异丙酚抑制血管紧张素Ⅱ诱导的大鼠心肌成纤维细胞增殖的机制

The mechanism of propofol inhibit the proliferation of cardiac fibroblasts in rats induced by angiotensinⅡ

目的探讨异丙酚(Propofol)抑制血管紧张素Ⅱ(AngⅡ)诱导的大鼠心肌成纤维细胞(CFb)增殖的机制。方法选取新生的Wistar大鼠,取其心肌成纤维细胞进行原代培养及分离纯化。采用AngⅡ建立诱导新生大鼠CFb纤维化模型。采用MTT法选取异丙酚抑制AngⅡ诱导心肌成纤维细胞增殖的最适宜浓度;将细胞分为3组:正常对照(Control)组、AngⅡ组和异丙酚(Propofol)处理组。采用流式细胞仪测定各组细胞的周期分布;采用ELISA法测定各组细胞中Ⅰ、Ⅲ型胶原含量;采用Western Blot法测定各组细胞中转化生长因子β1(TGF-β1)的蛋白表达量;采用免疫细胞化学化法测定各组细胞中细胞外信号调节蛋白激酶(ERK1/2)和经典型蛋白激酶Cα亚型(cPKCα)的蛋白表达。结果MTT实验结果显示,不同浓度的异丙酚对AngⅡ诱导的大鼠心肌成纤维细胞的增殖均具有一定的抑制作用,100μmol/L的异丙酚对AngⅡ诱导的大鼠心肌成纤维细胞的增殖抑制效果最明显(P<0.01),因此,后续实验选取100μmol/L的异丙酚作用于AngⅡ诱导的大鼠心肌成纤维细胞。流式细胞仪测定结果显示,与正常对照组相比,AngⅡ组细胞G0/G1期细胞百分率降低(P<0.01),S期和G2/M期细胞百分率升高(P<0.01);而异丙酚处理组细胞的G0/G1期细胞百分率高于AngⅡ组(P<0.01),S期和G2/M期细胞百分率低于AngⅡ组(P<0.01)。ELISA测定结果显示,与正常对照组相比,AngⅡ组大鼠的心肌成纤维细胞Ⅰ、Ⅲ型胶原含量增加(P<0.01);而异丙酚处理组大鼠的心肌成纤维细胞Ⅰ、Ⅲ型胶原含量均低于AngⅡ组(P<0.01)。Western Blot法测定结果显示,与正常对照组相比,AngⅡ组大鼠心肌成纤维细胞中TGF-β1的蛋白表达量升高(P<0.01),而异丙酚处理组大鼠心肌成纤维细胞中TGF-β1的蛋白表达量低于AngⅡ组(P<0.01)。免疫细胞组化的结果显示,与正常对照组相比,AngⅡ组大鼠心肌成纤维细胞中ERK1/2和cPKCα的阳性表达均增加(P<0.01);而异丙酚处理组大鼠心肌成纤维细胞中ERK1/2和cPKCα的阳性表达均低于AngⅡ组。结论异丙酚能够抑制AngⅡ诱导的大鼠心肌成纤维细胞的增殖和Ⅰ、Ⅲ型胶原含量的增加,推测其作用机制与其降低细胞中TGF-β1的蛋白表达和抑制ERK1/2、cPKCα通路有关。

Objective In this study,the mechanism of propofol inhibit the proliferation of cardiac fibroblasts in rats induced by angiotensinⅡwas investigated.Methods The myocardial fibroblasts of newborn Wistar rats were cultured and purified.The 1×10-6 mol/L AngⅡwas used to induce the newborn rat CFb fibrosis model.The MTT method was used to select the optimum concentration of Propofol to inhibit AngⅡinduced myocardial fibroblasts proliferation.The cells were divided into 3 groups:Control group,AngⅡgroup and Propofol treatment group.The periodic distribution of cells in each group was determined by flow cytometry.The contents of collagen typeⅠandⅢwere detected by ELISA method.Western Blot method was used to determine the protein expression of transformed growth factorβ1(TGF-β1)in cells of each group.The protein expressions of extracellular signal regulating protein kinase(ERK1/2)and transcriptional protein kinase Cαsubtype(cPKCα)in each group were determined by immunocytochemistry.Results The MTT results showed that different concentrations of propofol had certain inhibition on proliferation of cardiac fibroblasts induced by AngⅡ.And 100μmol/L of propofol had the most significant inhibiting effect on proliferation of cardiac fibroblasts induced by AngⅡ(P<0.01).Therefore,100μmol/L of propofol was selected to act on the cardiac fibroblasts induced by AngⅡin follow-up experiments.Flow cytometry results show that compared with control group,the percentage of cells in G0/G1 phase in AngⅡgroup was decreased(P<0.01),the percentage of cells in S phase and G2/M phase were increased(P<0.01).And the percentage of cells in G0/G1 phase in propofol group was higher than AngⅡgroup(P<0.01),the percentage of cells in S phase and G2/M phase were lower than AngⅡgroup(P<0.01).ELISA results show that compared with control group,the contents ofⅠ,Ⅲtype collagen in AngⅡgroup of rat cardiac fibroblasts were increased(P<0.01).And the contents ofⅠ,Ⅲtype collagen in propofol group of rat cardiac fibroblasts were lower than AngⅡgroup(P<0.01).Western Blot results showed that compared with control group,the protein expression of TGF-β1 in AngⅡgroup of rat cardiac fibroblasts was increased(P<0.01).And the protein expression of TGF-β1 in propofol group of rat cardiac fibroblasts was lower than AngⅡgroup(P<0.01).Immuno-histochemical results showed that compared with control group,the positive expressions of ERK1/2 and cPKCαin AngⅡgroup of rat cardiac fibroblasts were increased(P<0.01).And the positive expressions of ERK1/2 and cPKCαin propofol group of rat cardiac fibroblasts were lower than those in AngⅡgroup.Conclusion Propofol could inhibit the proliferation and the contents of collagen typeⅠ,Ⅲin rat cardiac fibroblasts induced by AngⅡ.It was speculated that the mechanism of inhibition was related to the reduction of the protein expression of TGF-β1 in cells and the inhibitions of ERK1/2 and cPKCαactivation pathways.