| Objective: To elucidate whether the TGF-β1/Notch-1/e IF3 a signaling pathway is involved in the process of epithelial-to-mesenchymal transition(EMT)of alveolar epithelial cells in pulmonary fibrosis.And through this research,we hope to provide a new theoretical basis for the pathogenesis of pulmonary fibrosis(PF)and new ideas for the search for anti-pulmonary fibrosis drugs.Method: 1.In vivo 30 male Sprague–Dawley(SD)rats were randomly divided into the control(CON)group and the bleomycin(BLM)group,15 rats in each group.The pulmonary fibrosis model was induced by intratracheal instillation of bleomycin(7500U/kg).The control group was injected with the same volume of normal saline(0.15 ml/100 g)in the same environment,and all rats continued feeding for four weeks after the finished of molding and sacrificed on day 29.Excised left lung was fixed in 10%neutral formalin solution for evaluation of morphology by HE staining and Masson staining.The freshly isolated right lung tissue samples were used for the determination the expression of collagen Ⅰ,collagen I,collagen III,Vimentin,alpha-smooth muscle actin(α-SMA),E-Cadherin,Zonula Occludens-1(ZO-1),transforming growth factor-β1(TGF-β1),Jagged-1,Notch-1,HES-1 and eukaryotic initiation factor 3a(e IF3a)by real-time PCR or Western blot.2.In vitro,part one: effect of notch-1 inhibitor DAPT on TGF-β1 induced EMT in alveolar epithelial cells.Rat alveolar type II epithelial(RLE-6TN)cells were cultured at 37 °C under 5% CO2 in RPMI 1640 medium containing 10% fetal bovine serum.And then the cells were divided into 4 groups: i)The control group,cells were incubated with double distilled water(TGF-β1 solvent)for 24 h;ii)TGF-β1 group: cells were incubated with TGF-β1(10 ng/m L)for 24 h;iii)DMSO group: cells were pre-treated with DMSO for 1 h,and then subjected to TGF-β1(10 ng/m L)for 24h;iv)DAPT group: cells were pre-treated with DAPT(1 μM)for 1 h,and then subjected to TGF-β1(10 ng/m L)for 24 h.Part two: effect of notch-1 si RNA on TGF-β1 induced EMT in alveolar epithelial cells.The cells were divided into 4groups: i)The control group,cells were incubated with double distilled water(TGF-β1solvent)for 24 h;ii)TGF-β1 group: cells were incubated with TGF-β1(10 ng/m L)for24 h;iii)Notch-1 si RNA negative control group(si RNA-NC): cells were pre-treated with Notch-1 si RNA scrambled(100 p M)for 24 h,and then subjected to TGF-β1(10ng/m L)for 24 h.;iv)Notch-1 si RNA group: cells were pre-treated with Notch-1 si RNA(100 p M)for 24 h,and then subjected to TGF-β1(10 ng/m L)for 24 h.The expression of collagen I,collagen III,α-SMA,Vimentin,E-Cadherin,Zonula ZO-1,Jagged-1,Notch-1,HES-1,e IF3 a m RNA and protein were measured by real time PCR and Western blot.Results: In vivo,1)HE staining showed that the alveolar structure of rat lung tissue in CON group was not significantly destroyed,and no obvious inflammatory response was present.Compared with the control group,the alveolar structure of the rats in the BLM group was severely destroyed,accompanied by inflammatory cell infiltration,and the lung tissue had a substantial lesion,the alveolar collapse,and the interval became significantly wider.2)Masson staining showed that there was no significant change in alveolar structure in the control group,but only a small amount of collagen was expressed in the interstitial lung.Compared with the CON group,the alveolar septum in the BLM group was significantly thickened and the pulmonary interstitial showed a large amount of collagen deposition.And compared with CON group,the expression of collagen I and collagen(both m RNA and protein)in lung tissues of rats were markedly increased in BLM group by real-time PCR and Western Blot analysis(P<0.01).3)Compared with CON group,the expression of Vimentin m RNA and protein and the expression of α-SMA protein were obviously increased(P<0.05 or P<0.01),meanwhile,the expression of E-cadherin m RNA and protein and the expression of ZO-1 protein were significantly decreased(P<0.05 or P<0.01)in BLM group.4)Compared with CON group,the expression of TGF-β1,e IF3 a m RNA and protein and the expression of Jagged-1,Notch-1,HES-1 proteins were obviously up-regulated(P<0.05 or P<0.01)in BLM group.In vitro,the first part: 1)Compared with control group,exposure of TGF-β1 significantly increased the expression of collagen I and collagen III(both m RNA and protein)(P<0.01).Compared with TGF-β1 group,the expression of collagen I and collagen III(both m RNA and protein)were obviously decreased(P<0.01)in DAPT group.2)Compared with control group,exposure of TGF-β1 significantly increased the expression of Vimentin(both m RNA and protein)and α-SMA protein(P<0.01)and significantly decreased the expression of E-Cadherin(both m RNA and protein)and ZO-1 protein(P<0.01).Compared with TGF-β1 group,the expression of Vimentin(both m RNA and protein)and α-SMA protein were obviously down-regulated(P<0.05 or P<0.01)and E-Cadherin(both m RNA and protein)and ZO-1 protein were obviously up-regulated(P<0.05 or P<0.01)in DAPT group.3)Compared with control group,exposure of TGF-β1 significantly increased the expression of Jagged-1,Notch-1,HES-1 proteins and e IF3a(both m RNA and protein)(P<0.01).Compared with TGF-β1 group,the expression of Notch-1,HES-1 proteins and e IF3a(both m RNA and protein)were obviously decreased(P<0.05 or P<0.01)in DAPT group,but there was no significant difference expression of Jagged-1(P>0.05).The second part: 1)Compared with control group,the expression of Jagged-1,Notch-1,HES-1 proteins and e IF3a(both m RNA and protein)were significantly increased(P<0.01)in TGF-β1 group.Compared with TGF-β1 group,there was no obvious change in si RNA-NC group(P>0.05),but the expression of Notch-1,HES-1proteins and e IF3a(both m RNA and protein)were obviously decreased(P<0.05 or P<0.01)in Notch-1 si RNA group,however,there was no significant difference expression of Jagged-1(P>0.05).2)Compared with control group,the expression of Vimentin(both m RNA and protein)and α-SMA protein were obviously increased(P<0.01)and the expression of E-Cadherin(both m RNA and protein)and ZO-1 protein were significantly decreased(P<0.01)in TGF-β1 group.Compared with TGF-β1 group,there was no obvious change in si RNA-NC group(P>0.05),but the expression of Vimentin(both m RNA and protein)and α-SMA protein were obviously decreased(P<0.05 or P<0.01)and E-Cadherin(both m RNA and protein)and ZO-1 protein were obviously increased(P<0.05 or P<0.01)in Notch-1 si RNA group.3)Compared with control group,the expression of collagen I and collagen III(both m RNA and protein)were significantly increased(P<0.01)in TGF-β1 group.Compared with TGF-β1 group,there was no obvious change in si RNA-NC group(P>0.05),but the expression of collagen I and collagen III(both m RNA and protein)were obviously decreased(P<0.01)in Notch-1 si RNA group.Conclusion: These results suggest that e IF3 a is involved in the EMT process of alveolar epithelial cells in rats with pulmonary fibrosis.The mechanism may be related to activation of the TGF-β1/Notch-1 signaling pathway. |
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