黄连素通过KLF4减轻缺血/再灌注损伤所致的大鼠心肌细胞凋亡的作用机制研究

Mechanism of the effect of berberine on KLF4-mediated alleviation of cardiomyocyte apoptosis induced by ischemia/reperfusion injury in rats

目的探讨黄连素(BBR)通过KRUPPEL样因子4(KLF4)减轻缺血/再灌注(I/R)损伤所致的大鼠心肌细胞凋亡的作用机制。方法将大鼠永久心肌细胞系H9C2细胞按随机数字表法分为4组,即对照组(正常培养4 h)、I/R组(诱导I/R模型)、BBR组(诱导模型期间给予50μmol/L BBR作用4 h)、KLF4小干扰RNA(siRNA)组(转染KLF4 siRNA后,诱导模型期间给予50μmol/L BBR作用4 h)。采用细胞计数试剂盒-8检测细胞活力,原位末端转移酶标记法染色和流式细胞术检测细胞凋亡情况,蛋白质印迹法检测细胞凋亡因子[包括B淋巴细胞瘤-2基因(Bcl-2)、Bcl-2相关X蛋白(Bax)]、KLF4蛋白表达水平,实时定量逆转录-聚合酶链反应检测KLF4 mRNA表达水平。结果BBR组、I/R组、对照组H9C2细胞活力、细胞凋亡因子蛋白表达水平、膜阳性面积百分比、细胞凋亡率以及KLF4蛋白、mRNA表达水平比较,差异均有统计学意义(均P<0.05);其中BBR组细胞活力、Bcl-2蛋白表达水平以及KLF4蛋白、mRNA表达水平均明显高于I/R组(均P<0.05),Bax蛋白表达水平、膜阳性面积百分比、细胞凋亡率均明显低于I/R组(均P<0.05)。KLF4 siRNA组细胞活力明显低于BBR组(P<0.05),膜阳性面积百分比和细胞凋亡率均明显高于BBR组(均P<0.05)。结论BBR可以通过KLF4依赖的方式保护心肌细胞免受I/R损伤所致的细胞凋亡。

Objective To investigate the mechanism of berberine(BBR)alleviating cardiomyocyte apoptosis induced by ischemia/reperfusion(I/R)injury in rats through Kruppel-like factor4(KLF4).Methods The H9C2 cells of rat permanent myocardial cell line were randomly divided into 4 groups according to random number table method,namely the control group(normal culture for 4 h),I/R group(induced I/R model),BBR group(50μmol/L BBR was given during the induction period for 4 h),KLF4 small interfering RNA(siRNA)group(transfected with KLF4 siRNA and given 50μmol/L BBR during the indection period for 4 h).The cell viability was detected by cell counting kit-8,and cell apoptosis was detected by TUNNEL staining and flow cytometry,and the expression levels of apoptosis factors including B lymphoblastoma-2 gene(Bcl-2),Bcl-2 associated X protein(Bax)and KLF4 protein were detected by western blotting.Real-time quantitative reverse transcription-polymerase chain reaction was used to detect KLF4 mRNA expression level.Results The H9C2 cell viability,apoptosis factor protein expression level,percentage of membrane positive area,cell apoptosis rate,protein and mRNA expression levels of KLF4 in BBR group,I/R group and control group were compared,and the differences were statistically significant(all P<0.05).The cell viability,protein expression level of Bcl-2,protein and mRNA expression levels of KLF4 of the BBR group were significantly higher than those of the I/R group(all P<0.05),while the protein expression level of Bax,percentage of membrane positive area and cell apoptosis rate of the BBR group were significantly lower than those of the I/R group(all P<0.05).The cell viability of KLF4 siRNA group was significantly lower than that of the BBR group(P<0.05),and the percentage of membrane positive area and cell apoptosis rate were significantly higher than those of the BBR group(all P<0.05).Conclusion BBR can protect cardiomyocytes from apoptosis induced by I/R injury in a KLF4-dependent manner.