PKCα对AngⅡ作用的心肌成纤维细胞增殖活性和NO分泌的影响

Effect of PKCα on the proliferation activity and NO level of myocardial fibroblasts after the action of AngⅡ

目的探讨蛋白激酶Cα(PKCα)对血管紧张素Ⅱ(AngⅡ)作用后的心肌成纤维细胞增殖活性和一氧化氮(NO)分泌的影响。方法分离培养乳鼠心肌成纤维细胞,分为对照组(正常培养)、AngⅡ组(AngⅡ处理)、实验组(PKCα抑制剂D4681和AngⅡ处理)。实时定量PCR和Western blot法测定细胞中的PKCαmRNA和蛋白水平。用PKCα抑制剂和AngⅡ共同处理心肌成纤维细胞,四甲基偶氮唑盐比色法(MTT)测定细胞增殖活性,硝酸还原法测定细胞分泌的NO水平,酶联免疫吸附(ELISA)法测定培养液中的诱导型一氧化氮合成酶(i NOS)含量,羟脯氨酸法测定细胞合成胶原蛋白水平,Western blot法测定各组细胞表达基质金属蛋白酶-1(MMP-1)、基质金属蛋白酶-2(MMP-2)、Ⅰ型胶原(COLLⅠ)、Ⅱ型胶原(COLLⅡ)水平。结果 AngⅡ组心肌成纤维细胞中的PKCαmRNA和蛋白水平均明显高于对照组(P<0.05)。AngⅡ组心肌成纤维细胞增殖活性升高,细胞分泌的NO含量和i NOS活性均下降,细胞合成胶原蛋白水平升高,细胞中的MMP-1、MMP-2蛋白水平下降,COLLⅠ、COLLⅡ蛋白水平升高,与对照组相比差异具有统计学意义(P<0.05)。实验组心肌成纤维细胞增殖活性降低,细胞分泌的NO含量和i NOS活性升高,胶原蛋白合成减少,细胞中MMP-1、MMP-2蛋白表达增多,COLLⅠ、COLLⅡ蛋白表达减少,与AngⅡ组相比差异具有统计学意义(P<0.05)。结论 PKCα在AngⅡ作用后的心肌成纤维细胞中表达升高,抑制PKCα可以减弱AngⅡ诱导的心肌成纤维细胞增殖作用,促进细胞分泌NO。

Objective To investigate the effect of PKCα on the proliferation activity and NO level of myocardial fibroblasts after the action of AngⅡ. Methods Myocardial fibroblasts of neonatal rats were isolated and cultured, and divided into control group （normal culture）, AngⅡ group （AngⅡ treatment）, experimental group （PKC alpha inhibitor D4681 and AngⅡ treatment）, the PKCα mRNA and protein levels in the cells were measured by real-time quantitative PCR and Western blot. Cardiac fibroblasts were treated with PKCα inhibitor and AngⅡ, MTT assay cell proliferation activity, the nitric acid reduction method were used to determine the level of NO secreted by cells, the ELISA method was used to determine the content of iNOS in the culture medium, the levels of MMP-1, MMP-2, COLL I and COLL II were measured by Western blot method. Results The levels of PKCα mRNA and protein in AngⅡ group were significantly higher than those in control group （ P 〈0.05）. The proliferation activity of myocardial fibroblasts increased in AngⅡ group, the content of NO and the activity of iNOS decreased in cell secretion, the level of collagen synthesis in cells was increased, the level of MMP-1 and MMP-2 protein in the cells decreased, the levels of COLL I and COLL II protein were elevated, the difference was statistically significant compared with control group （ P 〈0.05）. The proliferation activity of myocardial fibroblasts in experimental group was reduced, the content of NO and the activity of iNOS increased in cell secretion, the synthesis of collagen was reduced, the expression of MMP-1 and MMP-2 protein in the cells increased, the expression of COLL I and COLL II protein was reduced, compared with AngⅡ group, the difference was statistically significant （ P 〈0.05）. Conclusion The expression of PKCα is elevated in the cardiac fibroblasts after the action of AngⅡ, inhibition of PKCα can weaken the proliferation of AngⅡ induced myocardial fibroblasts, promote the secretion of NO.