| Osteoarthritis(OA)is a chronic proliferative inflammation of the articular bone system,also known as chronic osteoarthritis.Non-steroidal anti-inflammatory drugs are often used in veterinary practice to suppress pain but have many side effects.Therefore screening for effective,safe and efficient OA drugs is particularly important.Some studies suggest that oleanolic acid(OLA)of natural origin can inhibit cartilage degeneration,but the exact mechanism of action is unclear.It was found that articular cartilage degeneration and subchondral bone reconstruction play an important role in the development of OA and that the Wnt/β-catenin signaling pathway is abnormally activated in OA,which is a potential target for the treatment of OA.Based on this,this study established a rat OA model by Anterior cruciate ligament transection combined with partial meniscectomy method(ACLT+PMMx)to comprehensively evaluate the protective effect of OLA on OA in rats,and explore the mechanism of OLA action based on Wnt/β-catenin signaling pathway to provide theoretical basis for future OLA drug development and clinical application.The experiment was divided into two parts,in vivo and in vitro:(1)A rat OA model was established by ACLT+PMMx method with 50 mg/kg OLA by continuous gavage for 28 d.Knee,quadriceps and serum samples were taken.The pain level of rats was detected weekly by knee extension vocalization,Von-Frey mechanical pain and heat sensitivity test.The morphological and pathological changes of knee cartilage in rats were observed,and Pelletier and OARSI scores were performed respectively.micro-CT was performed to detect microstructural changes in bone tissue.ELISA was performed to detect levels of citrate synthase(CS)and myosin heavy chain(MHC)in muscle,serum levels of inflammatory factors(IL-1β and TNF-α),markers of cartilage metabolism(CTX-II and COMP),and levels of bone metabolism markers(OCN and CTX-Ⅰ).Immunohistochemistry was performed to detect Collagen II,β-catenin and Wnt3 a levels.Immunoblotting was performed to detect muscle Nrf2 antioxidant system-related protein levels.(2)An inflammatory injury model was established using 10 ng/m L IL-1β,and CCK8 screened for OLA intervention dose.Real-time fluorescence quantitative PCR was performed to detect the levels of genes related to Wnt/β-catenin signaling pathway(Gsk-3β,β-catenin and Wnt3a)and ECM metabolic enzymes(ADAMTS4,MMP3 and MMP13)in chondrocytes.Immunofluorescence observation of β-catenin nuclear translocation.Immunoblotting was performed to detect chondrocyte BMP-2,WISP1,Wnt/β-catenin signaling pathway and ECM metabolism-related proteins,as well as ALP,OCN and RUNX2 protein levels in MC3T3-E1 cells.osteogenic differentiation of MC3T3-E1 cells was observed by ALP staining with alizarin red staining.The results showed that(1)OLA was able to reduce OA pain score,reduce cartilage Pelletier and OARSI scores,significantly reduce serum levels of CTX-II,COMP,IL-1β and TNF-α,promote Collagen II expression,and inhibit β-catenin and Wnt3 a expression in rats.OLA activates the muscle Nrf2 antioxidant system and promotes CS and MHC expression.Bone volume fraction(BV/TV),bone mineral density(BMD)and mean bone trabecular thickness(Tb.Th)were significantly increased and OCN and CTX-I expression were significantly decreased after the OLA intervention.(2)In the IL-1β-induced inflammatory injury model,OLA inhibited chondrocyte ECM metabolic enzyme expression and promoted Collagen II expression.And OLA inhibited GSK-3β(Ser-9)phosphorylation and β-catenin nuclear translocation and decreased β-catenin,Wnt3 a,BMP-2 and WISP1 expression.the anti-matrix degradation effect of OLA was diminished after Wnt/β-catenin pathway activator SKL2001 treatment.in MC3T3-E1 cells,after OLA intervention ALP and alizarin red staining intensity was enhanced,and OCN,ALP and RUNX2 protein expression was increased.Conclusions:(1)OLA can inhibit joint pain response in OA rats.ola inhibits cartilage ECM degradation and improves cartilage pathological characteristics in OA rats,reduces muscle dysfunction,inhibits abnormal bone reconstruction in subchondral bone,and exerts chondroprotective effects on cartilage and subchondral bone.(2)OLA inhibits the Wnt/β-catenin signaling pathway in chondrocytes,reduces the expression of matrix metabolic enzymes,promotes Collagen II secretion,and thus inhibits cartilage degeneration.(3)OLA promotes ALP,RUNX2 and OCN protein expression in MC3T3-E1 cells,promotes osteoblast differentiation and inhibits subchondral bone resorption. |
