Effect Of Andrographolide On Pain And Inflammation In Osteoarthritis And Its Underlying Mechanism

THESIS : Effect of Andrographolide on pain and inflammation in osteoarthritis and its underlying mechanism SPECIALIZATION: Surgery POSTGRADUATE: Wang Rongliang MENTOR: Shi DongquanBackground: Osteoarthritis(OA)is a degenerative disease characterized by joint pain,swelling and stiffness.Its pathophysiological changes include local damage of cartilage in the load-bearing area of the joint,accompanied by osteophyte formation at the edge of the joint,subchondral bone change,and varying degrees of synovitis and joint capsule thickening.Traditionally,OA is considered as a pathological result of abnormal joint load and mechanics,but now it has been found that synovitis is also a common pathological reaction during OA process.It suggests that synovium plays an important role in the development of the disease.Synovium is the inner structure of articular capsule,and its functions include supplying cartilage nutrition and lubricating articular cavity structure.Among them,fibroblast-like synovial cells(FLSs)play a particularly important role.Previous studies have shown that the treatment response of OA synovial tissue has a stronger correlation with pain changes,and a variety of cytokines secreted by synovial cells can also harm the intra-articular structure and aggravate local inflammation.Tumor necrosis factor-α(TNF-α)contributes to intra-articular inflammation and it will transmit signals by binding to TNF receptor(TNFR).However,there are few reports about TNFR2 and downstream signal transduction mechanisms compared with well-reported TNFR1-related signal pathways.Therefore,in the process of therapeutic intervention of OA,whether TNFR2 can be used as a therapeutic target and its related downstream signal transduction is not clear to a large extent.Andrographolide(Andro)has strong anti-inflammatory activity,and its main anti-inflammatory mechanism is related to NF-κB signal pathway.A previous randomized,double-blind,placebo-controlled study found that Andro can relieve pain in patients with knee osteoarthritis,which suggested Andro could be used as a drug for the treatment and prevention of osteoarthritis.The latest research reports showed that Andro had an effect on the transport of receptors on the cell surface.Therefore,we speculate that the therapeutic effect of Andro on synovial inflammation and TNFR2 internalization resulted in attenuating OA progression and pain.Methods: FLSs were isolated from OA patients,and CCK8 was used to observe the effect of Andro on cell proliferation.The expression of inflammatory cytokines after Andro treatment and the anti-inflammatory effect of Andro under TNFα stimulation were measured by QPCR.In addition,the expression of TNFR2 and downstream NF-κ B was detected by Western Blot,and the distribution of TNFR2 on cell surface was analyzed by flow cytometry and immunofluorescence.According to the co-treatment of HCQ and Andro,the expressions of intracellular inflammatory cytokines,TNFR2 and downstream NF-κB signaling were observed as well.Finally,the OA rat model was used in vivo to observe the progress of articular cartilage and synovium under the treatment of Andro.At the same time,behavioral tests were used to reflect the progress of rat joint pain during drug treatment.Results: Andro can inhibit the proliferation of FLSs.However,even low concentration of Andro could inhibit the expression of ADAMTS4,ADAMTS5 and CCL2 in FLSs,and decreased the expression of TNFR2 in cells and on cell surface.In this study,we found that the degradation of TNFR2 induced by Andro is related to the function of lysosome.In addition,the decrease of TNFR2 expression caused by Andro will further lead to the decrease of downstream phosphorylation level of p65 of NF-κB signal pathway and the expression of inflammatory factors will be inhibited as well.Finally,in vivo results showed that Andro could reduce synovitis inflammation and OA progression.Andro can also significantly reduce the pain response,which is related to the sensitization and relief of spinal cord pain.Conclusions: Taken together,our results indicate that Andro suppressed synovial inflammation in vitro and in vivo via increased TNFR2 internalization and degradation.Andro may be helpful in the prevention of OA and pain.