TRV027在血管紧张素Ⅱ下调心肌细胞缝隙连接蛋白43表达中的作用

Role of TRV027 in angiotensinⅡmediated down-regulation of connexin 43 in cardiomyocytes

目的探讨TRV027是否通过影响AngⅡ并激活β-arrestin蛋白,从而影响心肌细胞Cx43的表达。方法通过培养大鼠心肌细胞(H9C2细胞),将其分为四组:对照组(无药物干预)、AngⅡ组(1×10^(-5)mol/L AngⅡ干预48h)、TRV027组(1×10^(-8)mol/L TRV027干预24h)、AngⅡ+TRV027组(1×10^(-5)mol/L AngⅡ干预24h后,1×10^(-8)mol/L TRV027干预24h)。采用蛋白质印迹(WB)法检测Cx43和β-arrestin的蛋白表达水平,PCR法检测Cx43和β-arrestin的mRNA表达水平,免疫荧光法观察上述两种蛋白在细胞内的表达情况。应用ImageJ分析图像,SPSS 22.0软件进行统计学分析。结果WB结果显示,与对照组相比,AngⅡ组H9C2细胞Cx43和β-arrestin的蛋白表达量明显降低(P<0.01);与AngⅡ组相比,AngⅡ+TRV027组和TRV027组H9C2细胞Cx43和β-arrestin的蛋白表达量明显升高(P<0.01)。PCR结果显示,与对照组相比,AngⅡ组H9C2细胞Cx43和β-arrestin的mRNA表达量降低(P<0.05);与AngⅡ组相比,AngⅡ+TRV027组和TRV027组H9C2细胞Cx43和β-arrestin的mRNA表达量明显升高(P<0.01)。免疫荧光结果显示,与对照组相比,AngⅡ组H9C2细胞Cx43和β-arrestin的荧光相对表达量明显降低(P<0.01);与AngⅡ组相比,AngⅡ+TRV027组和TRV027组H9C2细胞Cx43和β-arrestin的荧光相对表达量明显升高(P<0.01)。结论AngⅡ降低了H9C2细胞Cx43和β-arrestin的表达,而TRV027可以通过阻断AngⅡ的作用、激活β-arrestin来提高Cx43的表达。

Objective To investigate the relationship between angiotensinⅡ(AngⅡ)and connexin 43(Cx43)in cardiomyocytes,and whether TRV027 affect the expression of Cx43 in cardiomyocytes by influencing AngⅡand activatingβ-arrestin protein.Methods Rat cardiomyocytes(H9C2 cells)were cultured and divided into four groups:control group(absence of drug intervened),AngⅡgroup(1×10^(-5)mol/L AngⅡntervened for 48h),TRV027 group(1×10^(-8)mol/L TRV027 ntervened for 24h),AngⅡ+TRV027 group(1×10^(-5)mol/L AngⅡntervened for 24h,then 1×10^(-8)mol/L TRV027 ntervened for 24h).Western blotting(WB)method was used to detect the protein levels of Cx43,PCR method was used to detect the gene expression levels of Cx43 andβ-arrestin,and immunofluorescence method was used to observe the intracellular expression of the above two proteins.ImageJ was used to analyze images,and SPSS 22.0 software was used for statistical analysis.Results Compared with the control group,the expression levels of Cx43 andβ-arrestin in H9C2 cells in AngⅡgroup were significantly decreased(P<0.01);Compared with AngⅡgroup,the expression levels of Cx43 andβ-arrestin in H9C2 cells in AngⅡ+TRV027 group and TRV027 group were significantly increased(P<0.01).PCR results showed that mRNA expression levels of Cx43 andβ-arrestin in H9C2 cells in AngⅡgroup were decreased compared with control group(P<0.05);Compared with AngⅡgroup,mRNA expressions of Cx43 andβ-arrestin in H9C2 cells in AngⅡ+TRV027 group and TRV027 group were significantly increased(P<0.01).Compared with the control group,the relative fluorescence expressions of Cx43 andβ-arrestin in H9C2 cells in AngⅡgroup were significantly decreased(P<0.01);Compared with AngⅡgroup,the fluorescence relative expressions of Cx43 andβ-arrestin in H9C2 cells in AngⅡ+TRV027 group and TRV027 group were significantly increased(P<0.01).Conclusion AngⅡcan decrease the expression of Cx43 andβ-arrestin in rat cardiomyocytes,while TRV027 could improve Cx43 expression by blocking the action of AngⅡand activatingβ-arrestin pathway.