磷酸肌酸钠对血管紧张素Ⅱ诱导的乳鼠心肌成纤维细胞增殖和胶原合成的影响及机制研究

Effect of Phosphocreatine on Angiotensin II Induced Proliferation and Collagen Synthesis of Cardiac Fibroblasts in Neonatal Rats With its Mechanism

目的:观察磷酸肌酸钠对血管紧张素Ⅱ(AngⅡ)诱导的乳鼠心肌成纤维细胞(CF)增殖和胶原合成的影响,并初步探索磷酸肌酸钠抗心肌纤维化的作用机制。方法:将20只Wistar乳鼠取出心脏,体外原代、传代培养CF。实验分4组(每组n=3),对照组:无血清的DMEM培养液培养CF;AngⅡ组:含AngⅡ1×10-6mol/L的无血清DMEM培养液;磷酸肌酸钠组:含磷酸肌酸钠10mmol/L的无血清DMEM培养液;AngⅡ+磷酸肌酸钠组:含磷酸肌酸钠10 mmol/L加AngⅡ1×10-6mol/L的无血清DMEM培养液。采用流式细胞术测定细胞周期分布,Van Gieson(VG)氏染色法测定胶原含量,免疫细胞化学法检测磷酸化细胞外信号调节激酶(pERK1/2)蛋白的表达水平。结果:与对照组相比,AngⅡ组CF的S期细胞百分率明显增加,G0/G1期、G2/M期细胞百分率降低,胶原含量增加,pERK1/2蛋白表达增高,差异均有统计学意义(P均<0.01)。与对照组比较,磷酸肌酸钠组CF细胞周期、胶原含量和pERK1/2蛋白表达,差异均无统计学意义(P均>0.05)。与对照组比较,AngⅡ+磷酸肌酸钠组pERK1/2蛋白表达升高,差异有统计学意义(P<0.01),CF细胞周期和胶原含量差异均无统计学意义(P均>0.05)。与AngⅡ组比较,AngⅡ+磷酸肌酸钠组G0/G1期、G2/M期细胞百分率升高,S期百分率降低,胶原含量减少,pERK1/2蛋白表达降低,差异均有统计学意义(P均<0.01)。结论:磷酸肌酸钠可部分抑制AngⅡ诱导的CF增殖和胶原合成增加,其机制可能与抑制ERK1/2过度激活有关。这提示磷酸肌酸钠可以明显改善AngⅡ诱导的心肌纤维化。

Objective： To investigate the effect of phosphocreatine （PCr） on angiotensin Ⅱ （Ang Ⅱ） induced proliferation and collagen synthesis of cardiac ifbroblasts in neonatal rats with its mechanism. Methods： The cardiac ifbroblasts （CF） from neonatal rats were cultured in vitro and were divided into 4 groups.①Control group, the CF was cultured in non-serum DMEM,②Ang Ⅱ group, the CF was cultured with Ang Ⅱ at （1＆#215;10-6） mol/L,③PCr treated group, the CF was cultured with PCr at 10 mmol/L, and④Ang Ⅱ＋PCr group. The CF cell cycle percentage was detected by lfow cytometric assay, myocardial collagen content was observed by VG staining and protein expression of phosphorylated extracellular signal-regulated kinase （pERK1/2） was detected by immuneohistochemistry. Results： ① Compared with Control group, the CF in Ang Ⅱ group showed increased percentage of S phase and decreased percentage of G0/G1 and G2/M phases, increased collagen content and pERK1/2 protein expression, all P〈0.01.② The CF cell cycle, collagen content and pERK1/2 protein expression were similar between Control group and PCr treated group, all P〉0.05. ③ Compared with Control group, Ang Ⅱ ＋ PCr group had elevated pERK1/2 protein expression, P〈0.01, while the CF cell cycle and collagen content were similar with Control group, P〉0.05.④Compared with Ang Ⅱ group, the CF in Ang Ⅱ ＋ PCr group had increased percentage of G0/G1 and G2/M phases, decreased percentage of S phase, decreased collagen content and pERK1/2 protein expression, all P〈0.01. Conclusion： PCr may partially inhibit Ang Ⅱ induced CF proliferation and collagen synthesis which might be related to the inhibition of excessively activated ERK1/2. Therefore, PCr could improve Ang Ⅱ induced myocardial ifbrosis in neonatal rats.