The Molecular Mechanisms Of Bile Acids In The Pathogenesis Of Pulmonary Fibrosis

Objective This study aimed to explore the profibrotic effects of bile acids as well as the underlying mechanisms in vitro and in vivo,and to investigate the expression and function of the bile acid-activated nuclear farnesoid X receptor(FXR)in pulmonary fibrosis,so as to elucidate the biological role and molecular mechanisms of bile acids and FXR in pulmonary fibrosis.Contents and Methods 1.A rat model of chronic aspiration of bile acids was induced by weekly intratracheal instillation with the three major bile acid components: lithocholic acid(LCA),deoxycholic acid(DCA),and chenodeoxycholic acid(CDCA)(250μM,at dose of 0.5 ml/kg,for 6 weeks).Lung histopathology,fibrotic markers and fibrogenic mediators were then evaluated to assess the profibrotic effects of bile acid aspiration in vivo.2.Human alveolar epithelial cells(AECs)and lung fibroblasts cell lines were exposed to LCA,DCA and CDCA.The AEC injury indices,epithelial-mesenchymal transition(EMT),and lung fibroblast activation were evaluated.The critical mediators and signaling pathways possibly involved in bile-acid induced profibrotic effects were also evaluated by using Western blot and other techniques.3.FXR expression was measured by immunohistochemical staining in lung tissues from patients with IPF and rat model of chronic aspiration of bile acids,and the role of FXR in alveolar EMT and lung fibroblast activation was further elucidated.Furthermore,the molecular mechanisms underlying the contribution of FXR to pulmonary fibrosis were also investigated by using siRNA transfection,Western blot and other techniques.Results 1.The rat lungs from the bile-acid-treated group showed widening of the alveolar septa,fibrotic tissue and more inflammatory cell infiltrationin the lung interstitium.Compared with control group,the alveolitis and fibrosis scores increased significantly in rats from LCA,DCA or CDCA group(all P<0.05).In rat lungs from LCA,DCA or CDCA groups,the expression of α-SMA,collagen 1,fibrogenic mediators(TGF-β1,VEGF,CTGF,b-FGF and periostin)and p-Smad3 was remarkably increased,whereas E-cadherin expression was significantly decreased compared to controls.2.LCA,DCA,and CDCA reduced cell viability and induced intracellular reactive oxygen species(ROS)production and apoptosis in AECs.They all induced EMT,as shown by enhanced mesenchymal markers α-SMA and vimentin,decreased epithelial marker E-cadherin,and morphological transformation.LCA directly induced differentiation of lung fibroblasts to myofibroblasts as shown by its induction of α-SMA expression.All three bile acids promoted cellular migration but not proliferation of lung fibroblasts.The bile acids also indirectly induced differentiation of lung fibroblasts to myofibroblasts through stimulation of profibrotic factors from AECs,such as TGF-β1,b-FGF and CTGF etc.TGF-β1/Smad3 signaling was also involved in the bile acid-induced EMT and lung fibroblast differentiation.3.FXR was located in type II AECs,alveolar macrophages,bronchial epithelial cells,and a few mesenchymal cells in normal human and rat lung tissue.FXR expression was significantly increased in lung tissues from patients with IPF and rat model of chronic aspiration of bile acids.FXR agonist GW4064 induced alveolar EMT and lung fibroblast differentiation,whereas FXR antagonist z-Guggulsterone and FXR knockdown by specific siRNA significantly attenuated bile acid-induced EMT and lung fibroblast differentiation and migration.These results indicated that FXR contributed to bile acid-induced alveolar EMT and lung fibroblast activation.In addition,we found that the expression and activation of FXR could be induced by TGF-β1 in AECs and lung fibroblasts.Moreover,FXR mediated TGF-β1-induced profibrotic responses in AECs and lung fibroblasts,including alveolar EMT,lung fibroblasts activation and expression of profibrotic cytokines,possibly through regulating TGF-β receptor-activated Smad2/3 and non-Smad pathways.Conclusions Chronic aspiration of bile acids promotes the development and progression of pulmonary fibrosis via inducing alveolar epithelial cell injury,EMT and lung fibroblast activation.The overexpression and activation of FXR may contribute to in the fibrotic processes of IPF by regulating alveolar EMT and lung fibroblast activation.