Experimental Study On The Regulation Of Curcumin On IL-1?-induced Osteoarthritis Via NF-?B Signaling

Objective: To explore the possible molecular mechanism of curcumin in regulating the expression of type ? collagen,MMP-13 in IL-1?-induced osteoarthritis,and to clarify the therapeutic effect and the relative mechanisms of curcumin in treating osteoarthritis.Methods: 1.Articular cartilage was isolated,cultured and identified.2.The effect of IL-1b on the expression of type ? collagen and MMP-13.Chondrocytes were exposed to IL-1b for 24 h,RT-qPCR and Western Blotting were conducted to evaluate the expression of type ? collagen,MMP-13 at protein and mRNA levels.3.The effect of curcumin on down-regulation of collagen type ? and up-regulation of MMP-13 expression in IL-1?-induced chondrocytes.To acquire the optimum time point and concentration of curcumin for treatment of chondrocytes,IL-1b-pretreated chondrocytes were exposed to 50 mM curcumin for different time periods,chondrocytes were harvested at different time points,and Western Blotting was performed to measure the expression of type ? collagen and MMP-13 at protein level.IL-1b-pretreated chondrocytes were exposed to various concentrations of curcumin for 48 h,chondrocytes were harvested and Western Blotting was performed to measure the expression of type ? collagen and MMP-13 at protein level.4.The effects of curcumin on the cell proliferation activity of IL-1?-stimulated chondrocytes.chondrocytes were pretreated with IL-1b(10 ng/ml)for 24 h,and then co-treated with curcumin(50 mM)or PDTC(inhibitor of NF-?B activation,0.1 mmol/L),or curcumin plus PDTC.Cells in four groups were treated CCK-8 reagent at different time points,CCK-8 assays were performed to evaluate the differences of cell proliferation activity in in four groups.5.The possible molecular mechanism of curcumin in regulating the expression of type ? collagen,MMP-13 in IL-1?-induced chondrocytes.Chondrocytes were treated with IL-1b alone or together with curcumin for different time periods,then nuclear,cytoplasmic and total proteins were extracted at different time points,Western Blotting was carried out to evaluate the expression of IkBa,p-IkBa,and p65 at protein level.The cultured chondrocytes were divided into four groups: the blank control group,IL-1b group,IL-1b + Curcumin group,IL-1b + PDTC group,the expression of p65 inside and outside of chondrocytes in four groups were examined immunofluorescence.Results: 1.The chondrocytes were isolated successfully,and the phenotype of rat chondrocytes were identified by toluidine blue staining and immunohistochemical was used to identified type ? collagen.2.Western Blotting and RT-qPCR analyses showed that IL-1? markedly increased the expression of MMP-13 in rat chondrocytes and significantly decreased the expression of type ? collagen.3.Curcumin can reverses IL-1?-induced expression changes of type ? collagen,and MMP-13 in rat chondrocytes.The expression of MMP-13 was significantly decreased,while type ? collagen was markedly increased in chondrocytes after treatment with curcumin at the indicated time points.the decrease in MMP-13 peaked 36 h and the increase in type ? collagen peaked at 48 h.The optimum concentration of curcumin for treatment of chondrocytes: Curcumin markedly upregulated the expression of type ? collagen,and significantly inhibited the expression of MMP-13 in IL-1?-pretreated chondrocytes at each concentration.The optimum concentration of curcumin for regulation of type ? collagen and MMP-13 expression was 50 mM.4.Curcumin can block decreased proliferation activities of chondrocytes induced by IL-1?.The proliferation activities of IL-1?-treated chondrocytes were found to gradually increase after the pharmacologic interventions,while the proliferation activity of chondrocytes treated with IL-1? alone gradually decreased.Although the proliferation activity of each pharmacologic intervention group increased significantly,there were no significant differences in the cell proliferation activity among the groups treated with curcumin,PDTC or curcumin plus PDTC.5.Curcumin reversed IL-1?-induced expression changes of type ? collagen,MMP-13 via inhibition of NF-?B signaling in rat chondrocytes.The nuclear translocation of p65 was accumulated which was induced by IL-1?,curcumin can block IL-1?-induced nuclear translocation of p65 in rat chondrocytes,and increase the expression of p65 in the cytoplasm and decreased the expression of p65 in the nucleus.Curcumin had similar effects for treatment of IL-1?-stimulated chondrocytes with PDTC.What's more,curcumin can block IL-1?-induced IkBa phosphorylation,and inhibit the expression of p-IkBa.Conclusion: 1.IL-1? can increased the expression of MMP-13,and significantly decreased the expression of type ? collagen in rat chondrocytes.2.Curcumin can block IL-1?-induced changes in the expression of MMP-13 and type ? collagen.In our experiment,the suitable time point and concentration of curcumin for treatment of IL-1?-treated chondrocytes were 48 h and 50 mM.3.Curcumin can increase the proliferation activities of IL-1?-treated chondrocytes,and has no significant side effects in the experimental concentration.4.Curcumin can inhibit nuclear translocation of p65 and MMP-13 expression,upregulate type ? collagen expression through restraining IkBa phosphorylation.Curcumin had similar effects with PDTC for treatment of IL-1?-stimulated chondrocytes,suggesting that the regulation of chondrocytes by curcumin and PDTC might follow the same molecular mechanism.