| Idiopathic pulmonary fibrosis(IPF)is an interstitial fibrotic lung disease with unknown etiology.The early pathology is diffuse alveolitis,and the late stage shows a large number of fibroblast proliferation and differentiation.The external matrix deposits and replaces normal lung tissue.The prognosis of IPF is poor and the mortality rate is high.The pathogenesis of IPF has not been fully elucidated and there are no effective treatment measures.Modern studies have shown that low-activity autophagy has become an important cause of pulmonary fibrosis and plays an important role in the pathogenesis of pulmonary fibrosis.The PTEN/m TOR signaling pathway is the main regulatory pathway for autophagy in IPF.According to its symptoms,IPF corresponds to traditional medicines such as “Feiwei” and “Feibi”.In the long-term experimental research,our research group found that qi deficiency and blood stasis is an important pathogenesis of IPF,The use of Buyang Huanwu Decoction,a representative prescription for the treatment of IPF,is effective in intervening IPF.So we put forward the hypothesis,the intervention effect of Buyang Huanwu Decoction may be based on the autophagy-related PTEN/m TOR pathway.This study starts from “cell autophagy” and continues the PTEN/m TOR signaling pathway as the regulatory pathway.Exploring the mechanism of action of Buyang Huanwu Decoction on IPF intervention and providing a new method for the treatment of IPF.The experiment is divided into three parts:1.Experiment 1 Effect of Buyang Huanwu Decoction on Morphology of Lung Tissue inMice with Pulmonary Fibrosis Induced by BleomycinObjective: To observe the morphological changes of lung tissue in mice with fibrosis induced by bleomycin and the intervention effect of Buyang Huanwu Decoction.Methods: In this experiment,a mouse model of experimental fibrosis was established by injecting a bleomycin aqueous solution into the trachea.The experimental group was given high,medium and low doses of Buyang Huanwu Decoction for intervention.The positive control group was given prednisone for intervention.The mice were sacrificed on the 7th,14 th and 28 th day after modeling to separate the lung tissue.During the experiment,the general condition of the mice(including spirit,feeding,fur,weight,etc.)was observed.HE and Masson staining were performed on the lung tissue,and the Szapiel score of alveolitis and pulmonary fibrosis was performed to investigate the intervention effect of Buyang Huanwu Decoction on pulmonary fibrosis.Results: 1.General condition of the mice and gross morphology of the lung tissue: The mice in the sham operation group grew well.The mice in the model group were apathetic,slow-moving,reduced water intake,weight loss,hair loss,dull and dull skin,Shortness of breath,wheezing sound can be heard,increased urination,loose stools,residual feces in the perianal area,poor lung morphology.The general condition of the mice in the other administration groups and the morphology of the lung tissues were better than those in the model group.2.The effect of Buyang Huanwu Decoction on the degree of alveolitis and fibrosis in mice: Compared with the sham operation group,the alveolitis and pulmonary fibrosis scores in the model group were significantly increased at each time point,there was a significant difference(P<0.01).Compared with the model group,the scores of Buyang Huanwu Decoction in the high,middle and low dose groups and the prednisone group were significantly decreased at 7th,14 th,and 28 th days,and the difference was statistically significant(P<0.05).Compared with the other groups,the high dose group of Buyang Huanwu Decoction decreased at 7th,14 th,28th days,the difference was statistically significant(P<0.05).There was no statistically significant difference between the other groups.Conclusion: 1.Bleomycin tracheal injection can successfully replicate the mouse model of idiopathic pulmonary fibrosis.2.Buyang Huanwu Decoction can improve the general life condition and lung tissue morphology of bleomycin-induced pulmonary fibrosis mice,and have certain therapeutic effects on the early stage of alveolitis and the late stage of fibrosis in idiopathic pulmonary fibrosis.The therapeutic effect is optimal in high dose group of Buyang Huanwu Decoction.2.Experiment 2 The effect of Buyang Huanwu Decoction on PTEN/m TOR signaling pathway in bleomycin-induced pulmonary fibrosis in miceObjective: To investigate the mechanism of PTEN/m TOR signaling pathway in the pathogenesis of idiopathic pulmonary fibrosis in mice and the effect of Buyang Huanwu Decoction.Methods: Model preparation and grouping,drug administration,and material extraction methods are the same as Experiment 1.Western blot was used to detect the expression of PTEN protein,m TOR protein,ribosomal S6 protein and phosphorylated ribosomal S6 protein in mouse lung tissue.Immunohistochemistry(SABC)was used to detect m TOR protein and ribosomal S6 protein in mouse lung tissue.Results: 1.The effect of Buyang Huanwu Decoction on the expression of PTEN protein in lung tissue of mice: Compared with the sham operation group,the expression of PTEN in the lung tissue of the model group was significantly increased at 7th,14 th,28th days(P<0.01).The expression levels of PTEN in the high,middle and low dose groups of Buyang Huanwu Decoction and the prednisone group were significantly lower than those in the same model group(P<0.01).At 14 th days,The expression level of PTEN in the medium and low dose groups of Buyang Huanwu Decoction and the prednisone group was lower than that in the model group(P<0.05).the high dose group of Buyang Huanwu Decoction decreased compared with the prednisone group and the low dose group of Buyang Huanwu Decoction.(P<0.01).there was no significant difference between the low and middle dose group of Buyang Huanwu Decoction and the prednisone group(P>0.05).2.The effect of Buyang Huanwu Decoction on the expression of m TOR in mice lung tissue:(1)Western Blot: The effect of Buyang Huanwu Decoction on the expression of m TOR in mouse lung tissue:(1)Protein immunoblotting: Compared with the sham operation group,the expression level of m TOR was significantly increased in the lung tissue of the model group at 7th,14 th and 28 th days(P<0.01),the expression level of m TOR in the high,middle and low dose groups of Buyang Huanwu Decoction and the prednisone group was lower than that of the model group(P<0.01),Buyang Huanwu Decoction high-dose group were significantly lower than those of prednisone group and Buyang Huanwu Decoction in the middle and low dose groups(P<0.01).There was no statistical difference between the low dose group of Buyang Huanwu Decoction and the prednisone group(P>0.05).(2)Immunohistochemical method: Compared with the sham operation group,the expression level of m TOR in the lung tissue of the model group was significantly increased at 7th,14 th,28th days(P<0.01),The expression level of m TOR in the prednisone group and the high,medium and low dose groups of Buyang Huanwu Decoction was significantly lower than that in the model group(P<0.01).At 28 th day,The expression level of m TOR in the lung tissue of Buyang Huanwu Decoction low dose group and prednisone group was lower than that in the model group(P<0.05).At 7th day,compared with the other drug-administered groups,the high-dose group of Buyang Huanwu Decoction decreased significantly(P<0.01),At 14 th day the high-dose group of Buyang Huanwu Decoction decreased compared with the prednisone group(P<0.05).Compared with the middle and low dose groups,the decrease was significant(P<0.01).At 28 days,the time of the high-dose group of Buyang Huanwu Decoction was lower than that of the prednisone group in the same period,and the lower dose group of Buyang Huanwu Decoction decreased(P<0.05).There was no significant difference between the middle and low dose groups of Buyang Huanwu Decoction and the prednisone group(P>0.05).3.The effect of Buyang Huanwu Decoction on the expression of S6 in lung tissue of mice:(1)Western Blot: The effect of Buyang Huanwu Decoction on the expression of S6 in lung tissue of mice:(1)Protein immunoblotting: Compared with the sham operation group,the expression level of S6 was significantly increased in the lung tissue of the model group at 7th,14 th,28th days(P <0.01).The expression levels of S6 in the high,middle and low dose groups of Buyang Huanwu Decoction and the prednisone group were significantly lower than those in the same model group(P<0.01).At 7th day,Compared with the prednisone group,and low?middle dose group of Buyang Huanwu Decoction,the high-dose group of Buyang Huanwu Decoction was significantly decreased(P<0.01)..At 14 th day,compared with the prednisone group,and low dose group of Buyang Huanwu Decoction,the high-dose group of Buyang Huanwu Decoction had a significant decrease(P<0.01),compared with middle dose group of Buyang Huanwu Decoction,there was not significant(P>0.05).At 28 th day,Compared with the prednisone group,the high dose group of Buyang Huanwu Decoction decreased significantly(P<0.05).At 14 th day,compared with the prednisone group(P<0.05)and the low dose group of Buyang Huanwu Decoction(P<0.05),the middle dose group of Buyang Huanwu Decoction decrease significantly,compared with the other groups there was no significant difference(P>0.05).(2)Immunohistochemistry: Compared with the sham operation group,the expression level of S6 in the lung tissue of the model group was significantly increased at 7th,14 th,28th days(P<0.01).The expression level of S6 in the high,middle and low dose groups of Buyang Huanwu Decoction and the prednisone group was significantly lower than that of the model group(P<0.01).At 14 th days Compared with the model group,the high-dose group of Buyang Huanwu Decoction decreased(P<0.05),and the middle and low dose groups decreased significantly(P<0.01).At 7th days the high-dose group of Buyang Huanwu decoction group significantly decreased compared with the prednisone group,low and middle dose group of Buyang Huanwu Decoction(P<0.01),At 14 th days,the high-dose group of Buyang Huanwu Decoction decreased compared with the low and the middle dose group of Buyang Huanwu Decoction(P<0.05),and was not significant compared with the prednisone group(P>0.05).At 28 th days,the high dose group of Buyang Huanwu Decoction decreased significantly compared with the prednisone group(P=0.01),and decreased significantly compared with the middle and low dose groups(P<0.01).There was no statistical difference between the other groups(P>0.05).The effect of Buyang Huanwu Decoction on the expression of p-S6 in lung tissue of mice: Compared with the sham operation group,the expression level of p-S6 was significantly increased in the lung tissue of the model group at 7,14 and 28 days(P<0.01).The expression levels of p-S6 in the high,middle and low dose groups of Buyang Huanwu Decoction and the prednisone group were significantly lower than those in the same model group(P<0.01).Compared with the prednisone group and Buyang Huanwu Decoction group,the high dose group of Buyang Huanwu Decoction decreased at the 7th day(P<0.05),and decreased significantly compared with the low dose group of Buyang Huanwu Decoction(P<0.01).The high-dose group of Buyang Huanwu Decoction decreased significantly compared with the prednisone group,low and middle dose group of Buyang Huanwu Decoction at 14 th days and 28 th days(P<0.05).There was no significant difference between the other groups(P>0.05).Conclusion: 1.Activation of PTEN/m TOR signaling pathway is involved in the pathogenesis of idiopathic pulmonary fibrosis.2.Buyang Huanwu Decoction can effectively up-regulate the expression of PTEN protein in PTEN/m TOR pathway,down-regulate the expression of m TOR protein,S6 protein and p-S6 protein,indicating that Buyang Huanwu Decoction can inhibit the activation of PTEN/m TOR pathway.3.Buyang Huanwu Decoction exerts therapeutic effect on bleomycin-induced pulmonary fibrosis in mice by inhibiting PTEN/m TOR pathway.3.Experiment 3: Study on the Mechanism of Buyang Huanwu Decoction Intervening Pulmonary Fibrosis by Up-regulating AutophagyObjective: To investigate the mechanism of autophagy in the pathogenesis of idiopathic pulmonary fibrosis and the intervention of Buyang Huanwu Decoction.Methods: Model preparation and grouping,drug administration,and material extraction methods are the same as Experiment 1.Western blot was used to detect the expression of microtubule-associated protein 1 Ligjt chain 3(MAP1LC3-II)and selective autophagy adaptor protein p62(sequestosome 1)in mouse lung tissue.Autophagy condition of mouse lung tissue was observed under an electron microscope.Results:1.The effect of Buyang Huanwu Decoction on the expression of LC3-II protein in lung tissue of mice: Compared with the sham operation group,the expression level of LC3-II in the lung tissue of the model group was significantly increased at 7th day.(P<0.01).The expression level of LC3-II in lung tissue of the model group was increased at 14 th and 28 th days(P<0.05).The expression levels of LC3-II in the high,middle and low dose groups of Buyang Huanwu Decoction and the prednisone group were significantly lower than those in the model group(P<0.01).The high dose group of Buyang Huanwu Decoction decreased significantly compared with the middle and low dose groups of Buyang Huanwu Decoction and the prednisone group at 7th and 14 th days(P<0.01),at 28 days,Compared with the prednisone group,the low-dose group of Buyang Huanwu Decoction,the decrease was significant(P<0.01),and it was significantly lower than that of the middle-dose group of Buyang Huanwu Decoction(P<0.05).at 28 days,the middle-dose group of Buyang Huanwu Decoction was significantly lower than that in the prednisone group(P<0.05).There was no significant difference in the other groups(P>0.05).2.The effect of Buyang Huanwu Decoction on the expression of p62 protein in lung tissue of mice: Compared with the sham operation group,the expression of p62 was significantly increased in the lung tissue of the model group at 7th,14 th and 28 th days(P<0.01).The expression levels of p62 in the high,middle and low dose groups of Buyang Huanwu Decoction and the prednisone group were significantly lower than those in the same model group(P<0.01).The high-dose group of Buyang Huanwu Decoction decreased significantly at 7th and 28 th days compared with the prednisone group,the low and middle dose groups of Buyang Huanwu Decoction(P<0.01).At 14 th day,it was decreased significantly compared with the prednisone group,the low and middle dose groups of Buyang Huanwu Decoction(P<0.05).There was no significant difference in the other groups(P>0.05).3.The effect of Buyang Huanwu Decoction on autophagy in lung tissue of mice: autophagy in lung tissue of mice in each group at 7th day: the cell morphology of sham operation group and model group were best,the high dose group of Buyang Huanwu Decoction was the worst.The number of autophagosome was the highest in the high-dose group of Buyang Huanwu Decoction,and the difference between the other groups was not obvious.Autophagy in lung tissue of mice in each group at 14 th day:The morphology of the cells in the sham operation group was the best,and the high dose group of Buyang Huanwu Decoction was the worst.The number of autophagosome was the highest in the high dose group of Buyang Huanwu Decoction,and the middle dose group of Buyang Huanwu Decoction and the low dose group of Buyang Huanwu Decoction were the least.The difference between the other groups was not obvious.Autophagy in lung tissue of mice in each group at 28 th day: the cell morphology of the sham operation group was the best,and the high dose group of Buyang Huanwu Decoction was the worst.The number of autophagosomes in the sham operation group was the lowest,and the high dose group of Buyang Huanwu Decoction group was the most.Conclusion: 1.Lack of autophagy participates in the pathogenesis of idiopathic pulmonary fibrosis.2.The basic pathogenesis of IPF is qi deficiency and blood stasis,tonifying qi activating blood method is the fundamental method for treating IPF.3.Buyang Huanwu Decoction activate autophagy by inhibiting PTEN/m TOR signaling pathway to play a therapeutic role in bleomycininduced pulmonary fibrosis mice. |
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