二甲双胍调节AMPK/TGF-β1信号通路对百草枯中毒大鼠肺纤维化的保护作用

Protective effect of metformin regulating AMPK/TGF-β1 signaling pathway on pulmonary fibrosis in rats with paraquat poisoning

目的探讨MET通过激活AMPK信号通路对急性PQ中毒致大鼠肺纤维化的保护作用。方法健康成年SPF级雄性SD大鼠30只,随机分为五组,对照组(Control组)、PQ中毒组(PQ组),二甲双胍干预组(PQ+MET组),腺苷类似物组(PQ+AICAR组)和复合物C组(PQ+MET+CC组)。干预21 d后取大鼠肺组织匀浆分别检测Hyp、SOD、GSH-px和MDA的含量,Masson染色和透射电镜观察大鼠肺组织病理变化,Western-blotting观察大鼠肺组织Phospho-AMPK、TGF-β1、E-cadherin和α-SMA蛋白的表达。结果与PQ组比较,PQ+MET组和PQ+AICAR组明显降低了Hyp和MDA含量,差异有统计学意义(P<0.05),提高了SOD和GSH-px水平,差异有统计学意义(均P<0.05)。肺组织Masson染色和透射电镜结果示,PQ组21 d时可见肺泡壁增厚,肺泡腔挤压变形,基质增厚,支气管周围及肺泡间隔的胶原纤维增多,而PQ+MET组和PQ+AICAR组较PQ组明显减轻。Western-blotting结果示,与PQ组比较,PQ+MET组和PQ+AICAR组的TGF-β1和α-SMA蛋白含量明显减少,差异有统计学意义(P<0.05),而Phospho-AMPK和E-cadherin蛋白明显增高,差异有统计学意义(均P<0.05);使用AMPK抑制剂后,PQ+MET+CC组较PQ+MET组TGF-β1和α-SMA蛋白升高,差异有统计学意义(P<0.05),而Phospho-AMPK和E-cadherin蛋白降低,差异有统计学意义(P<0.05)。结论二甲双胍可以减轻PQ中毒大鼠肺纤维化,其机制与激活AMPK信号通路,下调TGF-β1来改善上皮-间质转化有关,而这一效应可以被AMPK抑制剂CC所抑制。

Objective To investigate the protective effect of Metformin(MET)on pulmonary fibrosis induced by acute Paraquat(PQ)poisoning in rats by activating AMPK signaling pathway.Methods Thirty healthy adult SPF male SD rats were randomly divided into 5 groups which were control group,PQ poisoning group,metformin intervention group(PQ+MET group),adenosine analogues group(PQ+AICAR group)and complex C group(PQ+MET+CC group).After 21 days of intervention,the contents of hydroxyproline(Hyp),superoxide dismutase(SOD),glutathione peroxidase(GSH-px)and malondialdehyde(MDA)were determined by homogenizing rat lung tissues.The pathological changes of lung tissues were observed by Masson staining and transmission electron microscopy.The expressions of Phospho-AMPK,TGF-β1,E-cadherin andα-SMA in rat lung were observed by western blotting(WB)method.Results Compared with PQ group,Hyp and MDA contents in PQ+MET group and PQ+AICAR group were significantly decreased(both P<0.05),increased SOD and GSH-px levels(both P<0.05).Masson staining and transmission electron microscopy of lung tissue showed that the alveolar wall was thickened,the alveolar cavity was compressed and deformed,the matrix was thickened,and the collagen fibers around the bronchus and alveolar septum were increased in the PQ+MET group and PQ+AICAR group,which were significantly reduced compared with the PQ group.WB results was shown that compared with PQ group,TGF-β1 andα-SMA protein contents in PQ+MET group and PQ+AICAR group were significantly decreased(both P<0.05)and significantly increased Phospho-AMPK and E-cadherin proteins(both P<0.05).TGF-β1 andα-SMA proteins in PQ+MET+CC group were higher than those in PQ+MET group after treatment with AMPK inhibitor(both P<0.05)and decreased Phospho-AMPK and E-cadherin proteins(both P<0.05).Conclusion Metformin can reduce pulmonary fibrosis in PQ-poisoned rats by activating AMPK signaling pathway and down-regulating TGF-β1 to improve epithelial-mesenchymal transformation,which can be inhibited by AMPK inhibitor CC.