| Objective: To explore whether metformin can inhibit epithelial-to-mesenchymal transition(EMT)of alveolar epithelial cells by transforming growth factor ?1(TGF-?1)related signaling pathway and then reduce the occurrence and development of pulmonary fibrosis so as to provide a theoretical basis for its clinical application.Methods: 1.In vivo: After one-week adaptive feeding,48 male Sprague–Dawley(SD)rats were randomly divided into four groups(n=12): normal control group,bleomycin(BLM)group,metformin,metformin low dose group(0.1g·kg-1)and metformin(100 mg·kg-1)group and metformin(300 mg·kg-1)group.The pulmonary fibrosis model of rats was induced by slowly injecting BLM(5mg·kg-1)into the gap of rat tracheal annular cartilage.Animals for drug treatment received daily drug via gavage 1 h for 28 days,the rat of control group and BLM group received equal amoant of saline with the same regimen(1ml·100g-1).After 28 days,lung tissue was extracted.HE staining and Masson staining were used to observe the primary histological changes and collagen deposition in lung tissue;immunohistochemistry was used to detect the expression of collagen I,collagen III and TGF-?1;The expression of TGF-?1,collagen I,collagen III,alpha-smooth muscle actin(?-SMA),Vimentin,E-Cadherin,zonula occludens-1(ZO-1)and p-ERK1/2,p-Smad2/3 were detected by Western blot;the expression of TGF-?1,collagen I,collagen III,?-SMA,Vimentin,E-Cadherin,ZO-1 m RNA were measured by real time PCR.2.In vitro: Rat alveolar type II epithelial cells(RLE-6TN)were randomly divided into 6 groups: Control group,TGF-?1(5ng·ml-1)group,TGF-?1 + metformin(1,10,100 ?mol·L-1)group and metformin 100 ?mol·L-1 alone group.The cells were pretreated with TGF-?1 for 1 h,and then treated with metformin for 48 h.Cell proliferation was detected by CCK8.Western blot method was used to detect the expression level of collagen I,collagen III,?-SMA,and Vimentin,E-Cadherin,ZO-1,and p-ERK1/2,p-Smad2/3 proteins;the expression of collagen I,collagen III,?-SMA,Vimentin,E-Cadherin and ZO-1 m RNA was measured by real time PCR.Results: 1.In vivo:(1)The staining result of HE and Masson staining showed that the alveolar structure of rats in BLM group were severely damaged,inflammatory cells infiltrated,a great amount of collagen fibers were proliferated,fibroblasts and epithelia fibroblasts increased significantly in the alveolar septum,whereas metformin administration of the two groups were significantly improved.(2)Compared with the control group,the expression of collagen I,collagen III and TGF-?1 were significantly increased in BLM group by immunohistochemistry.And compared with BLM group,the expression of collagen I,collagen III and TGF-?1 were markedly decreased in metformin(100,300 mg·kg-1)groups.(3)Compared with control group,the expression of TGF-?1,collagen I,collagen III,?-SMA and Vimentin(both m RNA and protein)were markedly increased and the expression of E-Cadherin and ZO-1(both m RNA and protein)were significantly decreased in BLM group(P<0.01).Compared with BLM group,the expression of TGF-?1,collagen I,collagen III,?-SMA and Vimentin(both m RNA and protein)were markedly decreased and the expression of E-Cadherin and ZO-1 were significantly increased in metformin(100,300 mg·kg-1)groups(P<0.05 or P<0.01).(4)Compared with control group,the levels of ERR1/2 and Smad2/3 phosphorylation were significantly increased in lung tissues of bleomycin-induced pulmonary fibrosis rats.Compared with BLM group,the levels of ERR1/2 and Smad2/3 phosphorylation were markedly decreased in metformin(100,300 mg·kg-1)groups(P<0.05 or P<0.01).2.In vitro:(1)TGF-?1 significantly induced proliferation of RLE-6TN cell,and metformin(1,10,100 ?mol·L-1)dramatically inhibited TGF-?1-induced proliferation of RLE-6TN cell(P<0.05 or P<0.01).(2)Compared with control group,the expression of TGF-?1,collagen I,collagen III,?-SMA and Vimentin(both m RNA and protein)were markedly increased and the expression of E-Cadherin and ZO-1(both m RNA and protein)were significantly decreased in TGF-?1 group(P<0.01).Compared with TGF-?1 group,the expression of TGF-?1,collagen I,collagen III,?-SMA and Vimentin(both m RNA and protein)were markedly decreased and the expression of E-Cadherin and ZO-1 were significantly increased in metformin(1,10,100 ?mol·L-1)groups(P<0.05 or P<0.01).But compared with control group,metformin 100 ?mol·L-1 group alone had no effect on the TGF-?1,collagen I,collagen III,?-SMA,Vimentin,E-Cadherin and ZO-1expression.(3)Compared with control group,the levels of ERR1/2 and Smad2/3 phosphorylation were significantly increased in TGF-?1 group(P<0.01).Compared with TGF-?1 group,the levels of ERR1/2 and Smad2/3 phosphorylation were markedly decreased in metformin(1,10,100 ?mol·L-1)groups(P<0.05 or P<0.01)Conclusion: These results suggest that metformin protects against bleomycin-induced pulmonary fibrosis in rats by inhibiting ERR1/2 and Smad2/3 phosphorylation,alleviating TGF-?1 induces alveolar EMT process. |
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