Study On The Effect And Mechanism Of Dehydroevocorine On Pulmonary Fibrosis

Objective: To explore the effect of dehydroevodiamine(DHED)on pulmonary fibrosis(PF)in mice.To investigate the effects of DHED on human transforming growth factor-β1(TGF-β1)induced human fetal lung fibroblast-1(HFL1)and the possible signaling pathways.Methods: Animal experiment: First,the mice PF model was induced by bleomycin(BLM),and DHED was treated with intraperitoneal injection,while pirfenidone(PFD)was given as positive control.The laboratory mice were randomly divided into four groups: Normal control group(NC group,n=6),BLM group(n=10),BLM+DHED group(n=10),and BLM+ PFD(n=10).Second,pathological changes in lung tissues of mice were detected by tianwolf scarlet staining,hematoxylin-eosin staining,and Masson trichromatic staining.Third,collagen content in mouse lung tissue was determined by acid hydrolyzation of hydroxyproline.Fourth,the expressions of Fibronectin,Collagen1,and α-SMA in mouse lung tissues were detected by Western Blot and RT-PCR.Cell experiment: First,HFL1 fibrosis was induced by TGF-β1 in vitro and treated with DHED.The cell experiment was divided into three groups:Control group(Dimethylsulfoxide,DMSO 0.5‰),DMSO(0.5‰)+TGF-β1(10ng/ml)group and TGF-β1(10 ng/ml)+DHED(10 μM)group.Second,the expressions of Fibronectin,Collagen1 and α-SMA in cells were detected by western blotting,immunofluorescence and reverse transcription-polymerase chain reaction.Third,Edu Cell proliferation Kit was used to measure the proliferation ability of cells,and cell migration was evaluated by cell scratch assay.Fourth,Western Blotting was used to detect the expression of classic fibrosis signaling pathway PI3K/AKT/mTOR was observed by fluoroscopic electron microscopy.Results: Animal experimental results: First,the mouse PF model was successfully established by one-time airway injection of BLM.Second,pathological staining results showed that: Compared with the NC group,lung inflammation,collagen deposition and lung structure damage were obvious in the BLM group,but the collagen content,the degree of pulmonary fibrosis and lung structure damage in the BLM+DHED group and the BLM+ PFD group was significantly lower than that in the BLM group(P < 0.001).Third,the expression of fibrosis proteins in the BLM group was significantly increased,but the expression of these proteins in the DHED group was significantly decreased(P < 0.001).Cell experiment results: First,compared with the control group,the expressions of Fibronectin,Collagen1 and α-SMA in the TGF-β1group were increased,but the expressions of these proteins in the DHED group were decreased compared with the TGF-β1 group.Second,HFL1 proliferation was significantly increased in the TGF-β1 group,but decreased in the DHED group(P < 0.001).Third,the results of cell scratch test indicated that TGF-β1could significantly stimulate HFL1 migration,but DHED treatment could significantly reduce HFL1 migration(P < 0.001).Fourth,Western Blot analysis of PI3K/AKT/mTOR signaling pathway showed that DHED treatment significantly reduced the level of phosphorylation of PI3K/AKT/mTOR signaling pathway,with the most significant inhibition at 3h(P < 0.01).Conclusions:First,dehydroevocorine can effectively improve the degree of bleomycin-induced pulmonary fibrosis in mice.Second,dehydroevocorine could restrain the differentiation,proliferation and migration of HFL1 cells induced by TGF-β1,possibly by inhibition of PI3K/AKT/mTOR signaling pathways.