KR101906808B1 - Rnf146-inducing pharmaceutical composition for treatment or prevention of degenerative brain disease - Google Patents

Abstract

The present invention relates to pharmaceutical compositions for the treatment or prevention of degenerative brain diseases including Rhododendrin, Liquiritigenin, Chlorogenic acid or Piperlonguminine, and to prevent or ameliorate degenerative brain diseases ≪ / RTI >

Description

TECHNICAL FIELD [0001] The present invention relates to an RNF146 expression inducing pharmaceutical composition for treating or preventing degenerative brain diseases,

The present invention relates to a method for the treatment or prevention of degenerative brain diseases including Rhododendrin, Liquiritigenin, Chlorogenic acid or Piperlonguminine, which induce the expression of the neuronal protection gene RNF146 A pharmaceutical composition, and a health functional food for preventing or improving degenerative brain diseases.

As the average life expectancy increases to enter an aging society, degenerative diseases that develop with aging are emerging as major medical and social problems. Among these degenerative diseases, especially degenerative brain diseases, occupies the first place in the ranking of diseases of geriatric diseases.

Degenerative brain diseases are typically Parkinson's disease, Alzheimer's disease, Huntington's disease, and amyotrophic lateral sclerosis (ALS), well known as Lou Gehrig's disease.

Parkinson's disease is a degenerative neurological disorder in which the muscles of the arms, legs, or body are spasmodic or rigid and not centralized. These symptoms are due to the problem of neuronal cells, the neurotransmitter, dopamine secretion, is abnormal, resulting in the gradual destruction of specific neurons in the brain. Typical symptoms of Parkinson's disease are movement disorders, including convulsions, muscle stiffness, and slowing motion. Parkinson's disease is slow to progress, so it is difficult to know the exact timing of the onset. When symptoms are found, the neurons in the brain's black areas are often highly denatured. In addition, facial muscles are stiffened and the pace of the walking pace is shortened, and depression and bowel obstruction may occur. Although there are many drugs that alleviate the symptoms of Parkinson's disease, the chronic use of drugs results in side effects that weaken the mind and body (Neurology, 1991, 41, 202-205) It is not. Although the exact cause of Parkinson 's disease is unknown, it has been found to be associated with environmental toxins, genetic factors, and mitochondrial dysfunction.

It has been extensively reported that hyperactivation of PARP1 (poly (ADP-ribose) polymerase-1) in neuronal cells in stroke leads to toxicity to neurons, and in dyskinesia patients with Parkinson's disease Overactivation of PARP1 has been reported. PARP1 causes cell apoptosis during hyperactivation, but is an enzyme that plays an important role in the regulation of various cell functions during normal activation. In this regard, inhibitors of PARP1 can be expected to protect neurons, but the inhibitors of several PARP1s developed have limitations in toxicity by long-term use. Therefore, there is an increasing demand for a therapeutic agent for degenerative brain diseases using a herbal medicine single substance which is relatively safe and expected to have a new therapeutic mechanism.

Korean Patent Application No. 10-2014-7022606

Degenerative brain diseases such as stroke and Parkinson's disease are known to be induced by activating a neuronal apoptosis mechanism characterized by hyperactivation of PARP1 by various cell stresses. RNF146 (IDUNA) does not inhibit PARP1 itself but E3 ubiquitin ligase, which selectively activates PARP1 and selectively cleaves ubiquitin to facilitate degradation through proteasome. Therefore, if the relationship between the expression of RNF146 and the protective effect of the nervous system including dopamine neurons is confirmed, it is expected that the compounds capable of inducing RNF146 expression can be selected as a therapeutic agent for degenerative brain diseases. However, there is a problem that a system capable of monitoring quantitatively the expression of RNF146 is not established at present.

Accordingly, a system capable of quantifying the expression of RNF146 with high sensitivity has been established in the present invention to induce the expression of RNF146 from natural herbal medicine and prevent neuronal cell death, thereby ultimately discovering compounds exhibiting the therapeutic effect of degenerative brain diseases . In particular, in the case of a herbal-based single substance, which is relatively safe and expected to have a new therapeutic mechanism, it was expected that the screening would lead to the discovery of a new therapeutic leader substance which inhibits neurotoxicity. In the academic field, And pathological mechanisms were expected to be possible.

However, the problems to be solved by the present invention are not limited to the above-mentioned problems, and other matters not mentioned can be clearly understood by those skilled in the art from the following description.

The first aspect of the present application provides a method of inducing expression of RNF146, which is E3 ubiquitin ligase, selected from the group consisting of Rhododendrin, Liquiritigenin, Chlorogenic acid and Piperlonguminine, A pharmaceutical composition for the treatment or prevention of a degenerative brain disease comprising as an active ingredient can be provided.

The second aspect of the present application provides a method of preventing degenerative brain diseases including natural extracts selected from the group consisting of Rhododendrin, Liquiritigenin, Chlorogenic acid and Piperlonguminine Or a health functional food for improvement can be provided.

The above-described task solution is merely exemplary and should not be construed as limiting the present invention. In addition to the exemplary implementations described above, there may be additional implementations and embodiments described in the drawings and detailed description of the invention.

Herein, it was confirmed that induction of the expression of RNF146, which is an E3 ligase inhibiting PARP1, which is hyperactivated, can inhibit cytotoxicity in a Parkinson's disease cell model. This suggests a novel therapeutic molecule target for degenerative brain disease at the present time when there is no drug capable of controlling dopamine neuronal cell death. In addition, by providing a luciferase expression search system capable of quantitatively measuring the promoter activity of RNF146, it has become possible to find a lead substance for controlling neurotoxicity.

Specifically, the present invention uniquely identifies RNF146 inducers by performing a luciferase screening of RNF146 using a self-developed luciferase expression screening system, biochemical studies at mRNA and protein levels for major candidate groups, Toxicity studies in the model suggested an efficient screening pipeline for identifying candidate neuroprotective agents.

In addition, Rhododendrin, Liquiritigenin, which is a herbal natural substance single compounds that induce the expression of RNF146, an E3 ligase enzyme that actually inhibits hyperactivation and cytotoxicity of PARP1 by performing luciferase screening of RNF146, , Chlorogenic acid and Piperlonguminine were excavated. In particular, it has been demonstrated at the molecular level that liquiritigenin promotes the expression of RNF146 by activating a molecular target called an estrogen receptor. This suggests an important signal transduction system in the basic study of RNF146 with neuroprotective effect. Through this research, not only a molecular mechanism of RNF146 expression but also a safe therapeutic agent for various degenerative brain diseases including Parkinson's disease .

FIGS. 1A and 1B are data obtained by comparing the cell viability of a human neuroblastoma cell line overexpressing RNF146 (IDUNA) according to one embodiment of the present invention.
2A is a schematic view of the structure and pre-treatment schedule of a luciferase construct for detecting RNF146 expression, and FIGS. 2B to 2D are RNF146 expression detection result data using the construct.
3A to 3F are RNF 146 induced natural herbal compound screening result data utilizing the RNF146 luciferase construct.
4A to 4E are data obtained by confirming the ability of Riquitigenin to express RNF146.
FIGS. 5A to 5H are data on the cell protection effect of liquitidin on a Parkinson's disease cell model.
FIGS. 6A to 6D are shRNA experimental data for confirming whether the cell protection effect by liquitigenin is mediated by induction of RNF146 expression.
FIGS. 7A to 7D are data obtained by analyzing the amount of protein expression of RNF146 and the protective effect against dopamine neuronal cell death on 6-OHDA injection in various tissues of the brain after administering liquitidin.

Hereinafter, embodiments of the present invention will be described in detail with reference to the accompanying drawings so that those skilled in the art can easily carry out the present invention. It should be understood, however, that the present invention may be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. In order to clearly explain the present invention in the drawings, portions not related to the description are omitted.

Throughout this specification, when an element is referred to as "including " an element, it is understood that the element may include other elements as well, without departing from the other elements unless specifically stated otherwise.

The terms "about "," substantially ", etc. used to the extent that they are used throughout the specification are intended to be taken to mean the approximation of the manufacturing and material tolerances inherent in the stated sense, Accurate or absolute numbers are used to help prevent unauthorized exploitation by unauthorized intruders of the referenced disclosure. The word " step (or step) "or" step "used to the extent that it is used throughout the specification does not mean" step for.

Throughout this specification, the term "combination thereof" included in the expression of the machine form means one or more combinations or combinations selected from the group consisting of the constituents described in the expression of the machine form, And the like.

Throughout this specification, the description of "A and / or B" means "A, B, or A and B".

Throughout the present specification, the term "degenerative brain disease" refers to a broad concept including all brain diseases that occur as degenerative changes occur in nerve cells of the central nervous system. Most of the degenerative brain diseases are unknown, but they are characterized by the selective involvement of the associated nervous system and the onset of the onset of the disease slowly. The present invention relates to the use of a compound according to the invention in the manufacture of a medicament for the treatment of Alzheimer's disease, mild cognitive impairment, stroke and vascular dementia, frontotemporal dementia, rheisome dementia, Creutzfeldt-Jakob disease, traumatic head injury, syphilis, acquired immunodeficiency syndrome and other viral infections, Alzheimer's disease, ischemic stroke, stroke, attention deficit hyperactivity disorder, schizophrenia, depression, bipolar disorder, post-traumatic stress disorder, spinal cord injury, dementia caused by metabolic diseases, hypoxia, Parkinson's disease, Lou Gehrig's disease, Huntington's disease, , Myelitis, and the like.

Hereinafter, a pharmaceutical composition for treating or preventing degenerative brain diseases of the present invention and a health functional food for preventing or improving degenerative brain diseases will be described in detail with reference to embodiments, examples and drawings. However, the present invention is not limited to these embodiments and examples and drawings.

The first aspect of the present invention relates to a pharmaceutical composition comprising E3 ubiquitin ligase expression inducing agent selected from the group consisting of Rhododendrin, Liquiritigenin, Chlorogenic acid and Piperlonguminine as an active ingredient The present invention provides a pharmaceutical composition for treating or preventing degenerative brain diseases. Herein, the E3 ubiquitin ligase may be RNF146.

The chemical structures of rhodendrine, liquitigenin, chlorogenic acid and piperuronminin are as follows.

Rhodendrine, liquitigenin, chlorogenic acid, and piperuronminin were selected from the herbal extract compound library (640 single compound) provided by the Korean Traditional Medicine Foundation. These compounds can control the neurotoxicity caused by degenerative brain diseases by increasing the promoter activity of E3 ubiquitin ligase such as RNF146. By this principle, the composition of the present invention shows a therapeutic or preventive effect of degenerative brain diseases such as Parkinson's disease But may not be limited thereto.

According to one embodiment of the present invention, the E3 ubiquitin ligase may be, but not limited to, RNF146 (Ring Finger Protein 146).

RNF146 is an E3 ubiquitin ligase that inhibits hyperactivation of the nuclear protein PARP1, which plays an important role in apoptosis, and is known to have a therapeutic effect on strokes associated with overactivation of PARP1. However, RNF146 has a protective effect against degenerative diseases such as Parkinson's disease Was not known. The pharmaceutical composition for treating or preventing degenerative brain diseases according to the present invention can inhibit neuronal cytotoxicity due to degenerative diseases such as Parkinson's disease at the cellular level by inducing the expression of RNF146.

According to one embodiment of the present invention, the pharmaceutical composition for treating or preventing the degenerative brain disease may be, but not limited to, inhibiting neuronal cell death.

According to one embodiment of the present invention, the pharmaceutical composition for the treatment or prevention of the degenerative brain disease selectively inhibits the hyper-activated poly (ADP-ribose) polymerase-1 (PARP1) But it may not be so limited.

PARP1 is known to exhibit neuronal cytotoxicity in stroke and hyperactivity, and hyperactivation of PARP1 has also been reported in Parkinson's disease accompanied by dopamine neuronal cell death. Thus, inhibitors of PARP1 have been proposed as therapeutic agents for neuronal apoptosis, but they were virtually unusable due to their cytotoxicity.

On the other hand, the E3 ubiquitin ligase contained in the pharmaceutical composition of the present invention selectively activates only PARP1 with ubiquitin labeling to decompose it through proteasome, and it is possible to suppress nerve cell death without exhibiting its own toxicity .

According to one embodiment, the degenerative brain disorder is selected from the group consisting of Alzheimer's disease, mild cognitive impairment, stroke, dementia, Creutzfeldt-Jakob disease, traumatic head injury, syphilis, acquired immunodeficiency syndrome, brain abscess, brain tumor, multiple sclerosis, hypoxia, Wherein the disorder is selected from the group consisting of: a disorder selected from the group consisting of Alzheimer's disease, Huntington's disease, Pick's disease, amyotrophic lateral sclerosis, epilepsy, ischemia, stroke, attention deficit hyperactivity disorder, schizophrenia, depression, manic depression, But may not be limited thereto. For example, the dementia may include, but is not limited to, vascular dementia, frontal temporal dementia, dementia due to rubicolitis, and dementia due to metabolic diseases.

According to one embodiment of the present invention, the pharmaceutical composition for treating or preventing the degenerative brain disease may be formulated into tablets, powders, hard or soft capsules, suspensions, injectable preparations, emulsions, powders or granules, But may not be limited.

The pharmaceutical composition of the present invention may be formulated into various forms such as tablets, powders, hard or soft capsules, suspensions, injectable preparations, emulsions, powders or granules according to a conventional method, Intravenous, intraperitoneal, subcutaneous, rectal, topical, and the like.

When the pharmaceutical composition of the present invention is a solid preparation, the composition may be formulated by mixing at least one or more excipients such as starch, calcium carbonate, sucrose, lactose, gelatin, etc., but may not be limited thereto.

Examples of the liquid preparation for oral administration of the pharmaceutical composition include suspensions, solutions, emulsions, syrups, and the like. In addition to water and liquid paraffin which are commonly used simple diluents, various excipients such as wetting agents, sweeteners, , Preservatives, and the like, but the present invention is not limited thereto.

Agents for parenteral administration of the pharmaceutical composition may include, but are not limited to, sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried preparations and suppositories. Examples of the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable esters such as ethyl oleate, etc. Injectables may contain solubilizers, isotonic agents, suspending agents, emulsifiers, stabilizers, But are not limited to, conventional additives such as solvents, preservatives, and the like.

The pharmaceutical compositions herein may vary widely, depending on various factors including the activity of the compound employed, age, weight, general health, sex, diet, time of administration, route of administration, rate of excretion, drug combination and the severity of the disease to be prevented or treated. The dosage of the pharmaceutical composition varies depending on the condition of the patient, the body weight, the severity of the disease, the drug form, the route of administration, and the period of time, but may be appropriately selected by a person skilled in the art, for example, 0.0001 to 50 mg / kg or 0.001 to 50 mg / kg. The administration may be carried out once a day or divided into several doses. The dose is not intended to limit the scope of the invention in any way.

The second aspect of the present application provides a method of preventing degenerative brain diseases including natural extracts selected from the group consisting of Rhododendrin, Liquiritigenin, Chlorogenic acid and Piperlonguminine Or a health functional food for improvement can be provided.

According to one embodiment of the present invention, the health functional food for preventing or ameliorating the degenerative brain disease may be in the form of a capsule, tablet, powder, granule, liquid, ring, flake, paste, syrup, gel, But may not be limited.

The amount of the natural product extract in the health functional food can be appropriately selected by a person skilled in the art in consideration of the purpose of consumption, the age, health condition, sex, body weight and the like of the consumer.

The health functional food may further contain various components in addition to the natural product extract, and may contain a flavoring agent or a natural carbohydrate as an additional ingredient. For example, natural carbohydrates include monosaccharides such as glucose, fructose, and the like; Disaccharides such as maltose, sucrose and the like; And polysaccharides such as, for example, conventional sugars such as dextrin, cyclodextrin and the like, and sugar alcohols such as xylitol, sorbitol, erythritol, and the like. Other flavors other than those mentioned above may include, but are not limited to, natural flavors and synthetic flavors.

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the following examples are for illustrative purposes only and are not intended to limit the scope of the present invention.

[ Example ]

Experimental Method

1. Cell culture and transfection

The experiment was carried out using SH-SY5Y cells, a human neuroblastoma cell line, in a DMEM medium containing 10% FBS and 1% antibiotic, at a CO _{2 of} 5%, a humidity of 95%, and 37 ° C. The DNA plasmids of each experimental group were mixed with Opti-MEM, and 'X-tremeGENE', a transfection reagent, was added to the cells for 15 to 30 minutes at room temperature, and then added to SH-SY5Y cells.

2. Western Blot

Cells cultured on a 6-well plate were washed once with PBS, and then 500 μl per well of 1% Nonidet P40 in PBS (Phosphate buffered saline) was added to each well. tube), and then lysed in ice for 30 minutes. Thereafter, vortexing was performed every 5 minutes for 30 minutes. After 30 minutes, it was rapidly frozen in dry ice and dissolved in ice water twice. After completely dissolved, it was placed in a 4 ° C centrifuge and centrifuged at a speed of 13,000 g for 15 minutes. After centrifugation for 15 minutes, the supernatant (protein) was transferred to a new E-tube. The purified protein was mixed 1: 1 with 2 × laemmli buffer (Bio-Rad) (+ β-mercaptoethanol, final 5% (v / v)) and boiled at 95 ° C. for 5 minutes. Subsequently, the sample was separated according to the protein size using SDS-PAGE gel (100 V, 1 hour 30 minutes). Subsequently, the proteins separated in the SDS-PAGE gel were transferred to a nitrocellulose membrane (100 V, 1 hour 30 minutes). The membrane from which the protein was transferred was blocked with 5% skim milk for 1 hour. After 1 hour, the skim milk was removed and the primary antibody (1: 5000) was added and the mixture was kept at 4 ° C for 16 hours in a shaker. After 16 hours, the primary antibody was removed and the secondary antibody was added to the nitrocellulose membrane at a ratio of 1: 1000 in 5% skim milk (2 hours at room temperature). After 2 hours, the secondary antibody was removed and an ECL solution (SuperSignal ™ West Pico Chemiluminescent Substrate, Thermo) was added, and the protein band was confirmed using an X-ray film in a dark room.

3. Lucifer Reyes  analysis

A. Induction of expression of RNF146 (IDUNA) (using H ₂ O ₂ )

PGL3-IDUNA-Luc and pRL-TK vector (Promega) were transfected into SH-SY5Y cells prepared on a 12-well plate. After 24 hours, cells transfected with 100 [mu] M H ₂ O ₂ prepared in DMEM without FBS were treated for 10 minutes. After 10 minutes, the H ₂ O ₂ was removed and DMEM containing 10% (v / v) FBS was fed. After 36 hours -24 hours -12 hours-6 hours, the medium was removed, washed once with PBS, and PLB (Passive Lysis Buffer, promega) was added to dissolve the cells in the shaker for 15 minutes. LARII (Promega) reagent was added to a white 96-well plate, and the lysed cells were added. Then, the wavelength was measured using a microplate luminometer, the plate was taken out, and the wavelength was again measured by adding Stop & Glo reagent (Promega). Then, the wavelength value obtained by measuring the LAR II reagent was added to the Stop & Glo reagent and the measured value was divided.

B. Induction of IDUNA expression (using natural substances)

PGL3-IDUNA-Luc and pRL-TK vector (Promega) were transfected into SH-SY5Y cells prepared in a 6-well plate. After 24 hours, all the cells were collected and transferred to a 96-well plate. After another 24 hours, 10 [mu] l of natural material was added to each well and incubated for 37 hours. After 37 hours, the medium was removed, washed with PBS, and PLB (Passive Lysis Buffer, promega) was added to dissolve the cells in the shaker for 15 minutes. The LAR II reagent was placed in a white 96-well plate and the lysed cells were added. After measuring the wavelength using a microplate spectrometer, the plate was taken out and the wavelength was again measured by adding a Stop & Glo reagent. The wavelength values measured by adding the LAR II reagent were divided into the values measured by adding the Stop & Glo reagent.

4. Real-time quantitation PCR (Real-time quantitative PCR )

Cells to be analyzed were washed with PBS. 1 ml of QIAzol lysis reagent (Lysis Reagent, cat # 79306, QIAGEN) was added to each well, and collected and transferred to an E-tube. Thereafter, it was inserted into ice and allowed to stand for 5 minutes with a medium vortex. 200 μl of chloroform was added to each tube, vortexed for 20 seconds, and left at room temperature for 3 minutes. After 3 minutes, the mixture was put into a centrifuge at 4 ° C and centrifuged at a speed of 12,000 g for 15 minutes. Then, only the separated RNA layer was transferred to a new E-tube, 500 μl of 2-propanol was added to the RNA supernatant, and the mixture was vortexed and left at room temperature for 10 minutes. It was then placed in a 4 ° C centrifuge and centrifuged for 10 minutes at a rate of 12,000 g. The supernatant was carefully removed, then the lid was opened and the supernatant, which had not been removed, was dried for 10 minutes. Then, 50 μl of the number of the nucleic acid digesting enzyme (Nuclease free water) was added and the RNA pellet was dissolved. After the concentration of RNA was measured, cDNA was prepared by using a first-strand cDNA synthesis kit (Biorad) (in this case, RNA was added at 1.5 μg). The synthesized cDNA was mixed with a specific mRNA primer, a SYBR Green PCR master mix, and then placed in a real-time PCR machine (QuantStudio 6 flex Real-Time PCR System, Applied Biosystems) to measure the mRNA level. cDNA, GAPDH primer, and SYBR Green PCR mastermix.

5. Cells Survival rate  analysis

A. Trypan blue analysis

All of the cells that had been tested by toxic reagents were collected in an E-tube and centrifuged at 300 g for 5 minutes. The supernatant was removed, washed once with PBS, and centrifuged at 300 g for 5 minutes. Next, the supernatant was removed, and 500 μl of PBS was added to each tube to release the cell pellet. After injecting 100 [mu] l of trypan blue into the new tube, 100 [mu] l of the cell pellet-resolved PBS was added and mixed. The cell viability (%) was measured using a Countess II Automated Cell Counter (Life Technologies) machine. Cell viability (%) was measured by adding 10 μl to the cell counting slide.

B. Measurement of ATP Level

The cells that had been tested by the toxic reagent were removed and transferred to 100 μl white 96-well plate. 100 [mu] l of Cell Titer reagent from CellTiter-Glo® Luminescent Cell Survival Assay Kit (promega) was added to each well. Thereafter, the mixture was shaken for 2 minutes and left at room temperature for 10 minutes. After 10 minutes, ATP levels were measured using a microplate luminometer.

6. IP injection - Fix (Fix)

* Concentration of liquiritigenin to be injected in a mouse: 10 mg / kg? 200? / 20 g (1?): 200? / 100?

1. Intraperitoneal injection of 100 μl (2 μg / μl) was given per mouse for 4 days.

On the fourth day, 6OHDA was injected into the brain striatum.

3. 100 μl (2 μg / μl) per mouse was injected intraperitoneally for 3 days again.

4. Finally, the day after the intraperitoneal injection, the brain was removed and fixed with 4% paraformaldehyde.

5. After 1 day, it was changed to 30% sucrose.

7. 6OHDA injection

1. The 2 ㎕ syringe was washed with PBS until there was no feeling of being caught on the needle.

2. I inserted the syringe into the fixture vertically and horizontally.

3. Because the injection needle hole was so narrow that it was easy to clog, it was confirmed that it was not clogged with PBS every time the mice were changed.

4. Anesthetics were intraperitoneally injected into the mice to be injected at about 50 to 60 μl. Anesthetics were injected into mice according to the weight of the mice.

5. After the anesthesia, the rat's tongue was removed, then the front teeth were tied, then both sides of the head were fixed, and the tail was pulled all the way to tape. The head of the rats was thoroughly rubbed with 10% H ₂ O ₂ , and the center of the head was cut about 1.5 cm with a surgical knife.

6. After cutting, the space was secured and the surface was scratched with a swab with H ₂ O ₂ until the bregma was visible.

7. After adjusting the syringe to the midpoint of the crown, this point was set to 0.0.0.

8. After reloading the syringe, first move to L: -2.0, AP: 0.5 of the striatum coordinates (L: -2.0, AP: 0.5, DV: -3.0) and remember the position of the syringe Then I put the syringe back up and pointed to the name pan.

9. Drill a hole at the point where the point was taken, and when the skull was pierced, the drill was immediately removed.

10. 2 μl of 6-OHDA (4 μg / μl) was added to the syringe and the syringe was lowered to the spot position (DV: -3.0 mm).

11. DV: When it reached -3.0 mm, it rose to 2.7 mm again.

12. Add 0.2 μL slowly for 1 min. 1.8 ㎕ of 2 ㎕ total was added in this way.

13. The last 0.2 μl and waited for 5 minutes. The following rats were anesthetized while waiting.

14. After 5 minutes, slowly raise the syringe, being careful not to bend the syringe needle.

15. I stitched my head with a needle / thread for sewing. The syringe was then washed with PBS and transferred to the next experimental mouse.

8. Brain sectioning

1. Dry ice was placed on a microtome plate to keep it cool.

2. The sucrose was discarded and the sample was placed on the ice sheet.

3. Ice plates were made with PBS on a microtome plate.

4. The thickness was set to 40 탆.

5. Place the brain to be cut flat on a cross section, PBS roll up, then placed on top of the brain level.

6. Place dry ice around, and when the brain turns white, dry ice is removed and it starts to cut.

7. Cut the brush with a brush with a continuous brush.

8. The hippocampus was harvested from both sides and placed in a 12-well plate pre-loaded with PBS.

9. After collecting, cross section was scraped and proceeded to the next brain.

9. TH staining

A. Day 1

1. Blocking solution (1XPBS, 0.2% Triton-X, 0.02% Na-azide, 10% goat serum) was prepared.

2. Place blocking solution in 12 well plates.

3. The brain sections collected in PBS were collected with a brush and transferred to a plate containing the blocking solution.

4. Leave the stirrer at room temperature for 30 minutes.

5. The TH antibody (Novus) was diluted 1: 3000 in the blocking solution and placed in a 12-well plate.

6. After 30 minutes, brain sections were collected and transferred to plates with TH antibody.

7. Place on a 4 ° C agitator for 16 hours.

B. Day 2

1. 1XPBS + 0.1% Triton-X was made and placed in two 12-well plates.

2. Brain sections were collected with a brush and placed in 1XPBS + 0.1% Triton-X and placed on a stirrer for 10 minutes. This was repeated twice.

3. Secondary antibody was prepared (1XPBS, 5% goat serum, 0.2% Triton-X, Biotin pAb (1: 1000))

4. The secondary antibody was placed in a 12-well plate.

5. At the end of the wash step, the brain section was transferred to the plate with the secondary antibody and placed on the agitator for 1 hour at room temperature.

6. After 1 hour, it was placed on a stirrer with 1XPBS + 0.1% Triton-X for 10 minutes. This was repeated twice.

7. Prepare the ABC kit (2 drops of solution A in 10 ml PBS, vortex in vortex, followed by 2 drops of solution B) (prepared in a 12-well plate)

8. At the end of the wash step, place in the ABC solution and leave at room temperature for 40 minutes.

9. After 40 minutes, it was placed on a stirrer with 1X PBS. This was washed twice repeatedly

10. Prepare a DAB solution (D4293, SIGMA) (1 x silver gold in 10 ml dH ₂ O and vortex until dissolved) (place in 12 well plates) Respectively.

11. At the end of the washing step, the brain sections were combined and collected, transferred to DAB solution at once and shaken to stain. When the color appeared and was clearly visible, it was collected and transferred to a PBS plate.

12. Put the DAB in the PBS.

13. Place slide (10 cm dish, slide, PBS, Triton-X) (place 1X PBS in 10 cm dish and put 2-3 drops of Triton-X) To the top of the slide, followed by drying at room temperature).

14. After rolling, DPX (06522, SIGMA) was dropped onto the tissue and slowly covered with a cover slide.

Experiment result

One. PARP1  An inhibitory protein RNF146  Proof of applicability to Parkinson's disease

A. Protective effect of RNF146 expression on Parkinson's disease-related toxicants

Oxidative stress (H ₂ O ₂ , 6-hydroxydopamine (6-OHDA)) or mitochondrial inhibitor (rotenone) was observed in the dopaminergic cell line SH-SY5Y cells. Respectively.

FIG. 1A shows the results of H ₂ O ₂ (1 mM), 6-OHDA (70 μM) and rotenone (20 μM) after overexpression of GFP or GFP-RNF146 (IDUNA) (24 hours), and cell viability was measured by trypan blue exclusion assay. Cytotoxicity after treatment with 3AB (10 μM, 4 hours before treatment with toxic substance) as a PARP inhibitor was also confirmed ( n = 6). Fig. 1b shows the results of measuring cytotoxicity by transfection (30 hours) with a combination of GFP-AIMP2 or GFP-IDUNA expressed in SH-SY5Y cells with the exception of trypan blue. 3AB (10 μM) was used as a PARP inhibitor.

As a result, cytotoxicity by H ₂ O ₂ , 6-OHDA, and rothenone was prevented by treatment with PARP1 inhibitor (3AB). That is, hyperactivation of PARP1 has been shown to be involved in apoptosis in the Parkinson's disease cell model. In addition, overexpression of RNF146, an E3 ligase that cleanses and activates PARP1, resulted in a similar level of cytoprotective effect (FIG. 1A).

B. Protective effect of RNF146 expression on Parkinson's disease gene model

AIMP2 is a substrate protein of Parkinson's disease gene, a toxin protein known to contribute to dopamine neuronal cell death, accumulation in the brain and accumulation in the mouse model of patients with Parkinson's disease. In this example, it was confirmed that overexpression of AIMP2 induces SH-SY5Y cytotoxicity, and at the same time, this cytotoxicity was effectively controlled by overexpression of 3AB or RNF146 (FIG. 1B). In other words, it was confirmed that PARP1 hyperactivation plays an important role in cytotoxicity by using two types of Parkinson's disease cell models, and that overexpression of RNF146 can be used as an effective therapeutic gene for nerve cell cytotoxicity of Parkinson's disease Experimental demonstration.

2. RNF146  The ability to measure promoter activity Lucifer Reyes  Build and validate constructs

To quantitatively and sensitively detect the expression of RNF146, the RNF146 (IDUNA) promoter (1.9 kb) was cloned into a pGL3 basic luciferase plasmid (Fig. 2a). The lower image of FIG. 2A is a simplified representation of the time schedule of various assays (luciferase assay, total RNA and protein preparation) after treatment with low concentration of H ₂ O ₂ (100 μM) for 10 minutes.

Actually, it was confirmed under the pretreatment condition that this luciferase construct (pGL3-IDUNA-Luc) can represent the condition in which IDUNA is expressed (Fig. 2a). Conventional studies have shown that weak H ₂ O ₂ It has been reported that stress-induced cell survival genes such as IDUNA are induced. Thus, pGL3-IDUNA-Luc and pRL-TK were transfected into SH-SY5Y cells and pretreated with H ₂ O ₂ at a low concentration for 24 hours. The IDUNA promoter activity was measured at every indicated time intervals through a Luciferase assay. And expressed as a relative value normalized based on the Luciferase value (Fig. 2B). In addition, the amount of IDUNA mRNA was measured by RTQ PCR for each time period, and the quantified value corrected by the amount of GAPDH was graphically displayed ( n = 3) (FIG. 2C). In addition, the amount of IDUNA per time zone was confirmed by Western blot using a specific antibody. beta -actin was used as a loading control (Figure 2d). The lower panel of Fig. 2d shows a quantitative graph of IDUNA western blot band intensities (band intensity of IDUNA corrected with β-actin) ( n = 3).

Cells were treated with 100 μM low-concentration H ₂ O ₂ for 10 minutes, and the activity of IDUNA promoter was measured. As a result, it was confirmed that the luciferase strength of pGL3-IDUNA-Luc was increased over 24 hours (FIG. This aspect was similarly observed in experiments confirming IDUNA mRNA and protein (Fig. 2c and 2d). These results demonstrate that the pGL3-IDUNA-Luc construct can be used as a useful tool to represent the actual activity and expression of IDUNA.

3. RNF146 Lucifer Reyes  Structured RNF146  Guided Natural Oriental Compound Screening

After transient transfection of pGL3-IDUNA-Luc DNA and renilla luciferase TK construct (pRL-TK vector, Promega) into SH-SY5Y neuroblastoma cell line, a total of 640 herbal single compounds (0.35 Mu] g / ml, for 37 hours) and DMSO were treated with a control to first sort compounds that increase IDUNA promoter activity.

Figure 3a shows a schematic diagram of high throughput screening of the IDUNA expression inducer. SH-SY5Y cells expressing pGL3-IDUNA-Luc and pRL-TK were cultured on a 96-well plate, treated with a library of 640 herbal natural products, and then subjected to luciferase assay after 37 hours. DMSO and low concentration H ₂ O ₂ were used as negative and positive control, respectively. FIG. 3B shows the results of the first-order high-speed luciferase screening, and a value obtained by normalizing the luciferase value of the DMSO-treated sample to 1 is represented by a bar graph. FIG. 3C shows the IDUNA promoter luciferase value ( n = 6) through repeated experiments on compounds (Rhododendrin, Liquiritigenin, Chlorogenic acid, Piperlonguminine) which increased IDUNA promoter activity in the first screening. FIG. 3D shows the results of quantitation of IDUNA mRNA level by RTQ PCR after treating each IDUNA expression inducing compound (10 μM) for 37 hours in SH-SY5Y cells ( n = 3) and the amount of GAPDH was measured as an internal loading control Respectively. FIG. 3E shows the results of Western blot analysis of IDUNA-specific protein after treatment of each IDUNA expression inducing compound (10 μM) in SH-SY5Y cells for 37 hours. Figure 3f shows the intensity of the IDUNA band on the western blot for each sample, and β-actin was used as a loading control ( n = 3).

A low dose H ₂ O ₂ -treated group was used as a positive control for each plate (FIGS. 3A and 3B). In the first screening, natural compounds that maximally increase the promoter activity of IDUNA were tested by repeated experiments. These compounds were Rhododendrin, Liquiritigenin, Chlorogenic acid and Piperlonguminine (Figure 3c). All of these compounds significantly increased the amount of actual mRNA and protein of IDUNA (FIGS. 3d and 3e). Finally, all of the four candidate compounds detected by IDUNA-induced compound screening with pGL3-IDUNA-Luc were found to actually increase IDUNA mRNA and protein. Thus, it has been experimentally proven that the IDUNA promoter luciferase construct used in this embodiment can be efficiently used for locating IDUNA-inducing compounds. In this example, more than 640 compounds were used, but it was expected that additional IDUNA inducing substances could be discovered using a large-scale library.

4. Liquiritigenin RNF146  Judo Mechanism  excavation

FIG. 4A shows the results of confirming the expression of IDUNA protein by Western blotting using antibodies to SH-SY5Y cells treated with 0.1, 1 and 10 μM of liquiritigenin for 48 hours, respectively. FIG. 4B is a graph ( n = 3) showing the amount of protein expression of IDUNA as the band intensity of western blot and calculated as a relative value to the control group after the correction by the β-actin band intensity, and β-actin was used as an internal loading control Respectively. FIG. 4C shows the results of Western blotting of the expression level of IDUNA after treatment with SH-SY5Y cells for 24 hours with tamoxifen (1 μM), which is an estrogen receptor antagonist, and β-actin was used as an internal loading control. 4D shows the result of quantifying the IDUNA western band intensity for the samples of panel C ( n = 3). FIG. 4E shows the results of quantitation of IDUNA mRNA by RTQ PCR after treating SR-SY5Y cells with liquiritigenin and tamoxifen for 48 hours ( n = 3), and GAPDH quantification was used to normalize the internal loading control . The amount of IDUNA mRNA was expressed relative to the DMSO-treated group.

Riquitigenin was the most consistently increased amount of IDUNA promoter activity, mRNA, and protein in the present example (FIGS. 3c-3f), showing increased expression of distinct IDUNA proteins at different concentrations (FIGS. 4a and 4b). Riquitigenin is known to act through the estrogen receptor beta (ERβ) as an acting molecule target. Using estrogen receptor antagonists, we first demonstrated for the first time that induction of the expression of IDUNA by liquiritigenin is mediated by estrogen receptor activation (Figs. 4a-4e). It has not been tested whether estrogen receptor is involved in the induction of IDUNA expression by the other three compounds, but it is expected that a new mechanism can be secured through further studies. Securing mechanisms at the molecular level of Riquitigenin-ERβ-IDUNA has important implications for proceeding with pre-clinical and clinical trials of Riquitigenin.

5. Liquiritigenin  Protective Effect of Dopaminergic Neurons and Validation of Mechanism

Riquiatinogen, an inducer of RNF146 (IDUNA) expression, was actually tested for its protective effect in a PD model. 5A shows a schematic diagram of the experimental schedule for the SH-SY5Y cell line, and the lower part shows the protective effect against H ₂ O ₂ toxicity by treatment with liquiritigenin in SH-SY5Y cells, except for trypan blue exclusion ) Essay ( n = 6). FIG. 5B shows the results of measuring the amount of ATP in cells ( n = 6) when H ₂ O ₂ (1 mM, 16 hours) was pretreated with SH-SY5Y cells after liquiritigenin (10 μM for 40 hours). Figure 5c The expression of IDUNA in SH-SY5Y cells pretreated with liqueitigin (10 μM, 40 hours total) and H ₂ O ₂ (1 mM, 24 hours) was confirmed by western blotting. As a result, Respectively. FIG. 5D is a graph showing the relative value of the β-actin band intensity after correction to the DMSO-treated control group as a result of quantifying the Western band intensity of IDUNA for the sample group of FIG. 5C ( n = 3). 5E shows the expression of poly (ADP-ribose) and PARP1 after SH-SY5 cells were pretreated with liquiritigenin (10 μM, 24 hours) and treated with H ₂ O ₂ (1 mM, 15 min) As a result of Western blotting using antibodies, β-actin was used as an internal loading control. FIG. 5F shows the band intensity quantification values of PAR and PARP1 on the western blot for the sample of FIG. 5E, which was expressed as a relative value to the control group after the correction with the? -Actin band intensity. Figure 5g shows cytotoxicity by SH-SY5Y cells treated with 6-OHDA (70 [mu] M, 24 hours) and rotenone (20 [mu] M for 24 hours) and cell protection by liquiritigenin pretreatment ( N = 6). The results were as follows. FIG. 5h shows the cytotoxic effect of SH-SY5Y cells induced by GFP and GFP-AIMP2 overexpression (30 hours) and cell protection effect by liquiritigenin ( n = 6).

As a result, cytotoxicity of SH-SY5Y at high concentration of H ₂ O ₂ (1 mM) was almost completely blocked by liquitigenin, and energy depletion (ATP level) and poly (ADP -Ribose) (PAR) could be inhibited by the expression of IDUNA by liquefactory genetin (Figs. 5A to 5F). In addition, cytotoxicity by rotenone and 6-OHDA, and cytotoxicity by overexpression of AIMP2, were also demonstrated (Fig. 5g and 5h).

In addition, shRNA experiments were conducted to demonstrate that the cell protection effect by liquitigenin is mediated by the induction of IDUNA expression. FIG. 6A shows the expression levels of PAR and PARP1 after SH-SY5Y cell line overexpression of shDsRed or shRNA-IDUNA (48 hours) and treatment with H ₂ O ₂ (1 mM) for 15 minutes by Western blot using a specific antibody, Riquitigenin was treated for 24 hours at a concentration of 10 [mu] M after 24 hours of transfection. beta -actin was used as an internal loading control. Figure 6b is the result of quantifying the band intensity of the PAR on the western blot for the sample group of Figure 6a, expressed as a relative value to the control group after correction to the beta -actin band intensity ( n = 3). Figure 6c SH-SY5Y cells, in which IDUNA was knocked down by shDsRed or shRNA-IDUNA transfection (total 72 hours), were treated with liqueitigin (10 μM, 48 hours) and high concentration H ₂ O ₂ ( N = 6). The results of this study are summarized as follows. Figure 6d is a process the requester utility genin to shDsRed or SH-SY5Y cells was knocked down the IDUNA to transform the shRNA-IDUNA injection (a total of 64 hours) (10 μM, 40 hours), and high-concentration _{H 2 O 2 (1 mM,} 16 Time ( n = 6) of the amount of intracellular ATP.

When IDUNA was knocked down via shRNA, inhibition of PAR by liqueitigin treatment and inhibition of cell death and ATP energy exhaustion disappeared (Figs. 6A to 6D). That is, it has been demonstrated that the cell protection effect of liquitigenin is entirely induced by the induction of IDUNA expression. These results indicate that IDUNA expression inducers can be applied to Parkinson 's disease, a neurodegenerative disease.

FIG. 7A is a graph showing changes in the expression of IDUNA in the brain (frontal cortex, CTX; striatum, STR, midbrain, VM) by a specific antibody at 4 days after intraperitoneal injection of 10 mg / kg / As a result of Western blotting, β-actin was used as an internal loading control. FIG. 7B is a graph showing the degree of expression of IDUNA in each brain region. The band intensity of IDUNA on the western blot was corrected by a β-actin band and compared with each group ( n = 3). Liquiritigenin has been reported to reach the brain upon administration to mice. In this example, the amount of protein expression of IDUNA was increased in various tissues of the brain upon administration to mice (FIGS. 7A and 7B). This indicates that the administration of liquiritigenin will have protective effects not only on the midbrain where Parkinson's disease occurs but also on the striatum and cerebral cortex where various degenerative diseases occur.

Figures 7c and 7d show that dopamine neuronal cell protection appears to be effective when liquiritigenin is administered in a mouse model of Parkinson's disease that induces dopamine neuronal cell death by injecting 6-OHDA into the striatum. FIG. 7c is a graph showing the results of immunohistochemical staining of a mouse model of Parkinson's disease in which 6-OHDA (8 μg, 4 days) was injected into striatum by intraperitoneal injection of DMSO or liquiritigenin (10 mg / FIG. 7D is a photograph of the results obtained by immunohistostaining and Nissl staining using a specific antibody against TH (tyrosine hydroxylase) on mouse brain tissue obtained from each treatment group, FIG. 7D is a photograph image of each mouse group in FIG. 7C The results of stereochemical counting of dopaminergic neurons stained with TH-specific antibodies in substantia nigra (using 6 mice per group).

All quantitative data are expressed as mean +/- standard error. Significance test between the two groups was done through unpaired student t-test, and comparison of three or more groups was analyzed by ANOVA, followed by Tukey's post hoc comparison between the groups. * P <0.05, ** P <0.01, *** P <0.001.

It will be understood by those of ordinary skill in the art that the foregoing description of the embodiments is for illustrative purposes and that those skilled in the art can easily modify the invention without departing from the spirit or essential characteristics thereof. It is therefore to be understood that the above-described embodiments are illustrative in all aspects and not restrictive. For example, each component described as a single entity may be distributed and implemented, and components described as being distributed may also be implemented in a combined form.

The scope of the present invention is defined by the appended claims rather than the detailed description, and all changes or modifications derived from the meaning and scope of the claims and their equivalents should be interpreted as being included in the scope of the present invention.

Claims (8)

A pharmaceutical composition for the treatment or prophylaxis of degenerative brain diseases comprising an E3 ubiquitin ligase expression inducing agent selected from the group consisting of Liquiritigenin and piperlonguminine as an active ingredient.
The method according to claim 1,
Wherein the E3 ubiquitin ligase is RNF146 (Ring Finger Protein 146).
The method according to claim 1,
A pharmaceutical composition for the treatment or prevention of degenerative brain diseases, which inhibits neuronal cell death.
The method of claim 3,
Wherein the compound inhibits neuronal cell death by selectively inhibiting hyperactivated poly (ADP-ribose) polymerase-1 (PARP1) enzyme in neurons.
The method according to claim 1,
Wherein the degenerative brain disease is selected from the group consisting of Alzheimer's disease, mild cognitive impairment, stroke, dementia, Creutzfeldt-Jakob disease, traumatic head injury, syphilis, acquired immunodeficiency syndrome, brain abscess, brain tumor, multiple sclerosis, hypoxia, Parkinson's disease, Wherein the treatment is selected from the group consisting of amyotrophic lateral sclerosis, amyotrophic lateral sclerosis, epilepsy, ischemia, stroke, attention deficit hyperactivity disorder, schizophrenia, depression, manic depression, post traumatic stress disorder, spinal cord injury, &Lt; / RTI &gt;
The method according to claim 1,
A pharmaceutical composition for the treatment or prevention of degenerative brain diseases, which is formulated into tablets, powders, hard or soft capsules, suspensions, injectable preparations, emulsions, powders or granules.
A natural product extract selected from the group consisting of Liquiritigenin and Piperlonguminine, for the prevention or amelioration of degenerative brain diseases.
8. The method of claim 7,
A health functional food for preventing or improving a degenerative brain disease which is in the form of a capsule, tablet, powder, granule, liquid, ring, flake, paste, syrup, gel or bar.

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