Experimental Study Of Astragaloside Alleviating Pulmonary Fibrosis By Regulating Autophagy Via ERK Pathway

Objective: Astragalus saponins(The main components of Chinese medicine Astragalus)were selected in this experiment,and animal experiments and cell experiments were used to explore whether the total astragalus saponins could alleviate pulmonary fibrosis by interfering with the Ras / Raf / MEK / ERK signaling pathway to regulate autophagy.The molecular mechanism of saponins in alleviating pulmonary fibrosis provides a theoretical basis for seeking multiple targets for preventing and treating pulmonary fibrosis from traditional Chinese medicine,and at the same time provides new ideas for clinical prevention and treatment of the disease.Methods: Animal experiment part: Replicate the mouse model of pulmonary fibrosis by bleomycin tracheal instillation,and give the astragalus saponin after gavage.Pathological sections made by HE staining and Masson staining were used to observe the inflammatory cell infiltration and collagen deposition in the lung tissue of the blank control group,model group and drug intervention group;alkaline hydrolysis method was used to detect the content of hydroxyproline;real-time fluorescent quantitative PCR(QPCR)method to detect the relative expression levels of collagen type I(Col-I)and collagen type III(Col-III);fluorescent dual-label method to detect the expression level of Col-I and Col-III protein;WB method to detect autophagy Protein expression levels of related genes LC3B-II and P62;Western blotting(WB)method was used to detect the activation level of Ras / Raf / MEK / ERK signaling pathway.Cell experiment part: MTS method is used to select the optimal concentration for cell experiment;TGF-?1 stimulates human lung fibroblasts to model;give Astragalus membranaceus saponin stimulation,and fluorescent dual-label method to detect Col-I and Col-III protein expression levels;Observe the changes in the number of autophagy fractions in each group of cells by transmission electron microscopy;WB method to detect the protein expression levels of autophagy-related genes LC3B-II and P62;WB method to detect the activation level of Ras / Raf / MEK / ERK signaling pathway si RNA-ATG7 detection of collagen-related indicators Col-I and Col-III after autophagy inhibition by fluorescent double labeling method;detection of collagen-related indicators Col by fluorescent double labeling method after using Ras / Raf / MEK / ERK signaling pathway inhibitors U0126 and Salilasib-I,Col-III;and observe the changes of the number of autophagosomes in each group of cells with transmission electron microscope,observe the number of autophagosomes under MDC staining fluorescence microscope;real-time fluorescent quantitative PCR method detects autophagy-related genes Beclin1,ATG5,ATG7 m RNA relative expression.Results: Animal experiment part: 1.After modeling with bleomycin tracheal instillation,the lung tissue of model mice showed obvious inflammatory cell infiltration and collagen deposition compared with the blank control group.After the intervention,the above symptoms were alleviated;after modeling,the content of hydroxyproline in the lung tissue of the model group was significantly higher than that of the blank group(P <0.05),and the total saponin of the astragalus was significantly reduced after intervention(P <0.05);The m RNA expression levels of type I and type III collagen and protein expression levels in the lung tissues of model group mice were significantly increased compared with the blank control group(P <0.05),and the total astragalus saponins were significantly reduced after intervention(P <0.05);2.The expression level of Beclin1 m RNA in the lung tissue of the model group was significantly lower than that of the blank control group(P <0.05),and the expression level of Beclin1 m RNA was significantly higher than that of the model group after the intervention of astragalus saponins(P <0.05);the expression level of autophagy-related protein LC3B-II in the model group was significantly lower than that in the blank control group(P <0.05),and the expression level of LC3B-II in the astragalus saponins group was significantly higher than that in the model group(P <0.05);the expression level of autophagy-related protein P62 in the model group was significantly increased compared with the blank control group(P <0.05),and the expression level of P62 was significantly reduced after the intervention of astragalus saponins(P <0.05);3.Compared with the blank group,the activation level of Ras / Raf / MEK / ERK-related protein in the lung tissue of the model group was significantly higher(P <0.05),and the activation level of Ras / Raf / MEK / ERK-related protein after the intervention of astragalus saponins Compared with the model group,it was significantly reduced(P<0.05);Cell experiment part: 1.After stimulating human lung fibroblasts with TGF-?1,the expression of type I and type III collagen in the model group cells was significantly increased compared with the blank control group(P <0.05);The expression level of Samsung collagen was significantly reduced(P <0.05);2.The number of autophagosomes and autophagolysosomes in the model group was significantly lower than that in the blank control group,and the expression level of autophagy-related protein LC3B-II was significant Decreased(P <0.05),the protein expression of P62 increased significantly(P<0.05);the number of autophagosomes and autolysosomes increased significantly after intervention with different concentrations of astragalus saponins,autophagy-related protein LC3B-II expression level was significantly increased(P <0.05),P62 protein expression was significantly reduced(P <0.05);extracellular collagen deposition was significantly increased after inhibiting autophagy related gene ATG7;3.Model group cells and blank control Compared with Ras / Raf / MEK / ERK,the activation level of Ras / Raf / MEK /ERK-related proteins was significantly increased(P <0.05);the activation level of Ras /Raf / MEK / ERK-related proteins was significantly inhibited after intervention with different concentrations of astragalus saponins(P < 0.05);after interfering with human lung fibroblasts with Ras / Raf / MEK / ERK pathway inhibitors,extracellular collagen deposition was significantly reduced,the number of autophagosomes and autophagolysosomes was significantly reduced,and the autophagy-related gene Beclin1 The m RNA expression levels of ATG5 and ATG7 were significantly reduced(P <0.05);conclusion:1.Ras / Raf / MEK / ERK signaling pathway activation level is negatively correlated with lung fibroblast autophagy level;positively correlated with extracellular collagen deposition2.The level of autophagy is negatively correlated with extracellular collagen deposition3.Astragalus total saponins can activate the autophagy of mouse lung tissue cells and relieve collagen deposition in the lung tissues of the lung fibrosis mice by inhibiting the activation of Ras / Raf / MEK / ERK signaling pathway in the lung tissues of lung fibrosis mice.