Effects Of Resveratrol On The Expression Of Caspase-8 And Flip Of Fibroblast-like Synoviocytes In Rats Induced By Type Ⅱ Collagen

Rheumatoid arthritis (RA) is a chronic inflammatory and autoimmune disease that mainly affects multiple synovial joints. The main pathological changes are over proliferation of synovial cells, growing of new blood vessel, infiltrating of macrophages and destruction of joint cartilage. Although the mechanisms of pathogensis are still unclear, there is growing evidence that activated fibroblast-like synoviocytes (FLS), as part of a complex cellular network, play an important role in the pathogenesis of rheumatoid arthritis. Inflammation of the synovial membrane is accompanied by an influx of immune-competent cells and by aberrant proliferation of resident fibroblast-like synoviocytes. Accretion of fibroblast-like synoviocytes directly contribute to joint destruction, through enhanced production of matrix-degrading enzymes, and indirectly, through excessive release of cytokines and chemotatic factors that boost the inflammation and the immune system. Targeting the proliferative fibroblast-like synoviocytes could facilitate regeneration of synovial joints. However, the cause and causative mechanism were unclear. Although there were a variety of drugs and treatment means, it couldn't cure or curb the progress effectively.Resveratrol, a polyphenolic compound which is abundantly present in the seeds and skin of grapes, has various biologic activities such as anti-inflammation, anti-oxidation, inducing cell cycle arrest, inducing cell apoptosis, anti-neovascularization, immunological regulation, anti-tumor, and so on. As well as the low virulence and abundant resource, resveratrol is generally considered to have extensive applicable prospect. Lots of studies have indicated that resveratrol could inhibit the proliferation of some malignant cells with overgrowth properties. Fibroblast-like synoviocytes in joints just like the growing tumor. Therefore, we hypothesized that resveratrol might have the similar effect on FLS in RA.Objectives: To investigate the effects of resveratrol on the expression of Caspase-8 and FLIP of FLS in rats induced by typeⅡcollagen, so as to study the concentration and the time effect.Methods: The experimental arthritis model was established by subcutaneous injection of typeⅡcollagen. The rats were randomly divided into two groups: the control group and the CIA group. The rats were immunized through the two sides of vertebral column with the emulsifying solution of typeⅡcollagen-acetic acid and Freund's incomplete adjuvant (FIA) and received a booster injection of this emulsion 21 days later. Controls were administered with an equal volume of Sodium Chloride at the same time. After 28 days the rats were killed and the synovial tissues of knee joint were taken. Synovial tissues were washed, sheared, digested, centrifugated, cultured and transferred in order to obtain the cells in generation 3 to 5. Morphological change of synovial cell was observed under the inverted microscope. Synoviocytes were cultured in vitro and were exposed to various concentrations of Resveratrol (50,100,200,400μmol/L) for duration of 24, 48 hours. And then we detected the expression of Caspase-8 and FLIP with Western-Bolt method. We carry out the semi-quantitative analysis of the experimental results with the automatic image analysis system. All statistical analyses were performed using SPSS 13.0 software.Results:1. Most rats caught arthritis in 2~3 weeks after collagen injection. The paws became swell and the movement of joint was limited.2. Two types of cells can be found from the primary cultured synoviocytes in Rats Induced by TypeⅡCollagen, the macrophagus-like synoviocytes and the fibroblast-like synoviocytes. We chose the third-generation to fifth- generation cells for the study. Because these cells shown lasting character, best growth, fibroblast-like cells in the main.3. The experiment by using different concentrations of Resveratrol (50,100,200,400μmol/L) deal with isolated and cultured in vitro rat fibroblast-like synovial cells in generation 3 to 5, after duration of 24 or 48 hours. The experimentral results with Western-Bolt method showed: both zymogen (55KD) and activated (20KD) caspase-8 can be detected in the control group and the Resveratrol group, and the expression of the caspase-8 increased in the latter group, and with the increasing concentration of expression gradually increase, and the expression increases along with the time extension in 24 or 48 hours.4. The experimentral results with Western-Bolt method showed: the expression of FLIP decreased in the Resveratrol group. And the expression decreased gradually with the increasing of Resveratrol concentration. The expression of FLIP decreased with the time extension in 24 or 48 hours.5. Through the correlation analysis indicated the FLIP expression and the caspase-8 expression were significantly negatively correlated.Conclusion:1. Resveratrol can increase the expression of caspase-8 of FLS in Rats Induced by TypeⅡCollagen, and the expression increases with the concentration of Resveratrol.2. Resveratrol can decrease the expression of FLIP of FLS in Rats Induced by TypeⅡCollagen, and the expression decrease with the concentration of Resveratrol.3. The expression of caspase-8 can be increased in Resveratrol through the decreasing of the expression of FLIP.