| Background:Idiopathic pulmonary fibrosis(IPF)is a chronic and refractory disease.One of its formation mechanisms is epithelial-mesenchymal transition(EMT).Recent studies have shown that the level of autophagy in pulmonary fibrosis is reduced,and activation of autophagy can inhibit epithelial-mesenchymal transition and alleviate pulmonary fibrosis.Feiwei Granules is an empirical Chinese medicine compound,which has been proved by multi-center clinical studies to effectively improve the clinical symptoms and quality of life of patients with idiopathic fibrosis.Feiwei Granules is a prescription for benefiting qi and promoting blood circulation.Studies have shown that qi deficiency and blood stasis are significantly related to the level of autophagy.Therefore,it is assumed that ccan regulate the level of autophagy,inhibit epithelial-mesenchymal transition,and improve pulmonary fibrosis.This study established an epithelial-mesenchymal transition model in vitro and a pulmonary fibrosis model in vivo to verify this hypothesisObjective:Using in vitro transforming growth factor ?1(Transforming growth factor.TGF-?1)induced A549 cell epithelial-mesenchymal transition model and in vivo bleomycin-induced C57BL/6N mouse lung fibrosis model to explore the effect of Feiwei Granules on pulmonary fibrosis,exploring the regulation of Feiwei Granules on epithelial mesenchymal transformation and autophagy activityMethods:? In vitro experimental cells used 10 ng/ml TGF-?1 to intervene in A549 cells to construct an epithelial-mesenchymal transition model.A549 cells were randomly divided into 4 groups:normal group,model group,positive control group,and experimental group.The normal group cells were cultured in normal cell culture medium,the model group cells were added 10 ng/ml TGF-?1 on the basis of the cell culture medium,and the positive control cells were added 10 ng/ml TGF-?1 and 10 ng/ml TGF-?1 rapamycin treatment on the basis of the cell culture medium.In the experimental group.10 ng/ml TGF-?1 and 1 mg/ml Feiwei Granules were added to the cell culture medium.Observe the indicators at the 24th hour after the cells were added.Observe the morphological changes of cells in each group through an inverted microscope.Immunofluorescence staining and Western Blotting were used to detect the expression levels of E-cadherin,?-SMA,vimentin and other epithelial-mesenchymal transition markers.The number of autophagosomes in each group was detected by transmission electron microscope.The expression levels of autophagy proteins mTOR and beclin-1 were detected by Western Blotting.?In vivo experiments used tracheal injection of 3 mg/Kg bleomycin to intervene in C57BL/6N mice induced lung interstitial fibrosis model.80 mice were randomly divided into 4 groups:sham operation group,model group,positive control group,and experimental group,with 20 mice in each group.The mice in the sham-operated group were injected with normal saline in the 0th weather tube,and the other three groups were given a single injection of 3 mg/Kg bleomycin in the trachea on the 0th day to establish a pulmonary fibrosis model.In the positive control group,mice were injected intraperitoneally with bleomycin and intraperitoneal injection of 4 mg/Kg rapamycin for 14 days.The experimental group was given intragastric injection of bleomycin by intragastric administration of 0.26ml/20g of Feiwei granules for 14 consecutive days.Material analysis was performed on the 14th day of treatment.H&E staining and Masson staining were used to observe the changes of lung inflammation and fibrosis in mice.The level of collagen in each group was detected by ELISA to determine the content of hydroxyproline.The expression levels of Snail in each group were detected by RT-PCR.and the expression levels of epithelial-mesenchymal transition surface markers in the lung tissue of mice in each group were detected by immunofluorescence staining and Western Blotting.The expression levels of autophagy proteins mTOR,beclin-1,LC3II and P62 were detected by Western Blotting.Results:?In vitro experiments,compared with the normal group,the cells in the model group had epithelial-mesenchymal transition.At 24 hours after TGF-?1 intervention in A549 cells,the cell morphology changed from an oval-shaped circle to an elongated spindle.Immunofluorescence staining and Western Blotting detection showed that the level of E-cadherin decreased,and the levels of ?-SMA and Vimentin increased.Transmission electron microscopy showed that autophagosomes were rare in the cells of the model group.Western Blotting detection showed that the level of autophagy protein mTOR increased and the level of Beclin-1 decreased.Compared with the model group,the experimental group Feiwei Granules can inhibit the change of cell epithelial-mesenchymal transition,inhibit the change of cell morphology from goose oval to fusiform,increase the roundness of the cell,up-regulate the expression of E-cadherin and down-regulate a-SMA And Vimentin's expression.Promotes the increase of autophagosomes,inhibits the expression of mTOR,and increases the expression of Beclin-1.? In the in vivo experiment,compared with the normal group,the lung tissue of the model group mice found fibrosis changes on the 14th day,and Masson staining showed that the model group had green collagen deposition.RT-PCR showed that the expression level of Snail increased,immunofluorescence staining and Western Blotting test showed that the level of E-cadherin decreased,and the level of?-SMA increased,suggesting that the lung tissue of the model group had epithelial-mesenchymal transition.Western Blotting detection showed that the expression levels of autophagy proteins mTOR and P62 in the model group increased,and the expression levels of Beclin-1 and LC3? decreased.Compared with the model group,Feiwei Granule can reduce the degree of bleomycin-induced pulmonary fibrosis in C57 BL/6N mice,inhibit the expression of Snail,up-regulate the expression level of E-cadherin.down-regulate the expression level of ?-SMA.inhibit mTOR.The expression of P62 increases the expression of Beclin-1 and LC3?.Conclusion:Feiwei Granules can inhibit TGF-?1-induced epithelial-mesenchymal transition in vivo and bleomycin-induced pulmonary fibrosis in vitro.This inhibition may be related to activated autophagy.It is suggested that insufficiency of autophagy is a potential mechanism of pulmonary fibrosis.Feiwei granules may promote autophagy to reduce the development of pulmonary fibrosis. |
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