Based On PI3K/Akt Signaling Pathway, Explore The Effect And Mechanism Of Buyang Huanwu Decoction Against Pulmonary Fibrosis

Objects Based on the theory of Yiqi Huoxue,Huayu Tongluo therapy for pulmonary sputum,and the effect of classical prescription Bu Yang Huan Wu Tang on pulmonary fibrosis,this study aims to explore the intervention of Buyang Huanwu Tang in pulmonary fibrosis through a series of in vitro and in vivo experiments.Mechanism of action and specific target,and verify whether Buyang Huanwu Tang exerts anti-pulmonary fibrosis through PI3K/Akt signaling pathway,in order to provide valuable ideas and data for pulmonary fibrosis treatment.Methods 1.144 SD male rats were randomly divided into 6 groups: blank group,model group,hormone group,Chinese medicine high-dose group,Chinese medicine middle-dose group,and traditional Chinese medicine low-dose group,24 groups in each group.Except the blank group,each group a rat pulmonary fibrosis model was established by injecting a bleomycin hydrochloride solution(5 mg/kg)into the trachea.On the 2nd day after model establishment,the stomach was intragastrically administered.The blank group was intragastrically administered with 1ml/100 g saline.The hormone group was intragastrically administered with 5mg/kg methylprednisolone solution.The high,medium and low doses of Chinese medicine were 10g/kg,5g/kg,2.5g/kg,Buyang Huanwu Tang was administered to the stomach,and the general state of the rats was observed for recording.The serum and lung tissues of rats were taken 7 days,14 days and 28 days after modeling.The pathological changes of rat lung tissues were observed.The content of hydroxyproline in serum was determined by enzyme-linked immunosorbent assay.Western blot,RT-q PCR was used to analyze the expression of Col?,Col?,CTGF protein,m RNA and p-Akt protein in rat lung tissue.The effect of Buyang Huanwu Tang on pulmonary fibrosis in rats and its possible mechanism of action were evaluated.2.Human lung fibrosis model was established by stimulation of TGF-?1 in human alveolar A549 epithelial cells at different times(24h,48 h,72h)After intervention of TGF-?1 stimulator cells,MTT assay was used to observe cell proliferation.The optimal concentration of Buyang Huanwu Tang was used to intervene TGF-?1-induced A549 cells,and the morphology of cells was observed by inverted phase contrast microscope for 24 hours.Vimentin and E were detected by immunofluorescence staining,Western blot and RT-q PCR.-cadherin,Col? 72 h positive staining rate,protein expression level and m RNA content at 24 h,in order to explore the effect of Buyang Huanwu Tang on the transformation of human alveolar epithelial cells to stromal cells in vitro.3.Intervention of TGF-?1-induced A549 cells with Bu Yang Huan Wu Tang at a concentration of 10 mg/m L.After 1 h,the protein levels of PI3 K,Akt,p-PI3 K and pAkt were detected.Further,10 mg/m L Buyang Huanwu Tang was used to intervene in A549 cells induced by IGF-I and SC79,respectively.The protein levels of p-PI3 K and p-Akt in the cells were detected at 1 h to investigate the inhibition of PI3K/ by Buyang Huanwu Tang.The Akt pathway interferes with the target of cell epithelialmesenchymal transition.Results 1.After Buyang Huanwu Tang in the rat model of pulmonary fibrosis induced by bleomycin,the rats were slightly slower and more mentally active than the model group at 7 days;the puncture of the lung surface was reduced;the alveolar wall Slightly thicker;the degree of pulmonary fibrosis decreased(P<0.05);the levels of hydroxyproline,Col? and Col? in serum decreased,and the expression of CTGF protein and m RNA in lung tissue decreased.The high and medium concentrations of Buyang Huanwu Tang group were the most obvious(P<0.05);the low concentration of Buyang Huanwu Tang group showed a significant decrease in p-Akt protein level(P<0.05).Compared with the model group,the reaction speed of the rats increased and the wheezing sound decreased.The lung surface of the rats was slightly rough and the sputum spots were reduced.The alveolar structure recovered,the inflammatory cells decreased,and the pulmonary fibrosis degree decreased(P<0.05).The contents of hydroxyproline,Col? and Col? protein in serum were significantly decreased(P<0.05).The expression levels of p-Akt and CTGF in lung tissue were down-regulated,the content of CTGF m RNA was decreased,and the high and medium concentration of Buyang Huanwu Tang group was the most obvious(P<0.05).Compared with the model group,the rats improved their spirits at 28 days,the purpura phenomenon was reduced and the body weight increased;the white nodules on the lung surface of rats decreased;the alveolar structure became normal,the fibroblasts decreased;the pulmonary fibrosis degree score decreased significantly(P<0.05);The contents of hydroxyproline,Col? and Col? protein in the serum of the rats were significantly decreased,and the middle and low concentrations of Buyang Huanwu Tang group were the most obvious(P<0.05);while the expression level of p-Akt protein in lung tissue was down-regulated.The CTGF m RNA gene content was significantly decreased(P<0.05),and the high and medium concentrations of Buyang Huanwu Tang group were the most obvious but higher than the blank group.There was no significant difference in the expression of CTGF protein between Buyang Huanwu Tang group.2.Buyang Huanwu Tang 1,5,10mg/m L is the best experimental concentration,which can promote TGF-?1-induced human alveolar epithelial A549 cell-like morphology to fusiform transformation,and the intercellular boundary is reduced;compared with the model group The levels of Col? and Vimentin protein,positive staining rate and m RNA content in Fuyang Huanwu Tang group were significantly decreased(P<0.05),and Ecadherin protein level,positive staining rate and m RNA content were significantly upregulated,and at 5,The 10 mg/m L concentration group was the most significant(P<0.05).3.The effect of Buyang Huanwu Tang 10mg/m L on human alveolar epithelial cells stimulated by TGF-?1,IGF-I and SC79 groups for 1h,compared with TGF-?1 model group,the expression of p-PI3 K and p-Akt protein Significantly down-regulated(P<0.05);compared with the IGF-I model group,the expression of p-PI3 K and p-Akt protein was significantly down-regulated(P<0.05);compared with the SC79 model group,the expression of p-Akt protein in the cells was comparable.Significantly downregulated(P<0.05).Conclusions 1.Buyang Huanwu Tang can effectively interfere with the pulmonary fibrosis process induced by bleomycin in rats.The mechanism may be related to the PI3K/Akt pathway.2.Buyang Huanwu Tang can effectively inhibit epithelial-mesenchymal transition and extracellular matrix deposition of A549 cells stimulated by TGF-?1,which may be through PI3K/Akt pathway in PI3K/Akt pathway,intervention of pulmonary fibrosis progression...