Resveratrol Reduces Autophagy Derived Mitochondrial Dysfunction And Apoptosis In Fibroblast-Like Synovial Cell

Background In the current clinical treatments of rheumatoid arthritis?RA?,most of drugs were treated with traditional anti-inflammatory and hormone drug.Due to the unable to treat the abnormal hyperplasia of synovial cells in the joint,the hyperplasia of the synovial tissue lead to the damage of articular cavity,cartilage,meniscus and ect.It eventually leads to the joints rigidity,deformity,and dysfunction.Therefore,there is a new idea to explore methods against proliferation of synovial cells.Autophagy,regulated by multiple autophagy related genes,was the process of degraded self-damag organelles and proteins by lysosomes.Cells produce reactive oxygen species?ROS?by aerobic metabolism,90%of the ROS is produced in the mitochondria.Many harmful stimulation breaks the balance of cell ROS production and scavenging system,thereby causing oxidative stress,which can lead to mitochondrial damage.Resveratrol?Res?,a kind of natural polyphenol compounds,mainly existing in the grape skins,giant knotweed rhizome etc,has showed many pharmacological activities,such as anti-tumor,antioxidant,anti-inflammatory etc.Studies have shown that Res regulates autophagy levels and ROS levels through multiple pathways.Cell autophagy or increased ROS levels causes the mitochondria damage,which eventually leads to cell damage.In this study,adjuvant arthritis?AA?rats and the original fibroid synovial cells was used as subjets.we explored the effect of resveratrol on the level of autophagy in synovicytes cells,and the relationship between mitochondrial dysfunction and apoptosis,provided a possible basis for clinical trials.Objective The aim of this study was to investigate the role of resveratrol in the reduction of autophagy induced the mitochondrial dysfunction during FLSs apoptosis.Methods The AA model was induced by Freund's Complete Adjuvant?FCA?.The model rats divided into five groups?n=10?:the model group,low dose of resveratrol group?5mg/kg?,medium dose of resveratrol group?15mg/kg?,high doses of resveratrol group?45 mg/kg?and Tripterygium group?100mg/kg?.Those rats were treated with the solvent or drugs every day by intragastric administration for 12 days from 18th day.The swelling degree and arthritis index score were recorded.After 12 days rats were killed,and the knee/ankle joints of rats were taken for pathological examination.The level of apoptosis and autophagy were studied in rat synovial tissue.The synovial tissue were obtained from the knee joint of RA patients,and the fibroblast-like synovial cells which identified by VCAM-I IHC were obtained from the primary cells which devived from synovial tissue.The FLSs cell viability was detected after incubates different concentrations of H₂O₂.Then we determine the follow-up experiment with 5?M H₂O₂ and different concentrations Res?40?M,80?M,160?M,320?M?.Cells viability was tested by CCK-8,and the cells apoptosis was detected using TUNEL staining.The expression of autophagy related protein was detected by immunofluorescence and Western blot.The changes of mitochondrial morphology were examined by electron microscopy.JC-1,calcium ion imaging and ATP kit was used to detect the changes of mitochondrial function.Results In rat experiment,the FCA could induce the establishment of AA rat model.Resveratrol decreased the toe swelling and arthritis index score in the other side of AA rats.Histopathological examination showed that resveratrol decreased the knee and ankle injury in AA rats.Immunohistochemical and Western blot results showed that resveratrol promoted the cell apoptosis in the synovial tissue,and decreased the autophagy of synovial tissues.In cell experiments,the primary cells was FLSs which were identified as positive by VCAM-I?vascular cell adhesion molecule-I?.The results of CCK-8?cell counting kit-8?indicated that the FLS cell viability?which pretreated with 5?M H₂O₂ group?was better compared with others.FLS cells was incubated with different concentrations resveratrol?40?M,80?M,160?M,320?M?after pretreatment with 5?M H₂O₂ solution.It was found cells apoptosis rate increased with the resveratrol concentration which were detected by TUNEL staining and Western blot.Western blot and immunofluorescence analysis indicated the expression level of autophagy related protein?LC3 B/A,ATG-5?decreased with the increase of resveratrol concentration.Moreover,mitochondrial function detection showed that with the increasing of resveratrol concentration,mitochondrial membrane potential gradually lost.The production of mitochondrial superoxide and the content of ATP inside FLS was positive,negatively correlated to resveratrol concentration.Simultaneously,the intracellular calcium released and calcium influx decreased gradually with the increase of resveratrol concentration.Conclusion?1?Resveratrol improved the inflammatory changes of AA rats which induced by Freund's complete adjuvant.?2?Resveratrol decreased the autophagy protein expression and increased the apoptosis protein expression in synovial tissue of knee joint?3?Resveratrol reduced the autophagy level of FLS,which derived the mitochondrial dysfunction,and promoted the apoptosis of FLS.