Celastrol Ameliorates Osteoarthritis Via Regulating TLR2/NF-κB Signaling Pathway

Objectives Osteoarthritis(OA)is a joint disease characterized by degeneration of joint cartilage and is a significant cause of severe joint pain,physical disability,and impaired quality of life in the aging population.Recent studies have shown that innate immunity plays a core role in the occurrence and development of osteoarthritis.Celastrol has a long history and has been shown to have therapeutic effects on various inflammatory diseases.This study aimed to investigate the effect of celastrol on OA as well as the mechanisms of celastrol in vivo and in vitro.Methods A rat knee OA model was established using medial collateral ligament transection(MCLT)+partial meniscectomy(MMT).At the 8th week postoperatively,celastrol(1mg/kg)was injected into the joint cavity for 8 weeks.The therapeutic effect of celastrol was evaluated by the following experiments:1)The joint pain of rats was evaluated by animal behavior experiment.2)The swelling and cartilage wear of knee joint were observed in general.3)The histopathological changes were observed by S&F and H&E staining.4)Micro-CT was used to evaluate osteophyte formation and subchondral bone mineral density of knee joint.5)Immunohistochemistry was used to detect the expression of MMP13 and COLⅡ in histologic sections.Cartilage was collected from osteoarthritis patients and healthy people.The histopathological changes of human cartilage were observed by S&F and H&E staining,and the expression of TLR2 was detected by immunohistochemistry.To further verify the results of animal experiments,chondrocytes were isolated and cultured from human and rat cartilage.The following experiments were carried out:1)Human chondrocytes were stimulated with IL-1β(lOng/mL)for 24h,and the expression of TLR2 was detected by WB and RT-PCR.2)Rat chondrocytes were stimulated by IL-1β(10ng/mL)for 24h.The expression of inflammatory factors IL6,PGE2 and NO in the supernatant of the chondrocytes was detected by ELISA.The expression of MMP13,COLⅡ,TLR2 and NF-κB were detected by WB and RT-PCR.Results Animal experimental studies showed that celastrol can effectively relieve osteoarthritis:1)Compared with the control group,the 5-minute walking distance and standing times of osteoarthritis group were significantly reduced,and the diameter of knee joint was enlarged.Compared with the osteoarthritis group,the 5-minute walking distance and standing times were increased and the knee diameter was shortened in the celastrol group.2)The articular cartilage of rats in the osteoarthritis group was severely worn and the articular surface was rough in general.Compared with osteoarthritis group,the joint surface of celastrol group was smooth without obvious wear.3)H&E and S&F staining showed that the morphology of chondrocytes in the osteoarthritis group was disorder,the number of chondrocytes decreased,and synovial inflammation was obvious.The morphology of chondrocytes in celastrol treatment group was regular and synovial inflammation was mild.4)Micro-CT showed that compared with the control group,osteophyte formation and subchondral osteoporosis were observed in the osteoarthritis group.Osteophytes were significantly reduced and subchondral osteoporosis improved in the celastrol group compared with the osteoarthritis group.5)Immunohistochemistry showed that the expression of MMP13 was up-regulated,the expression of COLⅡ was down-regulated,and extracellular matrix was degraded in the osteoarthritis group.Compared with the osteoarthritis group,the expression of MMP13 was down-regulated and the expression of COLⅡ was upregulated in the celastrol group,which promoted the synthesis of extracellular matrix.Cell experiments showed that the mechanisms of celastrol in the treatment of osteoarthritis were as follows:1)The expression of IL6,PGE2 and NO was significantly up-regulated in chondrocytes treated with IL-1β for 24h,and celastrol inhibited the up-regulation of inflammatory factors induced by IL-1β.2)IL-1β induced the expression of MMP13 in chondrocytes and down-regulated the expression of COLⅡ.Celastrol down-regulated the expression of MMP13 and increased the expression of COLⅡ,and reduced the degradation of extracellular matrix induced by IL-1β.3)IL1β stimulated chondrocytes to activate the TLR2/NF-κB signaling pathway and up-regulated the expression of TLR2 and p-NF-κB.Celastrol down-regulated the TLR2/NF-κB signaling pathway to inhibit inflammation and reduce degradation of extracellular matrix.Conclusions In summary,celastrol regulates TLR2/NF-κB signaling pathway,effectively alleviates cartilage wear,synovial inflammation and subchondral bone loss in osteoarthritis,delays various pathological progress of osteoarthritis.It provides innovative strategies for the treatment of osteoarthritis.