双苯氟嗪对血管紧张素Ⅱ诱导新生大鼠心肌成纤维细胞增殖和胶原合成的影响及其机制研究

目的研究双苯氟嗪(d ipfluzine,D ip)对血管紧张素Ⅱ(angiotensinⅡ,AngⅡ)诱导的新生大鼠心肌成纤维细胞(card iac fibrob lasts,CFB)增殖和胶原合成的影响,并探讨其抑制心肌纤维化的机制。方法用消化法培养新生SD大鼠CFB,建立AngⅡ诱导新生大鼠CFB纤维化模型。采用MTT法检测D ip对CFB增殖的影响;羟脯氨酸测定检测CFB胶原含量;流式细胞分析仪技术检测细胞周期及增殖细胞核抗原(PCNA)蛋白表达;RT-PCR检测Ⅰ型、Ⅲ型胶原蛋白和转化生长因子β1(TGF-β1)的基因表达;免疫组织化学染色方法,观察心肌成纤维细胞经典型蛋白激酶Cα亚型(cPKCα)和细胞外信号调节蛋白激酶(ERK1)蛋白的表达。结果①在一定浓度范围内,D ip能明显抑制AngⅡ诱导CFB的增殖和胶原合成(P<0.05或P<0.01)。②CFB细胞G0/G1期百分率随D ip浓度增加而增加,S期、G2/M期百分率和增殖指数随D ip浓度增加而减少,与AngⅡ组相比差异有显著性(P<0.05或P<0.01)。③D ip能降低PCNA蛋白表达,与AngⅡ组相比差异有显著性(P<0.01)。④D ip能够明显降低Ⅰ型、Ⅲ型胶原和TGF-β1mRNA的表达(P<0.05或P<0.01)。⑤免疫组化结果显示D ip(10、100μmol.L-1)组ERK1和cPKCα阳性表达低于AngⅡ组(P<0.05或P<0.01)。结论D ip通过抑制AngⅡ诱导的CFB增殖和胶原的增加而起到了抗心肌纤维化的作用,其抗纤维化作用与其降低TGF-β1基因表达以及抑制cPKCα、ERK1通路有关。

Effects of Dipfluzine on the proliferation and collagen synthesis of cardiac fibroblasts and mechanisms

Aim To investigate the effects of dipfluzine(Dip) on proliferation and collagen synthesis in cultured neonatal rat cardiac fibroblasts(CFB)stimulated by angiotensin Ⅱ(AngⅡ),and to explore the action mechanisms of dipfluzine.Methods CFB were treated with AngⅡ to induce fibrosis model.The effect of Dip on proliferation of CFB was observed by MTT coloricmetric assay;synthesis of collagen was observed by the hydroxyproline concentration detemined;cell cycle distribution and PCNA proein were determined with flow cytometer(FCM);The expression of collagenⅠmRNA,collagen Ⅲ mRNA and TGF-β_1 mRNA was examined using semi-quantitative RT-PCR analysis;The protein expression of cPKCα and ERK_1 in CFB was observed by immunohistochemical staining.Results ① Within a concentration coverage,Dip inhibited CFB proliferation and collagen synthesis(P<0.05,P<0.01,respectively).②The percentage of cells in G_0/G_1 phase in Dip groups was significantly higher than that in AngⅡ group(P<0.01)and the percentage of cells in S and G_2/M phases in Dip groups was significantly lower than that in AngⅡ group(P<0.05,P<0.01,respectively).③ Dip decreased the protein expression of PCNA stimulated by AngⅡ(P<0.01).④ Dip decreased the levels of CollagenⅠmRNA,Collagen Ⅲ mRNA and TGF-β_1 mRNA expression(P<0.05,P<0.01,respectively).⑤ The levels of cPKCα and ERK1 protein expression in Dip(10 μ,100 μmol·L~(-1)) groups were significantly lower than that in AngⅡ group(P<0.05,P<0.01,respectively).Conclusions Dip inhibited CFB proliferation and collagen synthesis stimulated by AngⅡ,which related to downregulation the levels of TGF-β_1 mRNA,the ERK1 and cPKCα protein expression.