Experimental Study On The Mechanism Of Astragaloside Iv In Osteoarthritis Through SDF-1/CXCR4 Signaling Pathway

Objective:Osteoarthritis(OA),known as "bone obstruction" or "bone atrophy" in traditional Chinese medicine(TCM),is a complex metabolic disease caused by metabolic imbalance including imbalance of synthesis and decomposition of articular cartilage and subchondral bone.In recent years,the incidence of OA is increasing year by year,which brings a heavy burden to the family and society.However,the pathogenesis of OA is still not clear,and there is a lack of radical treatment methods,so it is urgent to thoroughly study the pathogenesis of OA and find an effective treatment method for OA.In our previous study on traditional Chinese medicine "Sishen decoction",we found that Astragaloside Ⅳ(AST),one of its components,has a strong affinity with CXCR4 protein,suggesting that AST may play a role in treating OA through SDF-1/CXCR4 signaling pathway.Therefore,this project will study the efficacy of AST on OA,the effect of AST on SDF-1/CXCR4 signaling pathway,and the role of SDF-1/CXCR4 signaling pathway in OA.To verify whether AST can treat OA and whether its therapeutic effect on OA is achieved by its effect on SDF-1/CXCR4 signaling pathway.Methods:1.Establishment of ATDC5 cell OA model: The optimal concentration and time of IL-1β inducing OA model in ATDC5 cells were determined by cell proliferation rate using CCK-8 method,and the cell morphology was observed by toluidine blue staining and trypan blue staining.Western blot and q RT-PCR were used to detect the expression of OA markers such as MMP-3,MMP-9,MMP-13 and Collagen II in the cells to verify whether the model was successfully established.2.AST treatment of IL-1β-induced inflammatory response in chondrocytes: The optimal concentration and time of AST treatment for OA model ATDC5 cells were determined by cell proliferation rate using CCK-8 method,and the cell morphology was observed by toluidine blue staining and trypan blue staining.Western blot and q RT-PCR were used to detect the expression of OA markers such as MMP-3,MMP-9,MMP-13 and Collagen II in cells to observe the treatment effect.3.The effect of AST on SDF-1/CXCR4 signaling pathway: Western blot and q RT-PCR were used to detect the expression of CXCR4 and SDF-1 protein and gene in OA model group,normal group and AST treatment group to observe the effect of AST on SDF-1/CXCR4 signaling pathway.4.Inhibition of SDF-1/CXCR4 pathway by AMD3100: The optimal concentration of AMD3100 was determined by CCK-8 method.The protein and mrna expression levels of CXCR4 and SDF-1 in normal ATDC5 cells,OA cells and AMD3100 treated cells were detected by Western blot and q RT-PCR,respectively.To verify the inhibitory effect of AMD3100 on SDF-1/CXCR4 pathway.5.Role of SDF-1/CXCR4 pathway in OA The expression levels of MMP-3,MMP-9,MMP-13 and type II collagen in normal ATDC5 cells,OA cells and AMD3100 treated cells were detected by Western blot and q RT-PCR,respectively.To verify the role of SDF-1/CXCR4 signaling pathway in OA.Results:1.IL-1β can be used to induce ATDC5 to construct OA model: The optimal OA model of IL-1β-induced ATDC5 was induced by 100 μg/L IL-1β for 18 hours.After ATDC5 cells were treated with IL-1β,The proliferation rate decreased,cell morphology pyknosis,the expression of MMP-3,MMP-9 and MMP-13 increased significantly(P<0.05),and the expression of type II collagen decreased significantly(P<0.05),indicating that IL-1β could successfully induce inflammation of ATDC5 cells and cause OA-like symptoms.The results showed that IL-1β could be used to induce OA model.2.AST can relieve OA symptoms and play a role in the treatment of OA: The optimal treatment condition of AST in the treatment of OA was 5 μg/L AST for 18 hours.AST did not promote or inhibit the proliferation of normal cells,but it could significantly increase the activity of cells and improve the survival rate of cells in the IL-1β induced OA cell model.The results of Western blot and q RT-PCR showed that it could inhibit the expression of MMP-3,MMP-9,and MMP-13 induced by IL-1β and promote the expression of collagen type II(P<0.05),and play a role in relieving the symptoms of OA.3.AST has certain inhibitory effect on SDF-1/CXCR4 signaling pathway: In the study of the effect of AST on SDF-1/CXCR4 signaling pathway,the results of Western blot and q RT-PCR showed that IL-1β increased the expression of CXCR4 and SDF-1 in OA cells,while AST significantly inhibited the expression of CXCR4 and SDF-1 in OA cells(P<0.05).4.SDF-1/CXCR4 signaling pathway plays an important role in OA: The optimal concentration of AMD3100 is 5 μg/m L.Amd3100 can inhibit the expression of SDF-1 and CXCR4 protein and gene in normal cells and IL-1β-induced OA model cells.The expression levels of SDF-1 and CXCR4 were significantly decreased by about 30% after adding AMD3100(P<0.001),and the expression levels of SDF-1 and CXCR4 protein and gene were also reduced to similar levels to normal values in IL-1β-induced OA model cells.The results of Western blot and q RT-PCR showed that it had a certain inhibitory effect on the expression of MMP-3,MMP-9 and MMP-13(P<0.05),but it significantly increased the gene expression level of type II collagen(P<0.05),but did not increase its protein expression.Conclusions:1.SDF-1/CXCR4 signaling pathway plays an important role in OA.When inflammatory factors such as IL-1β cause OA symptoms,the protein and gene expression of related factors in this pathway can be observed to increase.The use of inhibitors such as AMD3100 to inhibit it can reduce the expression level of OA biomarkers such as MMPs and play a role in the treatment of OA.2.IL-1β could induce ATDC5 cells to develop OA symptoms and increase the expression of MMP-3,MMP-9,MMP-13 and decrease the expression of type II collagen.3.AST can inhibit the SDF-1/CXCR4 signaling pathway,inhibit the expression of MMP-3,MMP-9,MMP-13 and promote the expression of type II collagen,relieve the symptoms of OA,and play a therapeutic role in OA.4.The mechanism of AST and AMD3100 is not exactly the same,and inhibition of SDF-1/CXCR4 signaling pathway alone cannot promote the expression of type II collagen.Therefore,in addition to SDF-1/CXCR4 signaling pathway,AST may have other targets related to type II collagen,which is worthy of further study.