阿司匹林对TGF-β1诱导的心肌成纤维细胞增殖的抑制作用

Effect of aspirin in inhibiting TGF-β1 induced proliferation of cardiac fibroblasts

目的:研究阿司匹林(ASA)对转化生长因子β1(TGF-β1)诱导心肌成纤维细胞(CFs)异常增殖的干预作用并探讨其可能的作用机制。方法:通过组织块法以及差速贴壁法获得并培养CFs,免疫细胞化学法检测波形蛋白的表达用于鉴定CFs,建立TGF-β1诱导CFs增殖模型,实验分为对照组(无血清DMEM)、TGF-β1组(TGF-β1,20 ng·mL-1)、TGF-β1+ASA组(ASA,0.5、1、2μmol·L-1)。MTT法检测ASA对TGF-β1诱导CFs增殖的影响,羟脯氨酸含量试剂盒检测细胞内以及培养液上清中羟脯氨酸的含量,蛋白质免疫印迹法(Western blot)分析Smad2、Smad3蛋白表达的变化。结果:MTT结果显示,与对照组比较,TGF-β1显著诱导CFs增殖,与TGF-β1组相比,ASA可显著抑制TGF-β1诱导的CFs增殖;羟脯氨酸含量检测显示,与对照组比较,TGF-β1组羟脯氨酸含量明显升高,与TGF-β1组相比,ASA可降低其含量;Western blot结果显示,与对照组比较,TGF-β1显著诱导Smad2、Smad3蛋白水平表达升高,与TGF-β1组相比,ASA可显著降低Smad2、Smad3蛋白水平的表达。结论:ASA对TGF-β1诱导的CFs增殖具有显著的抑制作用,其作用机制可能与抑制TGF-β1/Smads信号通路下游蛋白Smad2、Smad3的表达相关。

OBJECTIVE To investigate the inhibitory effects of aspirin（ASA）on proliferation of cardiac fibroblasts（CFs）induced by transforming growth factor-β1（TGF-β1）,and clarify its possible mechanism.METHODS CFs were isolated and purified by differential adhesion.The CFs were identified by anti-vimentin immunocytochemistry.The model of CFs proliferation was induced by TGF-β1.The experiment was randomly divided into control group（serum-free DMEM）,TGF-β1 group（TGF-β1,20 ng·mL^-1）and TGF-β1＋ ASA group（ASA,0.5,1,2μmol·L^-1）.The inhibition of ASA on CFs proliferation was detected by MTT assay.The content of hydroxyproline in the cells was detected by hydroxyproline kit.The protein expressions of Smad2 and Smad3 were analyzed by Western blot.RESULTS The results of MTT showed that compared with the control group,TGF-β1 significantly induced the proliferation of CFs.Compared with TGF-β1 group,ASA significantly inhibited the proliferation of CFs induced by TGF-β1.The content of hydroxyproline increased in TGF-β1 group compared with the control group,but decreased after being co-cultured with ASA compared with TGF-β1 group.Western blot results showed that the protein expressions of Smad2 and Smad3 were significantly increased in TGF-β1 group,ASA could significantly suppress the protein expressions of Smad2 and Smad3（P〈0.05）.CONCLUSION ASA can significantly inhibit the content of hydroxyproline and proliferation of CFs induced by TGF-β1,and ASA ameliorates the cardiac fibrosis involving in inhibition of Smad2 and Smad3 expression.