Cx43沉默抑制机械应力诱导的小鼠软骨细胞凋亡

Cx43 silencing inhibits mechanical stress-induced apoptosis in mouse articular chondrocyte

目的:探讨缝隙连接蛋白43(connexin 43,Cx43)沉默对机械应力诱导的软骨细胞凋亡的影响。方法:将小鼠软骨细胞ATDC5分为空白组、机械应力组、Cx43 si RNA转染组、scramble si RNA转染组、机械应力+scramble组、机械应力+si Cx43组。用Flexcell FX-5000系统对体外培养的ATDC5细胞加载机械应力;用定量RT-PCR(quantitative RTPCR,RT-q PCR)和Western印迹分别检测细胞Cx43 m RNA和蛋白表达水平;用细胞计数试剂盒(cell counting kit-8,CCK-8)法检测细胞活性;用流式细胞术检测细胞凋亡情况;用比色法检测caspase-3活性;用Western印迹检测Bcl-2,Bax,p-JNK和JNK的蛋白表达。结果:经机械应力诱导后,ATDC5细胞Cx43m RNA和蛋白的表达显著升高(均P<0.05)。转染Cx43 si RNA显著降低细胞Cx43 m RNA和蛋白水平(均P<0.05)。在机械应力刺激下,ATDC5细胞活性显著降低,凋亡增多,caspase-3活性和Bax蛋白表达上调,Bcl-2蛋白表达和Bcl-2/Bax降低(均P<0.05)。而Cx43沉默显著增加机械应力下ATDC5细胞活性,减少凋亡,降低caspase-3活性和Bax蛋白表达,升高Bcl-2蛋白表达和Bcl-2/Bax(均P<0.05)。此外,Cx43 si RNA显著抑制机械应力诱导的p-JNK蛋白的表达(P<0.05)。结论:Cx43沉默可能通过调节JNK信号通路,抑制机械应力作用下小鼠软骨细胞凋亡。

Objective:To explore the effect of connexin 43(Cx43)silence on the apoptosis in mouse chondrocyte under mechanical stress.Methods:Mouse chondrocyte ATDC5 cells were divided into a control group,a mechanical stress group,a Cx43 siRNA transfection group,a scramble siRNA transfection group,a mechanical stress+scramble group,and a mechanical stress+siCx43 group.Flexcell FX-5000 system was used to produce mechanical stress on ATDC5 cells cultured in vitro.The mRNA and protein level of Cx43 was detected by quantitative RT-PCR(RT-qPCR)and Western blot.The cell activity and cell apoptosis was detected by cell counting kit-8(CCK-8)method and flow cytometry,respectively.Caspase-3 activity was detected by colorimetric assay.The protein expression of Bcl-2,Bax,p-JNK and JNK was detected by Western blot.Results:Mechanical stress upregulated the mRNA and protein expression of Cx43(both P<0.05).Transfection of Cx43 siRNA significantly decreased Cx43 mRNA and protein level(both P<0.05).After stimulation with mechanical stress,chondrocyte viability was significantly decreased,whereas cell apoptosis and caspase-3 activity were increased(both P<0.05).Mechanical stress obviously upregulated Bax protein level,and downregulated Bcl-2 protein expression and Bcl-2/Bax(both P<0.05).Cx43 siRNA transfection significantly increased cell viability,inhibited cell apoptosis and caspase-3 activity(both P<0.05).Cx43 siRNA also inhibited Bax expression,and increased the Bcl-2 protein expression and Bcl-2/Bax(both P<0.05).Furthermore,Cx43 siRNA significantly suppressed the p-JNK expression induced by mechanical stress(P<0.05).Conclusion:Cx43 silence inhibits mechanical stress-induced apoptosis in chondrocyte,which might be mediated by JNK signaling pathway.