Investigation On The Molecular Mechanisms Of Oxymatrine On The Degradation Of Cartilage And Subchondral Bone

Background and ObjectiveOsteoarthritis(OA)is a chronic degenerative disease.The articular cartilage and subchondral bone have undergone a complex process of remodeling,promote the degradation of cartilage matrix,induce hypertrophy of articular cartilage cells,apoptosis and necrosis,and eventually lead to cartilage degeneration.At the same time,the loss of subchondral bone mediated by osteoclasts resulting the surface articular cartilage loss its support from the subchondral bone.Oxymatrine(OMT)is a kind of natural small molecule compounds.To exploring the effects of OMT on OA cartilage degeneration and the subchondral bone remodeling is supposing to providing new drug selection for the treatment of OA,and is of great significance.Methods1.To investigate the protective effects of oxymatrine on human articular cartilage.The effects of oxymatrine on the inflammatory response and catabolism of chondrocytes induced by lipopolysaccharide were studied using real-time PCR and ELISA analysis.The protective effects of oxymatrine on human articular cartilage were examined using human articular cartilage ex vivo model.2.To study the role of oxymatrine in regulating the differentiation and maturation of osteoblasts and osteoclasts using TRAP staining,F-Actin ring formation assay and Alizarin Red staining.Osteoclast differentiation related genes were determined using real-time PCR and western blot analysis.3.The effects of OMT on activation of NF-kappa B signaling pathway and MAPK signaling pathway were detected using western blot.4.The effects of OMT on subchondral bone loss and cartilage degeneration were analyzed using the ACLT OA model.We applied tissue morphology and biological to analyze the effects of intraperitoneal injection of OMT on ACLT mice bone loss of protection and cartilage degeneration.Results1.The real-time PCR results showed that pretreatment with oxymatrine markedly prevented LPS-stimulated pro-inflammatory cytokine and MMP production in chondrocytes.ELISA analysis further validate the above results.The ex vivo results demonstrated that oxymatrine treatment significantly decreased the release of GAGs from LPS-stimulated human articular cartilage tissues into the culture supernatant and exerts a protective effects on OA articular cartilage.2.TRAP staining and f-actin staining showed that OMT could inhibit the differentiation and maturation of osteoclasts.The results of real-time PCR and western blot showed that OMT significantly inhibited the expression of the osteoclast gene.The Alizarin Red staining results shown that OMT had no significant effects on osteogenic differentiation of BMSC.3.Both western blot,immunofluorescence and nuclear separation analysis showed that OMT could significantly inhibit the activation of NF-?B and MAPK signaling pathways in chondrocytes and osteoclasts.4.The safranin O/fast green staining results showed that OMT abdominal injection could significantly delay the wear of articular cartilage in mice.Compared to the control mice,ACLT-operated mice exhibited a significant increase in the number of TUNEL-positive cells in the articular cartilage zone,while in the oxymatrine-treated group,there was less apoptosis in the cartilage chondrocytes.The percent of P65-positive chondrocytes in the ACLT with PBS group was substantially higher than that in the sham surgery group,while no change was observed in the low dose and high dose oxymatrine-treated groups.Similarly,the expression levels of MMP9 and MMP13 were coordinated with that of p65.The three-dimensional(3D)reconstruction of the ?CT results revealed extensive subchondral bone resorption in the ACLT group,which was observed as extensive bone loss in the subchondral bone,when compared with that of the sham control group.While intraperitoneal injections of OMT in ACLT mice dramatically attenuated the tibial subchondral bone loss.ACLT mice displayed an increased number of TRAP-positive multinucleated cells in the subchondral plate(SCP)and subchondral trabecular bone(STB),while oxymatrine treatment abolished those effects in the ACLT mice.Conclusion1.Oxymatrine inhibited catabolic events in LPS-stimulated human cartilage and substantially attenuated the degradation of articular cartilage.2.Oxymatrine inhibits osteoclast differentiation and has no obvious effect on osteogenic differentiation,which may be an attractive therapeutic agent for treating bone lose-related diseases.3.The inhibitory effect of oxymatrine on chondrocyte inflammation and osteoclastogenesis may be due to the attenuation of the NF-?B and MAPK signaling cascades.