【摘要】 目的:研究桑寄生提取物(SJS)联合微小RNA-375(miR-375)对骨关节炎软骨细胞活力和凋亡的影响及其作用机制。方法:将人原代骨性关节软骨细胞RPOC分为对照组(control)组、白细胞介素1β(IL-1β)组、IL-1β+SJS低剂量(SJS-L)组、IL-1β+SJS中剂量(SJS-M)组、IL-1β+SJS高剂量(SJS-H)组、IL-1β+anti-miR-NC组、IL-1β+anti-miR-375组、IL-1β+SJS-H+miR-NC组、IL-1β+SJS-H+miR-375组。MTT检测细胞活力,流式细胞术检测细胞凋亡,qPCR检测miR-375的表达,Western blot检测细胞周期蛋白D1(cyclin D1)、P21、Bcl-2、Bax和caspase-3蛋白的水平。结果:与control组比较,IL-1β组的RPOC活力(24 h、48 h和72 h)及cyclin D1和Bcl-2蛋白表达明显降低(P<0. 05),而P21、Bax和caspase-3蛋白的水平、细胞凋亡率及miR-375表达量均显著升高(P<0. 05)。与IL-1β组比较,IL-1β+SJS-M组和IL-1β+SJS-H组RPOC的活力(24 h、48 h和72 h)和cyclin D1表达显著升高(P<0. 05),P21表达量显著降低(P<0. 05);IL-1β+SJS-H组细胞的凋亡率、Bax及caspase-3蛋白水平、miR-375表达量明显减少(P<0. 05),Bcl-2蛋白水平显著提高(P<0. 05)。与IL-1β+anti-miR-NC组比较,IL-1β+anti-miR-375组RPOC中miR-375表达量、P21、Bax、caspase-3蛋白水平、细胞凋亡率明显下降(P<0. 05),cyclin D1、Bcl-2蛋白水平、细胞活力(24 h、48 h和72h)显著升高(P<0. 05)。过表达miR-375能逆转SJS对IL-1β作用的RPOC活力、凋亡的影响。结论:桑寄生提取物可以促进IL-1β作用的RPOC活力,并抑制其凋亡,其机制与调控miR-375表达有关。更多还原

【Abstract】 AIM:To study the effects of extracts of Herba Taxilli(Sangjisheng,SJS) on the viability and apoptosis of osteoarthritic chondrocytes and the underlying mechanism.METHODS:Human primary osteoarticular chondrocytes(RPOC) were divided into control group,interleukin-1 β(IL-1β group,IL-1β+low-dose extracts of SJS(SJS-L)group,IL-1 β+medium-dose extracts of SJS(SJS-M) group,IL-1β+high-dose extracts of SJS(SJS-H) group,IL-1β+antimiR-NC group,IL-1β+anti-miR-375 group,IL-1β+SJS-H+miR-NC group,IL-1β+SJS-H+miR-375 group.The cell viability was measured by MTT assay,apoptosis was analyzed by flow cytometry,miR-375 expression was detected by qPCR,and the protein levels of cyclin D1,P21,Bcl-2,Bax and caspase-3 were determined by Western blot.RESULTS:Compared with control group,the viability of RPOCat 24 h,48 h and 72 h and the protein expression levels of cyclin D1 and Bcl-2 were significantly decreased(P<0.05),the protein levels of P21,Bax and caspase-3,the apoptotic rate and the expression level of miR-375 were remarkably increased in IL-1β group(P<0.05).Compared with IL-1β group,the cell viability at 24 h,48 h and 72 h and the protein expression of cyclin D1 in the RPOC were greatly increased(P<0.05),while the expression of P21 was significantly decreased in IL-1β+SJS-M group and IL-1β+SJS-H group(P<0.05).The apoptotic rate,Bax,caspase-3 protein and miR-375 expression were obviously decreased(P<0.05),and Bcl-2 protein level was significantly increased in IL-1β+SJS-H group compared with IL-1β group(P<0.05).Compared with IL-1 β+anti-miR-NC group,the expression of miR-375,the protein levels of P21,Bax,caspase-3 and the apoptotic rate in the RPOC of IL-1β+anti-miR-375 group were markedly decreased(P<0.05),while the cell viability at 24 h,48 h and 72 h and the protein levels of cyclin D1 and Bcl-2 were significantly increased(P<0.05).Over-expression of miR-375 reversed the effects of extracts of SJS on the viability and apoptosis of RPOC with IL-1β stimulation.CONCLUSION:The extracts of Herba Taxilli promotes the viability and inhibits apoptosis of RPOC treated with IL-1β,which is related to the regulation of miR-375 expression.更多还原