Study On The Mechanism Of L-mimosine On Epithelial-mesenchymal Transition Of Alveolar Epithelial Cell In Pulmonary Fibrosis

Objective:This study aimed to assess whether L-mimosine（L-Mim）can against pulmonary fibrosis（PF）by inhibiting epithelial-mesenchymal transition（EMT）of alveolar epithelial cells through affecting the Notch1/Twist1/eukaryotic initiation factor3a（eIF3a）signaling pathway.Method:1.In vivo,Sixty male SD rats were randomly divided into control（CON）group,bleomycin（BLM）group,L-Mim（5 mg/kg）group and L-Mim（10 mg/kg）group,15 rats in each group.The PF rat model was induced by intratracheal injection of BLM（7500 U/kg）.Excised inferior lobe of left lung was fixed in 10%formalin for HE staining,Masson staining,Collagen I and Collagen III immunohistochemistry staining after BLM injection for four weeks.The mRNA and（or）proteins levels of transforming growth factor-beta 1（TGF-β1）,Collagen I,Collagen III,E-Cadherin,zonula occludens-1（ZO-1）,Vimentin,N-Cadherin,α-smooth muscle actin（α-SMA）,Notch1,Notch1 intracellular domain（NICD）,HES1,Twist1 and eIF3a were detected in lung tissue and type II alveolar epithelial cells by real-time PCR and（or）Western blotting.2.In vitro,The type II alveolar epithelial cells（RLE-6TN）were divided into six groups:Control group,TGF-β1（5 ng/mL）group,Dimethyl sulfoxide（DMSO）group,L-Mim 1μmol/L group,L-Mim 10μmol/L group and L-Mim 100μmol/L group.Each group was established nine holes.The cells were pre-treated with DMSO or L-Mim（1,10,100μmol/L）for 2 h,and then subjected to TGF-β₁（5 ng/mL）for 48 h.The ability of cell migration was applied by transwell assays and wound healing assay.The mRNA and（or）proteins levels of Collagen I,Collagen III,E-Cadherin,ZO-1,Vimentin,N-Cadherin,α-SMA,Notch1,NICD,HES1,Twist1 and eIF3a were detected in cells by real-time PCR and（or）Western blotting.Results:In vivo,1)Compared with the CON group,the alveolar atrophy,collapse and fusion occurred,alveolar septum widened significantly,and a large number of inflammatory cells infiltrated in the pulmonary interstitial of the rats in the BLM group.Compared with the BLM group,L-Mim significantly reduced bleomycin-induced pulmonary fibrosis in rats after four weeks of treatment.2)Compared with the CON group,BLM obviously increased collagen deposition and Collagen I and Collagen III expression（P<0.01）,but all these effects of bleomycin were significantly alleviated by treatment of rats with L-Mim treatment for four weeks.3)Compared with CON group,the expression of ZO-1 and E-Cadherin were decreased and the expression of Vimentin,N-Cadherin andα-SMA were increased in BLM group（P<0.01）.But compared with BLM group,the expression of ZO-1 and E-Cadherin were increased and the expression of Vimentin,N-Cadherin andα-SMA were decreased after L-Mim treatment for four weeks（P<0.05 or P<0.01）.4)Compared with CON group,the expression of TGF-β1,Notch1,NICD,HES1,Twist1 and eIF3a were increased in BLM group（P<0.01）.But compared with BLM group,the expression of TGF-β1,Notch1,NICD,HES1,Twist1and eIF3a were decreased after L-Mim treatment for four weeks（P<0.05 or P<0.01）.In vitro,1)Compared with control group,TGF-β1 treatment obviously increased Notch1,NICD,HES1,Twist1 and eIF3a expression（P<0.01）.Compared with TGF-β1 group,L-Mim（1,10,100μmol/L）treatment significantly inhibited Notch1,NICD,HES1,Twist1 and eIF3a expression induced by TGF-β₁（P<0.05 or P<0.01）.2)Compared with control group,TGF-β1 treatment obviously decreased ZO-1 and E-Cadherin expression and significantly increased Vimentin,N-Cadherin andα-SMA expression（P<0.01）.Compared with TGF-β1 group,L-Mim（1,10,100μmol/L）treatment reversed EMT of alveolar epithelial cell（the expression of E-Cadherin and ZO-1 were increased and the expression of Vimentin,N-Cadherin andα-SMA were decreased）induced by TGF-β₁（P<0.05 or P<0.01）.3)Compared with control group,TGF-β1 treatment obviously improved cell migration ability.Compared with TGF-β1 group,L-Mim（1,10,100μmol/L）treatment significantly reduced the cell migration ability.4)Compared with control group,TGF-β1 treatment obviously increased Collagen I and Collagen III expression（P<0.01）.Compared with TGF-β1 group,L-Mim（1,10,100μmol/L）treatment significantly inhibited Collagen I and Collagen III expression induced by TGF-β1（P<0.05 or P<0.01）.Conclusion:The results suggest that L-mimosine can reverse alveolar epithelial cells EMT and alleviate bleomycin-induced pulmonary fibrosis in rats,which may be involved in blocking Notch1 signaling pathway and inhibiting Twist1 and eIF3a expression.