KR101910733B1 - Composition of treating or preventing multiple sclerosis comprising piperlongumine as active ingredient - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for the prevention or treatment of multiple sclerosis comprising piperlongumine as an active ingredient. The present invention relates to a pharmaceutical composition for preventing or treating multiple sclerosis comprising piperlongumine as an active ingredient, wherein the piperoronmin is selected from the group consisting of nuclear factor kappa-light-chain-enhancer of activated B cells ), Inhibits the infiltration of T cells or macrophages into brain or spinal cord tissues, and inhibits demyelination in the brain or spinal cord, thereby being useful for preventing or treating multiple sclerosis.

Description

TECHNICAL FIELD [0001] The present invention relates to a composition for preventing or treating multiple sclerosis comprising Piper longin as an active ingredient,

The present invention relates to a composition for preventing or treating multiple sclerosis comprising piper longin as an active ingredient.

Multiple sclerosis is a demyelinating disease in which the myelin of the brain and spinal cord is withdrawn. Myelin sheath is an insulator that surrounds the axons of nerve cells. When a few seconds peel off, the nerve signal is disturbed. It occurs mainly in young people aged 20 to 40 years, and recurrence and relief are repeated, and as the recurrence is repeated, the symptoms are not completely reversed and the disability remains [1-2]. Approximately 2.3 million people worldwide suffer from the disease, but the disease is an unclear autoimmune disease, so it is suffering from the treatment of symptoms that alleviate symptoms at the onset. Multiple sclerosis is a disease characterized by autoimmune system abnormality that infiltrates lymphocytes and macrophages such as T cells in the white matter and spinal cord around the ventricles and causes the myelin basicity associated with oligodendrocyte, Myelin basic protein (MBP) and myelin oligodendrocyte glycoprotein (MOG) are destroyed by dehydration of brain and spinal cord [3].

NF-κB is a redox transcription factor important for regulation of inflammation and regulation of various autoimmune diseases [4-5]. It has been reported that the activity of NF-κB in cerebrospinal fluid of patients with multiple sclerosis is increased compared with that of the general population [9-11], and inactivated nitric oxide synthase (iNOS) and cyclo- The expression of oxigenase-2 (COX-2) is regulated by NF-κB activity because the NF-κB DNA sequence is conserved in each promoter [4-5]. Thus, regulation of NF-κB activity can be provided as a potential approach for managing multiple sclerosis through reduction of neuroinflammation [6-7].

Several evidence suggests that the inclusion of natural products is associated with a reduction in the risk of diseases such as cardiovascular disease, cancer, and inflammatory diseases [8]. Piperlongumine (PL) extracted from Piper longum from India has an excellent function in cancer treatment by inhibiting enzymes that remove active oxygen (which prevents normal cells from becoming cancer cells even when abnormal cells change). , Which was published on the online edition of the world scientific journal 'Nature' [9]. Piper longin has been shown to have antioxidant and anti-inflammatory properties. This compound has been reported to reduce NF-κB activity in several cell and animal models [10-12], but it has not been established how it can inhibit important processes in neurodegenerative diseases and autoimmune diseases.

[1] F.D. Lublin, S.C. Reingold, Defining the clinical course of multiple sclerosis: results of an international survey. Multiple Sclerosis, Neurology 46 (4) (1996) 907-11. [2] J. Goverman, Autoimmune T cell responses in the central nervous system, Nat Rev Immunol 9 (6) (2009) 393-407. [3] J. Patel, R. Balabanov, Molecular mechanisms of oligodendrocyte injury in multiple sclerosis and experimental autoimmune encephalomyelitis, Int J Mol Sci 13 (8) (2012) 10647-59. [4]: Yeo, S.J., J.G. Yoon, and A.K. Yi, Myeloid differentiation factor 88-dependent post-transcriptional regulation of cyclooxygenase-2 expression by CpG DNA: tumor necrosis factor-alpha receptor-associated factor 6, a diverging point in the Toll-like receptor 9-signaling. J Biol Chem, 2003. 278 (42): p. 40590-600. [5]: Kim, Y. M., et al., Upstream NF-kappaB site is required for the maximal expression of mouse inducible nitric oxide synthase gene in interferon-gamma plus lipopolysaccharide-induced RAW 264.7 macrophages. Biochem Biophys Res Commun, 1997. 236 (3): p. 655-60. [6] P.L. Vieira, H.C. Heystek, J. Wormmeester, E.A. Wierenga, M.L. Kapsenberg, Glatiramer acetate (copolymer-1, copaxone) promotes Th2 cell development and increased IL-10 production through modulation of dendritic cells, J Immunol 170 (9) (2003) 4483-8. [7] I. Hwang, D. Ha, DS. Kim, H. Joo, Y. Jee, Glatiramer acetate inhibits the activation of NFkappaB in the CNS of experimental autoimmune encephalomyelitis, Korean Journal of Veterinary Research 51 (3) (2011) 217-225. [8] Hu, N., et al., Nutrition and the risk of Alzheimer's disease. Biomed Res Int, 2013. 2013: p. 524820. [9] Rai, L., et al., Selective killing of cancer cells by a small molecule targeting stress response to ROS. Nature, 2011. 475 (7355): p. 231-234 [10] Son, D. J., et al., Piperlongumine inhibits atherosclerotic plaque formation and vascular smooth muscle cell proliferation bt suppressing PDGF receptor signaling. Biochemical and biophysical research communications, 2012, 427 (2), p.349-354 [11] Han, S. S., et al., Piperlongumin inhibits proliferation and survival of Burkitt lymphoma in vitro. Leukemia research, 2013. 37 (2): p.146-154 [12] Ginzburg, S., et al., Piperlongumine inhibits NF-κB activity and attenuates aggressive growth characteristics of prostate cancer cells. Prostate, 2014. 74 (2): p.177-186

An object of the present invention is to provide a pharmaceutical composition for preventing or treating multiple sclerosis comprising piperlongumine as an active ingredient.

It is still another object of the present invention to provide a food composition for preventing or ameliorating multiple sclerosis comprising piperlongumine as an active ingredient.

It is still another object of the present invention to provide a method for treating a cell, comprising: (a) activating a cell by treating CD3 / CD28 activator or macrophage with LPS (Lipopolysaccharide) to T cells; (b) contacting the activated T cell or macrophage with a candidate agent; (c) measuring the expression level of IκBα or p-IκBα in the cytoplasm of T cells or macrophages in which the candidate substance of (b) is contacted; And (d) measuring the expression level of p50 or p65 in the nucleus of the T cell or macrophage with which the candidate substance of (b) has been contacted. And a method for screening the same.

The present invention provides a pharmaceutical composition for preventing or treating multiple sclerosis comprising piperlongumine as an active ingredient.

In one embodiment of the present invention, the concentration of the Piper longummin may be 0.01 to 5 μM, preferably 0.1 to 3 μM, and more preferably 1 to 2.5 μM.

In one embodiment of the present invention, the concentration of Piper longummin may be 1 to 5 mg / kg / day, preferably 1.5 to 3 mg / kg / day.

In one embodiment of the present invention, the Piper longin is able to inhibit demyelination in the brain or spinal cord.

In one embodiment of the present invention, the Piper longin is able to inhibit the infiltration of T cells or macrophages into brain or spinal cord tissues.

In one embodiment of the present invention, the Piper longinmin may inhibit the activity of NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells).

The present invention also provides a food composition for preventing or ameliorating multiple sclerosis comprising piperlongumine as an active ingredient.

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The present invention relates to a method for inhibiting the activity of NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells) and inhibiting the infiltration of T cells or macrophages into brain or spinal cord tissues And can be useful for preventing or treating multiple sclerosis by inhibiting demyelination in the brain or spinal cord.

Figure 1 shows the mitigation effect on Piper longinum in an experimental autoimmune meningitis (EAE) model by MOG. (a) shows an administration schedule for Piper longinum in an animal model, (b) shows a clinical score, (c) shows a change in body weight, and (d) shows a clinical score.
FIG. 2 shows the result of confirming the extent of immune cell penetration in mouse spinal cord tissue. (a) is the result of H & E staining, (b) is the result of LFB staining, and (c) is the result of confirming the degree of destruction of MBP by immunofluorescence staining.
FIG. 3 shows the result of confirming the extent of immune cell penetration in mouse spinal cord tissue. (a) shows the result of confirming the infiltration of T cells through the expression of CD3, and (b) shows the result of confirming the infiltration of macrophages through the expression of F4 / 80.
FIG. 4 shows the effect of Piperlongmin on the activity of astrocytes and microglial cells through expression of GFAP and Iba1 in mouse spinal cord tissues. (a) is the result of immunofluorescence staining for GFAP, (b) is the result of immunofluorescence staining for Iba1, and (c) is the result of Western blotting for GFAP and Iba1.
FIG. 5 shows the results of confirming the effect of Piper longingmin through the expression of an inflammation-related gene. (a) shows immunostaining for iNOS expression in mouse spinal cord tissue, (b) immunostaining for expression of COX-2 in mouse spinal cord tissue, and (c) (D) shows the results of Western blotting of the expression levels of iNOS and COX-2 in RAW 264.7 cells. (E) shows the expression level of iNOS and COX-2 in Jurkat T The expression levels of iNOS and COX-2 in cells were determined by Western blotting.
Figure 6 shows the effect of Piperlongmin on inflammatory cytokines. (a) is the result of measuring the expression level of TNF-α in mouse spinal cord tissue, (b) is the result of measuring the expression level of IFN-γ in mouse spinal cord tissue, (c) (d) is the result of measurement of the expression level of IFN-? in RAW 264.7 cells, (e) is the result of measuring the expression level of TNF-? in Jurkat T cells, (d) is the result of measuring the expression level of IFN-y in Jurkat T cells.
Fig. 7 shows the results of confirming the effect of Piper longingmin on NF-kB activity. (a) shows the expression levels of IκB, p-IκB, p50 and p65 in mouse spinal cord tissues by Western blotting and (b) shows the expression levels of IκB, p-IκB, p50 and p65 in RAW 264.7 cells Western blotting, and (c) Western blotting of the expression levels of IκB, p-IκB, p50 and p65 in Jurkat T cells.
Fig. 8 shows the results of confirming the effect of the NF-κB inhibitor and the Piper longinmin on the concurrent treatment. (a) is the result of measurement of the mRNA expression level of TNF-α in RAW 264.7 cells by qPCR, (b) is the result of measuring the mRNA expression level of IFN-γ in RAW 264.7 cells by qPCR, and ) Shows the results of qPCR measurement of TNF-α mRNA expression level in Jurkat T cells, (d) mRNA expression level of IFN-γ in Jurkat T cells by qPCR, and (e) The expression level of iNOS and COX-2 protein in RAW 264.7 cells was determined by Western blotting. (F) The expression level of iNOS and COX-2 protein in Jurkat T cells was measured by Western blotting .

The term, "Piper long Resident (Piperlongumine)" of the present invention is 1 - [(2E) -3- ( 3,4,5-Trimethoxyphenyl) prop-2-enoyl] -5,6-dihydropyridin-2 (1 H) -one, and the structure thereof is as shown in the following formula (1).

[Chemical Formula 1]

The pharmaceutical composition according to the present invention can be used as it is or in the form of a pharmaceutically acceptable salt. The salt is not particularly limited as long as it is pharmaceutically acceptable so long as it is pharmaceutically acceptable and includes, for example, hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, hydrofluoric acid, hydrobromic acid, formic acid acetic acid, tartaric acid, lactic acid, citric acid, fumaric acid, , Benzenesulfonic acid, toluenesulfonic acid, and naphthalenesulfonic acid. In addition to acid addition salts, additional base salts such as sodium hydroxide, potassium hydroxide, triethylamine, tertiary-butylamine may also be used.

The pharmaceutical composition of the present invention may be preferably formulated into a pharmaceutical composition containing at least one pharmaceutically acceptable carrier in addition to the above-described effective ingredients for administration. When formulated in the form of a liquid solution, it can be sterilized and used as a carrier in the form of sterile water, sterile water, Ringer's solution, buffered saline, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, ethanol, If necessary, other conventional additives such as antioxidants, buffers, bacteriostats and the like may be added. Or it may be formulated into injectable formulations, pills, capsules, granules or tablets such as aqueous solutions, suspensions, emulsions and the like by additionally adding diluents, dispersants, surfactants, binders and lubricants.

In addition, the formulation of the pharmaceutical composition of the present invention and the pharmaceutically acceptable carrier are not particularly limited and may be appropriately selected according to known techniques in the art. An effective amount for the treatment or prevention of various immune related diseases is not particularly limited so long as the kind of the disease, the severity of the disease, the kind and amount of the active ingredient and the other ingredients contained in the composition, the kind of the formulation and the age, Sex and diet of the patient, the time of administration, the route of administration and the rate of administration of the composition, the duration of the treatment, the drugs used concurrently, and the like. For example, when the subject is an adult, the pharmaceutical composition of the present invention may be administered in a total dose of 0.01 to 10 mg / kg / day, preferably 1 to 5 mg / kg / day, Preferably 1.5 to 3 mg / kg / day, can be administered or administered in an effective amount. It should be apparent to those skilled in the art, however, that the dosage or dose of each active ingredient should be such that the content of each active ingredient is too high to cause adverse side effects.

The pharmaceutical composition of the present invention can be administered to mammals such as rats, mice, livestock, humans, and the like in various routes. All modes of administration may be expected, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intra-uterine or intracerebroventricular injections.

In addition, the food composition according to the present invention may contain various flavoring agents, natural carbohydrates, and the like as additional components, as well as conventional food compositions, in addition to containing the effective ingredient Piperlongmin.

Examples of such natural carbohydrates include monosaccharides such as glucose, fructose, and the like; Disaccharides such as maltose, sucrose and the like; And polysaccharides, for example, conventional sugars such as dextrin, cyclodextrin and the like, and sugar alcohols such as xylitol, sorbitol and erythritol. The flavors may be advantageously used as natural flavors (tau martin), stevia extracts (e.g., rebaudioside A, glycyrrhizin, etc.) and synthetic flavors (saccharin, aspartame, etc.).

The food composition of the present invention may be formulated in the same manner as the above-described pharmaceutical composition and used as a functional food or may be added to various foods. Foods to which the composition of the present invention can be added include, for example, beverages, meat, chocolates, foods, confectionery, pizza, ram noodles, other noodles, gums, candy, ice cream, alcoholic beverages, vitamin complexes, .

In addition, the food composition may contain various additives such as flavoring agents such as various nutrients, vitamins, minerals (electrolytes), synthetic flavors and natural flavors, colorants and aging agents (cheese, chocolate, etc.), pectic acid, Alginic acid and its salts, organic acids, protective colloid thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonating agents used in carbonated drinks, and the like. In addition, the food composition of the present invention may contain natural fruit juice and pulp for the production of fruit juice drinks and vegetable drinks.

Hereinafter, the present invention will be described in more detail with reference to examples. These examples are for further illustrating the present invention, and the scope of the present invention is not limited to these examples.

Example  1. Materials and Methods

1.1. material

Piper long cannum was purchased from INDOFINE Chemical Company. Piper longin was dissolved in 100% DMSO for in vivo experiments and stored at -20 ° C. Piper longin was dissolved in 100% 1: 19 = DMSO: PBS at -20 ° C for in vitro experiments. LPS was purchased from Sigma, dissolved in PBS and stored at -20 ° C. The CD3 / CD28 activator was purchased from STEMCELL ^TM Technologies and stored at 4 ° C.

1.2. EAE (experimental autoimmune  encephalomyelitis) induction and drug treatment

10-week-old female C57BL / 6 mice were used as experimental animals, and EAE was induced in the mice using EK-0115 kit (Hooke laboratories). Piper longons were administered once every two days with 50 μL at 1.5 or 3.0 mg / kg. Groups that did not treat Piper longingum received 50 μL of 100% DMSO once a day. The clinical score was measured daily from 0.5 to 0.5 points in accordance with the criteria provided by Hooke Laboratories.

1.3. Preparation of spinal cord tissue

After perfusion with PBS, the spine was separated and stored at -80 ° C or stored in 4% formaldehyde solution for 7 days, then in demineralized solution for 7 days, stored in 30% sucrose solution, Lt; / RTI >

1.4. Cell culture

Jurkat cells were cultured in RPMI (Roswell Park Memorial Institute) 1640 medium containing 10% FBS (fetal bovine serum) and 100 units / mL penicillin. RAW 264.7 cells were cultured in DMEM (Dulbecco's Modified Eagle Medium) medium containing 10% FBS and 100 units / mL penicillin. After treatment with Piper longinmin at 0.5, 1.0, and 2.5 μM concentrations, a further CD3 / CD28 activator (10 μL / mL) or LPS (1 μg / mL) was treated for 24 hours.

1.5. Tissue staining

The tissues stored in 30% sucrose solution were cut into 16 μm using a cryostat microtome and stained with H & E (hematoxylin and eosin) or Luxol fast blue (LFB) staining.

1.6. Immunochemical staining and Immunofluorescent staining

The tissues stored in 30% sucrose solution were cut into 16 μm using a cryostat microtome and washed with PBS. In order to inhibit endogenous peroxidase activity, the cells were reacted with 3% hydrogen peroxide for 20 minutes and 1% triton X-100 for 15 minutes for permeabilization. After washing with PBS, the cells were blocked with 5% bovine serum albumin (BSA) for 1 hour and then reacted with the primary antibody using an overnite. After washing with PBS, secondary antibody with IgG-HRP (horseradish peroxidase) or fluorescent secondary antibody was reacted at room temperature for 1 to 2 hours. After washing with PBS, the tissues reacted with secondary antibody with HRP were reacted with chromogen DAB, dehydrated with 70-100% ethanol stepwise, washed with xylene, and covered with cover slides. The tissue reacted with the fluorescent secondary antibody was stained with DAPI and covered with cover slides. The results were analyzed by observation under a microscope.

1.7. Western Blotting

The spinal cord tissues from the vertebrae were homogenized and the proteins in the cytoplasm and nucleus were extracted using PRO-PREP. Buffer A (50 mM HEPES, pH 7.4, 10 mM KCl, 1 mM EDTA, 1 mM EGTA, 1 mM dithiothretiol , Proteins were extracted from the cytoplasm using 0.1 mg / mL PMSF, 1 μg / mL pepstatin A, 1 mg / mL leupeptin, 10 μg / mL soybean trypsin inhibitor, 10 μg / mL aprotinin and 0.5% Nonidet P-40) And the protein in the nucleus was extracted with buffer C (buffer A + 10% glycerol + 400 mM KCl). After loading 20-40 μg of each protein in 8-15% SDS (sodium dodecyl sulfate) -Polyacrylamide gel, electrophoresis was performed and proteins were transferred to NC (nitrocellulose) membrane. After blocking with 5% skim milk for 1 hour, the antibody was reacted with the primary antibody by overnight, washed with TBST and reacted with secondary antibody with IgG-HRP (horseradish peroxidase) for 1-2 hours at room temperature . Enhanced chemiluminescence The protein bands were photographed using a Western blotting detection system and a Chemi-doc instrument to confirm the results.

1.8. Cytokine production measurement

Spinal cord tissues isolated from vertebrae were homogenized and proteins from cytoplasm and nucleus were extracted using PRO-PREP and assayed using ELISA Kits (R & D Systems) as described in the manual. For Jurkat cells and RAW 264.7 cells, RNA was extracted from the cells according to the manual using easy-BLUR total RNA extraction kit (intron), and cDNA was amplified by High Capacity cDNA Reverse Transcription Kits (Applied Biosystems) Were synthesized. The cDNA was synthesized using a Brilliant III Ultra-Fast Green QPCR Master Mix (Agilent Technologies, Inc.) along with primers suitable for the cytokine to be identified. The CT values were confirmed using the StepOnePlus Real-Time PCR System according to the manual. The final amount of mRNA expression was calculated according to the following formula.

[Equation 1]

ΔΔCT = (C _{T, target} C _{T, β -actin} ) experimental sample - (C _{T, target} C _{T, beta -actin} ) control sample

Example  2. MOG (myelin oligodendrocyte glycoprotein ) Induced EAE (experimental autoimmune  Inhibitory effect of Piper long canin on clinical score of encephalomyelitis model

The inventors of the present invention have found that the use of an experimental autoimmune encephalomyelitis (EAE) model (a mouse model in which dehydration-induced induction is induced by MOG _35-55 ), which is widely used as an animal model of multiple sclerosis, Experiments were conducted to examine the mitigation effects. Piper longin is administered intraperitoneally to the mouse model at a dose of 1.5 mg / kg / day or 3.0 mg / kg / day for 14 days (Fig. 1a). From the day of EAE induction, the degree of paralysis and weight of the mice for 29 days were observed daily.

As a result, no symptom of paralysis was observed in the control mice, whereas up to 2.38 ± 0.37 points in the EAE model was obtained based on the highest score of 5 points. The highest score was 1.69 ± 0.34 for the 1.5 mg / kg Piper longinmin group and the lowest was 1.57 ± 0.27 for the Piper longinmin 3.0 mg / kg group (Fig. 1b and 1d). In addition, the severity of paralysis increases the amount of body weight reduction, whereas in the control mice, the body weight of the MOG-treated group is generally decreased while the body weight is continuously increased from the start of the experiment. However, it was confirmed that the group to which Piper longummin was administered showed a lower amount of body weight as a whole than the group that only EAE induced (FIG. 1C).

Example  3. In the mouse spinal cord Piper on Long Kumin  by MOG - Induction Dehydrated candle (demyelination) inhibitory effect

Multiple sclerosis is a disease characterized by the destruction of myelin basic protein (MBP) or myelin oligodendrocyte glycoprotein (MOG) associated with oligodendrocyte cells, the cells of which immune cells are the myelin, Dehydration is progressing and is known to cause paralysis disease. Therefore, hematocylin and eosin (H & E) staining was performed to confirm the penetration of immune cells into the spinal cord. As a result, it was confirmed that a large number of immune cells were infiltrated into spinal cord tissues in EAE mice as compared with control mice , And the penetration of these cells was confirmed to be decreased in the piper longin-treated group (Fig. 2a).

In addition, when Luxol fast blue (LFB) staining was performed to confirm the degree of destruction of aquatic plant myelin, it was confirmed that a number of afferent nerves were destroyed in spinal cord tissues of EAE-induced mice compared with control mice, (Fig. 2 (b)).

Immunofluorescent staining of the myelin basic protein (MBP), which is a herbicide, showed that the myelin basic protein was significantly destroyed in the spinal cord tissue of the EAE-induced mouse compared to the control mouse. The destruction of the myelin basic protein (Fig. 2 (c)).

Example  4. In the mouse spinal cord Piper on Long Kumin  by MOG - Inhibitory effect of induction on immune cell penetration

The expression of CD3, a marker of T cells, was confirmed by immunofluorescence staining to confirm the extent of T cell infiltration in immune cells in mouse spinal cord tissue. It was confirmed that the expression of CD3 was significantly increased in EAE-induced mice compared to control mice, indicating that T cells infiltrated into the spinal cord tissues, and that the infiltration of these T cells was reduced by the administration of piperlongmin 3a).

The expression of F4 / 80, a macrophage marker, was confirmed by immunofluorescence staining to confirm the extent of macrophage infiltration in immune cells in mouse spinal cord tissues. The expression of F4 / 80 was significantly increased in EAE-induced mice compared to control mice, indicating that macrophages infiltrated into the spinal cord tissues, and that the infiltration of these macrophages was reduced by the administration of Piperlongmin (Fig. 3B).

Example  5. Piper on Long Kumin  Stimulant-induced glial cells ( glial  cell activity

It is known that activation and differentiation of glial cells are increased by neuronal inflammation during progression of multiple sclerosis. In order to investigate the protective effect of Piperlong canin against the activities of astrocytes and microglia, the inventors of the present invention have investigated the effects of GFAP (Glial fibrillary acidic protein), which is an astrocytic marker in mouse spinal cord tissue, and Iba1 Immunofluorescent staining and Western blotting were performed to confirm expression of binding adapter molecule 1.

As a result, it was confirmed that the activity of astrocytes and microglia was increased in the EAE-induced mouse compared to that of the control mice, and that the activity of the astrocytes and the microglia was decreased by the administration of the Piperlongmin (Figs. 4A and 4B). In addition, both of the differentiation of both cells were increased in EAE-induced mice compared to control mice, but it was confirmed that they were decreased by administration of piperlongmin (Fig. 4C).

Example  6. Piper on Long Kumin  Inhibitory effect of stimulant-induced inflammatory protein

To investigate the inhibitory effect of Piper longingmin on the MOG-induced paralysis by inhibition of neuroinflammation, the expression of inducible nitric oxide synthase (iNOS) and COX-2 (Cyclooxygenase 2), which are expressed in proportion to the inflammatory reaction, Blotting. The expression of iNOS and COX-2 was most increased in spinal cord tissues of EAE mice, which had the highest paralysis score compared with the control mice, and the expression level thereof was decreased by administration of Piper longum (Figs. 5A to 5C) .

In addition, iNOS and COX-2 increased when treated with lipopolysaccharide (LPS), which induced inflammation, in mouse cell line RAW 264.7 cells, one of the major causative cells of multiple sclerosis, (Fig. 5D). This could be confirmed in Jurkat T cells, which are human cell lines of T cells considered to be the major causative cells in multiple sclerosis. The increased expression of iNOS and COX-2 when increasing the activity of T cells with the CD3 / CD28 activator was reduced by treatment with Piper longinum (Fig. 5E).

Example  7. Piper on Long Kumin  Inhibitory Effect of Stimulant-Induced Pre-inflammatory Cytokines

To measure the degree of inflammatory cytokines TNF-α and IFN-γ, proteins were extracted from mouse spinal cord tissues and analyzed by ELISA. As a result, the cytokine was significantly increased in the EAE-induced mice compared to the control mice, and the cytokine was significantly decreased by the Piper longum administration as compared with the EAE-induced mice (FIGS. 6A and 6B).

In addition, the amount of mRNA expression was measured using qPCR to measure the degree of inflammatory cytokines TNF-a and IFN-y in immune cells. In RAW 264.7 cells treated or not treated with LPS and Piper longinum, the mRNA of TNF-α and IFN-γ was significantly increased by LPS and IFN-γ mRNA was significantly increased , But TNF-α mRNA was significantly reduced by Piper longin (FIGS. 6c and 6d).

In Jurkat T cells treated or not treated with CD3 / CD28 activator and Piper longin, the mRNA of TNF-α and IFN-γ was significantly increased by activator as in RAW 264.7 cells, and 1.0 μM of Piper longurmin or 2.5 mu M did not show a significant decrease in IFN-y mRNA, it was confirmed that TNF-alpha mRNA was significantly decreased by piper longin (Figs. 6E and 6F).

Example  8. Piper on Long Kumin  Stimulant-induced NF - κB (nuclear factor kappa-light-chain-enhancer of activated B cells)

NF-κB is an important transcription factor for the expression of inflammatory cytokines (TNF-α, IFN-γ, etc.) and inflammatory genes (iNOS, COX-2, etc.) and NF-κB is an NF-κB / IκBα complex IκBα is phosphorylated by an inflammatory substance such as LPS and is separated into NF-κB and p-IκBα. The isolated p-IκBα is degraded and NF-κB is transferred into the nucleus and then bound to DNA. Lt; RTI ID = 0.0 > expression < / RTI > Therefore, we examined the expression in the cytoplasm of IκBα and p-IκBα and the expression of the functional subunits p50 and p65 in the nucleus of the NF-κB complex by Western blotting.

As a result, in the cytoplasm of mouse spinal cord, RAW 264.7 cells and Jurkat T cells treated with Piper longingmin, the expression of IκBα was increased, but the expression of p-IκBα was decreased, compared with the group treated with each activator. The expression of p50 and p65 in the nucleus was found to be reduced in the group treated with piperlongmin compared to the group treated with each activator (Fig. 7).

Example  9. NF - κB  Inhibitors and Piper Long's Resident  Inhibitory effect of stimulant-induced inflammation by co-treatment

To further investigate whether Piper long-termin is involved in the inhibition of NF-κB, RAW 264.7 cells and Jurkat T cells were treated with a combination of PAO (phenylarsine oxide) and Piper longin, which are used as inhibitors of NF-κB, MRNA expression levels of related cytokines (TNF-a, IFN-y) were measured. TNF-α and IFN-γ mRNA increased by Activator decreased by Piper longin or PAO alone treatment. However, the effects of the treatment with Piper longingmin and PAO were significant only in the TNF-α mRNA levels in RAW 264.7 cells, and the levels of TNF-α and IFN-γ in IFN-γ mRNA and Jurkat T cells in RAW 264.7 cells mRNA was decreased but not significant (Figs. 8a to 8d). The degree of expression of iNOS and COX-2 protein was confirmed by Western blotting. INOS and COX-2 increased by activator in RAW 264.7 cells and Jurkat T cells were decreased by treatment with Piper longin or PAO alone, and iNOS and COX-2 (Fig. 8E and Fig. 8F).

Claims (10)

A pharmaceutical composition for preventing or treating multiple sclerosis comprising piperironumine as an active ingredient. The method according to claim 1,
Wherein the concentration of the piperonurmin is from 0.01 to 5 [mu] M. The method according to claim 1,
Wherein the concentration of Piper longummin is 1 to 5 mg / kg / day. The method according to claim 1,
Wherein the Piper longinmin inhibits demyelination in the brain or spinal cord. The method according to claim 1,
Wherein said Piper longinmin inhibits the infiltration of T cells or macrophages into brain or spinal cord tissue. The method according to claim 1,
Wherein the Piper longum inhibits the activity of NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells). A food composition for preventing or ameliorating multiple sclerosis comprising piperlongumine as an active ingredient. delete delete delete

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