Effect And Apoptosis Mechanism Of Resveratrol On H₂O₂-induced Of Fibroblast-like Synoviocytes And Part Of The Mechanism

Background Arthritis rheumatoid (RA) is a chronic, immune system disease, its pathogenesis has not yet fully understood. Treatment with non steroidal anti-inflammatory drugs, to some extent, it can effectively reduce the severity of the disease, but it can not change the structure of the joint destruction of RA patients in essence and solve the patient’s disease development. The excessive proliferation of synovial cells in patients with rheumatoid arthritis may be one of the important causes of the disease. From the point of view of physiological function, synovial membrane can produce synovial fluid to lubricate joints, joint function of nutrition supply, in addition, it has eliminate excess synovial cartilage debris, discharge off and other functions. Because of diseases caused by abnormal proliferation of synovial tissue, the abnormal tissue which is similar to the limitations of the invasive growth of tumor tissue will be continuing to destroy the joint osseous structure, and then the joint deformity, accompanied by varying degrees of dysfunction of the joint.Fibroblast like synoviocytes (FLS) is an important component of synovial tissues in the process of joint destruction, which plays an important role. It can secrete a variety of cytokines, proteases, peanut four acid metabolites, etc, and participate in and function in the whole process of destruction. A large number of studies have confirmed that the proliferation of FLS plays an important role in the pathogenesis and progression of RA. The FLS cells of RA patients are out of control and have abnormal proliferation and erosion characteristics. Studies have indicated that reactive oxygen species (ROS) is involved in the bone damage in the inflammatory lesions of the RA joint. Study confirmed that the forkhead box o (FoxOs) is has the species specificity of the forkhead transcription factor family, is sensitive to the redox reaction, involved in the control and regulation of cell proliferation, activation and oxidation process. Gene plays an important role in regulation of metabolism, cell proliferation and redox, and has a significant role in the maintenance of intracellular reactive oxygen species, and can increase the antioxidant capacity of the cells to protect the cells from oxidative damage. Previous studies show that localized in the mitochondria and endoplasmic reticulum of the anti apoptotic protein Bcl-2, Bax gene transcript exists widely in many tissues, Bax protein in the cytoplasm from the cytoplasmic translocation of apoptosis signal to the mitochondria membrane and activation of caspase and induction of apoptosis. Non-flavonoids polyphenolic compounds resveratrol is a kind of plant antitoxin, is more researched, has the function of anti-inflammatory, anti oxidative stress and anti tumor effect, for the therapeutic value of related metabolic diseases ---RA is hotspot of current medical research.Object To prepare the animal model of adjuvant arthritis rats (adjuvant arthritis, AA) was prepared, and to evaluate the model of AA rats. To observe the oxidative stress of AA rats, and the treatment of RES in vivo experiment; and with the cultured synovial cells, to explore the study of resveratrol alcohol (RES) on hydrogen peroxide (H2O2) treatment of rat adjuvant arthritis arthritis fibroblast like synoviocytes (FLS) apoptosis. Analyze each FLS FoxO1 and foxo4 gene level expression, and to explore the Fox genes in the process of apoptosis role.Methods 90 SD rats were randomly divided into normal group, model group, resveratrol group (low dose of 1 mg kg-1, medium dose of 5 mg kg-1, high dose mg kg-1), positive drug group (NAC30 mg kg-1),15 rats in each group. Using Freund’s complete adjuvant (Freund’s complete adjuvant, FCA), by subcutaneous to drug, rats injected with the toe position and induce SD rats to establish animal model of AA rats. After 14d model was made, the SD rats were fed with gastric lavage (Intragastric administration, ig). With the operation of 12D’ ingestion of resveratrol, through the joint swelling degree, multiple arthritis index, body weight and other aspects of the comprehensive indicators of changes, FCA induced AA model of the primary and secondary evaluation. In the twenty-sixth day, rats were killed by exsanguination, and blood samples were taken to detect the content of malondialdehyde (MDA) and superoxide dismutase (SOD) activity in the body of rats in each group.In vitro, the inflammatory side of synovial tissue, using tissue culture method to isolate and culture rat synovial cells cultured for third generations of FLS for experiment. (1) FLS isolation and identification:Using immunofluorescence staining, Rabbit anti mouse VCAM-1 polyclonal antibody was used to identify the synovial cells; (2) Cell proliferation test:To observe the effects of different concentration by CCK-8 method H2O2 on FLS proliferation; (3) Detection of apoptosis:Annexin V-FITC-PI flow cytometry to detect different concentrations of RES on H2O2 (20mol L-1) apoptosis treatment rate of FLS; (4) Mechanism discussion:Using RT-PCR detected in FLS FoxO1 and FoxO4 gene level expression and to explore the induction of apoptosis of synovial tissue internal relations; Western blot analysis in FLS of Bcl-2 and Bax protein expression changes, using the technology of cell biology and molecular biology, to further clarify the mechanism of resveratrol treatment of RA. Through the method of a variety of experiments, analyze the mechanism in treatment of RES AA rats from the perspective of cell apoptosis.Results In vivo, it has been proved that AA rats by hypodermic injection of FCA, toe parts of the injection induced arthritis 14 days later, secondary immune inflammatory reaction in AA rats with toe, experiments using res, can effectively reduce the AA rats secondary paw swelling, significantly reducing AA rats multiple arthritis index, to help mitigate the decrease in the body weight of rats in AA. By serological detection, the results show that rats in the model group compared with normal group rats in model group, SOD activity decreased, MDA levels were increased, compared with the model group, positive control group of res in the treatment group, and NAC can increase the level of SOD, and reduce the level of MDA, res in middle and high dose group with significant (P<0.05, P<0.01).In vitro experiments results show that CCK-8 observation showed that H2O2 (≤20μmol·L-1) in a certain range of concentration can promote the growth of FLS, and when H2O2 (>20μmol·L-1), the number of FLS apoptosis induced by H2O2 will be significantly increased, the low dose of H2O2 can promote the proliferation of FLS, resveratrol can promote the FLS apoptosis effect of low dose of H2O2 induced; Annexin V-FITC-PI Flow cytometry showed that the apoptosis rate of FLS increased with the increase of the concentration of RES under the stimulation of low concentration of H2O2 under oxidative stress; Using RT-PCR to detect the res significantly reduced the expression of FoxO1 and FoxO4 gene level, FLS apoptosis and some FoxOs gene expression related to the downregulation of res by decreases in FLS FoxO1 and foxo4 gene relative expression and apoptosis inducing effect. Western blot analysis showed that with the increasing of RES concentration, the expression of Bcl-2 was decreased, the expression of apoptotic protein Bax increased gradually.Conclusion Resveratrol can effectively improve the symptoms of arthritis in AA rats and reduce the serum level of AA in rats. The mechanism study showed that RES by down regulating FoxO1, FoxO4 gene, anti-apoptotic Bcl-2 protein, inhibit the expression of Bax protein induced apoptosis of synovial cells. RES can not only inhibit the proliferation of synovial cells in AA rats, but also can promote the apoptosis of synovial cells, to achieve the purpose of treatment of AA; RES can induce the apoptosis of synovial cells by regulating the relative expression of the apoptotic protein Bcl-2/Bax, At the same time, it can promote the role of FLS apoptosis by regulating the apoptosis protein, reducing the expression of Bcl-2 protein and increasing the expression of Bax may be one of the mechanisms of promoting apoptosis.