Based On The Regulation Of Autophagy Pathway, Explore The Mechanism Of Bushen Yifei Xiaozheng Recipe On IPF

Objective:Idiopathic pulmonary fibrosis(IPF)is a chronic,progressive,and fibrosis interstitial pneumonia.Globally,its incidence and prevalence are increasing year by year,and its mortality exceeds many malignant tumors.At present,there is still no effective medicine for the treatment of IPF.Chinese medicine has unique theoretical and method advantages,and has achieved fruitful results in the field of IPF disease research in recent years.According to the theory of traditional Chinese medicine and the characteristics of modern medical pathogenesis of idiopathic pulmonary fibrosis,the "Micro-zhengjia of lung collateral" is put forward.Based on this theory,the method of Bushen Yifei Xiaozheng has made positive research results in the clinical application and basic experimental research of prevention and treatment of idiopathic pulmonary fibrosis.Based on the previous study of our research group,we conducted this study to explore the theoretical origin of the formation of "Micro-zhengjia of lung collateral" summarize the clinical characteristics and rules of tutor in the treatment of idiopathic pulmonary fibrosis,and explore the effect of Bushen Yifei Xiaozheng formula on the epithelial to mesenchymal transition(EMT)and fibroblast transdifferentiation induced by transforming growth factor-?1(TGF-?1),to clarify the effect of this formula on PI3K/AKT/mTOR signal pathway and autophagy function in these two pathological processes,To explore the mechanism of Bushen Yifei Xiaozheng formula in the prevention and treatment of IPF,to provide basis for its clinical application,and to enrich the scientific connotation of the theory of "Micro-zhengjia of lung collateral".Method:1.Through the literature of traditional Chinese medicine to explore the theoretical origin of the formation of "Micro-zhengjia of lung collateral" summarize the research results under the guidance of the theory.2.Collect the outpatient medical records of the tutor,who treated IPF of traditional Chinese medicine from February 2016 to June 2019,select the prescription,and analyze the medication rule with the help of ancient and modern medical record cloud platform v2.1.3.The main components of lyophilized powder of Bushen Yifei Xiaozheng formula were analyzed by high performance liquid chromatography mass spectrometry(HPLC-MS).CCK-8 was used to screen two suitable concentrations of traditional Chinese medicine(TCM)and the appropriate concentration of nidanib for experiments.The A549 and MRC-5 cells induced by TGF-?1 were used as EMT and fibroblast transdifferentiation models respectively.The experimental intervention lasted 24h and were divided into seven groups:normal group,model group,TCM group 1,TCM group 2,control group,nidanib group and Rapamycin group.4.After the intervention,the morphology of A549 cells was observed by HE staining,the expression of ?-smooth muscle actin(?-SMA)and E-Cadherin in A549 cells was observed by immunofluorescence technique,and the collagen(Collagen,COL)1 and 3 in the supernatant of MRC-5 cells were detected by ELISA.The expression of PI3K/AKT/mTOR pathway and autophagy-related proteins were observed by Western-Blot method in both cells,autophagosomes and autophagolysosomes were observed by transmission electron microscopy.Result:1.The theory of "Micro-zhengjia of lung collateral" is based on the theoretical development of "lung collateral" and "zhengjia" in traditional Chinese medicine,combined with the characteristics of modern pathogenesis of idiopathic pulmonary fibrosis,and put forward the TCM pathogenesis theory for IPF,which has been verified by some clinical and basic experimental studies.2.91 effective formulas were obtained through screening.Among them,Rhizoma Pinelliae Preparata,Radix Glycyrrhizae preparata,Poria cocos and honey ephedra had the highest frequency.The highest frequency of drug efficacy is Eliminating Dampness and Phlegm.The mainly nature of medicinals were warm,flat and cold.The mainly nature of medicinals were pungent,sweet and bitter.The mainly channel tropism were lung,spleen,stomach,heart,liver and kidney.25 drug association rules were obtained by association analysis,and 5 clusters with drugs were extracted by cluster analysis.3.The results of HE staining showed that the cells in the normal group were oval or fusiform,the cell size was uniform,the cell morphology in the model group was poor,long strip cells and polygonal cells could be seen,the proportion of nucleoplasm was reduced,the cell morphology in the control group,the two TCM groups,the nedanib group and the rapamycin group showed a small number of long strip and polygonal changes,but the morphology in these groups was more uniform.Immunofluorescence showed that the expression of ?-SMA in the model group was higher than that in the normal group(P<0.05),and the expression of E-cadherin was significantly lower(P<0.01).Compared with the model group,the expression of ?-SMA decreased significantly in each experimental groups(P<0.05 or P<0.01),and E-cadherin increased significantly in each experimental groups(P<0.05 or P<0.01).4.To observe the activation of PI3K/AKT/mTOR signaling pathway in A549 cells,Western blot showed that the expression of PI3K,p-AKT/AKT,mTOR,p-mTOR/mTOR and p70(S6K)in the model group was significantly higher than that in the normal group(P<0.01).Compared with the model group,The expression or phosphorylation level of some proteins in the pathway were inhibited in different experimental groups.And the differences were statistically significant(P<0.05 or P<0.01).PI3K,p-mTOR/mTOR in TCM group 1,PI3K,p-AKT/AKT,p-mTOR/mTOR in TCM group 2 were significantly decreased;PI3K,p-AKT/AKT,mTOR,p-mTOR/mTOR and p70(S6K)were significantly decreased in the control group;PI3K and p-AKT/AKT were significantly decreased in nidanib group;PI3K,p-AKT/AKT,mTOR and p-mTOR/mTOR were significantly decreased in rapamycin group.There was no significant difference in AKT expression in all groups,and there was no significant difference in p70(S6K)expression in all groups except the control group,but they were lower than the model group.5.The autophagy level of A549 cells was observed,Western blot showed that compared with the normal group,the expression of p62 in the model group increased significantly(P<0.01),and the expression of Beclinl and LC3BII/LC3BI decreased(P<0.05).Compared with the model group,the expression of p62 decreased significantly in each experimental groups(P<0.01),Beclinl and LC3BII/LC3BI were increased in each experimental groups(P<0.05 or P<0.01).Electron microscopy results:autophagy was observed in all experimental groups,and autophagosomes were mostly found in cells of normal group and model group.Compared with model group,more autophagosomes and autophagolysosomes were observed in TCM groups,control group,nidanib group and rapamycin group.6.To observe the collagen secretion of fibroblasts,the result of ELISA showed that:compared with the normal group,the content of COL1 and COL3 in the supernatant of MRC-5 cells in the model group was significantly higher(P<0.01);compared with the model group,the content of COL1 and COL3 in the supernatant of cells in each experimental groups were significantly lower(P<0.01).7.The activation of PI3K/AKT/mTOR pathway in MRC-5 cells was observed.Western blot showed that compared with the normal group,the expression of PI3K,AKT,p-AKT/AKT,mTOR,p-mTOR/mTOR and p70(S6K)in the model group was significantly increased(P<0.01);Compared with the model group,each drug groups can inhibit the expression or phosphorylation of some proteins in the pathway,The differences were statistically significant(P<0.05 or P<0.01).The expression of PI3K and p70(S6K)in TCM groupl were significantly decreased.PI3K,p-AKT/AKT,mTOR,p-mTOR/mTOR and p70(S6K)were significantly reduced in TCM group2.In the control group,PI3K,AKT,p-AKT/AKT,mTOR,p-mTOR/mTOR and p70(S6K)were significantly decreased,PI3K,p-AKT/AKT,p70(S6K)were significantly decreased in nidanib group,while PI3K,AKT,p-AKT/AKT,mTOR,p-mTOR/mTOR,p70(S6K)were significantly decreased in rapamycin group.8.To observed the autophagy level of MRC-5 cells,Western blot showed that compared with the normal group,the expression of p62 in the model group was significantly higher(P<0.01),Beclinl and LC3BII/LC3BI were significantly lower(P<0.01 or P<0.05);compared with the model group,the expression of p62 in each experimental groups decreased significantly(P<0.01),and the Beclinl expression in each experimental groups increased significantly(P<0.01).LC3BII/LC3BI was significantly increased in the TCM group2,Control group,nidanib group and rapamycin group(P<0.05 or P<0.01).A small number of autophagosomes and autophagolysosomes were observed in the model group by transmission electron microscopy.Compared with the model group,autophagy was observed in the normal group,the TCM group 1,the TCM group 2,the control group,the nedanib group and the rapamycin group,more autophagosomes and autophagolysosomes were observed.Conclusion:1.The theory of "Micro-zhengjia of lung collateral" is supported by the basic theory of Traditional Chinese Medicine,which conforms to the modern pathogenesis characteristics of idiopathic pulmonary fibrosis,and is based on the results of clinical and basic experimental studies.It is a theoretical innovation of traditional Chinese medicine for the pathogenesis of IPF.2.In the clinical treatment of idiopathic pulmonary fibrosis,the tutor paid attention to the influence of "phlegm and blood stasis" on the disease.The medication was mainly pungent and warm.In the middle and early stage of the disease,the tutor paid special attention to regulating qi and phlegm,benefiting qi and promoting blood circulation,benefiting the pharynx,harmonizing the viscera,highlighting the main disease and grasping the main symptoms.While treating the lung,the tutor also gave consideration to strengthening the spleen and stomach,invigorating the liver and kidney.The prescriptions were mostly based on the classic prescription,with clear dialectical thinking and reasonable composition.3.Bushen Yifei Xiaozheng Formula can inhibit the overactivation of PI3K/AKT/mTOR signaling pathway and restore cell autophagy,inhibit the process of alveolar epithelial cell mesenchymal transition,and delay the pathological process of IPF.4.Bushen Yifei Xiaozheng Formula can inhibit fibroblast transdifferentiation,reduce collagen deposition and delay the pathological process of IPF by inhibiting the overactivation of PI3K/AKT/mTOR signaling pathway and restore cell autophagy function.