EL-7013促进Sirt1调控TGF-β1/Smad3通路抑制肺纤维化

EL-7013 inhibition of pulmonary fibrosis by promoting Sirt1 to regulate the TGF-β1/Smad3 signaling pathway

目的:对比EL-7013和吡非尼酮(pirfenidone,PFD)的抗纤维化作用及可能机制。方法:向C57BL/6小鼠气管内滴注3 mg/kg博来霉素(bleomycin,BLM)构建肺纤维化动物模型重组人TGF-β1(10 ng/mL)诱导人胚肺成纤维细胞(human embryonic lung fibroblasts,HLF-1)建立肺纤维化细胞模型,分别用EL-7013和PFD干预处理,机制探讨实验用EX-527抑制Sirt1表达及活性。HE、Masson染色评估肺组织病理变化,Western blot检测肺组织中COL1A1、COL3A1、α-SMA等纤维化指标和TGF-β1、Phospho-Smad1/Smad1、Phospho-Smad3/Smad3等通路指标表达,碱水法检测肺组织羟脯氨酸(hydroxyproline,HYP)含量。CCK-8检测细胞增殖活性,免疫荧光染色、Western blot、RT-qPCR检测细胞模型中纤维化及TGF-β1/Smad通路相关指标的蛋白和mRNA表达水平。结果:EL-7013明显减轻BLM诱导的肺组织结构破坏、胶原沉积,减轻TGF-β1及Phospho-Smad3蛋白表达(tBLM-300mg/kgEL-7013=11.149,P=0.008;tBLM-300mg/kgEL-7013=16.171,P=0.004)同时明显减轻TGF-β1诱导的细胞增殖(t=6.357,P=0.024),减少COL1A1、COL3A1、α-SMA、TGF-β1、Phospho-Smat3蛋白累积(t=66.136,P=0.000;t=13.726,P=0.005;t=10.230,P=0.009;t=9.263,P=0.011;t=14.809,P=0.005)。EL-7013可改善TGF-β1刺激导致的Sirt1表达及活性下降,特异性抑制Sirt1削弱了EL-7013的抗纤维化作用及对Phospho-Smad3的抑制作用(t=-6.009,P=0.027)。结论:EL-7013通过促进Sirt1从而调控TGF-β1/Smad3信号通路来抑制肺纤维化,为肺纤维的机制、新药研究提供新思路。

Objective:To compare the anti-fibrotic effect of EL-7013 and pirfenidone(PFD),and to explore their possible mechanism of action.Methods:An animal model of pulmonary fibrosis was established by intratracheal instillation of 3 mg/kg bleomycin(BLM)into C57 BL/6 mice,while a cell model of pulmonary fibrosis was established by inducing human embryonic lung fibroblasts(HLF-1)with recombinant human TGF-β1(10 ng/m L).Intervention with EL-7013 and PFD was then performed to investigate the mechanism of action of EX-527 in inhibiting the expression and activity of Sirt1.HE staining and Masson staining were used to evaluate the pathological changes of lung tissues.The expression of fibrosis markers(e.g.,COL1 A1,COL3 A1,andα-SMA)and pathway markers(e.g.,TGF-β1,Phospho-Smad1/Smad1,and Phospho-Smad3/Smad3)in lung tissues was measured by Western blot.And the content of hydroxyproline in lung tissues was also determined by alkaline water method.Furthermore,cell proliferative activity was evaluated by CCK-8 assay,and immunofluorescence,Western blot,and RT-q PCR were used to determine the protein and m RNA expression of fibrosis and TGF-β1/Smad pathway markers in cell model.Results:EL-7013 significantly mitigated BLM-induced lung histopathological changes,collagen deposition,and TGF-β1 and Phospho-Smad3 protein expression(tBLM-300 mg/kg EL-7013=11.149,P=0.008;tBLM-300 mg/kg EL-7013=16.171,P=0.004).Meanwhile,it significantly reduced the cell proliferation induced by TGF-β1(t=6.357,P=0.024),and decreased the accumulation of COL1 A1,COL3 A1,α-SMA,TGF-β1,and Phospho-Smad3 proteins(t=66.136,P=0.000;t=13.726,P=0.005;t=10.230,P=0.009;t=9.263,P=0.011;t=14.809,P=0.005).EL-7013 could improve the reduced expression and activity of Sirt1 induced by TGF-β1 stimulation.Specific inhibition of Sirt1 weakened the anti-fibrosis effect of EL-7013 and the inhibition of Phospho-Smad3(t=-6.009,P=0.027).Conclusion:EL-7013 inhibits pulmonary fibrosis by promoting Sirt1 to regulate the TGF-β1/Smad3 signaling pathway,which provides new ideas for the study on mechanism of pulmonary fibrosis and new drug for this disease.