3-吲哚丙酸抑制腹膜间皮细胞EMT和纤维化的机制研究

Study on Mechanism of 3-indolepropionic Acid Inhibiting EMT and Fibrosis in Peritoneal Mesothelial Cells

目的 评估3-吲哚丙酸(3-indoleacetic acid, IPA)对脂多糖(lipolyaccharide, LPS)诱导的小鼠腹膜间皮细胞上皮-间质转化(epithelial-mesenchymal transition, EMT)和纤维化的影响。方法 CCK-8法检测不同浓度(0.1、1.0和10μmol/L)的IPA对LPS处理前后的小鼠腹膜间皮细胞增殖活性的影响,确定IPA最佳使用剂量。将小鼠腹膜间皮细胞分成Control组、LPS组、LPS+IPA组、LPS+LY364947(TGF-β1/Smad3通路抑制剂)组、LPS+IPA+LY364947组和LPS+IPA+SRI-011381(TGF-β1/Smad3通路激活剂)组;CCK-8法检测细胞增殖活力,Transwell法检测细胞侵袭,ELISA检测培养上清中间质表型α-平滑肌肌动蛋白(α-smooth muscle actin, α-SMA)的浓度,Western blot检测细胞中α-SMA,EMT相关因子E-cadherin, TGF-β1/Smad3通路相关因子Smad3、p-Smad3的蛋白表达。结果 IPA的最佳使用剂量为1.0μmol/L。与Control组相比,LPS组细胞增殖活力、E-cadherin表达水平下降(均P<0.01),侵袭能力、α-SMA表达水平和p-Smad3/Smad3升高(均P<0.01)。与LPS组相比,LPS+IPA组和LPS+LY364947组细胞增殖活力、E-cadherin表达水平上升(均P<0.01),侵袭能力、α-SMA表达和p-Smad3/Smad3下降(均P<0.01)。与LPS+IPA组相比,LPS+IPA+LY364947组所测指标趋势更加显著,LPS+IPA+SRI-011381组所测指标变化趋势均被逆转。结论 IPA通过抑制TGF-β1/Smad3通路中Smad3蛋白的磷酸化,从而抑制细胞EMT进展,减轻LPS诱导的腹膜间皮细胞损伤。

Objective To evaluate the effects of 3-indoleacetic acid(IPA)on epithelial-mesenchymal transition(EMT)and fi-brosis in mice peritoneal mesoepithelial cells stimulated by lipopolysaccharide.Methods Mouse peritoneal mesothelial cells be-fore and after LPS treatment were interfered with IPA at different concentrations(o.1,1.0 and 10μmol/L),and the cell prolif-eration activity was examined by CCK-8 method to determine the optimal dose of IPA.Mice peritoneal mesothelium cells were divided into Control group,LPS group,LPS+IPA group,LPS+LY364947(TGF-β1 receptor inhibitor)group,LPS+IPA+LY364947 group and LPS+IPA+SRI-011381(TGF-β1 pathway activator)group.Cell proliferation viability was evaluated by CCK-8 assay and cell invasion was detected by Transwell assay.EMT-related factor(E-cadherin)and TGF-β1/Smad3 pathway-related factor(Smad3,p-Smad3)protein expression in cell supernatant was evaluated by Western blotting,and mesenchymal phe-notypeα-smooth muscle actin(α-SMA)protein content was detected by ELISA.Results The optimal dose of IPA was 1.0μmol/L.Compared with Control group,cell proliferation and E-cadherin expression level were decreased(all P<0.01).Invasivi-ty,α-SMA expression and p-Smad3/Smad3 were increased in LPS group(all P<0.01).Compared with LPS group,cell prolifer-ative activity and E-cadherin expression level were increased(all P<0.01),and invasivity,α-SMA expression and p-Smad3/Smad3 were decreased in LPS+IPA and LPS+LY364947 groups(all P<0.01).Compared with LPS+IPA group,LPS+IPA+LY364947 group showed a more significant trend.The trends of measured indexes were reversed after SRI-011381 treatment in LPS+IPA group.Conclusion IPA inhibits Smad3 phosphorylation,thereby inhibiting cell EMT progression and alleviating LPS-induced peritoneal mesothelial cell injury.