吡斯的明对人心肌成纤维细胞增殖、Ⅰ型胶原纤维表达及增殖信号通路的影响

The effects and mechanisms of pyridostigmine on cell proliferation of human cardiac fibroblasts, expression of collagen I and proliferation signal pathway

目的 探讨吡斯的明(PYR)对人心肌成纤维细胞(HCF)增殖、Ⅰ型胶原纤维(COL1)表达及增殖信号通路的影响.方法 采用转化生长因子β(TGFβ)刺激HCF.根据HCF的干预情况分为四组:对照组、PYR组、TGFβ组(TGFβ10 ng/ml)及TGFβ+PYR组.采用流式细胞仪分析细胞周期分布;羟脯氨酸法检测COL1含量;反转录酶-聚合酶链锁反应法(RT-PCR)测定结缔组织生长因子(CTGF)和Smad3的mRNA表达量;免疫印迹法测定CTGF、Smad3及pSmad3的蛋白表达量.结果 与对照组相比,TGFβ组的S/G2期细胞比例、COL1合成量、CTGF的mRNA和蛋白表达量、Smad3的mRNA和蛋白表达量及pSmad3蛋白表达量均增高(P均<0.05).采用TGFβ10 ng/ml与不同浓度PYR处理HCF后发现PYR 100μmol/L可使S/G2期细胞比例最低.与TGFβ组相比,TGFβ+PYR组(TGFβ10 ng/ml+PYR 100μmol/L)的S/G2期细胞比例(30.8%±1.4%比48.6%±3.5%)、COL1合成量(0.95±0.15比1.25±0.18)、CTGF mRNA(1.73±0.10比2.92±0.12)及CTGF蛋白表达量(1.23±0.42比2.36±0.62)均下降(P均<0.05).两组的Smad3 mRNA及Smad3、pSmad3蛋白表达量差异无统计学意义(P均>0.05).结论 PYR可减轻TGFβ诱导的HCF增殖和COL1表达,其机制可能与抑制CTGF的表达有关.

Objective To investigate the effects of pyridostigmine(PYR)on proliferation of human cardiac fibroblasts(HCF),expression of collagen I(COL1)and proliferation signal pathway.Methods HCF was cultured and divided into four groups according to the intervention:control group,PYR group,transforming growth factorβ(TGFβ)group(TGFβ10 ng/ml)and TGFβ+PYR group.Distribution of cell cycle were analysed by flow cytometry.The level of collagen I(COL1)was measured by hydroxyproline determination.The mRNA levels of connective tissue growth factor(CTGF)and Smad3 were examined by reverse transcription-polymerase chain reaction(RT-PCR)analysis.The protein expressions of CTGF,Smad3,and pSmad3 were examined by Western blot.Results Compared to HCF in control group,the proportion of S/G2 phase cells,extent of COL1,expressions of CTGF mRNA and protein,Smad3 mRNA and protein,and pSmad3 protein were higher than that in TGFβgroup(P<0.05).The distribution of cell cycle were observed after HCFs was treated with 10 ng/ml TGF-βand different level of PYR.As a result,PYR with the level of 100μmol/L was the most effective in reducing S/G2 phase cells.Compared to the TGFβgroup,the TGFβ+PYR group(TGFβ10 ng/ml+PYR 100μmol/L)had lower proportion of S/G2 phase cells(30.8%±1.4%vs.48.6%±3.5%),less synthesis of COL1(0.95±0.15 vs.1.25±0.18)and fewer expressions of CTGF mRNA(1.73±0.10 vs 2.92±0.12)and CTGF protein(1.23±0.42 vs.2.36±0.62)(P<0.05).However,the expressions of Smad3 mRNA,Smad3 protein and pSmad3 protein between TGFβgroup and TGFβ+PYR group were not significantly different(P>0.05).Conclusion Pyridostigmine significantly alleviated cell proliferation and collagen synthesis in HCFs induced by TGF-β,and its mechanism was possibly related to the inhibition of CTGF expression.