K31/4166—1,3-Diazoles having oxo groups directly attached to the heterocyclic ring, e.g. phenytoin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/47—Quinolines; Isoquinolines
    • A61K31/475—Quinolines; Isoquinolines having an indole ring, e.g. yohimbine, reserpine, strychnine, vinblastine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/54—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one sulfur as the ring hetero atoms, e.g. sulthiame
    • A61K31/542—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one sulfur as the ring hetero atoms, e.g. sulthiame ortho- or peri-condensed with heterocyclic ring systems
    • A61K31/545—Compounds containing 5-thia-1-azabicyclo [4.2.0] octane ring systems, i.e. compounds containing a ring system of the formula:, e.g. cephalosporins, cefaclor, or cephalexine
    • A61K31/546—Compounds containing 5-thia-1-azabicyclo [4.2.0] octane ring systems, i.e. compounds containing a ring system of the formula:, e.g. cephalosporins, cefaclor, or cephalexine containing further heterocyclic rings, e.g. cephalothin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding

Definitions

  • the present invention relates to a method for inhibiting cell death by inhibiting the synthesis or secretion of ABE-albumin in mononuclear phagocytes, an inhibitor of ABE-albumin synthesis, and a pharmaceutical composition for preventing or treating degenerative diseases and autoimmune diseases, including the same. It is about.
  • Apoptosis modulating therapy regulates abnormal cell growth and apoptosis by inducing apoptosis or inhibiting apoptosis.
  • the purpose of apoptosis control therapy is not only to block disease progression by abnormal cell death. By converting abnormal cells into cells of normal function, it is primarily for treating diseases.
  • cell survival-death reversible controlling technology is a next-generation key technology for the development of apoptosis control therapy.
  • Apoptosis control therapy which is being developed competitively in the world, can be applied to various senile and degenerative diseases such as leukemia, cancer, Alzheimer's disease, Parkinson's disease, AIDS and aging.
  • various senile and degenerative diseases such as leukemia, cancer, Alzheimer's disease, Parkinson's disease, AIDS and aging.
  • Apoptosis therapy has become the underlying technology that can be applied to the treatment of a wider range of diseases. .
  • Apoptosis therapy can be used to treat disease by blocking pathological growth of unregulated cells, such as cancer cells, or inhibiting the development of degenerative diseases due to excessive apoptosis of normal cells, such as in Alzheimer's or Parkinson's disease. have.
  • diseases such as cancer conventional extensive necrosis pharmacotherapy kills pathological cells and at the same time, cytotoxic enzymes (e.g., lysozyme) that leak out as the cell membranes of the pathological cells are destroyed are normal. It is cytotoxic to the cells, which inevitably accompanies excessive inflammation, so the side effects are serious.
  • Apoptosis therapy induces spontaneous killing of pathological cells or strongly inhibits the growth of these cells, thereby overcoming the inflammatory side effects induced by apoptosis of cancer cells.
  • the reason for this is that when cell growth is strongly inhibited, cancer cells that have superior cell division ability than normal cells show a relatively greater inhibitory effect, and when these effects are maximized and apoptosis is induced, various substances in cells with cytotoxicity During the killing process, most of the enzymes, called caspases, are cleaved and lose their function. At the same time, these substances are wrapped in the apoptotic body, which causes macrophage to process phagocytosis. Going through. As a result, the cytotoxic substances do not leak to the outside and exhibit no toxicity to the surrounding cells.
  • apoptosis is an active cell death process that requires energy and exhibits characteristic cell morphology.
  • apoptosis signal is transmitted, apoptosis is determined and carried out in the cell, and in the execution step, the cell shows characteristic biochemical and morphological changes.
  • the cells contract and fall off from adjacent cells, and the cell membrane becomes blebbing.
  • the nucleus is condensed and the DNA in the nucleus is cut into small oligonucleotide fragments. Will form.
  • the apoptosis thus formed undergoes a series of processes such as phagocytosis by macrophages, resulting in cell death.
  • Apoptosis is a complex intracellular process, and intracellular determination of apoptosis is not easy, but once it is determined, several substeps are performed in order for apoptosis to function properly. Most morphological changes are caused by caspases, the aspartic acid specific cysteine protease, an activating enzyme during cell death.
  • Albumin is the most abundant plasma protein with multifunctional properties, mainly synthesized in hepatocytes, and a major component of most extracellular fluids, including interstitial fluid, lymphatic fluid and cerebrospinal fluid. As albumin decreases in vivo, liver function decreases and nutrition becomes poor, so clinically, albumin has been widely used in a critical condition including vascular collapse in critically ill patients and cirrhosis patients. In addition, it has recently been suggested that albumin specifically binds to low molecular weight molecules, which are important diagnostic or prognostic indicators of disease.
  • albumin may imply Alzheimer's disease because it enters the brain through the blood-brain barrier by molecular diffusion and specifically binds and transports A ⁇ 1-42 to amyloid beta 1-42 (A ⁇ 1-42), an Alzheimer's trigger. It is reported.
  • the inventors found that albumin is synthesized in microglial cells of the human brain, a type of mononuclear phagocyte, and the synthesis and secretion of such albumin is increased by administration of A ⁇ 1-42.
  • Yearly journal [Ahn SM, Byun K, Cho K, Kim JY, Yoo JS, et al. (2008) Human Microglial Cells Synthesize Albumin in Brain. PLoS ONE 3 (7): e2829].
  • AGE advanced glycation end-product
  • AGE is a complex material that occurs constantly in the human body, mainly caused by the reaction of carbohydrates and free amino acids, and is a chemically very unstable and highly reactive substance. It is known as a molecule that promotes death.
  • the final glycation end products are reported to be increased in the brain of the elderly or aged animals, affecting all cells and biomolecules, causing aging and aging-related chronic diseases.
  • the final glycosylated product increases vascular permeability, inhibits vasodilation due to nitric oxide blockage, LDL oxidation, various types of cytokine secretion in macrophages or endothelial cells, and oxidative stress, thereby aging, Alzheimer's disease, kidney disease, It is known to be associated with adult diseases such as diabetes mellitus, diabetic vascular complications, diabetic retinal abnormalities and diabetic neurological abnormalities.
  • AGE-albumin is expressed in human microglial cells or macrophages of Alzheimer's model, stroke model, Parkinson's disease model, and rheumatoid arthritis model.
  • AGE-albumin Synthesized and secreted, AGE-albumin synthesis and secretion is due to oxidative stress, AGE-albumin induces aggregation of A ⁇ 1-42 in brain tissues of rats treated with A ⁇ 1-42 and brain tissues of Alzheimer's patients, AGE-albumin synthesized and secreted from human microglia or macrophages acts on primary human neurons or chondrocytes to increase the expression of RAGE, which activates the MAPK signaling system and increases the expression of Bax. Induction of calcium in the mitochondria, and finally confirmed to induce cell death, the present invention was completed.
  • the present invention provides a method for inhibiting cell death by inhibiting the synthesis or secretion of ABE-albumin in mononuclear phagocytes.
  • the present invention is to provide a pharmaceutical composition for the prevention or treatment of degenerative diseases and autoimmune diseases containing an inhibitor of the synthesis of AA-albumin as an active ingredient.
  • the present invention also provides a method for preventing or treating degenerative diseases and autoimmune diseases by administering to a subject a therapeutically effective amount of an ABR-albumin synthesis inhibitor.
  • the AGE-albumin of the present invention is synthesized and secreted in human microglia or macrophages of Alzheimer's model, stroke model, Parkinson's disease model, rheumatoid arthritis model, synthesis and secretion of AGE-albumin due to oxidative stress, AGE
  • the expression of RAGE is increased in primary human neurons or chondrocytes treated with albumin, which activates the MAPK signaling system and increases the expression of Bax, leading to an increase in calcium in the mitochondria and finally apoptosis. Induce.
  • the inhibitor of the synthesis of ABE-albumin of the present invention may be used for degenerative diseases such as Alzheimer's disease, stroke, Parkinson's disease, Lou Gehrig's disease, rheumatoid arthritis, diabetic retinopathy, AIDS, aging, pulmonary fibrosis or spinal cord injury and autoimmune diseases. It can be usefully used for diagnosis or treatment.
  • FIG. 1 is a schematic diagram showing a method for inhibiting cell death induction of cells around mononuclear phagocytes by inhibiting the synthesis or secretion of AGE-albumin in mononuclear phagocytes of the present invention.
  • Figure 2 shows the distribution and expression sites of AGE-albumin in human microglial cells and rat brain tissues prior to or after A ⁇ 1-42 treatment, and brain tissues of normal or Alzheimer's patients with antibodies, followed by laser confocal fluorescence microscopy. Observed.
  • Figure 3 shows the density of AGE-albumin measured in human microglial cells and rat brain tissues before or after A ⁇ 1-42 treatment, brain tissues of normal or Alzheimer's patients.
  • Figure 4 is a diagram observed by the laser confocal fluorescence microscopy after staining the distribution and expression position of AGE-albumin in the brain tissue of Alzheimer's patients using antibodies [AGE (red), albumin (green), microglial markers Phosphorus Iba1 (blue), astrocyte marker MBP (blue), oligodendrocyte marker Olig2 (blue), neuronal marker NeuroD (blue)].
  • AGE red
  • albumin green
  • microglial markers Phosphorus Iba1 blue
  • astrocyte marker MBP blue
  • oligodendrocyte marker Olig2 blue
  • neuronal marker NeuroD blue
  • 5 is a diagram confirming the synthetic amount of AGE-albumin in human microglia by co-immunoprecipitation method.
  • FIG. 6 is a diagram confirming the expression amount of AGE-albumin secreted into human microglial cells and culture medium using ELISA.
  • FIG. 7 is a diagram confirming the synthesis and secretion of AGE-albumin in human microglia by oxidative stress through immunoblotting analysis.
  • FIG. 8 is a graph illustrating the synthesis and secretion of AGE-albumin synthesized and secreted from microglia of brain tissue of rats treated with A ⁇ 1-42 and brain tissues of Alzheimer's patients using ThT fluorescence. .
  • FIG. 9 is a diagram showing the results of measuring the amount of A ⁇ 1-42 after administration of AGE-albumin to human microglia using ELISA assay.
  • FIG. 10 is a diagram showing the results of AGE-albumin administration to human microglial cells after the expression position and the amount of expression of BACE, ADAM10, APP using immunostaining chemistry (A) and immunoblotting (B), respectively. to be.
  • FIG. 11 shows the expression sites of RAGE, ERK1 / 2, pERK1 / 2, p38, pp38, SAPK / JNK, pSAPK / JNK, Bax after administration of AGE-albumin to primary human neurons using immunostaining chemistry. The results are shown.
  • FIG. 12 shows the expression levels of RAGE, ERK1 / 2, pERK1 / 2, p38, pp38, SAPK / JNK, pSAPK / JNK, Bax after administration of AGE-albumin to primary human neurons using an immunoblotting method.
  • Figure 1 shows the results.
  • Figure 13 is a diagram confirming the interaction between AGE and RAGE using cell lysate before or after the treatment of AGE-albumin to primary human neurons.
  • FIG. 14 is a diagram illustrating the change of calcium in mitochondria after administration of AGE-albumin to primary human neurons by laser confocal fluorescence microscopy.
  • FIG. 15 is a diagram illustrating cell viability using MTT assay after administration of AGE-albumin to primary human neurons.
  • Fig. 16 is a diagram illustrating the distribution and expression position of AGE-albumin in blood mononuclear cells of mice before or after A ⁇ 1-42 treatment using an antibody, followed by laser confocal fluorescence microscopy.
  • Figure 17 is treated with A ⁇ 1-42 alone or A ⁇ 1-42 and sRAGE (A ⁇ / sRAGE) in the rat brain and after 72 hours the number of neurons in the rat brain stained with cresyl violet It is a figure observed with the microscope after.
  • Figure 18 shows Iba1 (blue), albumin (green), and microglia cell markers Iba1 (blue) in brain tissues of rats treated with A ⁇ 1-42 alone or with A ⁇ 1-42 and sRAGE (A ⁇ / sRAGE). Color), and the distribution and expression positions of AGE-albumin after staining with an antibody and observed with a laser confocal fluorescence microscope (A) and the density (B) of AGE-albumin.
  • FIG. 19 shows the distribution and expression locations of RAGE, NeuN, DAPI, Bax and p-SAPK / JNK in the brain tissues of rats treated with A ⁇ 1-42 alone or A ⁇ 1-42 and sRAGE (A ⁇ / sRAGE) together. After staining with a laser confocal fluorescence microscope.
  • FIG. 20 is a diagram illustrating the distribution and expression position of AGE-albumin in brain tissue of a stroke patient after staining with an antibody, and then observed with a laser confocal fluorescence microscope.
  • FIG. 20 is a diagram illustrating the distribution and expression position of AGE-albumin in brain tissue of a stroke patient after staining with an antibody, and then observed with a laser confocal fluorescence microscope.
  • FIG. 21 shows the expression of hypoxia-induced factor (HIF-1 ⁇ ) in human microglia cells of hypoxia and glucose-deficient stroke models by immunohistochemistry (IHC) (A), PCR (B), immunoblotting (C). This is confirmed by the following.
  • FIG 22 shows immunohistochemistry (IHC) (A), PCR (B), immunoblotting (C) for the expression of high motility group protein B1 (HMGB1) in human microglial cells of hypoxia and glucose-deficient stroke models. This is confirmed by the following.
  • FIG 23 shows the immunohistochemistry (IHC) (A), immunoblotting (B, C) and ELISA (D) expression levels of AGE-albumin in human microglia cells of hypoxia and glucose-deficient stroke models. This is confirmed.
  • IHC immunohistochemistry
  • B, C immunoblotting
  • D ELISA
  • FIG. 24 shows the expression levels of AGE-albumin according to HMGB1 concentration (0, 50, 200, 500, 2000 ng / ml) in human microglial cells of hypoxia and glucose-deficient stroke models.
  • A ELISA
  • B ELISA
  • C immunoblotting
  • 25 shows the expression levels of AGE-albumin according to the concentration (0, 50, 200, 500, 2000 ng / ml) of glycyrrhizic acid, an HMGB1 inhibitor, in human microglial cells of hypoxia and glucose-deficient stroke models. This is confirmed by performing ELISA and immunoblotting.
  • Figure 26 is confirmed by performing an immunoblotting analysis whether the synthesis and secretion of AGE-albumin due to oxidative stress in human microglia cells of hypoxia and glucose-deficient stroke model.
  • FIG. 27 shows RAGE, ERK1 / 2, p-ERK1 / 2, p38, p-p38, SAPK / JNK, p-SAPK / JNK, Bax after administration of AGE-albumin to primary human neurons obtained from human brain tissue
  • FIG. 28 is a diagram showing the results of measuring cell viability using MTT assay after administration of AGE-albumin to primary human neurons (A) and the protective effect of sRAGE on neuronal cell death (B).
  • FIG. 29 is a diagram illustrating the distribution and expression positions of AGE-albumin in brain tissues of Parkinson's disease patients using an antibody, followed by laser confocal fluorescence microscopy.
  • FIG. 30 shows PCR expression of ⁇ -synuclein or TNF- ⁇ after treatment with rotenone or 6-hydroxydopamine (6-OHDA), a Parkinson's disease-causing agent, in human microglial cells.
  • 6-OHDA 6-hydroxydopamine
  • FIG. 31 is a diagram illustrating the synthesis and secretion of AGE-albumin using cell lysates and cell cultures after treatment of human microglial cells with 0 to 100 nM of rotenone, through ELISA and immunoblotting analysis.
  • FIG. 32 is a diagram showing the expression level of ⁇ -synuclein or AGE-albumin by immunoblotting analysis of human microglial cells after treatment with Parkinson's disease-causing rotenone followed by oxidative stress.
  • FIG. 33 shows RAGE, Bax, SAPK / JNK, pSAPK / JNK, p38, and ERK1 using cell lysate after treatment with or without sRAGE to dopamine neurons, and before or after AGE-albumin treatment to dopamine neurons. / 2, pERK1 / 2 expression level was confirmed by immunoblotting analysis.
  • Figure 34 is treated with PBS, rotenone, rotenone / sRAGE in the brain tissue of the mouse and observed in the microscope after staining the number of neurons in the brain tissue of the mouse with cresyl violet after one week and a month It is a degree.
  • FIG. 35 shows the distribution and expression of AGE-albumin, RAGE, and Bax in the brain tissues of mice after treatment with PBS, rotenone, and rotenone / sRAGE in the brain tissues of the mouse, and after laser fluorescence immunostaining.
  • Fig. Observed with a confocal fluorescence microscope.
  • FIG. 36 shows the expression level of TNF- ⁇ and IL-1 ⁇ and the synthesis and secretion of AGE-albumin after treatment with ⁇ 2-microglobulin in human macrophage line (U937) using cell lysates or cell cultures. Figures confirmed by blotting and ELISA.
  • FIG. 37 is a diagram illustrating cell viability using MTT assay after administration of AGE-albumin alone or AGE-albumin / sRAGE to chondrocytes.
  • FIG. 38 is a diagram showing the results of ELISA measurement of candidate substances selected from LOPAC 1280 compounds as candidates that inhibit the synthesis of AGE-albumin in human microglia of the Alzheimer's disease model.
  • FIG. 39 is a diagram showing the results of ELISA measurement of candidates by selecting candidate substances that inhibit the synthesis of AGE-albumin in human microglial cells of the Parkinson's disease model from LOPAC 1280 compounds.
  • FIG 40 shows the number of neurons in the brain tissue of mice after one week and one month after treatment with PBS, rotenone, rotenone / cephacller, and rotenone / cephalotin sodium in the brain tissue of the mouse. after staining with violet).
  • FIG. 41 shows the distribution of AGE-albumin, RAGE, and Bax in the brain tissues of mice one week and one month after treatment with PBS, rotenone, rotenone / sepacller, and rotenone / cephalotin sodium in the brain tissues of mice. After fluorescence immunostaining the expression site is a diagram observed by laser confocal fluorescence microscope.
  • the present invention provides a method for inhibiting cell death induction by inhibiting the synthesis or secretion of advanced glycation end-product (AbE) -albumin in mononuclear phagocytes.
  • AbE advanced glycation end-product
  • the present invention also provides an inhibitor for the synthesis of ABR-albumin containing a compound having the inhibitory activity for the synthesis of ABR-albumin.
  • the present invention also provides a pharmaceutical composition for the prevention or treatment of degenerative diseases and autoimmune diseases containing an inhibitor of the synthesis of ABE-albumin as an active ingredient.
  • the present invention also provides a method of preventing or treating a degenerative disease and an autoimmune disease by administering to a subject a therapeutically effective amount of an ABR-albumin synthesis inhibitor.
  • the method for inhibiting cell death induction according to the present invention is characterized by inhibiting the synthesis or secretion of AGE-albumin in mononuclear phagocytes, thereby inhibiting cell death induction of cells around the mononuclear phagocyte cells.
  • necrosis is the death of cells caused by stimuli such as burns, bruises, poisons and the like, which is known as accidental death of cells.
  • necrosis water is introduced from outside the cell, causing the cell to expand and destroy.
  • necrosis water is introduced from outside the cell, causing the cell to expand and destroy.
  • all cell deaths were considered necrosis.
  • cells have been known to have triggers for spontaneous death. This active cell death, controlled by genes, is apoptosis. Necrosis occurs disorderly over long periods of time, whereas apoptosis occurs in a short time and orderly. Apoptosis begins as cells shrink.
  • PCD programmed cell death
  • the mononuclear phagocytes are amyloid beta 1-42 (A ⁇ 1-42), HMGB1, rotenone or 6-hydroxydopamine (6-OHDA), ⁇ 2-microglobulin ( ⁇ 2-microglobulin), and the like. It is preferably activated by, but is not limited thereto.
  • the cell death cells are preferably cells surrounding mononuclear phagocytes, and the cells surrounding the mononuclear phagocytes include, but are not limited to, neurons, chondrocytes, lung cells and hepatocytes.
  • the inhibition of the synthesis or secretion of AGE-albumin can be inhibited using one selected from the group consisting of albumin siRNA, albumin antibody, AGE antibody, AGE-albumin antibody and AGE-albumin synthesis inhibitor.
  • Types of mononuclear phagocytes include microglia, blood mononuclear cells, alveolar macrophages (type II pneumocytes, dust cells), peritoneal macrophages, granulomatous macrophages, spleen macrophages, hepatic cooper cells, and joints. Synovial A cells, vascular epicardium cells, lymph node macrophages, skin Langerhans cells, and the like, but are not limited thereto.
  • FIG. 1 The method of inhibiting cell death induction of cells around mononuclear phagocytes by inhibiting the synthesis or secretion of AGE-albumin in the mononuclear phagocytes of the present invention is briefly shown in FIG. 1.
  • Albumin and AGE are stained and widely distributed in human microglial cells, macrophages, brain tissue or cartilage of Alzheimer's model, stroke model, Parkinson's disease model and rheumatoid arthritis model, and are widely distributed and expressed and density of AGE-albumin Is significantly higher than in normal brain tissue or cartilage, and the microglia marker Iba1 is also expressed at the same position as AGE-albumin, so it can be expected that most AGE-albumins are synthesized in human microglia or macrophages. have.
  • human microglia or macrophages of Alzheimer's model, stroke model, Parkinson's disease model, and rheumatoid arthritis model have amyloid beta 1-42 (A ⁇ 1-42), HMGB1, rotenone or 6-hydroxydopamine, ⁇ 2-microglobulin
  • Treatment with AGE-albumin increases the amount of expression.
  • hydrogen peroxide H 2 O 2
  • the expression amount of AGE-albumin increased as human hydrogen peroxide increased.
  • Treatment of glial cells with the antioxidant ascorbic acid significantly reduced the expression of AGE-albumin.
  • oxidative stress on human microglia accumulates A ⁇ 1-42, which increases the synthesis of AGE-albumin, suggesting that the synthesis and secretion of AGE-albumin in human microglia is due to oxidative stress. Can be.
  • AGE-albumin acts on primary human neurons or chondrocytes to increase the expression of RAGE, which activates the MAPK signaling system and increases the expression of Bax to induce an increase in calcium in the mitochondria, ultimately. Induce cell death.
  • RAGE reactive oxygen species
  • sRAGE sRAGE has a protective effect on cell death.
  • AGE-albumin is synthesized and secreted in human microglia or macrophages of Alzheimer's model, stroke model, Parkinson's disease model, rheumatoid arthritis model, and synthesis and secretion of AGE-albumin is due to oxidative stress In primary human neurons or chondrocytes treated with AGE-albumin, the expression of RAGE is increased, which activates the MAPK signaling system and increases the expression of Bax, leading to an increase in calcium in the mitochondria. Induce cell death
  • the selected inhibitors of the synthesis of AGE-albumin may be used for degenerative diseases such as Alzheimer's disease, stroke, Parkinson's disease, Lou Gehrig's disease, rheumatoid arthritis, diabetic retinopathy, AIDS, aging, pulmonary fibrosis or spinal cord injury, and autoimmune diseases. It can be usefully used for diagnosis or treatment.
  • composition of the present invention may contain one or more known active ingredients having a prophylactic or therapeutic effect of degenerative diseases and autoimmune diseases together with inhibitors of the synthesis of AGE-albumin.
  • composition of the present invention may be prepared by including one or more pharmaceutically acceptable carriers in addition to the above-described active ingredients for administration.
  • Pharmaceutically acceptable carriers may be used in combination with saline, sterile water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol and one or more of these components, if necessary, as an antioxidant, buffer And other conventional additives such as bacteriostatic agents can be added.
  • Diluents, dispersants, surfactants, binders and lubricants may also be added in addition to formulate into injectable formulations, pills, capsules, granules or tablets such as aqueous solutions, suspensions, emulsions and the like.
  • it may be preferably formulated according to each disease or component by a suitable method in the art or using a method disclosed in Remington's Pharmaceutical Science (Recent Edition), Mack Publishing Company, Easton PA.
  • composition of the present invention can be administered orally or parenterally (eg, applied intravenously, subcutaneously, intraperitoneally or topically) according to the desired method, and the dosage is based on the weight, age, sex and health of the patient. The range varies depending on the diet, the time of administration, the method of administration, the rate of excretion and the severity of the disease.
  • the daily dosage of the AGE-albumin synthesis inhibitor is about 0.1 to 10 mg / kg, preferably about 0.5 to 2 mg / kg, more preferably administered once to several times a day.
  • composition of the present invention can be used alone or in combination with methods using surgery, hormonal therapy, drug therapy and biological response modifiers for the prevention or treatment of degenerative diseases and autoimmune diseases.
  • the present invention can provide a method for preventing or treating degenerative diseases and autoimmune diseases by administering to a subject a therapeutically effective amount of an AGE-albumin synthesis inhibitor.
  • Example 1 Distribution and Expression Location of AGE-Albumin in Brain Tissues of Alzheimer's Patients
  • HMO6 human microglial cell line
  • DMEM Dulbecco's modified Eagle's medium, Gibco
  • FBS heat-inactivated FBS
  • HMO6 cells were maintained at 5% CO 2 , 37 ° C.
  • HMO6 cells were then treated with amyloid beta 1-42 (A ⁇ 1-42, Sigma-Aldrich) at 0-400 nM. Cells were harvested 6 hours after treatment with A ⁇ 1-42 for HMO6 cells for further analysis.
  • the primary antibody was washed three times with PBS and the slides were incubated with one of the secondary antibodies for one hour at room temperature: Alexa flour 633 anti-mouse IgG (1: 500, Invitrogen), Alexa flour 488 anti-rabbit IgG (1: 500, Invitrogen), or Alexa flour 555 anti-goat IgG (1: 500, Invitrogen).
  • Alexa flour 633 anti-mouse IgG 1: 500, Invitrogen
  • Alexa flour 488 anti-rabbit IgG 1: 500, Invitrogen
  • Alexa flour 555 anti-goat IgG 1: 500, Invitrogen
  • Brain tissues of normal or Alzheimer rats were obtained from the Brain Bank of Seoul National University Hospital and the Brain Bank of Niigata University Hospital. Specifically, brain tissues of normal or Alzheimer rats, and brain tissues of normal or Alzheimer's patients were fixed with 4% paraformaldehyde in 0.1 M neutral phosphate buffer solution and frozen overnight in 30% sucrose solution, followed by low temperature 10 ⁇ m sections were prepared with a holding device (cryostat, Leica CM 1900).
  • Paraffin-embedded brain tissue was cut into 4 ⁇ m thick sections, deparaffinized in xylene, and rehydrated with a series of grades of ethanol. Normal goat serum (10%) was used to block nonspecific protein binding. Tissue sections were incubated overnight at 4 ° C.
  • rabbit anti-AGE antibody (Abcam), mouse anti-human albumin antibody (1: 200, R & D System), goat anti-Iba1 antibody (1: 500, Abcam), anti-MBP antibody (1: 200, Chemicon), anti-Olig2 antibody (1: 100, R & D System), anti-NeuroD1 antibody (1: 200, R & D System), anti-BACE antibody (1: 50, SantaCruz), anti-ADAM10 antibody (1: 200, R & D System), anti-APP antibody (1: 200, Chemicon), anti-JNK antibody (1: 200, Cell Signaling), anti-p-JNK antibody ( 1: 200, Cell Signaling), and anti-Bax Antibody (1:50, Santa Cruz).
  • rabbit anti-AGE antibody (Abcam), mouse anti-human albumin antibody (1: 200, R & D System), goat anti-Iba1 antibody (1: 500, Abcam), anti-MBP antibody (1: 200, Chemicon), anti-Olig2 antibody (1: 100, R & D System), anti-NeuroD1
  • the cultured tissue sections were washed three times with PBS and washed with Alexa flour 633 anti-mouse IgG (1: 500, Invitrogen), Alexa flour 488 anti-rabbit IgG (1: 500, Invitrogen), or Alexa flour 555 anti-goat. Incubated with IgG (1: 500, Invitrogen) at room temperature for 1 hour. After washing the secondary antibody three times with PBS, the coverslip was mounted on a glass slide using Vectashield mounting medium (Vector Laboratories) and observed with a laser confocal fluorescence microscope (LSM-710, Carl Zeiss).
  • VSM-710 laser confocal fluorescence microscope
  • AGE red
  • albumin green
  • DAPI 6-diamidino-2-phenylindole
  • albumin green
  • AGE red
  • albumin is mostly glycated albumin.
  • albumin and AGE are widely distributed in human microglial cells treated with A ⁇ 1-42, rat brain (cerebral cortex), and brain tissue (cerebral cortex) of Alzheimer's patients, and AGE-albumin expression is significantly increased.
  • the microglia marker Iba1 was expressed at the same position as AGE-albumin, but the astrocyte marker MBP, the oligodendrocyte marker Olig2, and the neuronal marker NeuroD was not expressed at the same position as AGE-albumin.
  • the astrocyte marker MBP, the oligodendrocyte marker Olig2, and the neuronal marker NeuroD was not expressed at the same position as AGE-albumin.
  • cell lysates were prepared with a radioimmunoprecipitation assay (RIPA) buffer containing 1M Tris (pH 7.5), 5M NaCl, 10% NP-40, 10% deoxycholate and protease inhibitor cocktail (Calbiochem). Cell lysates (1 mg protein) were incubated overnight at 4 ° C. with 5 mg anti-AGE (Abcam) -bound Sepharose beads in 500 ml PBS.
  • RIPA radioimmunoprecipitation assay
  • Sepharose beads were precipitated by centrifugation at 14,000 rpm for 5 minutes and washed three times with 1 ml of wash buffer containing 50 mM Tris-Cl and 500 mM NaCl, pH 8.0. IgG-bound antigen-antibody complexes were separated by 4-12% polyacrylamide gel (Invitrogen) and immunoblotting assays were performed with anti-albumin antibodies (1: 1000, abcam).
  • the expression level of AGE-albumin increased as the concentration of A ⁇ 1-42 increased in human microglial cells.
  • the expression level of AGE-albumin secreted into the cell and culture medium was measured by ELISA. Specifically, human microglia were treated with 0-400 nM A ⁇ 1-42, and then measured using cell lysate (0.5 mg protein) and culture medium (0.1 mg protein). The amount of AGE-albumin was measured with rabbit anti-AGE antibody (1: 1000, Abcam) and mouse anti-human albumin antibody (1: 800, Abcam). HRP bound anti-mouse secondary antibody (1: 1000, Vector Laboratories) was added to each well. Each well was developed by adding TMB (3,3 ', 5,5'-tetramethylbenzidine) and stopped with an equal volume of 2M H 2 SO 4 . Absorbance was then measured at 450 nm using an ELISA plate reader (VERSA Max, Molecular Devices).
  • the amount of AGE-albumin in cell lysates of human microglial cells treated with A ⁇ 1-42 was significantly increased in cell lysates of human microglial cells not treated with A ⁇ 1-42.
  • the albumin antibody was reacted with the cell lysate, the amount of AGE-albumin was decreased, and when the albumin antibody and A ⁇ 1-42 were simultaneously treated with the cell lysate, the amount of AGE-albumin was increased. .
  • Example 4 Increased Synthesis and Secretion of AGE-Albumin by Oxidative Stress in Human Microglial Cells
  • a ⁇ 1-42 is known to accumulate by oxidative stress for a long time. Therefore, in this experiment, in order to confirm whether the synthesis and secretion of AGE-albumin in human microglia is caused by oxidative stress, hydrogen peroxide (H 2 O 2 ) of 0-1000 ⁇ M, which is an oxidative stress inducer in human microglia. After treatment, immunoblotting analysis was performed using the cell lysate. In addition, it was confirmed by immunoblotting whether the expression of AGE-albumin is reduced by treating the human microglia with antioxidant.
  • H 2 O 2 hydrogen peroxide
  • Example 5 Induction of A ⁇ 1-42 Aggregation of AGE-Albumin Synthesized and Secreted in Microglial Cells of Alzheimer's Model (ThT Fluorescence)
  • AGE-albumin and amyloid plaques were observed in the brain tissues of rats treated with A ⁇ 1-42 and the brain tissues of Alzheimer's patients (A), and albumin was administered to microglia. It was observed that aggregation of A ⁇ 1-42 was significantly increased when AGE-albumin was administered than when (B). Therefore, it can be seen that AGE-albumin induces aggregation of A ⁇ 1-42 in brain tissues of rats treated with A ⁇ 1-42 and brain tissues of Alzheimer's patients.
  • FIG. 9 The result of measuring the amount of A ⁇ 1-42 after administration of AGE-albumin to human microglia using ELISA is shown in FIG. 9, and after administration of AGE-albumin to human microglia, BACE, ADAM10, APP
  • FIG. 10 The expression position and the expression amount of were observed using immunostaining chemistry (A) and immunoblotting (B), respectively, are shown in FIG. 10.
  • the amount of A ⁇ 1-42 was increased when AGE-albumin was administered to human microglia.
  • Cell lysate before or after AGE-albumin treatment in primary human neurons was used to determine the expression location and expression levels of RAGE, ERK1 / 2, pERK1 / 2, p38, pp38, SAPK / JNK, pSAPK / JNK, and Bax. Immunohistochemistry and immunoblotting were used for the observation.
  • RAGE was increased in primary human neurons administered AGE-albumin, and ERK1 / 2, p38, pp38, SAPK / JNK, pSAPK / JNK were increased except for pERK1 / 2 and MAPK. Not only was activated but also the expression of Bax, a pro-apoptotic protein, was increased.
  • PLA was performed on primary human neurons and brain tissues to determine the interactions between AGE and RAGE. The degree was visualized.
  • target tissues are washed with cold PBS and mouse anti human-albumin antibody (1: 200, R & D system), rabbit anti-A ⁇ antibody (1: 100, Chemicon), or anti-RAGE antibody (1: 200). , Santa Cruz) and incubated overnight at 4 °C.
  • PLA and Hoechst staining were performed using the Duolink Detection Kit (O-link Bioscience) according to the manufacturer's protocol.
  • Tissue specimens were fixed in Vectashield mounting media (Vector Laboratories) and analyzed using confocal microscopy (LSM 710). The number of in-situ PLA signals per cell was counted using the semi-automated image analysis program BlobFinderV3.0.
  • Primary human neurons were seeded at 96 ⁇ well culture plates at 2 ⁇ 10 3 cells per well. After reaching 80% confluence, primary human neurons were harvested at various concentrations (0, 0.01, 0.1, 1, 10, 20 ⁇ g / ml) or at various concentrations (0, 0.5, 1, 5, 10 mg / ml) albumin. After 24 hours of treatment, the cells were washed with PBS, and cell viability was measured by MTT [3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyl tetrazolium bromide] assay. Absorbance of each well was measured at 540 nm using a 96-well plate reader (VERSA Max, Molecular Devices).
  • AGE-albumin acts on primary human neurons to increase the expression of RAGE, which activates the MAPK signaling system and then increases the expression of Bax to induce an increase in calcium in the mitochondria. It can be seen that induces cell death.
  • sRAGE soluble RAGE
  • Rats Sprague-Dawley rats weighing 230-350 g were used as experimental animals. Rats were maintained on a 12 hour light and dark cycle, freely consumed with food and water, and acclimated at least a week before use. All animal experiments were approved and humanely performed by the Institutional Animal Care and Use Committees (IACUC).
  • IACUC Institutional Animal Care and Use Committees
  • the experimental animals were anaesthetized with ketamine (0.75 mg / kg body weight) and lumpoon (0.2 mg / kg body weight) before surgery.
  • PBS, sRAGE was dissolved in sterile water at a concentration of 400 ⁇ M and kept at 4 ° C. until use.
  • the rat brain was fixed using a stereotaxic instrument, and then the center of the scalp skin was incised. Punch a 30 ⁇ l Hamilton syringe until a target area (depth, 4.5 mm) is reached in the cranial bregma with a biological electric drill (backward, 8.3 mm; laterally, 5.4 mm). Gauge) was lowered vertically.
  • the expression levels of AGE, albumin, microglia marker Iba1, and AGE-albumin were increased in brain tissues of rats injected with A ⁇ 1-42 alone, but A ⁇ 1-42 and sRAGE (A ⁇ / sRAGE). ) Decreased in the brain tissues of rats injected with). In addition, the density of AGE-albumin in brain tissues of rats injected with A ⁇ 1-42 alone was significantly higher than that of rats injected with both A ⁇ 1-42 and sRAGE (A ⁇ / sRAGE).
  • a ⁇ 1-42 induces neuronal death and sRAGE protects RAGE-mediated neuronal death in rat brain tissues
  • rats treated with A ⁇ 1-42 alone or in combination with A ⁇ 1-42 and sRAGE (A ⁇ / sRAGE) Immunohistochemistry (IHC) was performed by staining RAGE, NeuN, DAPI, Bax and p-SAPK / JNK in brain tissues. Then, their distribution and expression site were observed by laser confocal fluorescence microscopy.
  • Example 10 Distribution and location of AGE-albumin in brain tissue of stroke patients
  • IHC immunohistochemistry
  • Example 11 Expression of hypoxia-induced factors (HIF-1 ⁇ ) and HMGB1 (high motility group protein B1) in human microglial cells of stroke model
  • hypoxia induced factors HIF-1 ⁇
  • HMGB1 human microglia of the stroke model
  • Human microglial cells were cultured in a 37 ° C. incubator containing 5% CO 2 using DMEM medium (Gibco, 10% bovine serum (Gibco), 0.1% gentamycin (Gibco), high concentration of glucose).
  • DMEM medium Gibco, 10% bovine serum (Gibco), 0.1% gentamycin (Gibco), high concentration of glucose.
  • the prepared microglia line is exchanged with glucose-free DMEM medium and then in a chamber (Billups-Rothenberg, Del Mar, CA) that produces hypoxic states (5% CO 2 and 95% N 2 ). After culturing for 1 hour, the cells were extracted and used for the experiment.
  • hypoxia-inducing factor HIF-1 ⁇
  • HMGB1 hypoxia-inducing factor
  • IHC immunohistochemistry
  • hypoxia-inducing factor (HIF-1 ⁇ ) and HMGB1 in human microglia of hypoxia and glucose-deficient stroke models are shown in FIGS. 21 and 22, respectively.
  • hypoxia-inducing factor HIF-1 ⁇
  • HMGB1 hypoxia-inducing factor
  • HMGB1 concentrations (0, 50, 200, 500, 500) in human microglia cells of the stroke model were treated when HMGB1, which is known to be excreted in the brain during stroke and stimulated microglia, was applied to human microglia cells of the stroke model. 2000ng / mL)
  • the expression level of AGE-albumin was measured using immunohistochemistry (IHC), ELISA and immunoblotting.
  • the expression level of AGE-albumin increased significantly as the concentration of HMGB1 increased in human microglia of hypoxia and glucose-deficient stroke models.
  • HMGB1 inhibitor glycyrrhizic acid was treated with different concentrations (0, 50, 200, 500, 2000 ng / ml) in human microglial cells of the stroke model, followed by expression of AGE-albumin using ELISA and immunoblotting. The amount was measured.
  • the expression level of AGE-albumin decreased significantly as the concentration of HMGB1 inhibitor was increased in human microglial cells of hypoxia and glucose-deficient stroke models.
  • Example 13 Increased Synthesis and Secretion of AGE-Albumin by Oxidative Stress in Human Microglial Cells of Stroke Model
  • DMEM medium Gibco, 10% bovine serum (Gibco), 0.1% gentamycin (Gibco), high glucose concentration.
  • Cultured cells were treated with AGE-albumin (sigma, 10 ⁇ g / ml) for 24 hours before use in the following experiments.
  • AGE-albumin directly activates the MAPK signaling system and increases the expression of Bax in primary human neurons obtained from human brain tissue, prior to treatment with AGE-albumin to primary human neurons or The expression levels of RAGE, ERK1 / 2, p-ERK1 / 2, p38, p-p38, SAPK / JNK, p-SAPK / JNK, and Bax were observed using the subsequent cell lysates using an immunoblotting method.
  • RAGE was increased in primary human neurons administered AGE-albumin, and SAPK / JNK and p-SAPK / JNK were increased except for p-ERK1 / 2, p38, and p-p38.
  • the expression level of Bax was increased.
  • primary human neurons were seeded at 96 ⁇ well culture plates at 2 ⁇ 10 3 cells per well. After reaching 80% confluence, primary human neurons were treated with AGE-albumin at various concentrations (0, 0.1, 1, 10, 50 ⁇ g / ml). After 24 hours of treatment, the cells were washed with PBS, and the cell viability was measured at 540 nm using MTT [3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyl tetrazolium bromide] assay. It was.
  • Example 15 Distribution and location of AGE-albumin in brain tissue of Parkinson's disease patients
  • IHC immunohistochemistry
  • albumin green
  • AGE red
  • Iba1 microglia marker Iba1
  • Example 16 Expression of ⁇ -synuclein or TNF- ⁇ in human microglial cells of Parkinson's disease model
  • Human microglial cells were cultured in a 37 ° C. incubator containing 5% CO 2 using DMEM medium (Gibco, 10% bovine serum (Gibco), 0.1% gentamycin (Gibco), high concentration of glucose).
  • DMEM medium Gibco, 10% bovine serum (Gibco), 0.1% gentamycin (Gibco), high concentration of glucose.
  • the prepared microglia cells were treated with Parkinson's disease-producing rotenone (Sigma, 1nM) for 10 days or 6-hydroxydopamine (6-hydroxyda) for 24 hours. The cells were then extracted and used for the experiment.
  • ⁇ -synuclein or TNF- ⁇ is present at high levels in brain tissue of Parkinson's disease patients. Therefore, after treatment with Parkinson's disease-producing rotenone or 6-hydroxydopamine (6-OHDA) in human microglial cells, the expression level of ⁇ -synuclein or TNF- ⁇ was determined by PCR. It was confirmed by immunoblotting analysis.
  • Example 17 Synthesis and Secretion of AGE-Albumin in Human Microglial Cells of Parkinson's Disease Model
  • Example 18 Synthesis and Secretion of ⁇ -synuclein or AGE-Albumin by Oxidative Stress in Human Microglial Cells of Parkinson's Disease Model
  • human microglial cells were treated with rotenone, a Parkinson's disease-causing substance, and then oxidative Immunoblotting analysis was performed using cell lysates after treatment with hydrogen peroxide (H 2 O 2 ), a stress-inducing substance, 0-1000 ⁇ M.
  • H 2 O 2 hydrogen peroxide
  • Dopamine neurons were cultured in a 37 ° C. incubator containing 5% CO 2 using DMEM medium (Gibco, 10% bovine serum (Gibco), 0.1% gentamycin (Gibco), high concentration of glucose). Cultured cells were treated with AGE-albumin (sigma, 10 ⁇ g / ml) for 24 hours before use in the following experiments.
  • DMEM medium Gibco, 10% bovine serum (Gibco), 0.1% gentamycin (Gibco), high concentration of glucose.
  • Cultured cells were treated with AGE-albumin (sigma, 10 ⁇ g / ml) for 24 hours before use in the following experiments.
  • sRAGE was treated with or without sRAGE in the dopamine neurons.
  • the absorbance was measured at 540 nm to confirm the protective effect of water-soluble RAGE (sRAGE) on neuronal cell death.
  • mice CBL57 / bL6 mice weighing 20-25 g were used as experimental animals. Mice were maintained on a 12 hour light and dark cycle, freely fed food and water, and acclimatized at least one week before use. All animal experiments were approved and humanely performed by the Institutional Animal Care and Use Committees (IACUC).
  • IACUC Institutional Animal Care and Use Committees
  • Parkinson's disease animal models were prepared by inducing oral administration of rotenone to mice for one month to cause Parkinson's disease in mice.
  • Parkinson's disease animal model was anesthetized with ketamine (0.75 mg / kg body weight) and lumpoon (0.2 mg / kg body weight) prior to surgery.
  • phosphate buffer (PBS) sRAGE (10 ng / ⁇ l) was dissolved in sterile water at a concentration of 1 mM and kept at 4 ° C. until use.
  • the brain of the Parkinson's disease animal model was fixed using a stereotaxic instrument, and then the center of the scalp skin was incised.
  • a needle of a 10- ⁇ L Hamilton syringe was drilled with a biological electric drill (backward, 0.3 mm; laterally, 2 mm) and reached the target area (depth, 2.5 mm) (26). Gauge) was lowered vertically.
  • 3 ⁇ l of sRAGE was slowly injected into the olfactory cortex (EC) of the Parkinson's disease animal model at a rate of 1 ⁇ l per minute with an automatic microinjector. The syringe was then slowly removed and surgical wounds closed with a wound closure clip and topically treated with antibiotics.
  • PBS was injected into the olfactory cortex of normal mice.
  • mice were bred for one week and one month after injection of sRAGE. Rotenone continued to be administered orally during breeding. After breeding, all mice were anesthetized again in the same manner, perfused through the heart with heparinized saline with 100-200 ml of heparin at 18 ° C., followed by 4 in 0.1 M sodium phosphate buffer (pH 7.4). Perfusion was carried out with 400 ml of% paraformaldehyde-lysine periodate. The brain was removed, placed in the same fixative, fixed at 4 ° C. for 4 hours, and transferred to cold 0.1 M phosphate buffer solution (PBS) containing 20% sucrose. Brains were incised with a 10 ⁇ m thick cross section with a freezing microtome and stored at ⁇ 80 ° C. until use.
  • PBS cold 0.1 M phosphate buffer solution
  • PBS, rotenone, and rotenone / sRAGE were treated in mouse brain tissues, and one week and a month later, the number of neurons in the brain tissues of mice was stained with cresyl violet and observed under a microscope.
  • the expression levels of AGE, albumin, and microglia markers Iba1, AGE-albumin, RAGE, and Bax were increased in brain tissues of mice administered orally with rotenone, but were injected with rotenone / sRAGE. It was confirmed that the brain tissue of the decrease.
  • ⁇ 2-microglobulin is known to be abundant in cartilage in patients with rheumatoid arthritis.
  • the exact role of ⁇ 2-microglobulin in cartilage and its association with host macrophages or chondrocytes is not yet known. Therefore, we conducted experiments on whether ⁇ 2-microglobulin induces macrophage activity and synthesis of TNF- ⁇ and IL-1 ⁇ in macrophages.
  • Macrophage line (U937) from human lymphoma was 37 ° C containing 5% CO 2 using RPMI 1640 medium (Thermo, 10% bovine serum (Gibco), 0.1% gentamicin (Gibco), high concentration of glucose). Cultured in the incubator. In order to make an arthritis cell model, the prepared human macrophage line (U937) was treated with ⁇ 2-microglobulin (Sigma, 50 ⁇ g / ml) for 24 hours, and then cells were extracted and used for the experiment.
  • ⁇ 2-microglobulin Sigma, 50 ⁇ g / ml
  • Human chondrocytes were cultured in a 37 °C incubator containing 5% CO 2 using a medium containing chondrocyte growth medium (Promo cell) and chondrocyte growth medium supplementMix (Promo cell).
  • Cultured chondrocytes were seeded at 96 ⁇ well culture plates at 2 ⁇ 10 3 cells per well. After reaching 80% confluence, the cultured chondrocytes were treated with AGE-albumin (sigma, 10 ⁇ g / ml). After 24 hours of treatment, the cells were washed with PBS, and then absorbance was measured at 540 nm using MTT [3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyl tetrazolium bromide] assay.
  • chondrocytes were decreased after AGE-albumin treatment.
  • sRAGE and AGE-albumin were simultaneously treated with chondrocytes, the chondrocyte counts were similar to those of the control group.
  • sRAGE has a protective effect against chondrocyte death.
  • Example 23 Screening candidates for AGE-albumin synthesis inhibitor
  • Cells cultured on the plate were treated with A ⁇ 1-42 (sigma) at 2 ⁇ M for 6 hours, and then 1280 compounds (5 ⁇ M) included in LOPAC (sigma) were treated for 24 hours.
  • the cells were fixed with 100% methanol and reacted with AGE-albumin antibody (1: 10000, abcam). Then, the reaction was performed with a secondary antibody (1: 5000, Vector) containing peroxidase, followed by color development with TMB (sigma), and absorbance at 450 nm was measured with an ELISA reader. Then, candidate substances that inhibit the synthesis of AGE-albumin were selected.
  • Candidates that inhibit the synthesis of AGE-albumin in human microglia of the Alzheimer's disease model were selected from 42 compounds of LOPAC 1280, and the selected candidates are shown in Table 1 below. ELISA measurement results are shown in FIG.
  • the cells cultured on the plate were treated with a medium extracted from dopamine neurons treated with rotenone (sigma, 1 nM) for 10 days for 24 hours, and then 1280 kinds of compounds (5 ⁇ M) contained in LOPAC (sigma) were treated. Treatment was done for 24 hours. After incubation, the cells were fixed with 100% methanol and reacted with AGE-albumin antibody (1: 10000, abcam).
  • reaction was performed with a secondary antibody (1: 5000, Vector) containing peroxidase, followed by color development with TMB (sigma), and absorbance at 450 nm was measured with an ELISA reader. Then, candidate substances that inhibit the synthesis of AGE-albumin were selected.
  • the expression levels of AGE, albumin, and microglia markers Iba1, AGE-albumin, RAGE, and Bax were increased in the brain tissues of rotenone-orally administered mice, but rotenone / sepachlor, and rote. It was confirmed that the brain tissue of rats injected with non / cephalotin sodium decreased.
  • a tablet was prepared by a direct tableting method.
  • the powder was prepared by mixing the above components, the powder was filled in a hard capsule according to the conventional method for preparing a capsule to prepare a capsule.
  • the amount of the above-mentioned ingredient was prepared per ampoule (2 ml).
  • Each component was added and dissolved in purified water according to the conventional method for preparing a liquid, and lemon flavor was added appropriately, followed by mixing the above components. Then, purified water was added thereto to adjust the total volume to 100 ml, and then filled in a brown bottle and sterilized to prepare a liquid.

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Abstract

The present invention relates to a method for inhibiting the induction of cell death by inhibiting the synthesis or secretion of AGE-albumin in cells of the mononuclear phagocyte system, to an AGE-albumin synthesis inhibitor, and to a pharmaceutical composition comprising the AGE-albumin synthesis inhibitor for preventing or treating degenerative disease and autoimmune disease. The AGE-albumin of the present invention is synthesized and secreted in human microglia or human macrophages in an Alzheimer's model, stroke model, Parkinson's disease model and rheumatoid arthritis model. The AGE-albumin synthesis and secretion are caused by oxidative stress. The expression of RAGE increases in first-order human neurons or cartilage cells to which AGE-albumin is administered, whereupon a MAPK signaling pathway is activated and the expression of Bax increases to induce an increase in calcium in mitochondria, thus finally inducing cell death. Therefore, the AGE-albumin synthesis inhibitor of the present invention can be valuably used in the diagnosis or treatment of degenerative diseases or autoimmune diseases such as Alzheimer's disease, strokes, Parkinson's disease, amyotrophic lateral sclerosis, rheumatoid arthritis, diabetic retinopathy, AIDS, aging, pulmonary fibrosis, spinal cord injuries, etc.

Description

단핵식세포계 세포 내에서 ＡＧＥ-알부민의 합성 저해 또는 분비 저해에 의한 세포사 유도 저해 방법Method of inhibiting cell death by inhibiting synthesis or secretion of ABE-albumin in mononuclear phagocytes
본 발명은 단핵식세포계 세포 내에서 ＡＧＥ-알부민의 합성 저해 또는 분비 저해에 의한 세포사 유도 저해 방법, ＡＧＥ-알부민의 합성 저해제 및 이를 포함하는 퇴행성 질환 및 자가 면역 질환의 예방 또는 치료용 약학적 조성물 등에 관한 것이다.The present invention relates to a method for inhibiting cell death by inhibiting the synthesis or secretion of ABE-albumin in mononuclear phagocytes, an inhibitor of ABE-albumin synthesis, and a pharmaceutical composition for preventing or treating degenerative diseases and autoimmune diseases, including the same. It is about.
최근 연구에 의하면, 다양한 질병의 발병 원인은 근원적으로 세포사멸 신호전달 기구의 비정상적인 기능에 기인한다고 알려져 있다. 세포사멸 조절요법 (apoptosis modulating therapy)은 세포사멸 유도 또는 세포사멸 억제를 통하여 비정상적인 세포의 성장과 사멸을 조절하는 것으로, 세포사멸 조절요법의 목적은 비정상적인 세포의 사멸에 의한 질병의 진행을 차단할 뿐만 아니라 비정상적인 세포를 정상 기능의 세포로 전환시킴으로써 근원적으로 질병을 치료하는데 있다. 따라서, 세포성장-사멸의 가역적 제어기술(cell survival-death reversible controlling technology)은 세포사멸 조절요법 개발을 위한 차세대 핵심 기술이다.Recent studies suggest that the pathogenesis of various diseases is rooted in abnormal functioning of apoptosis signaling machinery. Apoptosis modulating therapy regulates abnormal cell growth and apoptosis by inducing apoptosis or inhibiting apoptosis. The purpose of apoptosis control therapy is not only to block disease progression by abnormal cell death. By converting abnormal cells into cells of normal function, it is primarily for treating diseases. Thus, cell survival-death reversible controlling technology is a next-generation key technology for the development of apoptosis control therapy.
전세계에서 경쟁적으로 개발되고 있는 세포사멸 조절요법은 백혈병을 비롯한 암, 알츠하이머병, 파킨슨병, AIDS 및 노화 현상 등의 각종 노인성 및 퇴행성 질환에 적용할 수 있다. 그러나, 최근 연구 결과에서 대부분 질병의 발병이 궁극적으로 세포사멸 신호전달 기구의 비정상적 기능에 기인한다는 사실이 밝혀짐에 따라 세포사멸 조절요법은 더욱 다양한 영역의 질환 치료에 적용될 수 있는 기반 기술이 되고 있다.Apoptosis control therapy, which is being developed competitively in the world, can be applied to various senile and degenerative diseases such as leukemia, cancer, Alzheimer's disease, Parkinson's disease, AIDS and aging. However, recent studies have shown that most disease outbreaks are ultimately due to abnormal functioning of apoptosis signaling mechanisms. Apoptosis therapy has become the underlying technology that can be applied to the treatment of a wider range of diseases. .
세포사멸 조절요법은 암세포와 같이 조절되지 않은 세포의 병리적 성장을 차단하거나, 알츠하이머병 또는 파킨슨병에서와 같이 정상 세포가 과도한 세포사멸을 일으켜 퇴행성 질환이 발발하는 것을 억제함으로써 질병 치료에 이용될 수 있다. 암과 같은 질병의 경우, 기존의 광범위한 세포괴사(necrosis) 약물요법은 병리 세포를 죽임과 동시에 병리 세포의 세포막이 파괴됨으로써 유출되는 세포 독성을 지닌 효소들(예를 들어, 리소자임)이 주변의 정상 세포에까지 세포 독성을 나타내어 과도한 염증 발현을 필연적으로 수반하게 됨으로 그 부작용이 심각하다. 반면, 세포사멸 조절요법은 병리 세포의 자발적인 사멸을 유도하거나 이러한 세포의 성장을 강하게 억제하기 때문에 암세포의 세포사멸에 의해 유도되는 염증성 부작용을 극복할 수 있다. 그 이유는 세포의 성장이 강력히 억제될 때는 세포 분열능이 정상세포보다 월등한 암세포가 상대적으로 더욱 큰 억제 효과를 보이며, 이러한 효과가 극대화되어 세포사멸이 유도되면 세포 독성을 지닌 세포내의 여러 물질들이 세포사멸 과정 동안 카스파제(caspase)라는 효소들에 의해 대부분 절단되어 그 기능을 상실함과 동시에 이러한 물질이 세포사멸체(apoptotic body)에 싸여 대식세포(macrophage)에 의해 식세포작용(phagocytosis)의 과정을 거치게 된다. 이로 인해, 세포 독성 물질들은 외부로 유출되지 않으며, 주변세포에 대한 독성을 나타내지 않게 된다.Apoptosis therapy can be used to treat disease by blocking pathological growth of unregulated cells, such as cancer cells, or inhibiting the development of degenerative diseases due to excessive apoptosis of normal cells, such as in Alzheimer's or Parkinson's disease. have. In diseases such as cancer, conventional extensive necrosis pharmacotherapy kills pathological cells and at the same time, cytotoxic enzymes (e.g., lysozyme) that leak out as the cell membranes of the pathological cells are destroyed are normal. It is cytotoxic to the cells, which inevitably accompanies excessive inflammation, so the side effects are serious. Apoptosis therapy, on the other hand, induces spontaneous killing of pathological cells or strongly inhibits the growth of these cells, thereby overcoming the inflammatory side effects induced by apoptosis of cancer cells. The reason for this is that when cell growth is strongly inhibited, cancer cells that have superior cell division ability than normal cells show a relatively greater inhibitory effect, and when these effects are maximized and apoptosis is induced, various substances in cells with cytotoxicity During the killing process, most of the enzymes, called caspases, are cleaved and lose their function. At the same time, these substances are wrapped in the apoptotic body, which causes macrophage to process phagocytosis. Going through. As a result, the cytotoxic substances do not leak to the outside and exhibit no toxicity to the surrounding cells.
예정된 세포사(programmed cell death; apoptosis)는 능동적인 세포사멸 과정으로 에너지를 필요로 하며, 특징적인 세포 형태를 나타낸다. 세포사멸 신호가 전달되면 세포내에서는 세포사멸을 결정하고, 이를 실행하는 단계로 진행되며 실행 단계에서는 세포가 특징적인 생화학적, 형태학적 변화를 나타낸다. 세포사멸이 진행되면 세포가 수축되면서 접하고 있던 주변세포와 떨어지게 되고 세포막이 기포 모양(blebbing)으로 변하게 되며, 더 나아가 핵이 응축(condensation)되고 핵 내의 DNA가 작은 올리고뉴클레오티드 절편으로 잘려지며 세포사멸체를 형성하게 된다. 이렇게 형성된 세포사멸체가 대식세포에 의해 식세포작용과 같은 일련의 과정을 거치면서 세포사멸이 일어나게 된다. 세포사멸은 복잡한 세포 내의 과정으로 세포사멸에 대한 세포 내에서의 결정은 쉽게 이루어지지 않지만, 일단 결정되면 세포사멸이 제대로 작동하기 위해 여러 하위 단계의 과정들이 진행된다. 대부분의 형태학적 변화는 세포사멸 과정에서 활성화 효소인 아스파라긴산 특이적 시스테인 프로테아제(aspartic acid specific cysteine protease)인 카스파제들에 의해 이루어진다.Programmed cell death (apoptosis) is an active cell death process that requires energy and exhibits characteristic cell morphology. When apoptosis signal is transmitted, apoptosis is determined and carried out in the cell, and in the execution step, the cell shows characteristic biochemical and morphological changes. As apoptosis progresses, the cells contract and fall off from adjacent cells, and the cell membrane becomes blebbing. Furthermore, the nucleus is condensed and the DNA in the nucleus is cut into small oligonucleotide fragments. Will form. The apoptosis thus formed undergoes a series of processes such as phagocytosis by macrophages, resulting in cell death. Apoptosis is a complex intracellular process, and intracellular determination of apoptosis is not easy, but once it is determined, several substeps are performed in order for apoptosis to function properly. Most morphological changes are caused by caspases, the aspartic acid specific cysteine protease, an activating enzyme during cell death.
한편, 알부민은 다기능 특성을 갖는 가장 풍부한 혈장 단백질로, 간세포에서 주로 합성되며, 간질액(interstitial fluid), 림프액 및 뇌척수액을 포함하는 대부분의 세포외액의 주요한 성분이다. 생체내에서 알부민이 감소되면 간기능 저하 및 영양상태가 불량하게 되므로, 임상학적으로 알부민은 중환자 및 간경변 환자의 혈관허탈(vascular collapse)을 포함하는 위독한 상태에서 광범위하게 사용되고 있다. 또한, 최근에는 알부민이 질병의 중요한 진단 또는 예후 지표인 저분자량 분자에 특이적으로 결합한다고 암시하고 있다. 예를 들어, 알부민은 분자 확산에 의해 혈뇌 장벽을 통해 뇌로 들어가고, 알츠하이머 유발 물질인 아밀로이드베타 1-42(Aβ1-42)에 특이적으로 결합하여 Aβ1-42를 수송하기 때문에 알츠하이머 질병을 암시할 수 있다고 보고되어 있다. 본 발명자들은 알부민이 단핵식세포계 세포의 한 종류인 인간 뇌의 미세아교세포(microglial cell)에서 합성되고, 이러한 알부민의 합성과 세포 밖으로의 분비가 Aβ1-42의 투여에 의해 증가된다는 연구결과를 2008년도 저널에 보고하였다[Ahn S-M, Byun K, Cho K, Kim JY, Yoo JS, et al. (2008) Human Microglial Cells Synthesize Albumin in Brain. PLoS ONE 3(7): e2829].Albumin, on the other hand, is the most abundant plasma protein with multifunctional properties, mainly synthesized in hepatocytes, and a major component of most extracellular fluids, including interstitial fluid, lymphatic fluid and cerebrospinal fluid. As albumin decreases in vivo, liver function decreases and nutrition becomes poor, so clinically, albumin has been widely used in a critical condition including vascular collapse in critically ill patients and cirrhosis patients. In addition, it has recently been suggested that albumin specifically binds to low molecular weight molecules, which are important diagnostic or prognostic indicators of disease. For example, albumin may imply Alzheimer's disease because it enters the brain through the blood-brain barrier by molecular diffusion and specifically binds and transports Aβ1-42 to amyloid beta 1-42 (Aβ1-42), an Alzheimer's trigger. It is reported. The inventors found that albumin is synthesized in microglial cells of the human brain, a type of mononuclear phagocyte, and the synthesis and secretion of such albumin is increased by administration of Aβ1-42. Yearly journal [Ahn SM, Byun K, Cho K, Kim JY, Yoo JS, et al. (2008) Human Microglial Cells Synthesize Albumin in Brain. PLoS ONE 3 (7): e2829].
또한, 최종당화산물(advanced glycation end-product; AGE)은 인체 내에서 끊임없이 발생하는 복합물질로 주로 탄수화물과 유리 아미노산의 반응에 의해 발생하며, 화학적으로 매우 불안정하고 반응성이 강한 물질이기 때문에 신경세포의 사멸을 촉진시키는 분자로 알려져 있다. 또한, 최종당화산물은 노인이나 노화된 동물의 뇌에서 증가되는 것으로 보고되어 있으며, 모든 세포와 생체 분자에 영향을 미쳐 노화 및 노화 관련 만성 질환의 원인이 된다. 즉, 최종당화산물은 혈관 투과성 증가, 산화 질소 방해에 의한 혈관 확장 억제, LDL 산화, 대식세포 또는 내피세포 등에서 여러 종류의 사이토카인 분비, 및 산화 스트레스를 증가시킴으로써, 노화, 알츠하이머병, 신장질환, 당뇨병, 당뇨병성 혈관 합병증, 당뇨병성 망막 이상 및 당뇨병성 신경 이상 등과 같은 성인병들과 관련이 있는 것으로 알려져 있다.In addition, advanced glycation end-product (AGE) is a complex material that occurs constantly in the human body, mainly caused by the reaction of carbohydrates and free amino acids, and is a chemically very unstable and highly reactive substance. It is known as a molecule that promotes death. In addition, the final glycation end products are reported to be increased in the brain of the elderly or aged animals, affecting all cells and biomolecules, causing aging and aging-related chronic diseases. In other words, the final glycosylated product increases vascular permeability, inhibits vasodilation due to nitric oxide blockage, LDL oxidation, various types of cytokine secretion in macrophages or endothelial cells, and oxidative stress, thereby aging, Alzheimer's disease, kidney disease, It is known to be associated with adult diseases such as diabetes mellitus, diabetic vascular complications, diabetic retinal abnormalities and diabetic neurological abnormalities.
상기한 바와 같이, AGE는 노인이나 노화된 동물의 뇌에서 증가된다고 알려져 있으므로, 신경세포의 사멸을 촉진하여 알츠하이머병 등의 퇴행성 질환에 영향을 미칠 수 있을 것이라고 많은 연구자들에 의해 제안되어 왔다. 그러나, 이러한 연구 결과에도 불구하고 AGE의 주요 합성 기전이나 주로 분비되는 곳에 대해서는 아직까지 규명되지 않고 있다.As mentioned above, since AGE is known to be increased in the brain of elderly and aged animals, many researchers have suggested that it may promote neuronal death and affect degenerative diseases such as Alzheimer's disease. However, despite these findings, the major synthetic mechanisms and major secretions of AGE have not been identified.
따라서, AGE의 주요 합성 기전과 기원에 대해 규명하면 세포사 유도를 저해할 수 있어 다양한 질병의 원인을 찾을 수 있을 것으로 생각된다. 따라서, 다양한 질병의 원인을 규명할 수 있는 AGE의 주요 합성 기전에 대한 연구의 필요성이 요구되고 있다.Therefore, it is thought that identifying the major synthetic mechanisms and origins of AGEs can inhibit the induction of cell death and find the causes of various diseases. Therefore, there is a need for a study of the major synthetic mechanism of AGE that can identify the causes of various diseases.
본 발명자들은 AGE의 주요 합성 기전에 대해 규명하여 세포사 유도를 저해하는 방법에 대해 연구하던 중, 알츠하이머 모델, 뇌졸중 모델, 파킨슨병 모델, 류마티스 관절염 모델의 인간 미세아교세포 또는 대식세포에서 AGE-알부민이 합성 및 분비되며, AGE-알부민의 합성 및 분비가 산화적 스트레스에 의한 것이고, Aβ1-42로 처리한 랫트의 뇌조직과 알츠하이머 환자의 뇌조직에서 AGE-알부민이 Aβ1-42의 응집을 유도하며, 인간 미세아교세포 또는 대식세포에서 합성 및 분비된 AGE-알부민이 일차 인간 신경세포 또는 연골세포 등에 작용하여 RAGE의 발현양을 증가시키고, 이것은 MAPK 신호전달체계를 활성화시킨 후 Bax의 발현양을 증가시켜 미토콘드리아에서 칼슘의 증가를 유도하고, 최종적으로 세포사를 유도하는 것을 확인하고, 본 발명을 완성하였다.While the present inventors have investigated the main synthetic mechanisms of AGE and studying the method of inhibiting cell death induction, AGE-albumin is expressed in human microglial cells or macrophages of Alzheimer's model, stroke model, Parkinson's disease model, and rheumatoid arthritis model. Synthesized and secreted, AGE-albumin synthesis and secretion is due to oxidative stress, AGE-albumin induces aggregation of Aβ1-42 in brain tissues of rats treated with Aβ1-42 and brain tissues of Alzheimer's patients, AGE-albumin synthesized and secreted from human microglia or macrophages acts on primary human neurons or chondrocytes to increase the expression of RAGE, which activates the MAPK signaling system and increases the expression of Bax. Induction of calcium in the mitochondria, and finally confirmed to induce cell death, the present invention was completed.
본 발명은 단핵식세포계 세포 내에서 ＡＧＥ-알부민의 합성 저해 또는 분비 저해에 의한 세포사 유도 저해 방법을 제공하고자 한다.The present invention provides a method for inhibiting cell death by inhibiting the synthesis or secretion of ABE-albumin in mononuclear phagocytes.
또한, 본 발명은 ＡＧＥ-알부민의 합성 저해 활성을 갖는 화합물을 포함하는 ＡＧＥ-알부민의 합성 저해제를 제공하고자 한다.It is another object of the present invention to provide an inhibitor for the synthesis of ABR-albumin containing a compound having the inhibitory activity for the synthesis of ABR-albumin.
또한, 본 발명은 ＡＧＥ-알부민의 합성 저해제를 유효성분으로 함유하는 퇴행성 질환 및 자가 면역 질환의 예방 또는 치료용 약학적 조성물을 제공하고자 한다.In addition, the present invention is to provide a pharmaceutical composition for the prevention or treatment of degenerative diseases and autoimmune diseases containing an inhibitor of the synthesis of AA-albumin as an active ingredient.
또한, 본 발명은 ＡＧＥ-알부민의 합성 저해제의 치료적 유효량을 개체에게 투여하여 퇴행성 질환 및 자가 면역 질환을 예방 또는 치료하는 방법을 제공하고자 한다.The present invention also provides a method for preventing or treating degenerative diseases and autoimmune diseases by administering to a subject a therapeutically effective amount of an ABR-albumin synthesis inhibitor.
본 발명의 AGE-알부민은 알츠하이머 모델, 뇌졸중 모델, 파킨슨병 모델, 류마티스 관절염 모델의 인간 미세아교세포 또는 대식세포에서 합성 및 분비되며, AGE-알부민의 합성 및 분비가 산화적 스트레스에 의한 것이고, AGE-알부민이 투여된 일차 인간 신경세포 또는 연골세포 등에서 RAGE의 발현양이 증가되고, 이것은 MAPK 신호전달체계를 활성화시킨 후 Bax의 발현양을 증가시켜 미토콘드리아에서 칼슘의 증가를 유도하고, 최종적으로 세포사를 유도한다. 따라서, 본 발명의 ＡＧＥ-알부민의 합성 저해제는, 알츠하이머병, 뇌졸중, 파킨슨병, 루게릭병, 류마티스 관절염, 당뇨성 망막변성, AIDS, 노화, 폐섬유증 또는 척수손상 등의 퇴행성 질환 및 자가 면역 질환의 진단 또는 치료에 유용하게 사용할 수 있다.The AGE-albumin of the present invention is synthesized and secreted in human microglia or macrophages of Alzheimer's model, stroke model, Parkinson's disease model, rheumatoid arthritis model, synthesis and secretion of AGE-albumin due to oxidative stress, AGE The expression of RAGE is increased in primary human neurons or chondrocytes treated with albumin, which activates the MAPK signaling system and increases the expression of Bax, leading to an increase in calcium in the mitochondria and finally apoptosis. Induce. Therefore, the inhibitor of the synthesis of ABE-albumin of the present invention may be used for degenerative diseases such as Alzheimer's disease, stroke, Parkinson's disease, Lou Gehrig's disease, rheumatoid arthritis, diabetic retinopathy, AIDS, aging, pulmonary fibrosis or spinal cord injury and autoimmune diseases. It can be usefully used for diagnosis or treatment.
도 1은 본 발명의 단핵식세포계 세포 내에서 AGE-알부민의 합성 저해 또는 분비 저해에 의한 단핵식세포계 세포 주변에 있는 세포의 세포사 유도 저해 방법을 간략히 나타낸 모식도이다.1 is a schematic diagram showing a method for inhibiting cell death induction of cells around mononuclear phagocytes by inhibiting the synthesis or secretion of AGE-albumin in mononuclear phagocytes of the present invention.
도 2는 Aβ1-42 처리 전 또는 후의 인간 미세아교세포 및 랫트의 뇌조직, 정상인 또는 알츠하이머 환자의 뇌조직에서 AGE-알부민의 분포 및 발현 위치를 항체를 이용하여 염색한 후 레이저 공초점 형광현미경으로 관찰한 도이다.Figure 2 shows the distribution and expression sites of AGE-albumin in human microglial cells and rat brain tissues prior to or after Aβ1-42 treatment, and brain tissues of normal or Alzheimer's patients with antibodies, followed by laser confocal fluorescence microscopy. Observed.
도 3은 Aβ1-42 처리 전 또는 후의 인간 미세아교세포 및 랫트의 뇌조직, 정상인 또는 알츠하이머 환자의 뇌조직에서 측정된 AGE-알부민의 밀도를 나타낸 도이다.Figure 3 shows the density of AGE-albumin measured in human microglial cells and rat brain tissues before or after Aβ1-42 treatment, brain tissues of normal or Alzheimer's patients.
도 4는 알츠하이머 환자의 뇌조직에서 AGE-알부민의 분포 및 발현 위치를 항체를 이용하여 염색한 후 레이저 공초점 형광현미경으로 관찰한 도이다[AGE(적색), 알부민(녹색), 미세아교세포 표지자인 Iba1(푸른색), 성상교세포 표지자인 MBP(푸른색), 희소돌기아교세포 표지자인 Olig2(푸른색), 신경세포 표지자인 NeuroD(푸른색)].Figure 4 is a diagram observed by the laser confocal fluorescence microscopy after staining the distribution and expression position of AGE-albumin in the brain tissue of Alzheimer's patients using antibodies [AGE (red), albumin (green), microglial markers Phosphorus Iba1 (blue), astrocyte marker MBP (blue), oligodendrocyte marker Olig2 (blue), neuronal marker NeuroD (blue)].
도 5는 인간 미세아교세포에서 AGE-알부민의 합성양을 공-면역침전법으로 확인한 도이다.5 is a diagram confirming the synthetic amount of AGE-albumin in human microglia by co-immunoprecipitation method.
도 6은 인간 미세아교세포 내와 배양 배지로 분비된 AGE-알부민의 발현양을 ELISA를 이용하여 확인한 도이다.6 is a diagram confirming the expression amount of AGE-albumin secreted into human microglial cells and culture medium using ELISA.
도 7은 인간 미세아교세포에서 AGE-알부민의 합성과 분비가 산화적 스트레스에 의한 것인지를 면역 블롯팅 분석을 통해 확인한 도이다.7 is a diagram confirming the synthesis and secretion of AGE-albumin in human microglia by oxidative stress through immunoblotting analysis.
도 8은 Aβ1-42로 처리한 랫트의 뇌조직과 알츠하이머 환자의 뇌조직의 미세아교세포에서 합성 및 분비된 AGE-알부민이 Aβ1-42의 응집을 유도하는지를 ThT 형광분석법을 이용하여 관찰한 도이다.FIG. 8 is a graph illustrating the synthesis and secretion of AGE-albumin synthesized and secreted from microglia of brain tissue of rats treated with Aβ1-42 and brain tissues of Alzheimer's patients using ThT fluorescence. .
도 9는 인간 미세아교세포에 AGE-알부민을 투여한 후 Aβ1-42의 양을 ELISA 분석법을 이용하여 측정한 결과를 나타낸 도이다.9 is a diagram showing the results of measuring the amount of Aβ1-42 after administration of AGE-albumin to human microglia using ELISA assay.
도 10은 인간 미세아교세포에 AGE-알부민을 투여한 후 BACE, ADAM10, APP의 발현 위치와 발현양을 각각 면역염색화학(A)과 면역 블롯팅(B)을 이용하여 관찰한 결과를 나타낸 도이다.10 is a diagram showing the results of AGE-albumin administration to human microglial cells after the expression position and the amount of expression of BACE, ADAM10, APP using immunostaining chemistry (A) and immunoblotting (B), respectively. to be.
도 11은 일차 인간 신경세포에 AGE-알부민을 투여한 후 RAGE, ERK1/2, pERK1/2, p38, pp38, SAPK/JNK, pSAPK/JNK, Bax의 발현 위치를 면역염색화학을 이용하여 관찰한 결과를 나타낸 도이다.11 shows the expression sites of RAGE, ERK1 / 2, pERK1 / 2, p38, pp38, SAPK / JNK, pSAPK / JNK, Bax after administration of AGE-albumin to primary human neurons using immunostaining chemistry. The results are shown.
도 12는 일차 인간 신경세포에 AGE-알부민을 투여한 후 RAGE, ERK1/2, pERK1/2, p38, pp38, SAPK/JNK, pSAPK/JNK, Bax의 발현양을 면역 블롯팅 방법을 이용하여 관찰한 결과를 나타낸 도이다.12 shows the expression levels of RAGE, ERK1 / 2, pERK1 / 2, p38, pp38, SAPK / JNK, pSAPK / JNK, Bax after administration of AGE-albumin to primary human neurons using an immunoblotting method. Figure 1 shows the results.
도 13은 AGE-알부민을 일차 인간 신경세포에 처리하기 전 또는 후의 세포 용해물을 이용하여 AGE와 RAGE의 상호작용을 확인한 도이다.Figure 13 is a diagram confirming the interaction between AGE and RAGE using cell lysate before or after the treatment of AGE-albumin to primary human neurons.
도 14는 일차 인간 신경세포에 AGE-알부민을 투여한 후 미토콘드리아에서 칼슘 변화를 레이저 공초점 형광현미경으로 관찰한 도이다.14 is a diagram illustrating the change of calcium in mitochondria after administration of AGE-albumin to primary human neurons by laser confocal fluorescence microscopy.
도 15는 일차 인간 신경세포에 AGE-알부민을 투여한 후 MTT assay를 이용하여 세포생존율을 측정한 도이다.FIG. 15 is a diagram illustrating cell viability using MTT assay after administration of AGE-albumin to primary human neurons.
도 16은 Aβ1-42 처리 전 또는 후의 마우스의 혈액단핵세포에서 AGE-알부민의 분포 및 발현 위치를 항체를 이용하여 염색한 후 레이저 공초점 형광현미경으로 관찰한 도이다.Fig. 16 is a diagram illustrating the distribution and expression position of AGE-albumin in blood mononuclear cells of mice before or after Aβ1-42 treatment using an antibody, followed by laser confocal fluorescence microscopy.
도 17은 랫트의 뇌에 Aβ1-42 단독으로 또는 Aβ1-42와 sRAGE(Aβ/sRAGE)를 함께 처리하고 72시간 후 랫트의 뇌에 있는 신경세포의 수를 크레실 바이올렛 (cresyl violet)으로 염색한 후 현미경으로 관찰한 도이다.Figure 17 is treated with Aβ1-42 alone or Aβ1-42 and sRAGE (Aβ / sRAGE) in the rat brain and after 72 hours the number of neurons in the rat brain stained with cresyl violet It is a figure observed with the microscope after.
도 18은 Aβ1-42 단독으로 또는 Aβ1-42와 sRAGE(Aβ/sRAGE)를 함께 처리한 랫트의 뇌조직에서 AGE(적색), 알부민(녹색), 미세아교세포(microglia cell) 표지자인 Iba1(푸른색), 및 AGE-알부민의 분포 및 발현 위치를 항체를 이용하여 염색한 후 레이저 공초점 형광현미경으로 관찰한 결과(A)와 AGE-알부민의 밀도(B)를 나타낸 도이다.Figure 18 shows Iba1 (blue), albumin (green), and microglia cell markers Iba1 (blue) in brain tissues of rats treated with Aβ1-42 alone or with Aβ1-42 and sRAGE (Aβ / sRAGE). Color), and the distribution and expression positions of AGE-albumin after staining with an antibody and observed with a laser confocal fluorescence microscope (A) and the density (B) of AGE-albumin.
도 19는 Aβ1-42 단독으로 또는 Aβ1-42와 sRAGE(Aβ/sRAGE)를 함께 처리한 랫트의 뇌조직에서 RAGE, NeuN, DAPI, Bax 및 p-SAPK/JNK의 분포 및 발현 위치를 항체를 이용하여 염색한 후 레이저 공초점 형광현미경으로 관찰한 도이다.19 shows the distribution and expression locations of RAGE, NeuN, DAPI, Bax and p-SAPK / JNK in the brain tissues of rats treated with Aβ1-42 alone or Aβ1-42 and sRAGE (Aβ / sRAGE) together. After staining with a laser confocal fluorescence microscope.
도 20은 뇌졸중 환자의 뇌조직에서 AGE-알부민의 분포 및 발현 위치를 항체를 이용하여 염색한 후 레이저 공초점 형광현미경으로 관찰한 도이다.FIG. 20 is a diagram illustrating the distribution and expression position of AGE-albumin in brain tissue of a stroke patient after staining with an antibody, and then observed with a laser confocal fluorescence microscope. FIG.
도 21은 저산소증과 포도당-결핍된 뇌졸중 모델의 인간 미세아교세포에서 저산소증 유발인자(HIF-1α)의 발현을 면역조직화학법(IHC)(A), PCR (B), 면역블롯팅(C)을 수행하여 확인한 도이다.Figure 21 shows the expression of hypoxia-induced factor (HIF-1α) in human microglia cells of hypoxia and glucose-deficient stroke models by immunohistochemistry (IHC) (A), PCR (B), immunoblotting (C). This is confirmed by the following.
도 22는 저산소증과 포도당-결핍된 뇌졸중 모델의 인간 미세아교세포에서 HMGB1(high motility group protein B1)의 발현을 면역조직화학법(IHC)(A), PCR(B), 면역블롯팅(C)을 수행하여 확인한 도이다.Figure 22 shows immunohistochemistry (IHC) (A), PCR (B), immunoblotting (C) for the expression of high motility group protein B1 (HMGB1) in human microglial cells of hypoxia and glucose-deficient stroke models. This is confirmed by the following.
도 23은 저산소증과 포도당-결핍된 뇌졸중 모델의 인간 미세아교세포에서 AGE-알부민의 발현양을 면역조직화학법(IHC)(A), 면역블롯팅(B,C) 및 ELISA(D)를 수행하여 확인한 도이다.Figure 23 shows the immunohistochemistry (IHC) (A), immunoblotting (B, C) and ELISA (D) expression levels of AGE-albumin in human microglia cells of hypoxia and glucose-deficient stroke models. This is confirmed.
도 24는 저산소증과 포도당-결핍된 뇌졸중 모델의 인간 미세아교세포에서 HMGB1의 농도(0, 50, 200, 500, 2000ng/㎖) 변화에 따른 AGE-알부민의 발현양을 면역조직화학법(IHC)(A), ELISA(B) 및 면역블롯팅(C)을 수행하여 확인한 도이다.FIG. 24 shows the expression levels of AGE-albumin according to HMGB1 concentration (0, 50, 200, 500, 2000 ng / ml) in human microglial cells of hypoxia and glucose-deficient stroke models. (A), ELISA (B) and immunoblotting (C) was performed to confirm the figure.
도 25는 저산소증과 포도당-결핍된 뇌졸중 모델의 인간 미세아교세포에 HMGB1 저해제인 글리시리진(glycyrrhizic acid)의 농도(0, 50, 200, 500, 2000ng/㎖) 변화에 따른 AGE-알부민의 발현양을 ELISA 및 면역블롯팅을 수행하여 확인한 도이다.25 shows the expression levels of AGE-albumin according to the concentration (0, 50, 200, 500, 2000 ng / ml) of glycyrrhizic acid, an HMGB1 inhibitor, in human microglial cells of hypoxia and glucose-deficient stroke models. This is confirmed by performing ELISA and immunoblotting.
도 26은 저산소증과 포도당-결핍된 뇌졸중 모델의 인간 미세아교세포에서 AGE-알부민의 합성과 분비가 산화적 스트레스에 의한 것인지 면역블롯팅 분석을 수행하여 확인한 도이다.Figure 26 is confirmed by performing an immunoblotting analysis whether the synthesis and secretion of AGE-albumin due to oxidative stress in human microglia cells of hypoxia and glucose-deficient stroke model.
도 27은 인간의 뇌조직으로부터 얻은 일차 인간 신경세포에 AGE-알부민을 투여한 후 RAGE, ERK1/2, p-ERK1/2, p38, p-p38, SAPK/JNK, p-SAPK/JNK, Bax의 발현양을 면역 블롯팅 방법을 이용하여 관찰한 결과를 나타낸 도이다.FIG. 27 shows RAGE, ERK1 / 2, p-ERK1 / 2, p38, p-p38, SAPK / JNK, p-SAPK / JNK, Bax after administration of AGE-albumin to primary human neurons obtained from human brain tissue Is a diagram showing the results of observing the expression amount of using an immunoblotting method.
도 28은 일차 인간 신경세포에 AGE-알부민을 투여한 후 MTT assay를 이용하여 세포생존율을 측정한 결과(A)와 신경세포사에 대한 sRAGE의 보호 효과(B)를 나타낸 도이다.28 is a diagram showing the results of measuring cell viability using MTT assay after administration of AGE-albumin to primary human neurons (A) and the protective effect of sRAGE on neuronal cell death (B).
도 29는 파킨슨병 환자의 뇌조직에서 AGE-알부민의 분포 및 발현 위치를 항체를 이용하여 염색한 후 레이저 공초점 형광현미경으로 관찰한 도이다.FIG. 29 is a diagram illustrating the distribution and expression positions of AGE-albumin in brain tissues of Parkinson's disease patients using an antibody, followed by laser confocal fluorescence microscopy.
도 30은 인간 미세아교세포에 파킨슨병 유발물질인 로테논(rotenone) 또는 6-히드록시도파민(6-hydroxydopamine; 6-OHDA)을 처리한 후 α-시누클레인 또는 TNF-α의 발현 정도를 PCR(A,B)과 면역블롯팅 분석(C,D)을 통해 확인한 도이다.FIG. 30 shows PCR expression of α-synuclein or TNF-α after treatment with rotenone or 6-hydroxydopamine (6-OHDA), a Parkinson's disease-causing agent, in human microglial cells. (A, B) and immunoblotting analysis (C, D) is a figure confirmed.
도 31은 인간 미세아교세포에 0~100nM의 로테논을 처리한 후 세포 용해물 및 세포 배양물을 이용하여 AGE-알부민의 합성 및 분비 여부를, ELISA 및 면역블롯팅 분석을 통해 확인한 도이다.FIG. 31 is a diagram illustrating the synthesis and secretion of AGE-albumin using cell lysates and cell cultures after treatment of human microglial cells with 0 to 100 nM of rotenone, through ELISA and immunoblotting analysis.
도 32는 인간 미세아교세포에 파킨슨병 유발물질인 로테논을 처리한 후 산화적 스트레스를 가하여 α-시누클레인 또는 AGE-알부민의 발현양을 면역블롯팅 분석을 통해 확인한 도이다.32 is a diagram showing the expression level of α-synuclein or AGE-albumin by immunoblotting analysis of human microglial cells after treatment with Parkinson's disease-causing rotenone followed by oxidative stress.
도 33은 도파민신경세포에 sRAGE를 처리하거나 처리하지 않은 다음, AGE-알부민을 도파민신경세포에 처리하기 전 또는 후의 세포 용해물을 이용하여 RAGE, Bax, SAPK/JNK, pSAPK/JNK, p38, ERK1/2, pERK1/2의 발현양을 면역 블롯팅 분석을 통해 확인한 도이다.FIG. 33 shows RAGE, Bax, SAPK / JNK, pSAPK / JNK, p38, and ERK1 using cell lysate after treatment with or without sRAGE to dopamine neurons, and before or after AGE-albumin treatment to dopamine neurons. / 2, pERK1 / 2 expression level was confirmed by immunoblotting analysis.
도 34는 마우스의 뇌조직에 PBS, 로테논, 로테논/sRAGE을 처리하고 1주일과 한달 후에 마우스의 뇌조직에 있는 신경세포의 수를 크레실 바이올렛(cresyl violet)으로 염색한 후 현미경으로 관찰한 도이다.Figure 34 is treated with PBS, rotenone, rotenone / sRAGE in the brain tissue of the mouse and observed in the microscope after staining the number of neurons in the brain tissue of the mouse with cresyl violet after one week and a month It is a degree.
도 35는 마우스의 뇌조직에 PBS, 로테논, 로테논/sRAGE을 처리하고 1주일과 한달 후에 마우스의 뇌조직에 있는 AGE-알부민, RAGE, Bax의 분포 및 발현 위치를 형광면역염색한 후 레이저 공초점 형광현미경으로 관찰한 도이다.FIG. 35 shows the distribution and expression of AGE-albumin, RAGE, and Bax in the brain tissues of mice after treatment with PBS, rotenone, and rotenone / sRAGE in the brain tissues of the mouse, and after laser fluorescence immunostaining. Fig. Observed with a confocal fluorescence microscope.
도 36은 인간 대식세포주(U937)에 β2-마이크로글로불린을 처리한 후 세포 용해물 또는 세포 배양물을 이용하여 TNF-α와 IL-1β의 발현 정도와 AGE-알부민의 합성 및 분비 여부를, 면역블롯팅 및 ELISA를 통해 확인한 도이다.36 shows the expression level of TNF-α and IL-1β and the synthesis and secretion of AGE-albumin after treatment with β2-microglobulin in human macrophage line (U937) using cell lysates or cell cultures. Figures confirmed by blotting and ELISA.
도 37은 연골세포에 AGE-알부민 단독으로 또는 AGE-알부민/sRAGE를 함께 투여한 후 MTT assay를 이용하여 세포생존율을 측정한 도이다.FIG. 37 is a diagram illustrating cell viability using MTT assay after administration of AGE-albumin alone or AGE-albumin / sRAGE to chondrocytes.
도 38은 알츠하이머병 모델의 인간 미세아교세포에서 AGE-알부민의 합성을 저해하는 후보물질을 LOPAC 1280종의 화합물로부터 선별하고, 후보물질의 ELISA 측정결과를 나타낸 도이다.FIG. 38 is a diagram showing the results of ELISA measurement of candidate substances selected from LOPAC 1280 compounds as candidates that inhibit the synthesis of AGE-albumin in human microglia of the Alzheimer's disease model.
도 39는 파킨슨병 모델의 인간 미세아교세포에서 AGE-알부민의 합성을 저해하는 후보물질을 LOPAC 1280종의 화합물로부터 선별하고, 후보물질의 ELISA 측정결과를 나타낸 도이다.FIG. 39 is a diagram showing the results of ELISA measurement of candidates by selecting candidate substances that inhibit the synthesis of AGE-albumin in human microglial cells of the Parkinson's disease model from LOPAC 1280 compounds.
도 40은 마우스의 뇌조직에 PBS, 로테논, 로테논/세파클러, 로테논/세팔로틴 나트륨을 처리하고 1주일과 한달 후에 마우스의 뇌조직에 있는 신경세포의 수를 크레실 바이올렛(cresyl violet)으로 염색한 후 현미경으로 관찰한 도이다.40 shows the number of neurons in the brain tissue of mice after one week and one month after treatment with PBS, rotenone, rotenone / cephacller, and rotenone / cephalotin sodium in the brain tissue of the mouse. after staining with violet).
도 41은 마우스의 뇌조직에 PBS, 로테논, 로테논/세파클러, 로테논/세팔로틴 나트륨을 처리하고 1주일과 한달 후에 마우스의 뇌조직에 있는 AGE-알부민, RAGE, Bax의 분포 및 발현 위치를 형광면역염색한 후 레이저 공초점 형광현미경으로 관찰한 도이다.FIG. 41 shows the distribution of AGE-albumin, RAGE, and Bax in the brain tissues of mice one week and one month after treatment with PBS, rotenone, rotenone / sepacller, and rotenone / cephalotin sodium in the brain tissues of mice. After fluorescence immunostaining the expression site is a diagram observed by laser confocal fluorescence microscope.
본 발명은 단핵식세포계 세포 내에서 ＡＧＥ(advanced glycation end-product; 최종당화산물)-알부민의 합성 저해 또는 분비 저해에 의한 세포사 유도 저해 방법을 제공한다.The present invention provides a method for inhibiting cell death induction by inhibiting the synthesis or secretion of advanced glycation end-product (AbE) -albumin in mononuclear phagocytes.
또한, 본 발명은 ＡＧＥ-알부민의 합성 저해 활성을 갖는 화합물을 포함하는 ＡＧＥ-알부민의 합성 저해제를 제공한다.The present invention also provides an inhibitor for the synthesis of ABR-albumin containing a compound having the inhibitory activity for the synthesis of ABR-albumin.
또한, 본 발명은 ＡＧＥ-알부민의 합성 저해제를 유효성분으로 함유하는 퇴행성 질환 및 자가 면역 질환의 예방 또는 치료용 약학적 조성물을 제공한다.The present invention also provides a pharmaceutical composition for the prevention or treatment of degenerative diseases and autoimmune diseases containing an inhibitor of the synthesis of ABE-albumin as an active ingredient.
또한, 본 발명은 ＡＧＥ-알부민의 합성 저해제의 치료적 유효량을 개체에게 투여하여 퇴행성 질환 및 자가 면역 질환을 예방 또는 치료하는 방법을 제공한다.The present invention also provides a method of preventing or treating a degenerative disease and an autoimmune disease by administering to a subject a therapeutically effective amount of an ABR-albumin synthesis inhibitor.
이하, 본 발명에 대해 상세히 설명한다.Hereinafter, the present invention will be described in detail.
본 발명에 따른 세포사 유도 저해 방법은, 단핵식세포계 세포 내에서 AGE-알부민의 합성 또는 분비를 저해하여 단핵식세포계 세포 주변에 있는 세포의 세포사 (cell death) 유도를 저해하는 것을 특징으로 한다.The method for inhibiting cell death induction according to the present invention is characterized by inhibiting the synthesis or secretion of AGE-albumin in mononuclear phagocytes, thereby inhibiting cell death induction of cells around the mononuclear phagocyte cells.
상기 세포사는 크게 괴사(necrosis)와 아폽토시스(apoptosis)로 나뉜다. 괴사는 화상, 타박, 독극물 등의 자극에 의해 일어나는 세포의 죽음으로, 일명 세포의 사고사라고 할 수 있다. 괴사의 경우에는 세포 밖에서 수분이 유입됨으로써 세포가 팽창하여 파괴된다. 종래에는, 세포의 죽음을 모두 괴사라고 생각하였다. 그러나, 최근 30여년 사이에 세포에는 자발적인 죽음을 일으키는 유발인자가 있다는 사실이 알려졌다. 유전자에 제어되는 이와 같은 능동적인 세포의 죽음이 아폽토시스이다. 괴사가 오랜 시간에 걸쳐 무질서하게 일어나는데 반해, 아폽토시스는 단시간에 질서있게 일어난다. 아폽토시스는 세포가 축소되면서 시작된다. 이후 인접하는 세포 사이에 틈새가 생기고, 세포 내에서는 DNA가 규칙적으로 절단되어 단편화 된다. 마지막에 세포 전체도 단편화되어 아폽토시스 소체로 된 후 가까이에 있는 세포에게 먹힘으로써 죽음에 이르게 된다. 아폽토시스는 발생 과정에서 몸의 형태 만들기를 담당하고, 성체에서는 정상적인 세포를 갱신하거나 이상이 생긴 세포를 제거하는 일을 담당하고 있다. 동물의 몸 안에서 일어나는 발생, 분화의 과정에서 유전적인 프로그램에 의해 일어나는 세포사를 예정된 세포사(PCD; programed cell death)라고 한다. 예정된 세포사는 발생의 어느 단계에서 치사 유전자가 움직이기 시작하여 그 세포가 죽은 경우 등이다. 사람의 경우에는 태아의 초기에 손이나 발은 주걱 모양을 하고 있어 발가락이나 손가락 사이가 벌어지지 않고 있다가 후기에 그 사이에 해당하는 부분에 있던 세포가 에정된 세포사 함으로써 손가락이나 발가락의 형태가 생긴다. 퇴행성 질환은 상기 두가지 형태의 세포를 동반한다고 알려져 있다.The cell death is largely divided into necrosis and apoptosis. Necrosis is the death of cells caused by stimuli such as burns, bruises, poisons and the like, which is known as accidental death of cells. In the case of necrosis, water is introduced from outside the cell, causing the cell to expand and destroy. Conventionally, all cell deaths were considered necrosis. However, over the past 30 years, cells have been known to have triggers for spontaneous death. This active cell death, controlled by genes, is apoptosis. Necrosis occurs disorderly over long periods of time, whereas apoptosis occurs in a short time and orderly. Apoptosis begins as cells shrink. Thereafter, gaps occur between adjacent cells, and within the cells, DNA is regularly cut and fragmented. Finally, the whole cell is also fragmented into apoptotic bodies and then eaten by nearby cells, leading to death. Apoptosis is responsible for shaping the body during development, and in adults it is responsible for renewing normal cells or removing abnormal cells. Cell death caused by genetic programs in the process of development and differentiation in an animal's body is called programmed cell death (PCD). Predicted cell death is when a lethal gene begins to move at some stage of development and the cell dies. In the case of humans, the hands or feet are shaped like a spatula in the early stages of the fetus, and there is no gap between the toes or fingers. . Degenerative diseases are known to accompany these two types of cells.
상기 단핵식세포계 세포는 아밀로이드베타 1-42(Aβ1-42), HMGB1, 로테논 (rotenone) 또는 6-히드록시도파민(6-hydroxydopamine; 6-OHDA), β2-마이크로글로불린(β2-microglobulin) 등에 의해 활성화되어 있는 것이 바람직하나, 이에 한정되지 않는다.The mononuclear phagocytes are amyloid beta 1-42 (Aβ1-42), HMGB1, rotenone or 6-hydroxydopamine (6-OHDA), β2-microglobulin (β2-microglobulin), and the like. It is preferably activated by, but is not limited thereto.
상기 세포사되는 세포는 단핵식세포계 세포 주변에 있는 세포가 바람직하며, 상기 단핵식세포계 세포 주변에 있는 세포는 신경세포, 연골세포, 폐세포 및 간세포 등을 포함하나, 이에 한정되지 않는다.The cell death cells are preferably cells surrounding mononuclear phagocytes, and the cells surrounding the mononuclear phagocytes include, but are not limited to, neurons, chondrocytes, lung cells and hepatocytes.
상기 AGE-알부민의 합성 저해 또는 분비 저해는 알부민 siRNA, 알부민 항체, AGE 항체, AGE-알부민 항체 및 AGE-알부민 합성 저해제로 이루어진 군으로부터 선택된 1종을 이용하여 저해될 수 있다.The inhibition of the synthesis or secretion of AGE-albumin can be inhibited using one selected from the group consisting of albumin siRNA, albumin antibody, AGE antibody, AGE-albumin antibody and AGE-albumin synthesis inhibitor.
상기 단핵식세포계 세포의 종류로는 뇌의 미세아교세포, 혈액단핵세포, 폐포대식세포(type Ⅱ pneumocyte, dust cell), 복강대식세포, 염증부위 육아종대식세포, 비장대식세포, 간의 쿠퍼세포, 관절활액막 A 세포(synovial A cell), 혈관외막세포, 림프절내 대식세포, 피부의 랑거한스 세포(Langehans cell) 등을 포함하나, 이에 한정되지 않는다.Types of mononuclear phagocytes include microglia, blood mononuclear cells, alveolar macrophages (type II pneumocytes, dust cells), peritoneal macrophages, granulomatous macrophages, spleen macrophages, hepatic cooper cells, and joints. Synovial A cells, vascular epicardium cells, lymph node macrophages, skin Langerhans cells, and the like, but are not limited thereto.
본 발명의 단핵식세포계 세포 내에서 AGE-알부민의 합성 저해 또는 분비 저해에 의한 단핵식세포계 세포 주변에 있는 세포의 세포사 유도 저해 방법은 도 1에 간략히 나타내었다.The method of inhibiting cell death induction of cells around mononuclear phagocytes by inhibiting the synthesis or secretion of AGE-albumin in the mononuclear phagocytes of the present invention is briefly shown in FIG. 1.
알츠하이머 모델, 뇌졸중 모델, 파킨슨병 모델, 류마티스 관절염 모델의 인간 미세아교세포, 대식세포, 뇌조직 또는 연골에서 알부민과 AGE가 같은 위치에서 염색되고, 넓게 분포되어 있으며, AGE-알부민의 발현양 및 밀도가 정상인의 뇌조직 또는 연골에서보다 현저히 증가하고, 미세아교세포 표지자인 Iba1도 AGE-알부민과 같은 위치에서 발현되므로, 대부분의 AGE-알부민이 인간 미세아교세포 또는 대식세포에서 합성된다는 것을 예상할 수 있다.Albumin and AGE are stained and widely distributed in human microglial cells, macrophages, brain tissue or cartilage of Alzheimer's model, stroke model, Parkinson's disease model and rheumatoid arthritis model, and are widely distributed and expressed and density of AGE-albumin Is significantly higher than in normal brain tissue or cartilage, and the microglia marker Iba1 is also expressed at the same position as AGE-albumin, so it can be expected that most AGE-albumins are synthesized in human microglia or macrophages. have.
또한, 알츠하이머 모델, 뇌졸중 모델, 파킨슨병 모델, 류마티스 관절염 모델의 인간 미세아교세포 또는 대식세포에 아밀로이드베타 1-42(Aβ1-42), HMGB1, 로테논 또는 6-히드록시도파민, β2-마이크로글로불린을 처리한 경우 AGE-알부민의 발현양이 증가한다. 또한, 알츠하이머 모델, 뇌졸중 모델, 파킨슨병 모델, 류마티스 관절염 모델의 인간 미세아교세포에 과산화수소(H2O2)를 처리한 경우 과산화수소의 농도가 증가할수록 AGE-알부민의 발현양이 증가하고, 인간 미세아교세포에 항산화제인 아스코르빈산을 처리한 경우 AGE-알부민의 발현양이 현저히 감소한다. 또한, 인간 미세아교세포에 산화적 스트레스를 가하면 Aβ1-42가 축적되고, 이로 인해 AGE-알부민의 합성이 증가됨으로, 인간 미세아교세포에서 AGE-알부민의 합성과 분비가 산화적 스트레스에 의한 것임을 알 수 있다.In addition, human microglia or macrophages of Alzheimer's model, stroke model, Parkinson's disease model, and rheumatoid arthritis model have amyloid beta 1-42 (Aβ1-42), HMGB1, rotenone or 6-hydroxydopamine, β2-microglobulin Treatment with AGE-albumin increases the amount of expression. In addition, when hydrogen peroxide (H 2 O 2 ) was treated to human microglial cells of Alzheimer's model, stroke model, Parkinson's disease model, and rheumatoid arthritis model, the expression amount of AGE-albumin increased as human hydrogen peroxide increased. Treatment of glial cells with the antioxidant ascorbic acid significantly reduced the expression of AGE-albumin. In addition, oxidative stress on human microglia accumulates Aβ1-42, which increases the synthesis of AGE-albumin, suggesting that the synthesis and secretion of AGE-albumin in human microglia is due to oxidative stress. Can be.
또한, AGE-알부민은 일차 인간 신경세포 또는 연골세포에 작용하여 RAGE의 발현양을 증가시키고, 이것은 MAPK 신호전달체계를 활성화시킨 후 Bax의 발현양을 증가시켜 미토콘드리아에서 칼슘의 증가를 유도하고, 최종적으로 세포사를 유도한다. 그러나, AGE-알부민과 sRAGE를 함께 투여한 경우 세포사가 감소한다. 따라서, sRAGE가 세포사에 대해 보호 효과를 갖는다는 것을 알 수 있다.In addition, AGE-albumin acts on primary human neurons or chondrocytes to increase the expression of RAGE, which activates the MAPK signaling system and increases the expression of Bax to induce an increase in calcium in the mitochondria, ultimately. Induce cell death. However, when a combination of AGE-albumin and sRAGE is administered, cell death is reduced. Thus, it can be seen that sRAGE has a protective effect on cell death.
상기한 바와 같이, AGE-알부민은 알츠하이머 모델, 뇌졸중 모델, 파킨슨병 모델, 류마티스 관절염 모델의 인간 미세아교세포 또는 대식세포에서 합성 및 분비되며, AGE-알부민의 합성 및 분비가 산화적 스트레스에 의한 것이고, AGE-알부민이 투여된 일차 인간 신경세포 또는 연골세포 등에서 RAGE의 발현양이 증가되고, 이것은 MAPK 신호전달체계를 활성화시킨 후 Bax의 발현양을 증가시켜 미토콘드리아에서 칼슘의 증가를 유도하고, 최종적으로 세포사를 유도한다.As mentioned above, AGE-albumin is synthesized and secreted in human microglia or macrophages of Alzheimer's model, stroke model, Parkinson's disease model, rheumatoid arthritis model, and synthesis and secretion of AGE-albumin is due to oxidative stress In primary human neurons or chondrocytes treated with AGE-albumin, the expression of RAGE is increased, which activates the MAPK signaling system and increases the expression of Bax, leading to an increase in calcium in the mitochondria. Induce cell death
따라서, 본 발명에서는 알츠하이머 모델, 뇌졸중 모델, 파킨슨병 모델, 류마티스 관절염 모델의 인간 미세아교세포 또는 대식세포에서 AGE-알부민의 합성 저해 활성을 갖는 물질을 LOPAC(sigma) 1280종의 화합물로부터 총 42종의 화합물을 선별하였으며, 이들 화합물의 AGE-알부민의 합성 저해 활성이 대조군과 유사하게 나타남을 확인하였다. 따라서, 상기 선별된 AGE-알부민의 합성 저해제는, 알츠하이머병, 뇌졸중, 파킨슨병, 루게릭병, 류마티스 관절염, 당뇨성 망막변성, AIDS, 노화, 폐섬유증 또는 척수손상 등의 퇴행성 질환 및 자가 면역 질환의 진단 또는 치료에 유용하게 사용할 수 있다.Therefore, in the present invention, 42 kinds of substances having inhibitory activity of AGE-albumin synthesis in human microglial cells or macrophages of Alzheimer's model, stroke model, Parkinson's disease model, and rheumatoid arthritis model were selected from 42 compounds of LOPAC (sigma) Was selected, and the inhibitory activity of the synthesis of AGE-albumin of these compounds appeared to be similar to the control. Accordingly, the selected inhibitors of the synthesis of AGE-albumin may be used for degenerative diseases such as Alzheimer's disease, stroke, Parkinson's disease, Lou Gehrig's disease, rheumatoid arthritis, diabetic retinopathy, AIDS, aging, pulmonary fibrosis or spinal cord injury, and autoimmune diseases. It can be usefully used for diagnosis or treatment.
본 발명의 조성물은 AGE-알부민의 합성 저해제와 함께 퇴행성 질환 및 자가 면역 질환의 예방 또는 치료 효과를 갖는 공지의 유효성분을 1종 이상 함유할 수 있다.The composition of the present invention may contain one or more known active ingredients having a prophylactic or therapeutic effect of degenerative diseases and autoimmune diseases together with inhibitors of the synthesis of AGE-albumin.
본 발명의 조성물은, 투여를 위해서 상기 기재한 유효성분 이외에 추가로 약학적으로 허용가능한 담체를 1종 이상 포함하여 제조할 수 있다. 약학적으로 허용 가능한 담체는 식염수, 멸균수, 링거액, 완충 식염수, 덱스트로오스 용액, 말토 덱스트린 용액, 글리세롤, 에탄올 및 이들 성분 중 1 성분 이상을 혼합하여 사용할 수 있으며, 필요에 따라 항산화제, 완충액, 정균제 등 다른 통상의 첨가제를 첨가할 수 있다. 또한 희석제, 분산제, 계면활성제, 결합제 및 윤활제를 부가적으로 첨가하여 수용액, 현탁액, 유탁액 등과 같은 주사용 제형, 환약, 캡슐, 과립 또는 정제로 제제화할 수 있다. 더 나아가 당분야의 적정한 방법으로 또는 Remington's Pharmaceutical Science(최근판), Mack Publishing Company, Easton PA에 개시되어 있는 방법을 이용하여 각 질환에 따라 또는 성분에 따라 바람직하게 제제화할 수 있다.The composition of the present invention may be prepared by including one or more pharmaceutically acceptable carriers in addition to the above-described active ingredients for administration. Pharmaceutically acceptable carriers may be used in combination with saline, sterile water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol and one or more of these components, if necessary, as an antioxidant, buffer And other conventional additives such as bacteriostatic agents can be added. Diluents, dispersants, surfactants, binders and lubricants may also be added in addition to formulate into injectable formulations, pills, capsules, granules or tablets such as aqueous solutions, suspensions, emulsions and the like. Furthermore, it may be preferably formulated according to each disease or component by a suitable method in the art or using a method disclosed in Remington's Pharmaceutical Science (Recent Edition), Mack Publishing Company, Easton PA.
본 발명의 조성물은 목적하는 방법에 따라 경구 투여하거나 비경구 투여(예를 들어, 정맥 내, 피하, 복강 내 또는 국소에 적용)할 수 있으며, 투여량은 환자의 체중, 연령, 성별, 건강상태, 식이, 투여시간, 투여방법, 배설율 및 질환의 중증도 등에 따라 그 범위가 다양하다. 상기 AGE-알부민의 합성 저해제의 일일 투여량은 약 0.1~10㎎/㎏, 바람직하게는 약 0.5~2㎎/㎏이며, 하루 일회 내지 수회에 나누어 투여하는 것이 더욱 바람직하다.The composition of the present invention can be administered orally or parenterally (eg, applied intravenously, subcutaneously, intraperitoneally or topically) according to the desired method, and the dosage is based on the weight, age, sex and health of the patient. The range varies depending on the diet, the time of administration, the method of administration, the rate of excretion and the severity of the disease. The daily dosage of the AGE-albumin synthesis inhibitor is about 0.1 to 10 mg / kg, preferably about 0.5 to 2 mg / kg, more preferably administered once to several times a day.
본 발명의 조성물은 퇴행성 질환 및 자가 면역 질환의 예방 또는 치료를 위하여 단독으로, 또는 수술, 호르몬 치료, 약물 치료 및 생물학적 반응 조절제를 사용하는 방법들과 병용하여 사용할 수 있다.The composition of the present invention can be used alone or in combination with methods using surgery, hormonal therapy, drug therapy and biological response modifiers for the prevention or treatment of degenerative diseases and autoimmune diseases.
본 발명은 AGE-알부민의 합성 저해제를 치료적 유효량으로 개체에게 투여하여 퇴행성 질환 및 자가 면역 질환을 예방 또는 치료하는 방법을 제공할 수 있다.The present invention can provide a method for preventing or treating degenerative diseases and autoimmune diseases by administering to a subject a therapeutically effective amount of an AGE-albumin synthesis inhibitor.
이하, 본 발명의 이해를 돕기 위하여 바람직한 실시예를 제시한다. 그러나 하기의 실시예는 본 발명을 보다 쉽게 이해하기 위하여 제공되는 것일 뿐, 실시예에 의해 본 발명의 내용이 한정되는 것은 아니다.Hereinafter, preferred examples are provided to aid in understanding the present invention. However, the following examples are merely provided to more easily understand the present invention, and the contents of the present invention are not limited by the examples.
실시예 1Example 1 : 알츠하이머 환자의 뇌조직에서 AGE-알부민의 분포 및 발현 위치 : Distribution and Expression Location of AGE-Albumin in Brain Tissues of Alzheimer's Patients
정상 또는 알츠하이머 모델의 미세아교세포, 정상인 또는 알츠하이머 환자의 뇌조직, 및 정상 또는 알츠하이머 동물모델의 뇌조직에서 AGE-알부민의 분포 및 발현 위치를 확인하기 위하여, 하기와 같은 실험을 수행하였다.In order to determine the distribution and expression position of AGE-albumin in the microglia of the normal or Alzheimer's model, the brain tissue of the normal or Alzheimer's patient, and the brain tissue of the normal or Alzheimer's animal model, the following experiment was performed.
1. 세포배양1. Cell Culture
in vitro 연구를 위해, 불멸화 인간 미세아교세포주(immortalized human microglial cell line; HMO6)를 사용하였다. HMO6 세포를 10% 열-불활성화된 FBS (fetal bovine serum, Gibco) 및 20㎎/㎖의 겐타마이신(Sigma-Aldrich)이 첨가된 고농도의 글루코오스를 함유한 DMEM(Dulbecco's modified Eagle's medium, Gibco)에서 성장시키고, HMO6 세포를 5% CO2, 37℃로 유지시켰다. 그 다음 HMO6 세포를 0~400nM의 아밀로이드베타 1-42(Aβ1-42, Sigma-Aldrich)로 처리하였다. 추가 분석을 위해 HMO6 세포를 Aβ1-42로 처리한 후 6시간째에 세포를 채취하였다.For in vitro studies, an immortalized human microglial cell line (HMO6) was used. HMO6 cells were cultured in DMEM (Dulbecco's modified Eagle's medium, Gibco) containing high concentrations of glucose supplemented with 10% heat-inactivated FBS (fetal bovine serum, Gibco) and 20 mg / ml gentamicin (Sigma-Aldrich). And HMO6 cells were maintained at 5% CO 2 , 37 ° C. HMO6 cells were then treated with amyloid beta 1-42 (Aβ1-42, Sigma-Aldrich) at 0-400 nM. Cells were harvested 6 hours after treatment with Aβ1-42 for HMO6 cells for further analysis.
2. 면역형광표지법(immunocytochemistry, ICC)2. Immunofluorescence Labeling (IMC)
세포를 Lab-Tek Ⅱ chamber slides(Nunc)에서 성장시키고, PBS로 세척한 다음, 메탄올에서 15분 동안 고정시키고 다시 PBS로 세척하였다. 슬라이드 챔버 위에 고정된 세포를 하기와 같은 일차 항체와 함께 각각 4℃에서 밤새도록 배양하였다: 토끼 항-AGE 항체(1:200, Abcam), 마우스 항 인간-알부민 항체(1:200, R&D system), 항-BACE 항체(1:50, Santa Cruz), 항-ADAM10 항체(1:200, R&D system), 항-APP 항체(1:200, Chemicon), 항-RAGE 항체(1:50, Santa Cruz), 또는 항-미토콘드리아 항체(1:50, abcam). 배양 후, 일차 항체를 PBS로 3번 세척하였고, 슬라이드를 하기와 같은 이차 항체 중 하나와 함께 실온에서 1시간 동안 배양하였다: Alexa flour 633 anti-mouse IgG(1:500, Invitrogen), Alexa flour 488 anti-rabbit IgG (1:500, Invitrogen), 또는 Alexa flour 555 anti-goat IgG(1:500, Invitrogen). 이차 항체를 10분 간격으로 PBS로 3번 세척한 후, 커버슬립을 Vectashield mounting medium(Vector Laboratories)을 사용하여 글라스 슬라이드 위에 설치하고, 레이저 공초점 형광현미경(LSM-710, Carl Zeiss)으로 관찰하였다.Cells were grown in Lab-Tek II chamber slides (Nunc), washed with PBS, then fixed in methanol for 15 minutes and washed again with PBS. Cells immobilized on the slide chamber were incubated overnight at 4 ° C. with primary antibodies as follows: rabbit anti-AGE antibody (1: 200, Abcam), mouse anti human-albumin antibody (1: 200, R & D system) , Anti-BACE antibody (1:50, Santa Cruz), anti-ADAM10 antibody (1: 200, R & D system), anti-APP antibody (1: 200, Chemicon), anti-RAGE antibody (1:50, Santa Cruz ), Or anti-mitochondrial antibody (1:50, abcam). After incubation, the primary antibody was washed three times with PBS and the slides were incubated with one of the secondary antibodies for one hour at room temperature: Alexa flour 633 anti-mouse IgG (1: 500, Invitrogen), Alexa flour 488 anti-rabbit IgG (1: 500, Invitrogen), or Alexa flour 555 anti-goat IgG (1: 500, Invitrogen). After washing the secondary antibody three times with PBS at 10 minute intervals, the coverslip was installed on a glass slide using a Vectashield mounting medium (Vector Laboratories) and observed with a laser confocal fluorescence microscope (LSM-710, Carl Zeiss). .
3. 면역조직화학(immunohistochemistry, IHC)3. Immunohistochemistry (IHC)
정상 또는 알츠하이머 랫트의 뇌조직, 및 정상인 또는 알츠하이머 환자의 뇌조직에서 면역조직화학을 수행하였다[S. M. Ahn et al., PLoS ONE 3, e2829 (2008)]. 정상인 또는 알츠하이머 환자의 뇌조직은 서울대학병원 뇌은행과 니가타 대학병원의 뇌은행으로부터 얻었다. 구체적으로는, 정상 또는 알츠하이머 랫트의 뇌조직, 및 정상인 또는 알츠하이머 환자의 뇌조직을 0.1M 중성 인산염완충용액 내 4% 파라포름알데히드로 고정시키고, 30% 수크로오스 용액에서 밤새도록 냉동보관한 다음, 저온유지장치(cryostat, Leica CM 1900)로 10㎛ 절편을 준비하였다. 파라핀-포매 뇌조직을 4㎛ 두께의 절편으로 절단하고, 자일렌에서 탈파라핀시킨 후, 일련의 등급 에탄올로 재수화하였다. 정상 염소 혈청(10%)을 사용하여 비특이적 단백질 결합을 차단하였다. 조직 절편을 하기 항체들 중 하나와 함께 4℃에서 밤새도록 배양하였다: 토끼 항-AGE 항체(Abcam), 마우스 항-인간 알부민 항체(1:200, R&D System), 염소 항-Iba1 항체(1:500, Abcam), 항-MBP 항체(1:200, Chemicon), 항-Olig2 항체(1:100, R&D System), 항-NeuroD1 항체(1:200, R&D System), 항-BACE 항체(1:50, SantaCruz), 항-ADAM10 항체(1:200, R&D System), 항-APP 항체(1:200, Chemicon), 항-JNK 항체(1:200, Cell Signaling), 항-p-JNK 항체(1:200, Cell Signaling), 및 항-Bax 항체(1:50, SantaCruz). 상기 배양된 조직 절편을 PBS로 3번 세척하고, Alexa flour 633 anti-mouse IgG(1:500, Invitrogen), Alexa flour 488 anti-rabbit IgG(1:500, Invitrogen), 또는 Alexa flour 555 anti-goat IgG (1:500, Invitrogen)와 함께 실온에서 1시간 동안 배양하였다. 이차 항체를 PBS로 3번 세척한 후, 커버슬립을 Vectashield mounting medium(Vector Laboratories)를 사용하여 글라스 슬라이드 위에 설치하고, 레이저 공초점 형광현미경(LSM-710, Carl Zeiss)으로 관찰하였다. 즉, Aβ1-42 처리 전 또는 후의 미세아교세포 및 랫트의 뇌조직, 정상인 또는 알츠하이머 환자의 뇌조직에서 AGE(적색), 알부민(녹색) 및 DAPI(4',6-diamidino-2-phenylindole)(푸른색), AGE-알부민을 염색하여 이들의 분포 및 발현 위치를 레이저 공초점 형광현미경으로 관찰하였고, AGE-알부민의 밀도를 측정하였다. 기타 염색 방법은 상기 면역형광표지법(ICC)과 동일하게 하였다.Immunohistochemistry was performed on the brain tissue of normal or Alzheimer rats and the brain tissue of normal or Alzheimer's patients [S. M. Ahn et al., PLoS ONE 3, e2829 (2008)]. Brain tissues of normal or Alzheimer's patients were obtained from the Brain Bank of Seoul National University Hospital and the Brain Bank of Niigata University Hospital. Specifically, brain tissues of normal or Alzheimer rats, and brain tissues of normal or Alzheimer's patients were fixed with 4% paraformaldehyde in 0.1 M neutral phosphate buffer solution and frozen overnight in 30% sucrose solution, followed by low temperature 10 μm sections were prepared with a holding device (cryostat, Leica CM 1900). Paraffin-embedded brain tissue was cut into 4 μm thick sections, deparaffinized in xylene, and rehydrated with a series of grades of ethanol. Normal goat serum (10%) was used to block nonspecific protein binding. Tissue sections were incubated overnight at 4 ° C. with one of the following antibodies: rabbit anti-AGE antibody (Abcam), mouse anti-human albumin antibody (1: 200, R & D System), goat anti-Iba1 antibody (1: 500, Abcam), anti-MBP antibody (1: 200, Chemicon), anti-Olig2 antibody (1: 100, R & D System), anti-NeuroD1 antibody (1: 200, R & D System), anti-BACE antibody (1: 50, SantaCruz), anti-ADAM10 antibody (1: 200, R & D System), anti-APP antibody (1: 200, Chemicon), anti-JNK antibody (1: 200, Cell Signaling), anti-p-JNK antibody ( 1: 200, Cell Signaling), and anti-Bax Antibody (1:50, Santa Cruz). The cultured tissue sections were washed three times with PBS and washed with Alexa flour 633 anti-mouse IgG (1: 500, Invitrogen), Alexa flour 488 anti-rabbit IgG (1: 500, Invitrogen), or Alexa flour 555 anti-goat. Incubated with IgG (1: 500, Invitrogen) at room temperature for 1 hour. After washing the secondary antibody three times with PBS, the coverslip was mounted on a glass slide using Vectashield mounting medium (Vector Laboratories) and observed with a laser confocal fluorescence microscope (LSM-710, Carl Zeiss). That is, AGE (red), albumin (green), and DAPI (4 ', 6-diamidino-2-phenylindole) in microglia and rat brain tissues before or after Aβ1-42 treatment and in brain tissues of normal or Alzheimer's patients ( Blue), AGE-albumin was stained and their distribution and expression site were observed by laser confocal fluorescence microscopy, and the density of AGE-albumin was measured. Other staining methods were the same as the above immunofluorescence labeling (ICC).
Aβ1-42 처리 전 또는 후의 인간 미세아교세포 및 랫트의 뇌조직, 정상인 또는 알츠하이머 환자의 뇌조직에서 AGE-알부민의 분포 및 발현 위치를 항체를 이용하여 염색한 후 레이저 공초점 형광현미경으로 관찰한 결과는 도 2에 나타내었으며, AGE-알부민의 밀도는 도 3에 나타내었다.The distribution and expression sites of AGE-albumin in human microglial cells and rat brain tissues before and after Aβ1-42 treatment and in normal or Alzheimer's patients were stained with antibodies and observed by laser confocal fluorescence microscopy. Is shown in FIG. 2, and the density of AGE-albumin is shown in FIG. 3.
도 2에 나타난 바와 같이, Aβ1-42 처리 전 또는 후의 인간 미세아교세포 및 랫트의 뇌조직, 정상인 또는 알츠하이머 환자의 뇌조직에서 알부민(녹색)과 AGE(적색)가 같은 위치에서 염색됨을 확인하였다. 이때, 알부민은 대부분 당화된 알부민이다. 또한, Aβ1-42를 처리한 인간 미세아교세포 및 랫트의 뇌조직(대뇌피질), 알츠하이머 환자의 뇌조직(대뇌피질)에서 알부민과 AGE가 넓게 분포되어 있으며, AGE-알부민의 발현양이 현저히 증가하는 것을 관찰하였다.As shown in FIG. 2, it was confirmed that albumin (green) and AGE (red) were stained at the same position in human microglial cells before and after Aβ1-42 treatment and in brain tissues of rats and brain tissues of normal or Alzheimer's patients. In this case, albumin is mostly glycated albumin. In addition, albumin and AGE are widely distributed in human microglial cells treated with Aβ1-42, rat brain (cerebral cortex), and brain tissue (cerebral cortex) of Alzheimer's patients, and AGE-albumin expression is significantly increased. Was observed.
또한, 도 3에 나타난 바와 같이, Aβ1-42를 처리한 미세아교세포 및 랫트의 뇌조직, 알츠하이머 환자의 뇌조직에서의 AGE-알부민의 밀도는 Aβ1-42를 처리하지 않은 인간 미세아교세포 및 랫트의 뇌조직, 정상인의 뇌조직에서의 AGE-알부민의 밀도보다 현저히 증가하였으며, 특히 알츠하이머 환자의 뇌조직에서의 AGE-알부민의 밀도는 정상인의 뇌조직에서의 AGE-알부민의 밀도보다 약 3.4배 이상 증가하는 것을 확인하였다.In addition, as shown in FIG. 3, the density of AGE-albumin in the brain tissues of Aβ1-42-treated rats and the brain tissues of rats, and the brain tissues of Alzheimer's patients, were not observed in human microglial cells and rats not treated with Aβ1-42. The density of AGE-albumin was significantly increased in the brain tissues of the normal and the brain tissues of normal subjects. It was confirmed to increase.
실시예 2Example 2 : 알츠하이머 환자의 뇌조직에서 AGE-알부민의 분포 및 발현 위치의 재확인 : Reconfirmation of AGE-Albumin Distribution and Expression Location in Brain Tissues of Alzheimer's Patients
AGE-알부민이 Aβ1-42를 처리한 랫트의 뇌조직 및 알츠하이머 환자의 뇌조직에서 증가하는 것을 확인한 후, AGE-알부민의 분포 및 발현 위치를 재확인하기 위하여 알츠하이머 환자의 뇌조직에서 면역조직화학(IHC)을 수행하였다. 즉, 알츠하이머 환자의 뇌조직에서 AGE(적색), 알부민(녹색), 미세아교세포(microglia cell) 표지자인 Iba1(푸른색), 성상교세포(astrocyte cell) 표지자인 MBP(푸른색), 희소돌기아교세포(oligodendrocyte cell) 표지자인 Olig2(푸른색), 신경세포(neuronal cell) 표지자인 NeuroD(푸른색), 및 AGE-알부민을 염색하여 이들의 분포 및 발현 위치를 레이저 공초점 형광현미경으로 관찰하였다.After confirming that AGE-albumin is increased in brain tissues of rats treated with Aβ1-42 and brain tissues of Alzheimer's patients, immunohistochemistry (IHC) in brain tissues of Alzheimer's patients to reconfirm AGE-albumin distribution and expression site ) Was performed. In other words, AGE (red), albumin (green), microglia cell marker Iba1 (blue), and astrocyte cell markers MBP (blue) and oligodendrocytes in brain tissues of Alzheimer's patients Cells (oligodendrocyte cell marker Olig2 (blue), neuronal cell marker NeuroD (blue), and AGE-albumin were stained and their distribution and expression site were observed by laser confocal fluorescence microscopy.
결과는 도 4에 나타내었다.The results are shown in FIG.
도 4에 나타난 바와 같이, 알츠하이머 환자의 뇌조직에서 미세아교세포 표지자인 Iba1은 AGE-알부민과 같은 위치에서 발현되었으나, 성상교세포 표지자인 MBP, 희소돌기아교세포 표지자인 Olig2, 및 신경세포 표지자인 NeuroD은 AGE-알부민과 같은 위치에서 발현되지 않았다. 따라서, 대부분의 AGE-알부민이 미세아교세포에서 합성된다는 것을 예상할 수 있다.As shown in FIG. 4, in the brain tissue of Alzheimer's patients, the microglia marker Iba1 was expressed at the same position as AGE-albumin, but the astrocyte marker MBP, the oligodendrocyte marker Olig2, and the neuronal marker NeuroD Was not expressed at the same position as AGE-albumin. Thus, one can expect that most AGE-albumins are synthesized in microglia.
실시예 3Example 3 : 인간 미세아교세포에서 AGE-알부민의 합성 및 분비 : Synthesis and Secretion of AGE-Albumin in Human Microglial Cells
인간 미세아교세포에서 AGE-알부민이 합성 및 분비되는지 확인하기 위하여, 공-면역침전법과 ELISA를 이용하여 AGE-알부민의 발현양을 측정하였다.In order to confirm the synthesis and secretion of AGE-albumin in human microglia, the expression level of AGE-albumin was measured by co-immunoprecipitation and ELISA.
1. 공-면역침전법1. Co-immunoprecipitation
인간 미세아교세포를 0~400nM의 Aβ1-42로 처리한 후 세포 용해물을 이용하여 면역블롯팅 분석을 수행하였다. 구체적으로는, 세포 용해물을 1M Tris(pH 7.5), 5M NaCl, 10% NP-40, 10% 데옥시콜레이트 및 프로테아제 저해제 칵테일 (Calbiochem)을 함유하는 RIPA(radioimmunoprecipitation assay) 완충용액으로 준비하였다. 세포 용해물(1㎎ 단백질)을 500㎖의 PBS에서 5㎎의 항-AGE(Abcam)-결합된 세파로오스 비드와 함께 4℃에서 밤새도록 배양하였다. 세파로오스 비드를 5분 동안 14,000rpm에서 원심분리하여 침전시키고, 50mM Tris-Cl 및 500mM NaCl(pH 8.0)을 함유한 세척 완충용액 1㎖로 3번 세척하였다. IgG-결합된 항원-항체 복합체를 4~12% 폴리아크릴아미드 겔(Invitrogen)로 분리하고, 항-알부민 항체(1:1000, abcam)로 면역블롯팅 분석을 수행하였다.Human microglia were treated with 0 ~ 400nM Aβ1-42 and immunoblot analysis was performed using cell lysates. Specifically, cell lysates were prepared with a radioimmunoprecipitation assay (RIPA) buffer containing 1M Tris (pH 7.5), 5M NaCl, 10% NP-40, 10% deoxycholate and protease inhibitor cocktail (Calbiochem). Cell lysates (1 mg protein) were incubated overnight at 4 ° C. with 5 mg anti-AGE (Abcam) -bound Sepharose beads in 500 ml PBS. Sepharose beads were precipitated by centrifugation at 14,000 rpm for 5 minutes and washed three times with 1 ml of wash buffer containing 50 mM Tris-Cl and 500 mM NaCl, pH 8.0. IgG-bound antigen-antibody complexes were separated by 4-12% polyacrylamide gel (Invitrogen) and immunoblotting assays were performed with anti-albumin antibodies (1: 1000, abcam).
결과는 도 5에 나타내었다.The results are shown in FIG.
도 5에 나타난 바와 같이, 인간 미세아교세포에서 Aβ1-42의 농도가 증가할수록 AGE-알부민의 발현양이 증가하였다.As shown in FIG. 5, the expression level of AGE-albumin increased as the concentration of Aβ1-42 increased in human microglial cells.
2. 세포내와 배양 배지로 분비된 AGE-알부민의 발현양(ELISA)2. Expression of AGE-Albumin Induced by Intracellular and Culture Media (ELISA)
이미 합성된 알부민을 알부민 항체로 제거한 후, 세포내와 배양 배지로 분비된 AGE-알부민의 발현양을 ELISA를 이용하여 측정하였다. 구체적으로는, 인간 미세아교세포를 0~400nM의 Aβ1-42로 처리한 후, 세포 용해물(0.5㎎ 단백질) 및 배양 배지(0.1㎎ 단백질)를 이용하여 측정하였다. AGE-알부민의 양은 토끼 항-AGE 항체 (1:1000, Abcam) 및 마우스 항-인간 알부민 항체(1:800, Abcam)로 측정하였다. HRP 결합된 항-마우스 이차 항체(1:1000, Vector Laboratories)를 각 웰에 첨가하였다. 각 웰에 TMB(3,3',5,5'-테트라메틸벤지딘)를 가하여 발색시키고, 같은 부피의 2M H2SO4로 정지시켰다. 그 다음 ELISA 플레이트 리더(VERSA Max, Molecular Devices)를 이용하여 450㎚에서 흡광도를 측정하였다.After removing the already synthesized albumin with albumin antibody, the expression level of AGE-albumin secreted into the cell and culture medium was measured by ELISA. Specifically, human microglia were treated with 0-400 nM Aβ1-42, and then measured using cell lysate (0.5 mg protein) and culture medium (0.1 mg protein). The amount of AGE-albumin was measured with rabbit anti-AGE antibody (1: 1000, Abcam) and mouse anti-human albumin antibody (1: 800, Abcam). HRP bound anti-mouse secondary antibody (1: 1000, Vector Laboratories) was added to each well. Each well was developed by adding TMB (3,3 ', 5,5'-tetramethylbenzidine) and stopped with an equal volume of 2M H 2 SO 4 . Absorbance was then measured at 450 nm using an ELISA plate reader (VERSA Max, Molecular Devices).
결과는 도 6에 나타내었다.The results are shown in FIG.
도 6에 나타난 바와 같이, Aβ1-42를 처리한 인간 미세아교세포의 세포 용해물에서 AGE-알부민의 양은 Aβ1-42를 처리하지 않은 인간 미세아교세포의 세포 용해물에서 보다 현저히 증가하였다. 그러나, 세포 용해물에 알부민 항체를 반응시킨 경우 AGE-알부민의 양이 감소하였고, 세포 용해물에 알부민 항체와 Aβ1-42를 동시에 처리한 경우는 AGE-알부민의 양이 정상보다 증가하는 것을 관찰하였다.As shown in FIG. 6, the amount of AGE-albumin in cell lysates of human microglial cells treated with Aβ1-42 was significantly increased in cell lysates of human microglial cells not treated with Aβ1-42. However, when the albumin antibody was reacted with the cell lysate, the amount of AGE-albumin was decreased, and when the albumin antibody and Aβ1-42 were simultaneously treated with the cell lysate, the amount of AGE-albumin was increased. .
상기 결과에 의해, AGE-알부민은 인간 미세아교세포에서 합성 및 분비된다는 것을 확인할 수 있었다.As a result, it was confirmed that AGE-albumin is synthesized and secreted in human microglia.
실시예 4Example 4 : 인간 미세아교세포에서 산화적 스트레스에 의해 AGE-알부민의 합성과 분비 증가 : Increased Synthesis and Secretion of AGE-Albumin by Oxidative Stress in Human Microglial Cells
Aβ1-42는 오랜 기간 동안 산화적 스트레스에 의해 축척되는 것으로 알려져 있다. 따라서, 본 실험에서는 인간 미세아교세포에서 AGE-알부민의 합성과 분비가 산화적 스트레스에 의한 것인지 확인하기 위하여, 인간 미세아교세포에 산화적 스트레스 유도물질인 0~1000μM의 과산화수소(H2O2)를 처리한 후 세포 용해물을 이용하여 면역블롯팅 분석을 수행하였다. 또한, 인간 미세아교세포에 항산화제를 처리하여 AGE-알부민의 발현양이 감소하는지를 면역블롯팅 분석을 통해 확인하였다.Aβ1-42 is known to accumulate by oxidative stress for a long time. Therefore, in this experiment, in order to confirm whether the synthesis and secretion of AGE-albumin in human microglia is caused by oxidative stress, hydrogen peroxide (H 2 O 2 ) of 0-1000 μM, which is an oxidative stress inducer in human microglia. After treatment, immunoblotting analysis was performed using the cell lysate. In addition, it was confirmed by immunoblotting whether the expression of AGE-albumin is reduced by treating the human microglia with antioxidant.
결과는 도 7에 나타내었다.The results are shown in FIG.
도 7에 나타난 바와 같이, 인간 미세아교세포에 과산화수소(H2O2)를 처리한 경우 과산화수소의 농도가 증가할수록 AGE-알부민의 발현양이 증가하였다. 또한, 인간 미세아교세포에 항산화제인 아스코르빈산을 처리한 경우 AGE-알부민의 발현양이 현저히 감소하였으며, Aβ1-42와 아스코르빈산을 동시에 처리한 경우에도 AGE-알부민의 발현양이 감소하였다.As shown in FIG. 7, when the human microglia treated with hydrogen peroxide (H 2 O 2 ), the expression amount of AGE-albumin increased as the concentration of hydrogen peroxide increased. In addition, the expression of AGE-albumin was significantly decreased when the ascorbic acid, an antioxidant, was treated in human microglia, and the expression of AGE-albumin was also decreased when Aβ1-42 and ascorbic acid were simultaneously treated.
상기 결과에 의해, 인간 미세아교세포에 산화적 스트레스를 가하면 Aβ1-42가 축적되고, 이로 인해 AGE-알부민의 합성이 증가됨으로, 인간 미세아교세포에서 AGE-알부민의 합성과 분비가 산화적 스트레스에 의한 것임을 확인할 수 있었다.As a result, when oxidative stress is applied to human microglial cells, Aβ1-42 accumulates, thereby increasing the synthesis of AGE-albumin, and thus the synthesis and secretion of AGE-albumin in human microglia is induced by oxidative stress. It could be confirmed that.
실시예 5Example 5 : 알츠하이머 모델의 미세아교세포에서 합성 및 분비된 AGE-알부민의 Aβ1-42 응집 유도 (ThT 형광분석법) : Induction of Aβ1-42 Aggregation of AGE-Albumin Synthesized and Secreted in Microglial Cells of Alzheimer's Model (ThT Fluorescence)
알부민은 아밀로이드베타 1-42(Aβ1-42)의 중합체 형성을 방지하는 것으로 보고되어 있으며, 뇌척수액으로부터 분리된 아밀로이드 억제제 활성의 60%를 보이고 있는 것으로 알려져 있다. 하지만 현재까지 알츠하이머 모델의 미세아교세포에서 합성 및 분비된 AGE-알부민의 아밀로이드 플라크(plaque)에 대한 양성적인 관계에 대해서는 보고되어 있지 않다. 따라서, 본 실험에서는 ThT(thioflavin T) 분석법을 이용하여 알츠하이머 모델의 미세아교세포에서 합성 및 분비된 AGE-알부민과 Aβ1-42의 응집(aggregation)의 상호작용을 관찰하였다.Albumin has been reported to prevent polymer formation of amyloid beta 1-42 (Aβ 1-42) and is known to exhibit 60% of amyloid inhibitor activity isolated from cerebrospinal fluid. To date, however, no positive relationship has been reported for amyloid plaques of AGE-albumin synthesized and secreted in microglia of the Alzheimer's model. Therefore, in this experiment, the interaction of Aβ1-42 with the aggregation of AGE-albumin synthesized and secreted in microglia of the Alzheimer's model was observed using a thioflavin T (ThT) assay.
구체적으로는, Aβ1-42로 처리한 랫트의 뇌조직과 알츠하이머 환자의 뇌조직에 알부민 또는 AGE-알부민을 각각 10mM씩 가하고 37℃에서 2.5시간 동안 일정하게 흔들면서 Perkin-Elmer 형광분광 광도계(LS-55)를 이용하여 ThT 형광을 483㎚(450㎚ 여기)에서 측정하였다. 알부민 또는 AGE-알부민을 처리한 세포의 형광값은 DMSO-처리된 음성대조군으로 표준화하였으며, 상대 형광 백분율로 나타내었다.Specifically, 10 mM of albumin or AGE-albumin were added to brain tissues of rats treated with Aβ1-42 and brain tissues of Alzheimer's patients, respectively, and shaken at 37 ° C. for 2.5 hours. 55) was used to measure ThT fluorescence at 483 nm (450 nm excitation). Fluorescence values of cells treated with albumin or AGE-albumin were normalized to DMSO-treated negative controls and expressed as relative fluorescence percentages.
결과는 도 8에 나타내었다.The results are shown in FIG.
도 8에 나타난 바와 같이, Aβ1-42로 처리한 랫트의 뇌조직과 알츠하이머 환자의 뇌조직에서 AGE-알부민과 아밀로이드 플라크가 같은 위치에 존재하는 것을 관찰하였고(A), 미세아교세포에 알부민을 투여하였을 때보다 AGE-알부민을 투여하였을 때 Aβ1-42의 응집이 현저히 증가하는 것을 관찰하였다(B). 따라서, Aβ1-42로 처리한 랫트의 뇌조직과 알츠하이머 환자의 뇌조직에서 AGE-알부민이 Aβ1-42의 응집을 유도한다는 것을 알 수 있다.As shown in FIG. 8, AGE-albumin and amyloid plaques were observed in the brain tissues of rats treated with Aβ1-42 and the brain tissues of Alzheimer's patients (A), and albumin was administered to microglia. It was observed that aggregation of Aβ1-42 was significantly increased when AGE-albumin was administered than when (B). Therefore, it can be seen that AGE-albumin induces aggregation of Aβ1-42 in brain tissues of rats treated with Aβ1-42 and brain tissues of Alzheimer's patients.
실시예 6Example 6 : AGE-알부민이 인간 미세아교세포에서 Aβ1-42의 합성 유도 : AGE-Albumin Induces Aβ1-42 Synthesis in Human Microglial Cells
AGE-알부민이 인간 미세아교세포에서 Aβ1-42의 합성을 유도하는지 확인하기 위하여, 인간 미세아교세포에 AGE-알부민을 투여한 후 Aβ1-42의 양을 ELISA 분석법을 이용하여 측정하였고, BACE, ADAM10, APP의 발현 위치와 발현양을 면역염색화학과 면역 블롯팅을 이용하여 관찰하였다.To determine whether AGE-albumin induced the synthesis of Aβ1-42 in human microglial cells, the amount of Aβ1-42 after AGE-albumin administration to human microglial cells was measured using an ELISA assay. BACE, ADAM10 , The expression location and expression level of APP were observed using immunostaining chemistry and immunoblotting.
인간 미세아교세포에 AGE-알부민을 투여한 후 Aβ1-42의 양을 ELISA 분석법을 이용하여 측정한 결과는 도 9에 나타내었고, 인간 미세아교세포에 AGE-알부민을 투여한 후 BACE, ADAM10, APP의 발현 위치와 발현양을 각각 면역염색화학(A)과 면역 블롯팅(B)을 이용하여 관찰한 결과는 도 10에 나타내었다.The result of measuring the amount of Aβ1-42 after administration of AGE-albumin to human microglia using ELISA is shown in FIG. 9, and after administration of AGE-albumin to human microglia, BACE, ADAM10, APP The expression position and the expression amount of were observed using immunostaining chemistry (A) and immunoblotting (B), respectively, are shown in FIG. 10.
도 9에 나타난 바와 같이, 인간 미세아교세포에 AGE-알부민을 투여하였을 때 Aβ1-42의 양이 증가하였다.As shown in FIG. 9, the amount of Aβ1-42 was increased when AGE-albumin was administered to human microglia.
또한, 도 10에 나타난 바와 같이, 인간 미세아교세포에 AGE-알부민을 투여하였을 때 BACE의 발현양은 증가하였으나, ADAM10과 APP의 발현양은 유사하게 나타났다.In addition, as shown in Figure 10, when the AGE-albumin administered to human microglia, the expression of BACE increased, but the expression of ADAM10 and APP was similar.
상기 결과에 의해, AGE-알부민은 인간 미세아교세포에서 Aβ1-42의 합성을 유도한다는 것을 알 수 있다.The results indicate that AGE-albumin induces the synthesis of Aβ1-42 in human microglia.
실시예 7Example 7 : 일차 인간 신경세포에서 AGE-알부민에 의한 신경세포사 유도 Induction of Neuronal Death by AGE-Albumin in Primary Human Neurons
스트레스에 의해 활성화된 MAPK(Mitogen-Activated Protein Kinase)가 신경세포사를 유도한다고 보고되어 있다. 따라서, 본 실험에서는 일차 인간 신경세포에서 AGE-알부민이 직접적으로 MAPK 신호전달체계를 활성화시키고, Bax의 발현양을 증가시키는지 확인하기 위하여, 하기와 같은 실험을 수행하였다.It is reported that stress-activated mitogen-activated protein kinase (MAPK) induces neuronal death. Therefore, in this experiment, to determine whether AGE-albumin directly activates the MAPK signaling system and increases the expression level of Bax in primary human neurons, the following experiment was performed.
1. 일차 인간 신경세포 배양(primary human neuronal cell culture)Primary human neuronal cell culture
일차 인간 신경세포는 사람의 뇌 조직으로부터 얻었다. 뇌조직 수집과 사용은 서울대학교 의과대학 윤리심의위원회(Ethics Committee of the Seoul National University College of Medicine, Seoul, Korea)에 의해 승인받았다. 인간 두뇌 피질의 작은 조각을 0.25% 트립신과 40㎎/㎖ DNase I을 함유하는 인산염완충용액 (PBS)에서 37℃에서 30분 동안 배양하였다. 분리 배양된 세포를 5% FBS, 5% HS (horse serum), 20㎎/㎖의 겐타마이신 및 2.5㎎/㎖의 암포테리신 B가 첨가된 DMEM (배양배지)에 현탁시키고, 10㎝ 배양접시에 1×106 cells/㎖ (10㎖)로 플레이트한 다음, 5% CO2/95% 대기 하의 배양기에서 37℃로 유지하였다. in vitro 배양 2~3주 후, 배양접시에 떠있는 부유세포를 수집하고, 면역형광표지법용 Lab-Tek Ⅱ Chamber Slide System(2×104 cells/wells, Nunc)으로 세포를 다시 플레이팅하여 미세아교세포-풍부 세포를 준비하였다. 남아있는 신경세포는 AGE-알부민으로 처리한 후 아폽토시스-관련 특성을 위해 사용하였다.Primary human neurons were obtained from human brain tissue. The collection and use of brain tissue was approved by the Ethics Committee of the Seoul National University College of Medicine, Seoul, Korea. Small pieces of human brain cortex were incubated at 37 ° C. for 30 minutes in phosphate buffer solution (PBS) containing 0.25% trypsin and 40 mg / ml DNase I. The isolated cultured cells are suspended in DMEM (culture medium) to which 5% FBS, 5% HS (horse serum), 20 mg / ml gentamycin and 2.5 mg / ml amphotericin B are added, and a 10 cm dish. Were plated at 1 × 10 6 cells / ml (10 mL) and then maintained at 37 ° C. in an incubator under 5% CO 2 /95% atmosphere. After 2-3 weeks of in vitro culture, the floating cells floating in the culture dish are collected, and the cells are replated with Lab-Tek II Chamber Slide System (2 × 10 4 cells / wells, Nunc) for immunofluorescence labeling. Glue-rich cells were prepared. The remaining neurons were used for apoptosis-related properties after treatment with AGE-albumin.
2. 면역조직화학과 면역 블롯팅2. Immunohistochemistry and Immunoblotting
AGE-알부민을 일차 인간 신경세포에 처리하기 전 또는 후의 세포 용해물을 이용하여 RAGE, ERK1/2, pERK1/2, p38, pp38, SAPK/JNK, pSAPK/JNK, Bax의 발현 위치와 발현양을 면역조직화학과 면역 블롯팅 방법을 이용하여 관찰하였다.Cell lysate before or after AGE-albumin treatment in primary human neurons was used to determine the expression location and expression levels of RAGE, ERK1 / 2, pERK1 / 2, p38, pp38, SAPK / JNK, pSAPK / JNK, and Bax. Immunohistochemistry and immunoblotting were used for the observation.
일차 인간 신경세포에 AGE-알부민을 투여한 후 RAGE, ERK1/2, pERK1/2, p38, pp38, SAPK/JNK, pSAPK/JNK, Bax의 발현 위치와 발현양을 각각 면역염색화학과 면역 블롯팅 방법을 이용하여 관찰한 결과는 각각 도 11 도 12에 나타내었다.Immunohistochemistry and immunoblotting method for expression and expression of RAGE, ERK1 / 2, pERK1 / 2, p38, pp38, SAPK / JNK, pSAPK / JNK, and Bax after primary AGE-albumin administration The results observed using are shown in FIGS. 11 and 12, respectively.
도 11 및 도 12에 나타난 바와 같이, AGE-알부민을 투여한 일차 인간 신경세포에서 RAGE가 증가하였으며, pERK1/2를 제외한 ERK1/2, p38, pp38, SAPK/JNK, pSAPK/JNK가 증가하여 MAPK가 활성화되는 것을 관찰하였을 뿐만 아니라 세포사멸 유발(pro-apoptotic) 단백질인 Bax의 발현양이 증가하는 것을 관찰하였다.As shown in FIGS. 11 and 12, RAGE was increased in primary human neurons administered AGE-albumin, and ERK1 / 2, p38, pp38, SAPK / JNK, pSAPK / JNK were increased except for pERK1 / 2 and MAPK. Not only was activated but also the expression of Bax, a pro-apoptotic protein, was increased.
3. 일차 인간 신경세포에서 AGE와 RAGE의 상호작용 : PLA(proximity ligation assay)3. Interaction between AGE and RAGE in primary human neurons: PLA (proximity ligation assay)
AGE-알부민을 일차 인간 신경세포에 처리하기 전 또는 후의 세포 용해물을 이용하여 AGE와 RAGE의 상호작용을 확인하기 위하여, 일차 인간 신경세포와 뇌조직에서 PLA를 수행하여 AGE와 RAGE의 상호작용의 정도를 시각화하였다.To confirm the interaction between AGE and RAGE using cell lysates before or after treatment with AGE-albumin in primary human neurons, PLA was performed on primary human neurons and brain tissues to determine the interactions between AGE and RAGE. The degree was visualized.
구체적으로는, 표적 조직을 차가운 PBS로 세척하고, 마우스 항 인간-알부민 항체(1:200, R&D system), 토끼 항-Aβ 항체(1:100, Chemicon), 또는 항-RAGE 항체 (1:200, Santa Cruz)와 함께 4℃에서 밤새도록 배양하였다. PLA 및 Hoechst 염색을 Duolink Detection Kit(O-link Bioscience)를 이용하여 제조자의 프로토콜에 따라 수행하였다. 조직 표본을 Vectashield mounting media(Vector Laboratories)에 고정시키고, 공초점 현미경(LSM 710)을 이용하여 분석하였다. 세포 당 in-situ PLA 신호 수는 반자동 영상 분석 프로그램 BlobFinderV3.0을 이용하여 세었다.Specifically, target tissues are washed with cold PBS and mouse anti human-albumin antibody (1: 200, R & D system), rabbit anti-Aβ antibody (1: 100, Chemicon), or anti-RAGE antibody (1: 200). , Santa Cruz) and incubated overnight at 4 ℃. PLA and Hoechst staining were performed using the Duolink Detection Kit (O-link Bioscience) according to the manufacturer's protocol. Tissue specimens were fixed in Vectashield mounting media (Vector Laboratories) and analyzed using confocal microscopy (LSM 710). The number of in-situ PLA signals per cell was counted using the semi-automated image analysis program BlobFinderV3.0.
AGE-알부민을 일차 인간 신경세포에 처리하기 전 또는 후의 세포 용해물을 이용하여 AGE와 RAGE의 상호작용을 확인한 결과는 도 13에 나타내었다.The results of confirming the interaction between AGE and RAGE using cell lysate before or after treatment with AGE-albumin in primary human neurons are shown in FIG. 13.
도 13에 나타난 바와 같이, AGE-알부민을 투여한 일차 인간 신경세포에서 AGE와 RAGE의 상호작용이 증가하는 것을 확인하였다.As shown in Figure 13, it was confirmed that the interaction between AGE and RAGE in primary human neurons administered AGE-albumin.
4. 세포 내 칼슘 변화 측정4. Measurement of intracellular calcium changes
일차 인간 신경세포를 Lab-Tek Ⅱ glass slide chambers(Nunc)에서 성장시켰다. 세포 배양 2일 후, 세포를 4μM의 Fluo-3 염료(Invitrogen)로 염색한 후 37℃에서 40분 동안 배양하였다. 배양 후, 슬라이드 챔버에 100ng/㎖의 AGE-알부민 (AGE-ALB)을 조심스럽게 가하고 실시간 이미징을 통해 20분 동안 세포 내 칼슘의 변화를 레이저 공초점 형광현미경(LSM 710, Carl Zeiss)으로 관찰하였다.Primary human neurons were grown in Lab-Tek II glass slide chambers (Nunc). After 2 days of cell culture, cells were stained with 4 μM Fluo-3 dye (Invitrogen) and incubated at 37 ° C. for 40 minutes. After incubation, 100 ng / ml AGE-albumin (AGE-ALB) was carefully added to the slide chamber and the change in intracellular calcium was observed by laser confocal fluorescence microscopy (LSM 710, Carl Zeiss) for 20 minutes through real-time imaging. .
결과는 도 14에 나타내었다.The results are shown in FIG.
도 14에 나타난 바와 같이, 일차 인간 신경세포에 AGE-알부민을 투여한 후 미토콘드리아에서 칼슘이 증가하는 것을 확인하였다.As shown in FIG. 14, it was confirmed that calcium was increased in mitochondria after administration of AGE-albumin to primary human neurons.
5. 세포생존율(MTT assay) 측정5. Measurement of MTT assay
AGE-알부민을 투여한 일차 인간 신경세포에서 Bax의 발현양의 증가가 미토콘드리아의 칼슘을 증가시키고, 궁극적으로 AGE-알부민이 세포사를 유도하는지 확인하기 위하여, 하기와 같은 실험을 수행하였다.In order to confirm whether the increase in the expression of Bax in the primary human neurons administered AGE-albumin increased the calcium of mitochondria and ultimately AGE-albumin induced cell death, the following experiment was performed.
일차 인간 신경세포를 96-웰 배양 플레이트에 웰당 2×103 세포로 접종하였다. 80% 융합(confluence)에 도달한 후, 일차 인간 신경세포를 여러 농도(0, 0.01, 0.1, 1, 10, 20㎍/㎖)의 AGE-알부민 또는 여러 농도(0, 0.5, 1, 5, 10㎎/㎖)의 알부민으로 처리하였다. 처리 24시간 후, 세포를 PBS로 세척한 다음, 세포생존율을 MTT[3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] assay를 이용하여 측정하였다. 각 웰의 흡광도는 96-웰 플레이트 리더(VERSA Max, Molecular Devices)를 이용하여 540㎚에서 측정하였다.Primary human neurons were seeded at 96 × well culture plates at 2 × 10 3 cells per well. After reaching 80% confluence, primary human neurons were harvested at various concentrations (0, 0.01, 0.1, 1, 10, 20 μg / ml) or at various concentrations (0, 0.5, 1, 5, 10 mg / ml) albumin. After 24 hours of treatment, the cells were washed with PBS, and cell viability was measured by MTT [3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyl tetrazolium bromide] assay. Absorbance of each well was measured at 540 nm using a 96-well plate reader (VERSA Max, Molecular Devices).
결과는 도 15에 나타내었다.The results are shown in FIG.
도 15에 나타난 바와 같이, 일차 인간 신경세포에 AGE-알부민을 처리한 경우 AGE-알부민의 농도가 증가할수록 세포생존율이 감소하여 세포사가 유도됨을 확인하였다. 반면, 일차 인간 신경세포에 알부민을 처리한 경우 알부민의 농도에 상관없이 세포생존율이 거의 변화가 없어 세포사가 유도되지 않음을 확인하였다.As shown in FIG. 15, it was confirmed that when AGE-albumin was treated to primary human neurons, cell viability decreased as the concentration of AGE-albumin increased, leading to cell death. On the other hand, when albumin was treated to primary human neurons, it was confirmed that cell death was not induced due to almost no change in cell viability regardless of albumin concentration.
상기 결과에 의하여, AGE-알부민은 일차 인간 신경세포에 작용하여 RAGE의 발현양을 증가시키고, 이것은 MAPK 신호전달체계를 활성화시킨 후 Bax의 발현양을 증가시켜 미토콘드리아에서 칼슘의 증가를 유도하고, 최종적으로 세포사를 유도함을 알 수 있다.As a result, AGE-albumin acts on primary human neurons to increase the expression of RAGE, which activates the MAPK signaling system and then increases the expression of Bax to induce an increase in calcium in the mitochondria. It can be seen that induces cell death.
실시예 8Example 8 : 마우스의 혈액단핵세포에서 AGE-알부민의 분포 및 발현 위치 : Distribution and Expression Location of AGE-Albumin in Blood Mononuclear Cells of Mice
마우스의 혈액단핵세포에서 AGE-알부민의 분포 및 발현 위치를 확인하기 위하여, Aβ1-42 처리 전 또는 후의 마우스의 혈액단핵세포에서 알부민(녹색), AGE(적색) 및 DAPI(푸른색), AGE-알부민을 염색하여 이들의 분포 및 발현 위치를 레이저 공초점 형광현미경으로 관찰하였다.To determine the distribution and expression location of AGE-albumin in the blood mononuclear cells of the mouse, albumin (green), AGE (red) and DAPI (blue), AGE- in the blood mononuclear cells of the mouse before or after Aβ1-42 treatment. Albumin was stained and their distribution and expression site were observed by laser confocal fluorescence microscopy.
결과는 도 16에 나타내었다.The results are shown in FIG.
도 16에 나타난 바와 같이, Aβ1-42 처리 전 또는 후의 마우스의 혈액단핵세포에서 알부민(녹색)과 AGE(적색)가 같은 위치에서 염색됨을 확인하였다. 또한, Aβ1-42를 처리한 마우스의 혈액단핵세포에서 알부민과 AGE가 넓게 분포되어 있으며, AGE-알부민의 발현양이 Aβ1-42 처리 전의 마우스의 혈액단핵세포에서보다 증가하는 것을 관찰하였다.As shown in FIG. 16, it was confirmed that albumin (green) and AGE (red) were stained at the same position in blood mononuclear cells of mice before or after Aβ1-42 treatment. In addition, it was observed that albumin and AGE were widely distributed in blood mononuclear cells of Aβ1-42-treated mice, and the expression level of AGE-albumin was increased in blood mononuclear cells of mice before Aβ1-42 treatment.
실시예 9Example 9 : Aβ1-42 매개된 신경세포사에 대한 수용성 RAGE(sRAGE)의 보호 효과 :  Protective Effect of Soluble RAGE (sRAGE) on Aβ1-42 Mediated Neuronal Death in vivoin vivo 실험 Experiment
Aβ1-42 매개된 신경세포사에 대한 수용성 RAGE(sRAGE)의 보호 효과를 확인하기 위하여, 랫트의 뇌조직에 Aβ1-42 단독으로 또는 Aβ1-42와 sRAGE(Aβ/sRAGE)를 함께 주입한 후 in vivo 실험을 수행하였다.To determine the protective effect of soluble RAGE (sRAGE) on Aβ1-42 mediated neuronal cell death, rats were injected with Aβ1-42 alone or Aβ1-42 and sRAGE (Aβ / sRAGE) in vivo. The experiment was performed.
1. 동물모델1. Animal Model
실험동물로 체중 230~350g의 Sprague-Dawley 랫트를 사용하였다. 랫트를 12시간 명암 주기로 유지시켰으며, 식량과 물을 자유로이 섭취시켰고, 적어도 사용하기 일주일 전에 순응시켰다. 모든 동물 실험은 실험동물사용관리위원회(IACUC; Institutional Animal Care and Use Committees)에 의해 승인되고 인도적으로 수행되었다.Sprague-Dawley rats weighing 230-350 g were used as experimental animals. Rats were maintained on a 12 hour light and dark cycle, freely consumed with food and water, and acclimated at least a week before use. All animal experiments were approved and humanely performed by the Institutional Animal Care and Use Committees (IACUC).
실험동물을 수술 전에 케타민(0.75 ㎎/㎏ 체중)과 럼푼(0.2　㎎/㎏ 체중)으로 마취시켰다. in vivo 처리를 위해, PBS, sRAGE를 400μM의 농도로 멸균수에 용해시키고, 사용할 때까지 4℃로 유지하였다. 랫트의 뇌를 뇌심부고정장치(stereotaxic instrument)를 이용하여 고정한 후, 두피 피부의 중앙을 절개하였다. 두개골 브레그마(bregma)에서 생물학적 전기 드릴로 구멍을 뚫고(뒤쪽으로, 8.3㎜; 측면으로, 5.4㎜), 표적 면적(깊이, 4.5㎜)에 도달할 때까지 5-㎕ 해밀톤 주사기의 바늘(30 게이지)을 수직으로 하강시켰다. 5㎕의 200μM Aβ1-42, 5㎕의 200μM Aβ1-42/sRAGE 또는 5㎕의 인산염완충용액(PBS)을 자동미세주입기(automatic microinjector)로 분당 1㎕의 속도로 랫트의 내후각뇌피질(entorhinal cortex, EC)에 천천히 주입하였다. 그 다음, 주사기를 천천히 제거하고 수술 창상(surgical wounds)을 상처봉합용 클립으로 봉합하고 항생제로 국소 처리하였다.The experimental animals were anaesthetized with ketamine (0.75 mg / kg body weight) and lumpoon (0.2 mg / kg body weight) before surgery. For in vivo treatment, PBS, sRAGE was dissolved in sterile water at a concentration of 400 μM and kept at 4 ° C. until use. The rat brain was fixed using a stereotaxic instrument, and then the center of the scalp skin was incised. Punch a 30 μl Hamilton syringe until a target area (depth, 4.5 mm) is reached in the cranial bregma with a biological electric drill (backward, 8.3 mm; laterally, 5.4 mm). Gauge) was lowered vertically. 5 μl 200 μM Aβ 1-42, 5 μl 200 μM Aβ 1-42 / sRAGE or 5 μl phosphate buffer solution (PBS) with an automatic microinjector at 1 μl per minute to enterohinal rats cortex, EC). The syringe was then slowly removed and surgical wounds closed with a wound closure clip and topically treated with antibiotics.
대부분의 랫트는 주사 후 3일 동안 회복되었다. 완전히 회복한 후, 모든 랫트를 동일한 방법으로 다시 마취시키고, 18℃에서 100~200㎖의 헤파린이 첨가된 증류수(heparinized saline)로 심장을 통해 관류하고, 이어서 0.1M 소듐 포스페이트 완충용액(pH 7.4) 내 4% 파라포름알데히드-리신 페리오데이트(paraformaldehyde-lysine periodate)의 400㎖로 관류시켰다. 뇌를 제거하고 동일한 고정액에 넣은 다음 4℃에서 4시간 동안 고정하고, 20% 수크로오스를 함유하는 차가운 0.1M 인산염완충용액(PBS)으로 옮겼다. 뇌를 동결박편제작기(freezing microtome)로 10㎛ 두께로 횡단면을 절개하고, 사용할 때까지 -80℃에서 저장하였다.Most rats recovered for 3 days after injection. After complete recovery, all rats were anesthetized again in the same manner, perfused through the heart with heparinized saline with 100-200 ml of heparin at 18 ° C., followed by 0.1 M sodium phosphate buffer (pH 7.4). Perfusion was performed with 400 ml of 4% paraformaldehyde-lysine periodate in. The brain was removed, placed in the same fixative, fixed at 4 ° C. for 4 hours, and transferred to cold 0.1 M phosphate buffer solution (PBS) containing 20% sucrose. Brains were incised with a 10 μm thick cross section with a freezing microtome and stored at −80 ° C. until use.
2. 랫트의 뇌조직에 있는 신경세포의 수2. Number of neurons in rat brain tissue
랫트의 뇌조직에 Aβ1-42 단독으로 또는 Aβ1-42와 sRAGE(Aβ/sRAGE)를 함께 처리하고 72시간 후 랫트의 뇌조직에 있는 신경세포의 수를 크레실 바이올렛 (cresyl violet)으로 염색한 후 현미경으로 관찰하였다.After treatment with Aβ1-42 alone or Aβ1-42 and sRAGE (Aβ / sRAGE) in rat brain tissue, 72 hours later, the number of neurons in rat brain tissue was stained with cresyl violet. It was observed under a microscope.
결과는 도 17에 나타내었다.The results are shown in FIG.
도 17에 나타난 바와 같이, 랫트의 뇌조직에 Aβ1-42와 sRAGE(Aβ/sRAGE)를 함께 처리한 경우 Aβ1-42를 단독으로 처리하였을 때보다 신경세포의 수가 급증하였다.As shown in Figure 17, when treated with Aβ1-42 and sRAGE (Aβ / sRAGE) to the brain tissue of rats, the number of neurons increased more than when treated with Aβ1-42 alone.
3. 랫트의 뇌조직에서 AGE-알부민의 분포 및 발현 위치3. Distribution and Expression Location of AGE-Albumin in Brain Tissues of Rats
Aβ1-42 단독으로 또는 Aβ1-42와 sRAGE(Aβ/sRAGE)를 함께 처리한 랫트의 뇌조직에서 AGE-알부민의 분포 및 발현 위치를 확인하기 위하여, 랫트의 뇌조직에서 AGE(적색), 알부민(녹색), 미세아교세포(microglia cell) 표지자인 Iba1(푸른색), 및 AGE-알부민을 염색하여 면역조직화학(IHC)을 수행하였다. 그 다음, 이들의 분포 및 발현 위치를 레이저 공초점 형광현미경으로 관찰하고, AGE-알부민의 밀도를 측정하였다.To determine the distribution and expression of AGE-albumin in brain tissues of rats treated with Aβ1-42 alone or with Aβ1-42 and sRAGE (Aβ / sRAGE), AGE (red), albumin ( Immunohistochemistry (IHC) was performed by staining green), microglia cell marker Iba1 (blue), and AGE-albumin. Then, their distribution and expression site were observed by laser confocal fluorescence microscopy and the density of AGE-albumin was measured.
결과는 도 18에 나타내었다.The results are shown in FIG.
도 18에 나타난 바와 같이, AGE, 알부민, 미세아교세포 표지자인 Iba1, 및 AGE-알부민의 발현양은 Aβ1-42를 단독으로 주입한 랫트의 뇌조직에서 증가하였으나, Aβ1-42와 sRAGE(Aβ/sRAGE)를 함께 주입한 랫트의 뇌조직에서는 감소하였다. 또한, Aβ1-42를 단독으로 주입한 랫트의 뇌조직에서의 AGE-알부민의 밀도는 Aβ1-42와 sRAGE(Aβ/sRAGE)를 함께 주입한 랫트의 뇌조직에서보다 현저히 증가하였다.As shown in FIG. 18, the expression levels of AGE, albumin, microglia marker Iba1, and AGE-albumin were increased in brain tissues of rats injected with Aβ1-42 alone, but Aβ1-42 and sRAGE (Aβ / sRAGE). ) Decreased in the brain tissues of rats injected with). In addition, the density of AGE-albumin in brain tissues of rats injected with Aβ1-42 alone was significantly higher than that of rats injected with both Aβ1-42 and sRAGE (Aβ / sRAGE).
4. 랫트의 뇌조직에서 RAGE, NeuN, DAPI, Bax 및 p-SAPK/JNK의 분포 및 발현 위치4. Distribution and Expression Location of RAGE, NeuN, DAPI, Bax, and p-SAPK / JNK in Rat Brain Tissue
랫트의 뇌조직에서 Aβ1-42가 신경세포사를 유도하고 sRAGE가 RAGE-매개된 신경세포사를 보호하는지 확인하기 위하여, Aβ1-42 단독으로 또는 Aβ1-42와 sRAGE(Aβ/sRAGE)를 함께 처리한 랫트의 뇌조직에서 RAGE, NeuN, DAPI, Bax 및 p-SAPK/JNK를 염색하여 면역조직화학(IHC)을 수행하였다. 그 다음, 이들의 분포 및 발현 위치를 레이저 공초점 형광현미경으로 관찰하였다.To determine whether Aβ1-42 induces neuronal death and sRAGE protects RAGE-mediated neuronal death in rat brain tissues, rats treated with Aβ1-42 alone or in combination with Aβ1-42 and sRAGE (Aβ / sRAGE) Immunohistochemistry (IHC) was performed by staining RAGE, NeuN, DAPI, Bax and p-SAPK / JNK in brain tissues. Then, their distribution and expression site were observed by laser confocal fluorescence microscopy.
결과는 도 19에 나타내었다.The results are shown in FIG. 19.
도 19에 나타난 바와 같이, RAGE, NeuN, DAPI, Bax 및 p-SAPK/JNK의 발현양은 Aβ1-42를 단독으로 주입한 랫트의 뇌조직에서 증가하였으나, Aβ1-42와 sRAGE(Aβ/sRAGE)를 함께 주입한 랫트의 뇌조직에서는 감소하였다. 따라서, Aβ1-42가 신경세포사를 유도하고, sRAGE가 RAGE-매개된 신경세포사를 보호한다는 것을 알 수 있다.As shown in FIG. 19, the expression levels of RAGE, NeuN, DAPI, Bax and p-SAPK / JNK were increased in brain tissues of rats injected with Aβ1-42 alone, but Aβ1-42 and sRAGE (Aβ / sRAGE) were increased. It was decreased in the brain tissues of rats injected together. Thus, it can be seen that Aβ1-42 induces neuronal death and sRAGE protects RAGE-mediated neuronal death.
실시예 10Example 10 : 뇌졸중 환자의 뇌조직에서 AGE-알부민의 분포 및 발현 위치 : Distribution and location of AGE-albumin in brain tissue of stroke patients
뇌졸중 환자의 뇌조직에서 AGE-알부민의 분포 및 발현 위치를 확인하기 위하여, 뇌졸중 환자의 뇌조직에 AGE, 알부민 및 AGE-알부민을 염색하여 면역조직화학 (IHC) 실험을 수행하고, 이들의 분포 및 발현 위치를 레이저 공초점 형광현미경으로 관찰하였다.In order to confirm the distribution and expression position of AGE-albumin in the brain tissues of stroke patients, immunohistochemistry (IHC) experiments were performed by staining AGE, albumin and AGE-albumin in the brain tissues of stroke patients, and their distribution and Expression sites were observed by laser confocal fluorescence microscopy.
결과는 도 20에 나타내었다.The results are shown in FIG.
도 20에 나타난 바와 같이, 뇌졸중 환자의 뇌조직에서 알부민(녹색)과 AGE (적색)가 같은 위치에서 염색되고 넓게 분포되어 있으며, AGE-알부민이 넓게 분포되어 있는 것을 관찰하였다.As shown in FIG. 20, it was observed that albumin (green) and AGE (red) were stained and widely distributed at the same position, and AGE-albumin was widely distributed in the brain tissue of the stroke patient.
실시예 11Example 11 : 뇌졸중 모델의 인간 미세아교세포에서 저산소증 유발인자(HIF-1α)와 HMGB1(high motility group protein B1)의 발현 : Expression of hypoxia-induced factors (HIF-1α) and HMGB1 (high motility group protein B1) in human microglial cells of stroke model
뇌졸중 모델의 인간 미세아교세포에서 저산소증 유발인자(HIF-1α)와 HMGB1의 발현 정도를 확인하기 위하여, 하기와 같은 실험을 수행하였다.In order to confirm the expression level of hypoxia induced factors (HIF-1α) and HMGB1 in human microglia of the stroke model, the following experiment was performed.
1. 세포배양 및 뇌졸중 세포 모델의 제작1. Cell Culture and Construction of Stroke Cell Model
인간 미세아교세포주는 DMEM 배지(Gibco, 10% 소혈청(Gibco), 0.1% 겐타마이신(Gibco), 고농도의 포도당)를 이용하여 5% CO2가 포함되어 있는 37℃ 배양기에서 배양하였다. 뇌졸중 세포 모델을 만들기 위하여, 준비된 미세아교세포주를 포도당이 없는 DMEM 배지로 교환한 후, 저산소 상태(5% CO2와 95% N2)를 만들어주는 챔버 (Billups-Rothenberg, Del Mar, CA)에서 1시간 동안 배양한 다음 세포들을 추출하여 실험에 사용하였다.Human microglial cells were cultured in a 37 ° C. incubator containing 5% CO 2 using DMEM medium (Gibco, 10% bovine serum (Gibco), 0.1% gentamycin (Gibco), high concentration of glucose). To create a stroke cell model, the prepared microglia line is exchanged with glucose-free DMEM medium and then in a chamber (Billups-Rothenberg, Del Mar, CA) that produces hypoxic states (5% CO 2 and 95% N 2 ). After culturing for 1 hour, the cells were extracted and used for the experiment.
2. 뇌졸중 모델의 인간 미세아교세포에서 저산소증 유발인자(HIF-1α)와 HMGB1의 발현 확인2. Expression of Hypoxia Inducer (HIF-1α) and HMGB1 in Human Microglial Cells of Stroke Model
저산소증과 포도당-결핍된 뇌졸중 모델의 인간 미세아교세포에서 저산소증 유발인자(HIF-1α)와 HMGB1의 발현을 확인하기 위하여, 면역조직화학법(IHC), PCR, 면역블롯팅을 수행하였다.In order to confirm the expression of hypoxia-inducing factor (HIF-1α) and HMGB1 in human microglia of hypoxia and glucose-deficient stroke models, immunohistochemistry (IHC), PCR, and immunoblotting were performed.
저산소증과 포도당-결핍된 뇌졸중 모델의 인간 미세아교세포에서 저산소증 유발인자(HIF-1α)와 HMGB1의 발현 정도는 각각 도 21 및 도 22에 나타내었다.The expression levels of hypoxia-inducing factor (HIF-1α) and HMGB1 in human microglia of hypoxia and glucose-deficient stroke models are shown in FIGS. 21 and 22, respectively.
도 21 및 도 22에 나타난 바와 같이, 저산소증과 포도당-결핍된 뇌졸중 모델의 인간 미세아교세포에서 저산소증 유발인자(HIF-1α)와 HMGB1의 발현이 증가하는 것을 확인하였다.As shown in FIG. 21 and FIG. 22, it was confirmed that the expression of hypoxia-inducing factor (HIF-1α) and HMGB1 was increased in human microglia of hypoxia and glucose-deficient stroke models.
실시예 12Example 12 : 뇌졸중 모델의 인간 미세아교세포에서 AGE-알부민의 합성 및 분비 : Synthesis and Secretion of AGE-Albumin in Human Microglial Cells of Stroke Model
1. 뇌졸중 모델의 인간 미세아교세포에서 AGE-알부민의 발현양 측정1. Measurement of AGE-Albumin Expression in Human Microglial Cells of Stroke Model
저산소증과 포도당-결핍된 뇌졸중 모델의 인간 미세아교세포에서 AGE-알부민이 합성 및 분비되는지 확인하기 위하여, 면역조직화학법(IHC), 면역블롯팅 및 ELISA를 이용하여 AGE-알부민의 발현양을 측정하였다.To determine the synthesis and secretion of AGE-albumin in human microglial cells of hypoxia and glucose-deficient stroke models, the expression level of AGE-albumin was measured using immunohistochemistry (IHC), immunoblotting and ELISA. It was.
결과는 도 23에 나타내었다.The results are shown in FIG.
도 23에 나타난 바와 같이, 저산소증과 포도당-결핍된 뇌졸중 모델의 인간 미세아교세포에서 AGE-알부민의 발현양이 현저히 증가하였다.As shown in FIG. 23, the expression level of AGE-albumin was markedly increased in human microglial cells of hypoxia and glucose-deficient stroke models.
2. 뇌졸중 모델의 인간 미세아교세포에서 HMGB1의 농도 변화에 따른 세포내와 배양 배지로 분비된 AGE-알부민의 발현양2. Expression of AGE-Albumin Induced by Intracellular and Culture Media of HMGB1 Levels in Human Microglial Cells of Stroke Model
뇌졸중 시에 가장 많이 뇌에서 분비되어 미세아교세포를 흥분시킨다고 알려진 HMGB1를 뇌졸중 모델의 인간 미세아교세포에 처리하였을 때, 뇌졸중 모델의 인간 미세아교세포에서 HMGB1의 농도(0, 50, 200, 500, 2000ng/㎖) 변화에 따른 AGE-알부민의 합성 및 분비 여부를 확인하기 위하여, 면역조직화학법(IHC), ELISA 및 면역블롯팅을 이용하여 AGE-알부민의 발현양을 측정하였다.HMGB1 concentrations (0, 50, 200, 500, 500) in human microglia cells of the stroke model were treated when HMGB1, which is known to be excreted in the brain during stroke and stimulated microglia, was applied to human microglia cells of the stroke model. 2000ng / mL) In order to confirm the synthesis and secretion of AGE-albumin according to the change, the expression level of AGE-albumin was measured using immunohistochemistry (IHC), ELISA and immunoblotting.
결과는 도 24에 나타내었다.The results are shown in FIG.
도 24에 나타난 바와 같이, 저산소증과 포도당-결핍된 뇌졸중 모델의 인간 미세아교세포에서 HMGB1의 농도가 증가할수록 AGE-알부민의 발현양이 현저히 증가하였다.As shown in FIG. 24, the expression level of AGE-albumin increased significantly as the concentration of HMGB1 increased in human microglia of hypoxia and glucose-deficient stroke models.
3. 뇌졸중 모델의 인간 미세아교세포에서 HMGB1 저해제의 농도 변화에 따른 세포내와 배양 배지로 분비된 AGE-알부민의 발현양3. Expression of AGE-Albumin Induced by Intracellular and Culture Media According to Changes in HMGB1 Inhibitor Concentration in Human Microglial Cells of Stroke Model
뇌졸중 모델의 인간 미세아교세포에 HMGB1 저해제인 글리시리진 (glycyrrhizic acid)을 농도별(0, 50, 200, 500, 2000ng/㎖)로 처리한 다음, ELISA 및 면역블롯팅을 이용하여 AGE-알부민의 발현양을 측정하였다.HMGB1 inhibitor glycyrrhizic acid was treated with different concentrations (0, 50, 200, 500, 2000 ng / ml) in human microglial cells of the stroke model, followed by expression of AGE-albumin using ELISA and immunoblotting. The amount was measured.
결과는 도 25에 나타내었다.The results are shown in FIG.
도 25에 나타난 바와 같이, 저산소증과 포도당-결핍된 뇌졸중 모델의 인간 미세아교세포에서 HMGB1 저해제의 농도가 증가할수록 AGE-알부민의 발현양이 현저히 감소하였다.As shown in FIG. 25, the expression level of AGE-albumin decreased significantly as the concentration of HMGB1 inhibitor was increased in human microglial cells of hypoxia and glucose-deficient stroke models.
실시예 13Example 13 : 뇌졸중 모델의 인간 미세아교세포에서 산화적 스트레스에 의해 AGE-알부민의 합성과 분비 증가 : Increased Synthesis and Secretion of AGE-Albumin by Oxidative Stress in Human Microglial Cells of Stroke Model
뇌졸중 모델의 인간 미세아교세포에서 AGE-알부민의 합성과 분비가 산화적 스트레스에 의한 것인지 확인하기 위하여, 뇌졸중 모델의 인간 미세아교세포에 산화적 스트레스 유도물질인 0~1000μM의 과산화수소(H2O2)를 처리한 후 세포 용해물을 이용하여 면역블롯팅 분석을 수행하였다. 또한, 뇌졸중 모델의 인간 미세아교세포에 항산화제인 아스코르빈산을 처리하여 AGE-알부민의 발현양이 감소하는지 면역블롯팅 분석을 통해 확인하였다.To determine whether AGE-albumin synthesis and secretion are due to oxidative stress in the human microglia of the stroke model, 0-1000 μM hydrogen peroxide (H 2 O 2) , an oxidative stress inducer, in human microglia of the stroke model ) And immunoblotting analysis using cell lysates. In addition, it was confirmed by immunoblotting whether the expression of AGE-albumin decreased by treating ascorbic acid, an antioxidant, to human microglia of the stroke model.
결과는 도 26에 나타내었다.The results are shown in FIG.
도 26에 나타난 바와 같이, 뇌졸중 모델의 인간 미세아교세포에 과산화수소 (H2O2)를 처리한 경우 과산화수소의 농도가 증가할수록 AGE-알부민의 발현양이 증가하였다. 반면, 뇌졸중 모델의 인간 미세아교세포에 항산화제인 아스코르빈산을 처리한 경우 저산소증과 포도당-결핍에 상관없이 AGE-알부민의 발현양이 현저히 감소하였다.As shown in FIG. 26, when hydrogen peroxide (H 2 O 2 ) was treated to human microglial cells of the stroke model, the expression amount of AGE-albumin increased as the concentration of hydrogen peroxide increased. On the other hand, when the ascorbic acid, an antioxidant, was treated to human microglia in the stroke model, the expression level of AGE-albumin was significantly reduced regardless of hypoxia and glucose-deficient.
실시예 14Example 14 : 일차 인간 신경세포에서 AGE-알부민에 의한 신경세포사 유도 Induction of Neuronal Death by AGE-Albumin in Primary Human Neurons
일차 인간 신경세포에서 AGE-알부민이 신경세포사를 유도하는지 확인하기 위하여, 하기와 같은 실험을 수행하였다.To determine whether AGE-albumin induces neuronal death in primary human neurons, the following experiments were performed.
1. 일차 인간 신경세포 배양(primary human neuronal cell culture)Primary human neuronal cell culture
일차 인간 신경세포는 DMEM 배지(Gibco, 10% 소혈청(Gibco), 0.1% 겐타마이신(Gibco), 고농도의 포도당)를 이용하여 5% CO2가 포함되어 있는 37℃ 배양기에서 배양하였다. 배양된 세포에 AGE-알부민(sigma, 10㎍/㎖)을 24시간 동안 처리한 후 하기 실험에 사용하였다.Primary human neurons were cultured in a 37 ° C. incubator containing 5% CO 2 using DMEM medium (Gibco, 10% bovine serum (Gibco), 0.1% gentamycin (Gibco), high glucose concentration). Cultured cells were treated with AGE-albumin (sigma, 10 μg / ml) for 24 hours before use in the following experiments.
2. 면역조직화학과 면역 블롯팅2. Immunohistochemistry and Immunoblotting
인간의 뇌조직으로부터 얻은 일차 인간 신경세포에서 AGE-알부민이 직접적으로 MAPK 신호전달체계를 활성화시키고, Bax의 발현양을 증가시키는지 확인하기 위하여, AGE-알부민을 일차 인간 신경세포에 처리하기 전 또는 후의 세포 용해물을 이용하여 RAGE, ERK1/2, p-ERK1/2, p38, p-p38, SAPK/JNK, p-SAPK/JNK, Bax의 발현양을 면역 블롯팅 방법을 이용하여 관찰하였다.To determine whether AGE-albumin directly activates the MAPK signaling system and increases the expression of Bax in primary human neurons obtained from human brain tissue, prior to treatment with AGE-albumin to primary human neurons or The expression levels of RAGE, ERK1 / 2, p-ERK1 / 2, p38, p-p38, SAPK / JNK, p-SAPK / JNK, and Bax were observed using the subsequent cell lysates using an immunoblotting method.
결과는 도 27에 나타내었다.The results are shown in FIG. 27.
도 27에 나타난 바와 같이, AGE-알부민을 투여한 일차 인간 신경세포에서 RAGE가 증가하였으며, p-ERK1/2, p38, p-p38을 제외한 SAPK/JNK, p-SAPK/JNK가 증가하여 MAPK가 활성화되는 것을 관찰하였을 뿐만 아니라 세포사멸 유발(pro-apoptotic) 단백질인 Bax의 발현양이 증가하는 것을 관찰하였다.As shown in FIG. 27, RAGE was increased in primary human neurons administered AGE-albumin, and SAPK / JNK and p-SAPK / JNK were increased except for p-ERK1 / 2, p38, and p-p38. In addition to being activated, the expression level of Bax, a pro-apoptotic protein, was increased.
3. 세포생존율(MTT assay) 측정 및 신경세포사에 대한 수용성 RAGE(sRAGE)의 보호 효과3. Effect of Soluble RAGE (sRAGE) on MTT Assay and Neuronal Death
AGE-알부민을 투여한 일차 인간 신경세포에서 Bax의 발현양의 증가가 신경세포사를 유도하는지 확인하기 위하여, 일차 인간 신경세포를 96-웰 배양 플레이트에 웰당 2×103 세포로 접종하였다. 80% 융합(confluence)에 도달한 후, 일차 인간 신경세포를 여러 농도(0, 0.1, 1, 10, 50㎍/㎖)의 AGE-알부민으로 처리하였다. 처리 24시간 후, 세포를 PBS로 세척한 다음, 세포생존율을 MTT[3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] assay를 이용하여 540㎚에서 흡광도를 측정하였다.To determine whether increased expression of Bax induces neuronal death in primary human neurons administered AGE-albumin, primary human neurons were seeded at 96 × well culture plates at 2 × 10 3 cells per well. After reaching 80% confluence, primary human neurons were treated with AGE-albumin at various concentrations (0, 0.1, 1, 10, 50 μg / ml). After 24 hours of treatment, the cells were washed with PBS, and the cell viability was measured at 540 nm using MTT [3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyl tetrazolium bromide] assay. It was.
또한, 신경세포사에 대한 수용성 RAGE(sRAGE)의 보호 효과를 확인하기 위하여, 인간 미세아교세포에 sRAGE 단독, AGE-알부민 단독, 또는 sRAGE/AGE-알부민을 함께 처리한 후 540㎚에서 흡광도를 측정하였다.In addition, in order to confirm the protective effect of water-soluble RAGE (sRAGE) on neuronal cell death, absorbance was measured at 540 nm after treatment with sRAGE alone, AGE-albumin alone, or sRAGE / AGE-albumin. .
결과는 도 28에 나타내었다.The results are shown in FIG.
도 28에 나타난 바와 같이, 일차 인간 신경세포에 AGE-알부민을 처리한 경우 AGE-알부민의 농도가 증가할수록 세포생존율이 감소하여 세포사가 유도됨을 확인하였으며(A), 일차 인간 신경세포에 sRAGE와 AGE-알부민을 동시에 처리한 경우 세포생존율이 증가하여 세포사가 감소됨을 확인하였다(B). 따라서, sRAGE가 신경세포사에 대해 보호 효과를 갖는다는 것을 알 수 있다.As shown in FIG. 28, when AGE-albumin was treated to primary human neurons, the cell survival rate decreased as the concentration of AGE-albumin increased (A) and sRAGE and AGE to primary human neurons. -Simultaneous treatment with albumin increased cell viability and reduced cell death (B). Thus, it can be seen that sRAGE has a protective effect against neuronal cell death.
실시예 15Example 15 : 파킨슨병 환자의 뇌조직에서 AGE-알부민의 분포 및 발현 위치 : Distribution and location of AGE-albumin in brain tissue of Parkinson's disease patients
파킨슨병 환자의 뇌조직에서 AGE-알부민의 분포 및 발현 위치를 확인하기 위하여, AGE, 알부민, 미세아교세포 표지자인 Iba1 및 AGE-알부민을 염색하여 면역조직화학(IHC) 실험을 수행하고, 이들의 분포 및 발현 위치를 레이저 공초점 형광현미경으로 관찰하였다.In order to confirm the distribution and position of expression of AGE-albumin in brain tissue of Parkinson's disease patients, immunohistochemistry (IHC) experiments were performed by staining AGE, albumin, and microglia markers Iba1 and AGE-albumin. Distribution and expression site were observed by laser confocal fluorescence microscopy.
결과는 도 29에 나타내었다.The results are shown in FIG.
도 29에 나타난 바와 같이, 파킨슨병 환자의 뇌조직에서 알부민(녹색)과 AGE (적색)가 같은 위치에서 염색되고 넓게 분포되어 있으며, 미세아교세포 표지자인 Iba1도 AGE-알부민과 같은 위치에서 염색되고 넓게 분포되어 있는 것을 관찰하였다.As shown in FIG. 29, albumin (green) and AGE (red) are stained and widely distributed in the brain tissue of Parkinson's disease patients, and microglia marker Iba1 is also stained at the same position as AGE-albumin. It was observed to be widely distributed.
실시예 16Example 16 : 파킨슨병 모델의 인간 미세아교세포에서 α-시누클레인(α-synuclein) 또는 TNF-α의 발현 : Expression of α-synuclein or TNF-α in human microglial cells of Parkinson's disease model
파킨슨병 모델의 인간 미세아교세포에서 α-시누클레인(α-synuclein) 또는 TNF-α의 발현 정도를 확인하기 위하여, 하기와 같은 실험을 수행하였다.In order to confirm the expression level of α-synuclein or TNF-α in human microglial cells of the Parkinson's disease model, the following experiment was performed.
1. 세포배양 및 파킨슨병 세포 모델의 제작1. Cell Culture and Construction of Parkinson's Disease Cell Model
인간 미세아교세포주는 DMEM 배지(Gibco, 10% 소혈청(Gibco), 0.1% 겐타마이신(Gibco), 고농도의 포도당)를 이용하여 5% CO2가 포함되어 있는 37℃ 배양기에서 배양하였다. 파킨슨병 세포 모델을 만들기 위하여, 준비된 미세아교세포주에 파킨슨병 유발물질인 로테논(rotenone; Sigma, 1nM)을 10일 또는 6-히드록시도파민(6-hydroxydopamine; 6-OHDA)을 24시간 동안 처리한 후 세포들을 추출하여 실험에 사용하였다.Human microglial cells were cultured in a 37 ° C. incubator containing 5% CO 2 using DMEM medium (Gibco, 10% bovine serum (Gibco), 0.1% gentamycin (Gibco), high concentration of glucose). To prepare a Parkinson's disease cell model, the prepared microglia cells were treated with Parkinson's disease-producing rotenone (Sigma, 1nM) for 10 days or 6-hydroxydopamine (6-hydroxyda) for 24 hours. The cells were then extracted and used for the experiment.
2. 파킨슨병 모델의 인간 미세아교세포에서 α-시누클레인(α-synuclein) 또는 TNF-α의 발현 확인2. Confirmation of α-synuclein or TNF-α expression in human microglial cells of Parkinson's disease model
파킨슨병 환자의 뇌조직에서 α-시누클레인 또는 TNF-α이 높은 수준으로 존재한다고 알려져 있다. 따라서, 인간 미세아교세포에 파킨슨병 유발물질인 로테논 (rotenone) 또는 6-히드록시도파민(6-hydroxydopamine; 6-OHDA)을 처리한 후 α-시누클레인 또는 TNF-α의 발현 정도를 PCR과 면역블롯팅 분석을 통해 확인하였다.It is known that α-synuclein or TNF-α is present at high levels in brain tissue of Parkinson's disease patients. Therefore, after treatment with Parkinson's disease-producing rotenone or 6-hydroxydopamine (6-OHDA) in human microglial cells, the expression level of α-synuclein or TNF-α was determined by PCR. It was confirmed by immunoblotting analysis.
결과는 도 30에 나타내었다.The results are shown in FIG.
도 30에 나타난 바와 같이, 인간 미세아교세포에 파킨슨병 유발물질인 로테논 또는 6-히드록시도파민을 처리한 후 α-시누클레인 또는 TNF-α의 발현이 증가하는 것을 확인하였다. 따라서, 인간 미세아교세포가 활성화됨을 알 수 있다.As shown in FIG. 30, it was confirmed that the expression of α-synuclein or TNF-α was increased after treatment with Parkinson's disease-producing rotenone or 6-hydroxydopamine in human microglia. Thus, it can be seen that human microglial cells are activated.
실시예 17Example 17 : 파킨슨병 모델의 인간 미세아교세포에서 AGE-알부민의 합성 및 분비 : Synthesis and Secretion of AGE-Albumin in Human Microglial Cells of Parkinson's Disease Model
인간 미세아교세포에 0~100nM의 로테논을 처리한 후 세포 용해물 및 세포 배양물을 이용하여 AGE-알부민의 합성 및 분비 여부를, ELISA 및 면역블롯팅 분석을 통해 확인하였다.After treating human microglial cells with 0 to 100 nM rotenone, the synthesis and secretion of AGE-albumin using cell lysates and cell cultures were confirmed by ELISA and immunoblotting analysis.
결과는 도 31에 나타내었다.The results are shown in FIG.
도 31에 나타난 바와 같이, 인간 미세아교세포에 1nM의 로테논을 처리하였을 때 AGE-알부민의 합성 및 분비가 증가하는 것을 확인하였다.As shown in FIG. 31, it was confirmed that the synthesis and secretion of AGE-albumin increased when human microglial cells were treated with 1 nM rotenone.
실시예 18Example 18 : 파킨슨병 모델의 인간 미세아교세포에서 산화적 스트레스에 의해 α-시누클레인 또는 AGE-알부민의 합성과 분비 증가 : Synthesis and Secretion of α-synuclein or AGE-Albumin by Oxidative Stress in Human Microglial Cells of Parkinson's Disease Model
파킨슨병 모델의 인간 미세아교세포에서 α-시누클레인 또는 AGE-알부민의 합성과 분비가 산화적 스트레스에 의한 것인지 확인하기 위하여, 인간 미세아교세포에 파킨슨병 유발물질인 로테논을 처리한 다음 산화적 스트레스 유도물질인 0~1000μM의 과산화수소(H2O2)를 처리한 후 세포 용해물을 이용하여 면역블롯팅 분석을 수행하였다. 또한, 인간 미세아교세포에 파킨슨병 유발물질인 로테논을 처리한 다음 α-시누클레인 저해제인 사이토칼라신 D(cytochalasin D) 또는 항산화제인 아스코르빈산을 처리하여 α-시누클레인 또는 AGE-알부민의 발현양이 감소하는지 면역블롯팅 분석을 통해 확인하였다.In order to determine whether the synthesis and secretion of α-synuclein or AGE-albumin in human microglial cells of Parkinson's disease model is caused by oxidative stress, human microglial cells were treated with rotenone, a Parkinson's disease-causing substance, and then oxidative Immunoblotting analysis was performed using cell lysates after treatment with hydrogen peroxide (H 2 O 2 ), a stress-inducing substance, 0-1000 μM. In addition, human microglia treated with Parkinson's disease-causing rotenone and then treated with α-synuclein inhibitor cytocalasin D or antioxidant ascorbic acid to treat α-synuclein or AGE-albumin. It was confirmed by immunoblotting analysis whether the amount of expression is reduced.
결과는 도 32에 나타내었다.The results are shown in FIG.
도 32에 나타난 바와 같이, 인간 미세아교세포에 로테논을 처리한 다음 과산화수소(H2O2)를 처리한 경우 과산화수소의 농도가 증가할수록 α-시누클레인과 AGE-알부민의 발현양이 증가하였다. 반면, 인간 미세아교세포에 로테논을 처리한 다음 α-시누클레인 저해제인 사이토칼라신 D 또는 항산화제인 아스코르빈산을 처리한 경우 α-시누클레인과 AGE-알부민의 발현양이 현저히 감소하였다.As shown in FIG. 32, when the human microglia treated with rotenone and then treated with hydrogen peroxide (H 2 O 2 ), the expression levels of α-synuclein and AGE-albumin increased as the concentration of hydrogen peroxide increased. On the other hand, when the human microglia were treated with rotenone and then treated with α-synuclein inhibitor cytocalin D or the antioxidant ascorbic acid, the expression levels of α-synuclein and AGE-albumin significantly decreased.
실시예 19Example 19 : 도파민신경세포에서 AGE-알부민에 의한 신경세포사 유도 Induction of Neuronal Cell Death by AGE-Albumin in Dopamine Neurons
도파민신경세포에서 AGE-알부민이 신경세포사를 유도하는지 확인하기 위하여, 하기와 같은 실험을 수행하였다.To determine whether AGE-albumin induces neuronal death in dopamine neurons, the following experiment was performed.
1. 도파민신경세포 배양(dopamine neuronal cell culture)1. Dopamine neuronal cell culture
도파민신경세포는 DMEM 배지(Gibco, 10% 소혈청(Gibco), 0.1% 겐타마이신 (Gibco), 고농도의 포도당)를 이용하여 5% CO2가 포함되어 있는 37℃ 배양기에서 배양하였다. 배양된 세포에 AGE-알부민(sigma, 10㎍/㎖)을 24시간 동안 처리한 후 하기 실험에 사용하였다.Dopamine neurons were cultured in a 37 ° C. incubator containing 5% CO 2 using DMEM medium (Gibco, 10% bovine serum (Gibco), 0.1% gentamycin (Gibco), high concentration of glucose). Cultured cells were treated with AGE-albumin (sigma, 10 μg / ml) for 24 hours before use in the following experiments.
2. 면역조직화학과 면역 블롯팅2. Immunohistochemistry and Immunoblotting
인간의 뇌조직으로부터 얻은 도파민신경세포에서 AGE-알부민이 직접적으로 MAPK 신호전달체계를 활성화시키고, Bax의 발현양을 증가시키는지 확인하기 위하여, 도파민신경세포에 sRAGE를 처리하거나 처리하지 않은 다음, AGE-알부민을 도파민신경세포에 처리하기 전 또는 후의 세포 용해물을 이용하여 RAGE, Bax, SAPK/JNK, pSAPK/JNK, p38, ERK1/2, pERK1/2의 발현양을 면역 블롯팅 방법을 이용하여 관찰하였다. 또한, 신경세포사에 대한 수용성 RAGE(sRAGE)의 보호 효과를 확인하기 위하여, 540㎚에서 흡광도를 측정하였다.To determine whether AGE-albumin directly activates the MAPK signaling system and increases the expression of Bax in dopamine neurons obtained from human brain tissue, sRAGE was treated with or without sRAGE in the dopamine neurons. Expression of RAGE, Bax, SAPK / JNK, pSAPK / JNK, p38, ERK1 / 2, pERK1 / 2 using immunoblotting using cell lysate before or after treatment with albumin to dopamine neurons Observed. In addition, the absorbance was measured at 540 nm to confirm the protective effect of water-soluble RAGE (sRAGE) on neuronal cell death.
결과는 도 33에 나타내었다.The results are shown in FIG. 33.
도 33에 나타난 바와 같이, 도파민신경세포에 sRAGE를 처리하지 않고 AGE-알부민을 투여한 경우 RAGE가 증가하였으며, p-ERK1/2, p38, p-p38을 제외한 SAPK/JNK, pSAPK/JNK가 증가하여 MAPK가 활성화되는 것을 관찰하였을 뿐만 아니라 세포사멸 유발(pro-apoptotic) 단백질인 Bax의 발현양이 증가하는 것을 관찰하였다. 또한, 도파민신경세포에 sRAGE를 처리한 다음 AGE-알부민을 투여한 경우 RAGE, SAPK/JNK, pSAPK/JNK, Bax의 발현양은 대조군과 유사하게 나타났다. 따라서, sRAGE가 신경세포사에 대해 보호 효과를 갖는다는 것을 알 수 있다.As shown in FIG. 33, when AGE-albumin was administered to dopamine neurons without sRAGE treatment, RAGE was increased, but SAPK / JNK and pSAPK / JNK were increased except for p-ERK1 / 2, p38, and p-p38. Not only did MAPK be activated, but the expression of Bax, a pro-apoptotic protein, was increased. In addition, when dopamine neurons were treated with sRAGE followed by AGE-albumin, the expression levels of RAGE, SAPK / JNK, pSAPK / JNK, and Bax were similar to those of the control group. Thus, it can be seen that sRAGE has a protective effect against neuronal cell death.
실시예 20Example 20 : 로테논-매개된 신경세포사에 대한 수용성 RAGE(sRAGE)의 보호 효과 :  Protective Effect of Soluble RAGE (sRAGE) on Rotenone-Mediated Neuronal Death in vivoin vivo 실험 Experiment
로테논-매개된 신경세포사에 대한 수용성 RAGE(sRAGE)의 보호 효과를 확인하기 위하여, 마우스의 뇌조직에 로테논 단독 또는 로테논/sRAGE을 주입한 후 in vivo 실험을 수행하였다.In order to confirm the protective effect of water-soluble RAGE (sRAGE) on rotenone-mediated neuronal cell death, in vivo experiments were performed after injection of rotenone alone or rotenone / sRAGE into mouse brain tissue.
1. 동물모델1. Animal Model
실험동물로 체중 20~25g의 CBL57/bL6 마우스를 사용하였다. 마우스를 12시간 명암 주기로 유지시켰으며, 식량과 물을 자유로이 섭취시켰고, 적어도 사용하기 일주일 전에 순응시켰다. 모든 동물 실험은 실험동물사용관리위원회(IACUC; Institutional Animal Care and Use Committees)에 의해 승인되고 인도적으로 수행되었다.CBL57 / bL6 mice weighing 20-25 g were used as experimental animals. Mice were maintained on a 12 hour light and dark cycle, freely fed food and water, and acclimatized at least one week before use. All animal experiments were approved and humanely performed by the Institutional Animal Care and Use Committees (IACUC).
로테논을 마우스에 한달 동안 경구 투여하여 마우스에 파킨슨병을 유발시켜 파킨슨병 동물 모델을 제작하였다. 파킨슨병 동물 모델을 수술 전에 케타민(0.75 ㎎/㎏ 체중)과 럼푼(0.2　㎎/㎏ 체중)으로 마취시켰다. in vivo 처리를 위해, 인산염 완충용액(PBS), sRAGE(10ng/㎕)를 1mM의 농도로 멸균수에 용해시키고, 사용할 때까지 4℃로 유지하였다. 파킨슨병 동물 모델의 뇌를 뇌심부고정장치(stereotaxic instrument)를 이용하여 고정한 후, 두피 피부의 중앙을 절개하였다. 두개골 브레그마(bregma)에서 생물학적 전기 드릴로 구멍을 뚫고 (뒤쪽으로, 0.3㎜; 측면으로, 2㎜), 표적 면적(깊이, 2.5㎜)에 도달할 때까지 10-㎕ 해밀톤 주사기의 바늘(26 게이지)을 수직으로 하강시켰다. 3㎕의 sRAGE을 자동미세주입기로 분당 1㎕의 속도로 파킨슨병 동물 모델의 내후각뇌피질(entorhinal cortex, EC)에 천천히 주입하였다. 그 다음, 주사기를 천천히 제거하고 수술 창상(surgical wounds)을 상처봉합용 클립으로 봉합하고 항생제로 국소 처리하였다. 양성대조군으로는 정상 마우스의 내후각뇌피질에 PBS를 주입하였다.Parkinson's disease animal models were prepared by inducing oral administration of rotenone to mice for one month to cause Parkinson's disease in mice. Parkinson's disease animal model was anesthetized with ketamine (0.75 mg / kg body weight) and lumpoon (0.2 mg / kg body weight) prior to surgery. For in vivo treatment, phosphate buffer (PBS), sRAGE (10 ng / μl) was dissolved in sterile water at a concentration of 1 mM and kept at 4 ° C. until use. The brain of the Parkinson's disease animal model was fixed using a stereotaxic instrument, and then the center of the scalp skin was incised. In a cranial bregma, a needle of a 10-μL Hamilton syringe was drilled with a biological electric drill (backward, 0.3 mm; laterally, 2 mm) and reached the target area (depth, 2.5 mm) (26). Gauge) was lowered vertically. 3 μl of sRAGE was slowly injected into the olfactory cortex (EC) of the Parkinson's disease animal model at a rate of 1 μl per minute with an automatic microinjector. The syringe was then slowly removed and surgical wounds closed with a wound closure clip and topically treated with antibiotics. As a positive control, PBS was injected into the olfactory cortex of normal mice.
대부분의 마우스는 sRAGE의 주사 후 1주일과 한달 동안 사육하였다. 사육 동안에도 로테논은 계속 경구투여하였다. 사육 후, 모든 마우스를 동일한 방법으로 다시 마취시키고, 18℃에서 100~200㎖의 헤파린이 첨가된 증류수(heparinized saline)로 심장을 통해 관류하고, 이어서 0.1M 소듐 포스페이트 완충용액(pH 7.4) 내 4% 파라포름알데히드-리신 페리오데이트(paraformaldehyde-lysine periodate)의 400㎖로 관류시켰다. 뇌를 제거하고 동일한 고정액에 넣은 다음 4℃에서 4시간 동안 고정하고, 20% 수크로오스를 함유하는 차가운 0.1M 인산염완충용액(PBS)으로 옮겼다. 뇌를 동결박편제작기(freezing microtome)로 10㎛ 두께로 횡단면을 절개하고, 사용할 때까지 -80℃에서 저장하였다.Most mice were bred for one week and one month after injection of sRAGE. Rotenone continued to be administered orally during breeding. After breeding, all mice were anesthetized again in the same manner, perfused through the heart with heparinized saline with 100-200 ml of heparin at 18 ° C., followed by 4 in 0.1 M sodium phosphate buffer (pH 7.4). Perfusion was carried out with 400 ml of% paraformaldehyde-lysine periodate. The brain was removed, placed in the same fixative, fixed at 4 ° C. for 4 hours, and transferred to cold 0.1 M phosphate buffer solution (PBS) containing 20% sucrose. Brains were incised with a 10 μm thick cross section with a freezing microtome and stored at −80 ° C. until use.
2. 마우스의 뇌조직에 있는 신경세포의 수2. Number of nerve cells in brain tissue of mouse
마우스의 뇌조직에 PBS, 로테논, 로테논/sRAGE을 처리하고 1주일과 한달 후에 마우스의 뇌조직에 있는 신경세포의 수를 크레실 바이올렛(cresyl violet)으로 염색한 후 현미경으로 관찰하였다.PBS, rotenone, and rotenone / sRAGE were treated in mouse brain tissues, and one week and a month later, the number of neurons in the brain tissues of mice was stained with cresyl violet and observed under a microscope.
결과는 도 34에 나타내었다.The results are shown in FIG.
도 34에 나타난 바와 같이, 마우스의 뇌조직에 로테논을 처리한 경우 로테논을 처리하지 않은 경우에 비해 세포사가 많이 일어나는 것을 확인하였다. 또한, 마우스의 뇌조직에 로테논/sRAGE을 처리한 경우 세포사가 회복되는 것을 관찰하였으며, PBS로 처리하였을 때보다 신경세포의 수가 급증하였다. 특히, sRAGE을 주입한 부분(오른쪽 화살표)이 주입하지 않은 부분(왼쪽 화살표)보다 더 많이 회복되는 것을 확인하였으며, 흑색질(substantia nigra) 부분이 더 많이 회복되었다.As shown in FIG. 34, when the rotenone was treated in the brain tissue of the mouse, it was confirmed that a lot of cell death occurred compared to the case where the rotenone was not treated. In addition, it was observed that cell death was recovered when rotenone / sRAGE was treated in the brain tissue of the mouse, and the number of neurons increased rapidly than when treated with PBS. In particular, it was confirmed that the portion injected with sRAGE (right arrow) recovered more than the portion not injected (left arrow), and the part of black matter (substantia nigra) recovered more.
3. 마우스의 뇌조직에서 AGE-알부민의 분포 및 발현 위치3. Distribution and Expression Location of AGE-Albumin in Brain Tissues of Mice
마우스의 뇌조직에 PBS, 로테논, 로테논/sRAGE을 처리하고 1주일과 한달 후에 마우스의 뇌조직에 있는 AGE-알부민, RAGE, Bax의 분포 및 발현 위치를 형광면역염색한 후 레이저 공초점 형광현미경으로 관찰하였다.After treatment with PBS, rotenone, and rotenone / sRAGE in the brain tissues of the mouse, fluorescence immunostaining of the distribution and expression sites of AGE-albumin, RAGE, and Bax in the brain tissues of the mouse was observed after laser confocal fluorescence. It was observed under a microscope.
결과는 도 35에 나타내었다.The results are shown in FIG. 35.
도 35에 나타난 바와 같이, AGE, 알부민, 미세아교세포 표지자인 Iba1, AGE-알부민, RAGE, Bax의 발현양은 로테논을 경구투여한 마우스의 뇌조직에서 증가하였으나, 로테논/sRAGE를 주입한 랫트의 뇌조직에서는 감소함을 확인하였다.As shown in FIG. 35, the expression levels of AGE, albumin, and microglia markers Iba1, AGE-albumin, RAGE, and Bax were increased in brain tissues of mice administered orally with rotenone, but were injected with rotenone / sRAGE. It was confirmed that the brain tissue of the decrease.
실시예 21Example 21 : 류마티스 관절염 모델의 대식세포에서 AGE-알부민의 합성 및 분비 : Synthesis and Secretion of AGE-Albumin in Macrophages in Rheumatoid Arthritis Model
β2-마이크로글로불린(β2-microglobulin)은 류마티스 관절염 환자의 연골에서 풍부하다고 알려져 있다. 그러나, 연골에서 β2-마이크로글로불린의 정확한 역할과 숙주 대식세포 또는 연골세포와의 관련성은 아직까지 밝혀지지 않았다. 따라서, 본 실험에서는 β2-마이크로글로불린이 대식세포의 활성과 대식세포 내에서 TNF-α와 IL-1β의 합성을 유도하는지에 대해서 실험을 수행하였다.β2-microglobulin is known to be abundant in cartilage in patients with rheumatoid arthritis. However, the exact role of β2-microglobulin in cartilage and its association with host macrophages or chondrocytes is not yet known. Therefore, we conducted experiments on whether β2-microglobulin induces macrophage activity and synthesis of TNF-α and IL-1β in macrophages.
1. 세포배양 및 관절염 세포 모델의 제작1. Cell Culture and Construction of Arthritis Cell Model
인간 림프종에서 채취한 대식세포주(U937)는 RPMI 1640 배지(Thermo, 10% 소혈청(Gibco), 0.1% 겐타마이신(Gibco), 고농도의 포도당)를 이용하여 5% CO2가 포함되어 있는 37℃ 배양기에서 배양하였다. 관절염 세포 모델을 만들기 위하여, 준비된 인간 대식세포주(U937)에 β2-마이크로글로불린(Sigma, 50㎍/㎖)을 24시간 동안 처리한 후 세포들을 추출하여 실험에 사용하였다.Macrophage line (U937) from human lymphoma was 37 ° C containing 5% CO 2 using RPMI 1640 medium (Thermo, 10% bovine serum (Gibco), 0.1% gentamicin (Gibco), high concentration of glucose). Cultured in the incubator. In order to make an arthritis cell model, the prepared human macrophage line (U937) was treated with β2-microglobulin (Sigma, 50 µg / ml) for 24 hours, and then cells were extracted and used for the experiment.
2. 류마티스 관절염 모델의 대식세포에서 TNF-α, IL-1β, AGE-알부민의 발현 확인2. Expression of TNF-α, IL-1β, and AGE-Albumin in Macrophage Cells in Rheumatoid Arthritis Model
인간 대식세포주(U937)에 β2-마이크로글로불린(0㎍, 12.5㎍, 25㎍, 50㎍)을 처리한 후 세포 용해물 또는 세포 배양물을 이용하여 TNF-α와 IL-1β의 발현 정도와 AGE-알부민의 합성 및 분비 여부를, 면역블롯팅 및 ELISA를 통해 확인하였다.Treatment of β2-microglobulin (0 μg, 12.5 μg, 25 μg, 50 μg) with human macrophage line (U937), followed by expression of TNF-α and IL-1β using cell lysates or cell cultures Synthesis and secretion of albumin was confirmed by immunoblotting and ELISA.
결과는 도 36에 나타내었다.The results are shown in FIG. 36.
도 36에 나타난 바와 같이, 인간 대식세포주(U937)에 β2-마이크로글로불린을 처리한 후 TNF-α, IL-1β 및 AGE-알부민의 발현이 증가하였으며, β2-마이크로글로불린의 농도가 증가할수록 세포밖으로 분비되는 AGE-알부민의 양이 증가하는 것을 확인하였다.As shown in FIG. 36, the expression of TNF-α, IL-1β and AGE-albumin increased after treatment with β2-microglobulin in human macrophage line (U937), and as the concentration of β2-microglobulin increased, It was confirmed that the amount of AGE-albumin secreted increases.
실시예 22Example 22 : 연골세포에서 AGE-알부민에 의한 연골세포사 유도 Induction of Chondrocyte Death by AGE-Albumin in Chondrocytes.
연골세포에서 AGE-알부민이 연골세포사를 유도하는지 확인하기 위하여, 하기와 같은 실험을 수행하였다.To determine whether AGE-albumin induces chondrocyte death in chondrocytes, the following experiment was performed.
1. 연골세포 배양(chondrocyte cell culture)Chondrocyte Cell Culture
인간 연골세포는 연골세포 성장배지(Promo cell)와 연골세포 성장배지 supplementMix(Promo cell)를 혼합한 배지를 사용하여 5% CO2가 포함되어 있는 37℃ 배양기에서 배양하였다.Human chondrocytes were cultured in a 37 ℃ incubator containing 5% CO 2 using a medium containing chondrocyte growth medium (Promo cell) and chondrocyte growth medium supplementMix (Promo cell).
2. 세포생존율(MTT assay) 측정2. Measurement of MTT assay
배양된 연골세포를 96-웰 배양 플레이트에 웰당 2×103 세포로 접종하였다. 80% 융합(confluence)에 도달한 후, 배양된 연골세포를 AGE-알부민(sigma, 10㎍/㎖)으로 처리하였다. 처리 24시간 후, 세포를 PBS로 세척한 다음, MTT[3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] assay를 이용하여 540㎚에서 흡광도를 측정하였다.Cultured chondrocytes were seeded at 96 × well culture plates at 2 × 10 3 cells per well. After reaching 80% confluence, the cultured chondrocytes were treated with AGE-albumin (sigma, 10 μg / ml). After 24 hours of treatment, the cells were washed with PBS, and then absorbance was measured at 540 nm using MTT [3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyl tetrazolium bromide] assay.
결과는 도 37에 나타내었다.The results are shown in FIG.
도 37에 나타난 바와 같이, 연골세포에 AGE-알부민을 처리한 후 연골세포 수가 감소하였다. 그러나, 연골세포에 sRAGE와 AGE-알부민을 동시에 처리한 경우 연골세포 수가 대조군과 유사하게 나타났다. 따라서, sRAGE가 연골세포사에 대해 보호 효과를 갖는다는 것을 알 수 있다.As shown in FIG. 37, the chondrocytes were decreased after AGE-albumin treatment. However, when sRAGE and AGE-albumin were simultaneously treated with chondrocytes, the chondrocyte counts were similar to those of the control group. Thus, it can be seen that sRAGE has a protective effect against chondrocyte death.
실시예 23Example 23 : AGE-알부민 합성 저해제의 후보물질 선별 : Screening candidates for AGE-albumin synthesis inhibitor
인간 미세아교세포에서 AGE-알부민의 합성을 저해하는 물질을 발굴하기 위하여, LOPAC(sigma)으로부터 후보물질을 선별하는 실험을 하기와 같이 수행하였다.In order to identify substances that inhibit the synthesis of AGE-albumin in human microglia, experiments were performed to select candidates from LOPAC (sigma) as follows.
1. 알츠하이머병 모델의 인간 미세아교세포에서 AGE-알부민의 합성 저해제의 선별Screening of AGE-Albumin Synthesis Inhibitors in Human Microglial Cells of Alzheimer's Disease Model
인간 미세아교세포를 96-웰 플레이트에 1×104 /200㎕의 농도로 분주하고 24시간 동안 배양시켰다. 플레이트에 배양된 세포에 Aβ1-42(sigma)를 2μM로 6시간 동안 처리한 후, LOPAC(sigma)에 포함되어 있는 1280종의 화합물(5μM)을 24시간 동안 처리하였다. 배양이 끝난 세포들을 100% 메탄올로 고정시킨 후, AGE-알부민 항체(1:10000, abcam)를 이용하여 반응시켰다. 그 다음, 퍼옥시다제가 포함되어 있는 이차 항체(1:5000, Vector)와 반응을 시킨 후 TMB(sigma)로 발색시키고, ELISA 리더로 450㎚에서 흡광도를 측정하였다. 그리고, AGE-알부민의 합성을 저해하는 후보물질을 선별하였다.Dispensed at a concentration of 1 × 10 4 / 200㎕ human microglial cells in a 96-well plate and incubated for 24 hours. Cells cultured on the plate were treated with Aβ1-42 (sigma) at 2 μM for 6 hours, and then 1280 compounds (5 μM) included in LOPAC (sigma) were treated for 24 hours. After incubation, the cells were fixed with 100% methanol and reacted with AGE-albumin antibody (1: 10000, abcam). Then, the reaction was performed with a secondary antibody (1: 5000, Vector) containing peroxidase, followed by color development with TMB (sigma), and absorbance at 450 nm was measured with an ELISA reader. Then, candidate substances that inhibit the synthesis of AGE-albumin were selected.
알츠하이머병 모델의 인간 미세아교세포에서 AGE-알부민의 합성을 저해하는 후보물질은 LOPAC 1280종의 화합물 중에서 총 42종의 화합물이 선별되었으며, 선별된 후보물질은 하기 표 1에 나타내었고, 후보물질의 ELISA 측정결과는 도 38에 나타내었다.Candidates that inhibit the synthesis of AGE-albumin in human microglia of the Alzheimer's disease model were selected from 42 compounds of LOPAC 1280, and the selected candidates are shown in Table 1 below. ELISA measurement results are shown in FIG.
[표 1]TABLE 1
Figure PCTKR2011003147-appb-I000001
Figure PCTKR2011003147-appb-I000001
도 38에 나타난 바와 같이, 알츠하이머병 모델의 인간 미세아교세포에서 후보물질의 AGE-알부민의 합성 저해 활성이 대조군과 유사하게 나타남을 확인하였다.As shown in Figure 38, it was confirmed that the inhibitory activity of the synthesis of AGE-albumin of the candidate in the human microglia of the Alzheimer's disease model is similar to the control.
2. 파킨슨병 모델의 인간 미세아교세포에서 AGE-알부민의 합성 저해제의 선별2. Screening of AGE-Albumin Synthesis Inhibitors in Human Microglial Cells of Parkinson's Disease Model
인간 미세아교세포를 96-웰 플레이트에 1×104 /200㎕의 농도로 분주하고 24시간 동안 배양시켰다. 플레이트에 배양된 세포에, 로테논(sigma, 1nM)을 10일 동안 처리한 도파민신경세포에서 추출한 배지를 24시간 동안 처리한 후, LOPAC (sigma)에 포함되어 있는 1280종의 화합물(5μM)을 24시간 동안 처리하였다. 배양이 끝난 세포들을 100% 메탄올로 고정시킨 후, AGE-알부민 항체(1:10000, abcam)를 이용하여 반응시켰다. 그 다음, 퍼옥시다제가 포함되어 있는 이차 항체(1:5000, Vector)와 반응을 시킨 후 TMB(sigma)로 발색시키고, ELISA 리더로 450㎚에서 흡광도를 측정하였다. 그리고, AGE-알부민의 합성을 저해하는 후보물질을 선별하였다.Dispensed at a concentration of 1 × 10 4 / 200㎕ human microglial cells in a 96-well plate and incubated for 24 hours. The cells cultured on the plate were treated with a medium extracted from dopamine neurons treated with rotenone (sigma, 1 nM) for 10 days for 24 hours, and then 1280 kinds of compounds (5 μM) contained in LOPAC (sigma) were treated. Treatment was done for 24 hours. After incubation, the cells were fixed with 100% methanol and reacted with AGE-albumin antibody (1: 10000, abcam). Then, the reaction was performed with a secondary antibody (1: 5000, Vector) containing peroxidase, followed by color development with TMB (sigma), and absorbance at 450 nm was measured with an ELISA reader. Then, candidate substances that inhibit the synthesis of AGE-albumin were selected.
파킨슨병 모델의 인간 미세아교세포에서 AGE-알부민의 합성을 저해하는 후보물질은 LOPAC 1280종의 화합물 중에서 총 9종의 화합물이 선별되었으며, 선별된 후보물질은 하기 표 2에 나타내었고, 후보물질의 ELISA 측정결과는 도 39에 나타내었다.As a candidate for inhibiting the synthesis of AGE-albumin in human microglial cells of Parkinson's disease model, a total of nine compounds were selected from 1280 compounds of LOPAC, and the selected candidates are shown in Table 2 below. ELISA measurement results are shown in FIG. 39.
[표 2]TABLE 2
Figure PCTKR2011003147-appb-I000002
Figure PCTKR2011003147-appb-I000002
도 39에 나타난 바와 같이, 파킨슨병 모델의 인간 미세아교세포에서 후보물질의 AGE-알부민의 합성 저해 활성이 대조군과 유사하게 나타남을 확인하였다.As shown in Figure 39, it was confirmed that the inhibitory activity of the synthesis of AGE-albumin of the candidate in the human microglia cells of the Parkinson's disease model similar to the control.
실시예 24Example 24 : AGE-알부민의 합성 저해제가 로테논-매개된 신경세포사에 미치는 영향  : Effect of AGE-Albumin Synthesis Inhibitor on Rotenone-Mediated Neuronal Death
AGE-알부민의 합성 저해제가 로테논-매개된 신경세포사에 미치는 영향을 확인하기 위하여, 마우스의 뇌조직에 상기 실시예 23에서 선별된 AGE-알부민의 합성 저해제 중 C-20(세파클러), C-21(세팔로틴 나트륨)을 주입한 후 실시예 20과 동일하게 실험을 수행하여, 마우스의 뇌조직에 있는 신경세포의 수와 AGE-알부민, RAGE, Bax의 분포 및 발현 위치를 관찰하였다.In order to confirm the effect of AGE-albumin synthesis inhibitors on rotenone-mediated neuronal cell death, C-20 (sepacller), C, among the inhibitors of the synthesis of AGE-albumin selected in Example 23 on the brain tissues of mice Experiments were carried out in the same manner as in Example 20 after injecting -21 (cephalotin sodium) to observe the distribution of AGE-albumin, RAGE, and Bax, and the number of neurons in the brain tissue of the mouse.
마우스의 뇌조직에 PBS, 로테논, 로테논/세파클러, 로테논/세팔로틴 나트륨을 처리하고 1주일과 한달 후에 마우스의 뇌조직에 있는 신경세포의 수를 크레실 바이올렛(cresyl violet)으로 염색한 후 현미경으로 관찰한 결과는 도 40에 나타내었고, 마우스의 뇌조직에 PBS, 로테논, 로테논/세파클러, 로테논/세팔로틴 나트륨을 처리하고 1주일과 한달 후에 마우스의 뇌조직에 있는 AGE-알부민, RAGE, Bax의 분포 및 발현 위치를 형광면역염색한 후 레이저 공초점 형광현미경으로 관찰한 결과는 도 41에 나타내었다.After 1 week and a month of treatment with PBS, rotenone, rotenone / sephackler, and rotenone / cephalotin sodium in the brain tissue of the mouse, the number of neurons in the brain tissue of the mouse was changed to cresyl violet. After staining, the results of observation under a microscope are shown in FIG. 40, and the brain tissues of the mice were treated with PBS, rotenone, rotenone / sephackler, and rotenone / cephalotin sodium. 41 shows the results of fluorescence immunostaining of AGE-albumin, RAGE, and Bax in fluorescence immunostaining and laser confocal fluorescence microscopy.
결과는 도 40에 나타내었다.The results are shown in FIG. 40.
도 40에 나타난 바와 같이, 마우스의 뇌조직에 로테논을 처리한 경우 로테논을 처리하지 않은 경우에 비해 세포사가 많이 일어나는 것을 확인하였다. 또한, 마우스의 뇌조직에 로테논/세파클러, 로테논/세팔로틴 나트륨을 처리한 경우 세포사가 회복되는 것을 관찰하였으며, PBS로 처리하였을 때보다 신경세포의 수가 급증하였다. 특히, 세파클러, 세팔로틴 나트륨을 주입한 부분(오른쪽 화살표)이 주입하지 않은 부분(왼쪽 화살표)보다 더 많이 회복되는 것을 확인하였으며, 흑색질 (substantia nigra) 부분이 더 많이 회복되었다. 또한, 세팔로틴 나트륨이 세파클러보다 효율이 더 좋음을 확인하였다.As shown in Figure 40, when treated with the rotenone in the brain tissue of the mouse was confirmed that a lot of cell death occurs compared to the case without the treatment with rotenone. In addition, it was observed that the cell death was recovered when the mouse brain tissues were treated with rotenone / cephacller and rotenone / cephalotin sodium. In particular, it was confirmed that the portion injected with cephacller and cephalotin sodium (right arrow) recovered more than the portion not injected (left arrow), and the black portion (substantia nigra) recovered more. In addition, cephalotin sodium was found to be more efficient than Sephacller.
또한 도 41에 나타난 바와 같이, AGE, 알부민, 미세아교세포 표지자인 Iba1, AGE-알부민, RAGE, Bax의 발현양은 로테논을 경구투여한 마우스의 뇌조직에서 증가하였으나, 로테논/세파클러, 로테논/세팔로틴 나트륨을 주입한 랫트의 뇌조직에서는 감소함을 확인하였다.As shown in FIG. 41, the expression levels of AGE, albumin, and microglia markers Iba1, AGE-albumin, RAGE, and Bax were increased in the brain tissues of rotenone-orally administered mice, but rotenone / sepachlor, and rote. It was confirmed that the brain tissue of rats injected with non / cephalotin sodium decreased.
하기에 본 발명의 조성물을 위한 제제예를 예시한다.Examples of preparations for the compositions of the present invention are illustrated below.
제제예 1Formulation Example 1 : 산제의 제조 : Preparation of powder
AGE-알부민의 합성 저해제 0.1 g0.1 g of inhibitor of synthesis of AGE-albumin
유당 1.5 gLactose 1.5 g
탈크 0.5 gTalc 0.5 g
상기의 성분들을 혼합하고 기밀포에 충진하여 산제를 제조하였다.The above ingredients were mixed and filled in airtight cloth to prepare a powder.
제제예 2Formulation Example 2 : 정제의 제조 : Preparation of Tablet
AGE-알부민의 합성 저해제 0.1 g0.1 g of inhibitor of synthesis of AGE-albumin
락토오스 7.9 gLactose 7.9 g
결정성 셀룰로오스 1.5 g1.5 g of crystalline cellulose
마그네슘 스테아레이트 0.5 g0.5 g of magnesium stearate
상기의 성분들을 혼합한 후 직타법(direct tableting method)으로 정제를 제조하였다.After mixing the above components, a tablet was prepared by a direct tableting method.
제제예 3Formulation Example 3 : 캡슐제의 제조: Preparation of Capsule
AGE-알부민의 합성 저해제 0.1 g0.1 g of inhibitor of synthesis of AGE-albumin
옥수수전분 5 g5 g of corn starch
카르복시 셀룰로오스 4.9 g4.9 g of carboxy cellulose
상기의 성분들을 혼합하여 분말을 제조한 후, 상기 분말을 통상의 캡슐제의 제조방법에 따라 경질 캡슐에 충전하여 캡슐제를 제조하였다.After the powder was prepared by mixing the above components, the powder was filled in a hard capsule according to the conventional method for preparing a capsule to prepare a capsule.
제제예 4Formulation Example 4 : 주사제의 제조 : Preparation of Injection
AGE-알부민의 합성 저해제 0.02~0.2 g0.02-0.2 g inhibitor of synthesis of AGE-albumin
주사용 멸균 증류수 적량Appropriate sterile distilled water for injection
pH 조절제 적량pH adjuster
안정화제 적량Stabilizer
통상의 주사제의 제조방법에 따라 1 앰플 당(2㎖) 상기의 성분 함량으로 제조하였다.According to the conventional method for preparing an injection, the amount of the above-mentioned ingredient was prepared per ampoule (2 ml).
제제예 5Formulation Example 5 : 액제의 제조 : Manufacture of liquid
AGE-알부민의 합성 저해제 0.1 g0.1 g of inhibitor of synthesis of AGE-albumin
이성화당 10 g10 g of isomerized sugar
만니톨 5 g5 g of mannitol
정제수 적량Purified water
통상의 액제의 제조방법에 따라 정제수에 각각의 성분을 가하여 용해시키고, 레몬향을 적량 가한 다음 상기의 성분을 혼합하였다. 그 다음 정제수를 가하여 전체 100㎖로 조절한 후 갈색병에 충전하고 멸균시켜 액제를 제조하였다.Each component was added and dissolved in purified water according to the conventional method for preparing a liquid, and lemon flavor was added appropriately, followed by mixing the above components. Then, purified water was added thereto to adjust the total volume to 100 ml, and then filled in a brown bottle and sterilized to prepare a liquid.

Claims (9)

  1. 단핵식세포계 세포 내에서 ＡＧＥ(advanced glycation end-product; 최종당화산물)-알부민의 합성 저해 또는 분비 저해에 의한 세포사(cell death) 유도 저해 방법.A method of inhibiting cell death induction by inhibiting synthesis or secretion of advanced glycation end-product (ABE) -albumin in mononuclear phagocytes.
  2. 제 1항에 있어서, 상기 세포사되는 세포는 단핵식세포계 세포 주변에 있는 세포인 것을 특징으로 하는, 세포사 유도 저해 방법.The method of claim 1, wherein the cell death cells are cells surrounding mononuclear phagocytes.
  3. 제 2항에 있어서, 상기 단핵식세포계 세포 주변에 있는 세포는 신경세포, 연골세포, 폐세포 및 간세포로 이루어진 군으로부터 선택된 1종 이상을 포함하는 것을 특징으로 하는, 세포사 유도 저해 방법.The method of claim 2, wherein the cells surrounding the mononuclear phagocytes comprise one or more selected from the group consisting of neurons, chondrocytes, lung cells, and hepatocytes.
  4. 제 1항에 있어서, 상기 AGE-알부민의 합성 저해 또는 분비 저해는 알부민 siRNA, 알부민 항체, AGE 항체, AGE-알부민 항체 및 AGE-알부민 합성 저해제로 이루어진 군으로부터 선택된 1종을 이용하여 저해되는 것을 특징으로 하는, 세포사 유도 저해 방법.The method according to claim 1, wherein the inhibition or secretion of AGE-albumin synthesis is inhibited using one species selected from the group consisting of albumin siRNA, albumin antibody, AGE antibody, AGE-albumin antibody and AGE-albumin synthesis inhibitor. Cell death induction inhibition method.
  5. 제 1항에 있어서, 상기 단핵식세포계 세포는 뇌의 미세아교세포, 혈액단핵세포, 폐포대식세포(type Ⅱ pneumocyte, dust cell), 복강대식세포, 염증부위 육아종대식세포, 비장대식세포, 간의 쿠퍼세포, 관절활액막 A 세포(synovial A cell), 혈관외막세포, 림프절내 대식세포, 및 피부의 랑거한스 세포(Langehans cell)로 이루어진 군으로부터 선택된 것을 특징으로 하는, 세포사 유도 저해 방법.According to claim 1, wherein the mononuclear phagocyte cells are microglial cells, blood mononuclear cells, alveolar macrophages (type II pneumocyte, dust cell), peritoneal macrophages, inflammatory site granuloma macrophages, splenic macrophages, Cooper of the liver Cells, synovial A cells, synovial A cells, endocardium cells, lymph node macrophages, and Langerhans cells of the skin, characterized in that selected from the group consisting of cell death induction.
  6. ＡＧＥ-알부민의 합성 저해 활성을 갖는 하기 C-1 내지 C-42의 화합물로 이루어진 군으로부터 선택된 1종 이상의 화합물을 포함하는 ＡＧＥ-알부민의 합성 저해제.An inhibitor of the synthesis of ABE-albumin comprising at least one compound selected from the group consisting of the compounds of the following C-1 to C-42 having a synthesis inhibitory activity of ABE-albumin.
    Figure PCTKR2011003147-appb-I000003
    Figure PCTKR2011003147-appb-I000003
  7. 제 6항의 ＡＧＥ-알부민의 합성 저해제를 유효성분으로 함유하는 퇴행성 질환 및 자가 면역 질환의 예방 또는 치료용 약학적 조성물.A pharmaceutical composition for the prevention or treatment of degenerative diseases and autoimmune diseases, comprising the inhibitor of the synthesis of ABE-albumin according to claim 6 as an active ingredient.
  8. 제 7항에 있어서, 상기 퇴행성 질환 및 자가 면역 질환은 알츠하이머병, 뇌졸중, 파킨슨병, 루게릭병, 류마티스 관절염, 당뇨성 망막변성, AIDS, 노화, 폐섬유증 및 척수손상으로 이루어진 군으로부터 선택된 1종 이상을 포함하는 것을 특징으로 하는, 조성물.8. The method of claim 7, wherein the degenerative disease and autoimmune disease are at least one selected from the group consisting of Alzheimer's disease, stroke, Parkinson's disease, Lou Gehrig's disease, rheumatoid arthritis, diabetic retinopathy, AIDS, aging, pulmonary fibrosis and spinal cord injury. Characterized in that it comprises a.
  9. 제 6항의 ＡＧＥ-알부민의 합성 저해제의 치료적 유효량을 개체에게 투여하여 퇴행성 질환 및 자가 면역 질환을 예방 또는 치료하는 방법.A method for preventing or treating a degenerative disease and an autoimmune disease by administering to a subject a therapeutically effective amount of the AB-albumin synthesis inhibitor.
PCT/KR2011/003147 2010-05-11 2011-04-28 Method for inhibiting the induction of cell death by inhibiting the synthesis or secretion of age-albumin in cells of the mononuclear phagocyte system WO2011142545A2 (en)

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Non-Patent Citations (5)

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AHN, S-M. ET AL.: 'Human Microglial Cells Synthesize Albumin in Brain' PLOS ONE vol. 3, no. ISS.7, 30 July 2008, page E2829 *
CAMERON, N.E. ET AL.: 'Inhibitors of Advanced Glycation End Product Formation and Neurovascular Dysfunction in Experimental Diabetes' ANN, N.Y. ACAD. SCI. vol. 1043, 2005, pages 784 - 792 *
DUKIC-SKTEFANOVIC, S ET AL.: 'AGES in Brain Ageing: AGE-Inhibitors as Neuroprotective and Anti-Dementia Drugs?' BIOGERONTOLOGY vol. 2, 2001, pages 19 - 34 *
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