Cx43沉默对机械应力作用下调控的软骨细胞自噬介导软骨基质合成的影响

Effects of Cx43 silencing on chondrocyte matrix synthesis mediated by autophagy in mouse chondrocytes under mechanical stress

目的:探究机械应力作用下通过沉默缝隙连接蛋白43(Cx43)调控软骨细胞自噬对软骨细胞的基质代谢的影响。方法:对小鼠成软骨细胞系ATDC5加载机械应力,采用q PCR和Western blot法检测ATDC5细胞中Cx43的mRNA和蛋白表达。将细胞分为空白对照组、机械应力组、Cx43 siRNA转染组、scramble siRNA转染组、机械应力+scramble siRNA转染组、机械应力+Cx43 siRNA转染组和机械应力+Cx43 siRNA转染+3-MA组,CCK-8法检测细胞活力,q PCR检测软骨细胞Ⅱ型胶原mRNA的表达,1,9-二甲基亚甲蓝(DMMB)染色法检测细胞糖胺多糖(GAG)含量,Western blot法检测自噬相关蛋白beclin 1和LC3的表达。结果:机械应力刺激下,ATDC5细胞Cx43表达上调,细胞活性显著降低,II型胶原mRNA表达降低,GAG含量减少,beclin 1和LC3-II/LC3-I蛋白表达降低(P <0. 05)。转染Cx43 siRNA可下调Cx43的mRNA和蛋白的表达。机械应力作用下,Cx43沉默显著增强软骨细胞活力,提高II型胶原的mRNA表达和GAG含量,上调beclin 1和LC3-II/LC3-I的蛋白表达(P <0. 05)。此外,自噬抑制剂3-MA处理后,Cx43沉默对机械应力下软骨细胞的保护作用受到抑制。结论:Cx43沉默通过促进机械应力作用下小鼠软骨细胞自噬,从而促进软骨细胞的合成代谢,减轻软骨细胞在机械应力下的损伤。

AIM:To investigate the effect of autophagy regulated by connexin 43(Cx43)silencing on the matrix metabolism of chondrocyte under mechanical stress.METHODS:After stimulation of mechanical stress on mouse chondrogenic cell line ATDC5,qPCR and Western blot were used to detect the mRNA and protein levels of Cx43.The cells were divided into control group,mechanical stress group,Cx43 siRNA transfection group,scramble siRNA transfection group,mechanical stress+scramble siRNA group,mechanical stress+Cx43 siRNA group and mechanical stress+Cx43 siRNA+3-MA group.The cell viability was measured by CCK-8 assay.The mRNA expression of collagen II in the ATDCS cells was detected by qPCR and the glycosaminoglycan(GAG)content was measured by 1,9-dimethylmethylene blue(DMMB)staining.The protein expression of autophagy related proteins beclin 1 and LC3 was determined by Western blot.RESULTS:The expression of Cx43 at mRNA and protein levels was over-expressed in the ATDC5 cells under mechanical stress(P<0.05).Transfection of Cx43 siRNA significantly down-regulated Cx43 expression at mRNA and protein levels(P<0.05).Under mechanical stress,the viability of chondrocytes was significantly decreased,while the mRNA expression of collagen II,GAG content,the protein expression of beclin 1 and LC3-II/LC3-I were down-regulated(P<0.05).Under mechanical stress,Cx43 siRNA transfection obviously increased the cell viability,GAG content,and the mRNA level of collagen II(P<0.05).Cx43 siRNA also up-regulated the the protein expression of beclin 1 and LC3-II/LC3-I(P<0.05).Moreover,autophagy inhibitor 3-MA reversed the protective effect of Cx43 silencing on the chondrocytes under mechanical stress.CONCLUSION:Cx43 silencing promotes autophagy of chondrocytes in mice under mechanical stress,thus promoting the synthesis and metabolism of chondrocytes,protecting chondrocytes from injury under mechanical stress.