KR20110117788A - Composition for treating activated-fibroblast-like synoviocytes containing melittin - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for treating rheumatoid arthritis containing melittin, which is an active ingredient of bee venom that can inhibit the activity of fibroblast-like synovial cells, which plays an important role in rheumatoid arthritis. Treatment with human fibroblast-like synovial cells activated with the cytokine IL-6 / sIL-6R reduced the p65 and STAT3 activities of the transcription factors NF-κB during apoptosis with mitochondria. In addition, the expression of apoptosis inhibitors bcl-2 and mitochondrial cytochrome c were inhibited, and the expression of caspase-3, Apaf-1 and cytoplasmic cytochrome c were increased. As a result, melittin has an effect of inducing cell death mechanisms associated with mitochondria in inhibiting activation of activated human fibroblast-like synovial cells.

Description

Composition for treating activated-fibroblast-like synoviocytes containing melittin

The present invention relates to a composition for inhibiting the activity of fibroblast-like synovial cells containing melittin, and more specifically, the melittin induces cell death by regulating the apoptosis mechanism associated with mitochondria and the activity of transcription factors related thereto. It relates to a pharmaceutical composition for the treatment of rheumatoid arthritis by inhibiting the activity of fibroblast-like synovial cells.

Rheumatoid arthritis is a representative disease of inflammatory arthritis characterized by chronic synovitis of the peripheral joint and is known to occur in about 0.5 to 1% of the adult population. Arthritis affects psychological and social health in addition to the physical function of the patient and leads to increased mortality and morbidity through decreased mobility. In addition, the incidence of heart disease, infection and cancer is high in arthritis patients. The degree of arthritis can lead to joint destruction and deformation, resulting in movement disorders if the joint inflammation, which changes over time, is poorly controlled. The cause of the disease is not known yet, but it may be related to an infection such as Mycoplasma, Epstcin-Barr virus, cytomegalovirus, or rubella virus, or the body's immune response. Therefore, rheumatoid arthritis can be treated and prevented by reducing joint pain and inflammation, increasing joint function, and preventing deformation and destruction of joints. Treatment methods can be divided into drug therapy and non-drug therapy. As a therapeutic agent, COX-2 inhibitors and anti-TNF preparations have been actively conducted, but the cost is very expensive, and research on biological agents such as Etanercept is being actively conducted.

Bee venom is made up of about 40 substances contained in the bee venom sac and has been used to treat human diseases since BC. Studies on bee venom are currently active in many countries, including the United States, China, Russia and Northern Europe. Although studies have been conducted on the effects and mechanisms of bee venom components, hypersensitivity and toxicity of bee venom, immunotherapy and arthritis, herpes simplex, multiple sclerosis, and treatment of tumors, etc. There is not much scientific research.

Conventional techniques using bee venom include treatment of arthritis in pigs using live bee venom as an animal treatment method (Korean Patent Application No. 10-1999-0005472), and treatment of mastitis in pigs using live bee venom (Korean Patent Application No. 10-1999-0005477) ), Treatment of soybean-free sows using live bee venom (Korean Patent Application No. 10-1999-0005474), treatment of diarrheal disease of pigs using raw bee venom (Korean Patent Application No. 10-1999-0005475), sow using live bee venom Treatment of MMA syndrome in Korea (Korean Patent Application No. 10-2000-0046411), treatment of diarrheal disease of calf using live bee venom (Korean Patent Application No. 10-2000-0046412), treatment of endometritis milk cow using raw bee venom (Korea Patent Application No. 10-2001-0071220).

In addition, bee venom and its main component, melittin, in Korean Patent Registration No. 10-0668229 "Nuclear Factor-Kappabi Activity Inhibitor" has an activity of inhibiting NF-κB activation and regulating DNA binding ability, thereby inhibiting NF-κB activity. Or it can be used as a transcription inhibitor for regulating the transcription of NF-κB involved, and the bee venom hexane extraction in Korean Patent No. 10-0483496 "anti-inflammatory and analgesic composition containing bee venom water soluble" A bee venom fraction having an anti-inflammatory and analgesic effect is disclosed, which is composed of a substance having a molecular weight of 10 KDa or less obtained from a water-soluble fraction obtained by removing the hexane solubles and then extracting it with ethyl acetate again to remove the ethyl acetate solubles.

In addition, the Republic of Korea Patent No. 10-0844886 "Pharmaceutical composition for treating vitiligo containing bee venom as an active ingredient and a method for screening inhibitors of pigmentation induced by bee venom" drug for treating vitiligo that can easily treat vitiligo A method is disclosed that can screen in a bulk / highthroughput manner a pharmaceutical composition and a substance that inhibits pigmentation induced by bee venom.

However, there has been no research on the therapeutic activity of melittin, an active ingredient of bee venom associated with rheumatoid arthritis.

The present inventors isolated the synovial cells from the synovial tissue of patients with rheumatoid arthritis, and then produced fibroblast-like synovial cells, activated them using IL-6 / sIL-6R, and treated melittin to identify mechanisms related to cell death. It was confirmed that it is effective in the treatment of rheumatoid arthritis.

Accordingly, an object of the present invention is to provide a pharmaceutical composition for treating arthritis as an inhibitor of activation of fibroblast-like synovial cells containing melittin as an active ingredient.

The purpose of the present invention as described above is to treat the melanin to human fibroblast-like synovial cells activated with the inflammatory cytokine IL-6 / sIL-6R and p65 of the transcription factor NF-κB in the process of apoptosis associated with mitochondria By confirming the effect of melittin by confirming the expression levels of cytochrome c of bcl-2 and mitochondria and the expression of caspase-3, Apaf-1, and cytochrome c of cytoplasm c, which are STAT3 activity and apoptosis inhibitors Was achieved.

The present invention provides a pharmaceutical composition for treating arthritis as an inhibitor of the activation of fibroblast-like synovial cells containing melittin as an active ingredient.

In the present invention, the term "active ingredient" refers to a substance or a group of substances (a pharmacologically active ingredient or the like, which is expected to express directly or indirectly the efficacy and effect of the drug by intrinsic pharmacological action. It means containing a main component).

In addition, the pharmaceutical composition may contain melittin and other carriers or excipients, and may be provided in a form obvious to those skilled in the art such as tablets, pills, granules, liquids, capsules, and the like. It is a pharmaceutical composition for treating arthritis as an inhibitor of the activation of fibroblast-like synovial cells.

The melittin according to the present invention was treated to human fibroblast-like synovial cells activated with the inflammatory cytokine IL-6 / sIL-6R to confirm the mechanism related to apoptosis.

Treatment of melittin with activated human fibroblast-like synovial cells reduced the activity of p65 and STAT3 of the transcription factors NF-κB during the apoptosis process associated with mitochondria.

In addition, melittin inhibited the expression of apoptosis inhibitors bcl-2 and mitochondrial cytochrome c, increased the expression of caspase-3 and Apaf-1, apoptosis inducing factors, and increased the expression level of cytoplasmic cytochrome c. I was.

As a result, melittin has an effect of inducing cell death mechanisms associated with mitochondria in inhibiting activation of activated human fibroblast-like synovial cells.

Melitin inhibits the activation of fibroblast-like synapses that play an important role in rheumatoid arthritis by inducing apoptosis mechanisms of fibroblast-like synapses.

As a result of treatment of melittin according to the present invention to human fibroblast-like synovial cells activated with IL-6 / sIL-6R, p65 of the transcription factor NF-κB and a transcription factor in the process of apoptosis associated with mitochondria, which are common in rheumatoid arthritis, The activity of STAT3 was decreased, and the expression level of cytochrome c of apoptosis inhibitors bcl-2 and mitochondria was decreased, the expression level of caspase-3 and Apaf-1, apoptosis inducing factor, and the expression level of cytochrome c of cytoplasm. With the effect of increasing, the melittin of the present invention is a very effective invention for the treatment of rheumatoid arthritis.

1 is a diagram showing the effect of melittin on the expression of STAT3 in activated fibroblast-like synovial cells.
2 is a diagram showing the effect of melittin on the apoptosis mechanism in activated fibroblast-like synovial cells.
Figure 3 shows the effect of melittin on the expression of transcription factor NF-κB p65 during apoptosis in activated fibroblast-like synovial cells.
4 is a diagram showing the mechanism by which melittin induces apoptosis associated with mitochondria in a rheumatoid arthritis cell model.

Hereinafter, the present invention will be described in detail with reference to specific embodiments, but the scope of the present invention is not limited only to these embodiments.

Example

In an embodiment of the present invention, the synovial cells were isolated from the synovial tissue of a patient with rheumatoid arthritis, and then treated with collagenase A (1 mg / ml) for 2 hours to prepare fibroblast-like synovial cells. Fibroblast-like synovial cells were activated by treatment with 100 ng / ml of IL-6 / sIL-6R for 30 minutes. Melitin was treated for 24 hours by concentration (1, 2 μg / ml).

The analysis results of the experiments were expressed using mean and standard deviation (mean ± SD) for each group, and all data were processed using the SPSS program (Statistical Package for Social Science, SPSS Inc., Chicago, IL, USA). The significance level was confirmed at p <0.05.

Example  1: protein isolation and Weston Blot  analysis

Fibroblast-like synovial cells were added at 5 ° C. at 4 ° C. after addition of cytoplasmic buffer A (100 mM HEPES, pH8.0, 1.5 mM MgCl ₂ , 10 mM KCl, 0.5 mM dithiothreitol, 0.1% NP-40, 0.5 mM PMSF). The reaction was carried out for 1 minute and centrifuged at 12,000 rpm for 10 minutes. Cytoplasmic extract (supernatant) was used after storage at -80 ℃. Mitochondrial extraction was performed using pellets centrifuged. Cell pellets for nuclear extraction were added with buffer B (20 mM HEPES, pH8.0, 20% glycerol, 100 mM KCl, 100 mM NaCl, 0.5 mM dithiothreitol, 0.5 mM PMSF), followed by reaction at 4 ° C. for 15 minutes. And centrifuged at 12,000 rpm for 10 minutes. The extracted protein was quantified by the Bradford method (Bio-Rad Laboratories, CA, USA), and the quantified protein was electrophoresed with 10% SDS-PAGE and the NC membrane (Bio-Rad, Hercules, CA, USA) Moved to. The membrane was reacted with the primary antibody anti-STAT3, anti-phosphorylated STAT3, anti-bcl-2, anti-caspase-3, anti-Apaf-1 and NF-κB p65 and washed twice with TBS-T for 15 minutes before The reaction was carried out for 2 hours with a secondary antibody. Expression analysis for the antibodies was compared using the ECL enhanced chemiluminescence Western Blot Analysis (Amersham, Braunschweig, Germany) system kit expressed by a horseradish peroxidase-linked secondary antibody.

Example  2: experimental results

 Fibroblast-like synovial cells activated with IL-6 / sIL-6R were found to have been shown to have been shown to have been shown to be particularly active when activated with IL-6 / sIL-6R for 5 minutes, 30 minutes and 120 minutes. Stimulation for 30 minutes showed the highest activity. Therefore, the fibroblast-like synovial cells treated with IL-6 / sIL-6R for 30 minutes were found to have a concentration-dependent decrease in the expression level of p-STAT3 due to the treatment of melittin (see FIG. 1).

As shown in Figure 2, the activated fibroblast-like synovial cells were treated with melittin and then the proteins were separated to confirm the expression level of proteins related to the apoptosis mechanism. Treatment with melittin decreased the expression levels of apoptosis inhibitors bcl-2 and mitochondrial cytochrome c and increased the expression of caspase-3 and Apaf-1 and cytoplasmic cytochrome c cytoplasmic induction factors. This example confirmed the effect of melittin through apoptosis mechanism.

Activated fibroblast-like synovial cells confirmed the expression of NF-κB p65, a transcription factor, during apoptosis-related apoptosis by melittin treatment. It was confirmed that the expression level of NF-κB p65 in the cytoplasm was increased by melittin, and the expression level of NF-κB p65 in the mitochondria was effectively reduced. This result confirmed that melittin inhibits the introduction of NF-κB p65 into the nucleus into the nucleus (see FIG. 3).

As shown in FIG. 4, melittin was treated to IL-6 / sIL-6R activated fibroblast-like synovial cells to reduce the activity of the transcription factors NF-κB p65 and STAT-3 in apoptotic mechanisms associated with mitochondria. I was. The expression of apoptosis inducing factors, bcl-2 and mitochondrial cytochrome c, was decreased, and the expression of caspase-3, apopt-1, and cytoplasmic cytochrome c was increased.

Claims (1)

A pharmaceutical composition for treating arthritis as an inhibitor of activation of fibroblast-like synovial cells containing melittin as an active ingredient.

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