杜仲苷对骨关节炎软骨细胞增殖和凋亡的影响及其分子机制

Effect of aucubin on proliferation and apoptosis of osteoarthritis chondrocytes and its molecular mechanism

目的杜仲苷影响软骨细胞增殖和凋亡的分子机制尚未完全清楚。文中旨在研究杜仲苷对骨关节炎软骨细胞增殖、凋亡的影响及分子机制。方法取对数生长期软骨细胞,分别用终浓度为10、100、500、1000μmol/L的杜仲苷处理,分别为杜仲苷10μmol/L组、杜仲苷100μmol/L组、杜仲苷500μmol/L组、杜仲苷1000μumol/L组,对照组为正常培养的细胞。检测不同浓度杜仲苷对骨关节炎软骨细胞增殖的影响,并选出最佳杜仲苷浓度作后续实验,为杜仲苷组。取对数生长期软骨细胞,无血清重悬后进行转染,分为pcDNA组(转染peDNA)、pcDNA-Prdx5组(转染peDNA-Prdx5 mimics)、杜仲苷+si-NC组(转染si-NC后用500 μmol/L杜仲苷处理)、杜仲苷+si-Prdx5组(转染si-Prdx5后用500 μmol/L杜仲苷处理)。qRT-PCR检测骨关节炎软骨细胞中Prdx5mRNA的表达水平;Westernblot检测Prdx5蛋白表达;CCK-8法检测细胞增殖;流式细胞术检测细胞凋亡。结果与对照组比较,随着杜仲苷浓度的升高,骨关节炎软骨细胞的活性逐渐升高(P<0.05),选用500μmoVL杜仲苷作后续实验。流式细胞仪检测结果显示,杜仲苷组骨关节炎软骨细胞的凋亡率[(7.89±1.03)%]较对照组[(18.76±1.24)%]显著降低(P<0.05)。qRT-PCR和Western blot检测结果显示,与对照组比较,杜仲苷组Bel-2、Prdx5的mRNA和蛋白表达水平显著升高,Bax mRNA和蛋白的表达水平显著降低(P<0.05)。peDNA-Prdx5组Prdx5蛋白的表达水平、骨关节炎软骨细胞的活性显著高于pcDNA组(P<0.05),骨关节炎软骨细胞的凋亡率显著低于pcDNA组(P<0.05)。与杜仲苷+si-NC组Prdx5蛋白的表达水平比较,杜仲苷+si-Prdx5组显著降低(P<0.05)。CCK-8法检测结果显示,与对照组骨关节炎软骨细胞的活性比较.杜仲苷组显著升高(P<0.05);与杜仲苷+si-NC组骨关节炎软骨细胞的活性比较,杜仲苷+si-Prdx5组显著降低(P<0.05)。流式细胞仪检测结果显示,与对照组骨关节炎软骨细胞的凋亡率比较,杜仲苷组显著降低(P<0.05);与杜仲苷+si-NC组骨关节炎软骨细胞的凋亡率比较,杜仲苷+si-Prdx5组显著升高(P<0.05)。结论杜仲苷可能通过上调Prdx5促进骨关节炎软骨细胞增殖,抑制其凋亡。

Objective The molecular mechanism of aucubin on chondrocyte proliferation and apoptosis hasn't completely known.The aim of this study was to investigate the effects of aucubin on proliferation and apoptosis of osteoarthritis chondrocytes and its molecular mechanism.Methods Chondrocytes at logarithmic growth stage were treated with aucubin(of 10μmol/L,100μmol/L,500μmol/L and 1000μmol/L respectively).Cells in the control group were cultured normally.The effects of different concentrations of aucubin on the proliferation of osteoarthritis chondrocytes were detec-ted,and the optimal concentration of aucubin was selected for subsequent experiments,which was the aucubin group.Logarithmic growth chondrocytes were taken and transfected after serum-free resuspended.They were divided into pcDNA group(transfected peD-NA),peDNA-Prdx5 group(transfected peDNA-Prdx5 mimics),aucubin+siNC group(transfected siNC with 500μmol/L aueubin),and aucubin+siPrdx5 group(transfected siPrdx5 with 500μmol/L aucubin).QRTPCR was used to detect the expression of Prdx5 mRNA in osteoarthritis chondrocytes.Prdx5 protein expression was detected by Westem blot.Cell prolifeation was dected by CCK8 method.Apoplosis was detected by flow cytometry.Results Compared with the control group,the activity of osteoarthritis chondro-cytes increased gradually with the increase of aucubin concentration(P<0.05).500 umol/L aucubin was selected for the fllowup ex-periment.The results of flow cytometry showed that the apoptosis rate of osteoarthritis chondrocytes in aucubin group[(7.89±1.03)%]was significantly lower than that in control group[(18.76±1.24)%](P<0.05).QRTPCR and W estern blot results showed that compared with the control group,the mRNA and protein expression levels of Bcl2 and Prdx5 in aucubin group were significantly in-creased,while the Bax mRNA and protein expression levels in aucubin group were significantly decreased(P<0.05).The expression level of Prdx5 protein and the activity of osteoarthritis chondrocytes in pcDNA-Prdx5 group were signifcantly higher than those in peD-NA group(P<0.05),and the apoptosis rate of osteoarthritis chondrocytes in peDNA-Prdx5 group was significantly lower than that in peDNA group(P<0.05).Prdx5 protein expression in aucubin+siPrdx5 group was signifcantly decreased compared with that in aucu-bin+siNC group(P<0.05).CCK8 assay showed that compared with the control group,the activity of chondrocytes in aucubin group was signifcantly increased(P<0.05).Compared with aucubin+siNC group,the activity of osteoarthritis chondrocytes in aucubin+siPrdx5 group was significantly decreased(P<0.05).The results of flow cytometry showed that compared with the control group,the apoptosis rate of chondrocytes in aucubin group was signifcantly decreased(P<0.05).Compared with aucubin+siNC group,the ap-optosis rate of osteoarthritis chondrocytes in aucubin+siPrdx5 group was significantly increased(P<0.05).Conclusion Aucubin may promote the proliferation and inhibit the apoptosis of osteoarhritis chondrocytes by up-regulating Prdx5.