Effect And Mechanism Of Nintedanib On Epithelial-mesenchymal Transition Of Alveolar Epithelial Cells

Objective: To study whether nitedanib can restrain epithelial-to-mesenchymal transition(EMT)of alveolar epithelial cells and whether these effects are achieved via inhibiting VEGF/ TGF-β1/ Notch1 signal pathway.And then reduce the occurrence and development of pulmonary fibrosis so as to provide a theoretical basis for its clinical application.Method: 52 male Sprague–Dawley(SD)rats were randomly divided into four groups,with twelve rats per group,as follows: The control group,the bleomycin(BLM)and the BLM treated with nitedanib(50,100 mg/kg)group.The pulmonary fibrosis model of rats was induced by by intratracheal instillation of bleomycin(5000 U/kg).Nintedanib was dissolved in 0.9% physiologic saline and administered via oral gavage daily from day 1 to day 28 after BLM treatment and all rats were sacrificed with exsanguinations on day 29.HE staining and Masson’s 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,alpha-smooth muscle actin(α-SMA)and transforming growth factor-β1(TGF-β1).Rat alveolar type II epithelial(RLE-6TN)cells were cultured at 37 °C under 5% CO2 in Roswell Park Memorial Institute(RPMI)1640 medium containing 10% fetal bovine serum.And then the cells were divided into 5 groups:i)The control group,cells were incubated with double distilled water(VEGF solvent)for 48 h;ii)vascular endothelial growth factor(VEGF)group,cells were incubated with VEGF(10 ng/mL)for 48 h;iii-v)+ nitedanib(1,10,100 nmol/L): cells were pre-treated with nitedanib(1,10,100 nmol/L)for 1 h,and then subjected to VEGF(10 ng/mL)for 48 h.incubated with double distilled water for 24 h.The expression of collagen I,collagen III,α-SMA,Vimentin,E-Cadherin,zonula occludens-1(ZO-1)and(or)VEGF,TGF-β1,Notch1 mRNA and protens were measured by real time PCR and Western blot.Result: In vivo: 1)After 4 weeks of exposure to bleomycin induced rat pulmonary fibrosis,as shown by a significant disturbed alveolar structure,markedthickening of the interalveolar septa and dense interstitial infiltration by inflammatory cells and fibroblasts with HE staining.All these effects of bleomycin were significantly alleviated by treatment of rats with nitedanib.2)Masson’s trichrome staining of lung specimens demonstrated that bleomycin instillation induced severe distortion of lung structure and accumulation of collagen fiber(blue)in rat lungs,whereas a well-alveolized normal histology was seen in rats treated with saline.And compared with control group,the expression of collagen I and collagen(both mRNA and protein)were significantly increased in lungs of rats.All these effects of bleomycin were significantly alleviated by treatment of rats with nitedanib(P<0.05 or P<0.01).3)Compared with control group,bleomycin dramatically increased the expression of α-SMA and Vimentin(both mRNA and protein)and obviously decreasesd the expression of E-Cadherin and ZO-1(both mRNA and protein)in lungs of rats.All these effects of bleomycin were significantly reversed by treatment of rats with nitedanib(P<0.05 or P<0.01).4)Compared with control group,the expression of VEGF,TGF-β1,and Notch1(both mRNA and protein)were significantly increased in lungs of rats.And Compared with BLM group,the levels of VEGF,TGF-β1,and Notch1 expression were markedly decreased in nitedanib(50,100 mg/kg)groups(P<0.05 or P<0.01).In vitro: 1)Exposure of VEGF(10 ng/mL)for 48 h significantly increased TGF-β1 and Notch1 expression in cultured RLE-6TN cells,whereas nitedanib(1,10,100 nmol/L)obviously inhibited the expression of TGF-β1 and Notch1(both mRNA and protein)(P<0.05 or P<0.01).2)Exogenous VEGF markedly up-regulated the expression of α-SMA and Vimentin and significantly down-regulated the expression of E-Cadherin and ZO-1 in cultured RLE-6TN cells.But nitedanib(1,10,100 nmol/L)obviously reversed VEGF-induced up-regulation of α-SMA and Vimentin expression and down-regulation of E-Cadherin and ZO-1 expression(both mRNA and protein)(P<0.05 or P<0.01).3)Compared with control group,the expression of collagen I and collagen III(both mRNA and protein)was markedly increased in VEGF group.Compared with VEGF group,the expression of collagen I and collagen III(both mRNA and protein)was markedly decreased in nitedanib(1,10,100 nmol/L)groups(P<0.05 or P<0.01).Conclusion: These results suggest that Nintedanib inhibit alveolar epithelial cell EMT process and alleviate bleomycin-induced pulmonary via inhibiting VEGF/ TGF-β1/ Notch1 signal pathway.