Anti-pulmonary Fibrosis Effect And Mechanism Of Ampelopsin Based On PI3K/AKT-mTOR Autophagy Pathway

Objective:This study construct a pulmonary fibrosis model in vivo and in vitro to research Ampelopsis（APS）how to exert the effect of anti-pulmonary fibrosis,and explore the mechanism of anti-pulmonary fibrosis under the condition of autophagy pathway,to help we find anti-pulmonary fibrosis drugs as basic theoretical research.Method:1.In vivo,we constructed a pulmonary fibrosis model by bleomycin in mice,the species of mice is BALB/c.Intratracheal injection of bleomycin solution（3mg/kg）,a total of two injections,fourteen days after the second injection,model construction time was 28 days.Additionally,we set up dexamethasone group（5mg/kg）and drug treatment group（200、100、50mg/kg）.28 days later,we collected the relevant indicators of mice,include respiratory function（airway resistance,dynamic lung compliance,maximal ventilator volume）,lung coefficient,indexes of oxidative damage（SOD,MDA）,level of hydroxyproline and transforming growth factor-β₁,and used Western Blot method to detect the protein expression level（Akt,m TOR,LC3）,and histopathological examination.2.In vitro,we constructed a fibrosis-like model by transforming growth factor-β₁ to in A549 cell.Cell culture in DMEM high sugar medium.First,we started the experiment of model construction,to decide the time and concentration of structural model.And then,we started the experiment of cell viability by A549,to decide Non-toxic concentration of APS.After the completion of cytopharmacotherapy,we collected the relevant indicators,included morphological examination of cells,level of hydroxyproline,we used Western Blot method to detect the protein expression level（Akt,m TOR,LC3）,and observation of Autophagosomes in Cells by Electron Microscope.Results:1.In animal model experiments,after 28 days,the model group mice had sparse hair,Shortness of breath,and cough.But other groupsreduced this phenomenon.Respiratory function was detected after 28 days,the model group mice had severe respiratory resistance,dynamic lung compliance and maximal ventilator volume was decreased,the high dose group could enhance respiratory function than model group（P<0.05）.Lung coefficients were lower in the drug group than in the model group（P<0.05）.The oxidation index of the drug group was less than that of the model group（P<0.05）.The hydroxyproline and transforming growth factor-β₁ of the drug group was less than that of the model group（P<0.05）.In the model group,the PI3K/Akt-m TOR autophagy pathway was inhibited,and drug group was activated.Histopathological examination showed that the degree of fibrosis increased in the model group（P<0.05）,but the drug group was decreased（P<0.05）.2.In cell model experiments,the concentration of transforming growth factor-β₁ was 5ng/kg,Molding time was 48h,Non-toxic concentration of APS was under the 12μg/m L,half maximal inhibitory concentration(IC₅₀)was17.42μg/m L,so we chose 12、6、3μg/m L these three concentrations.After completion of drug treatment,we observed under a microscope that the cell morphology was elongated of the model group,but the drug group was returned to normal.The hydroxyproline of the drug group was less than that of the model group（P<0.05）.In autophagy pathway,we found the PI3K/Akt-m TOR autophagy pathway was activated,and drug group was inhibited.The increase of autolysosomes in model group was observed under electron microscope,but the drug group was reduced.Conclusion（s）:In bleomycin TGF-β₁induced animal fibrosis model,APS inhibited PI3K/Akt-m TOR autophagy pathway to produce anti-pulmonary fibrosis effectin in vitro and activated autophagy.In TGF-β₁induced cell fibrosis-like model,APS activated PI3K/Akt-m TOR autophagy pathway to produce anti-pulmonary fibrosis effectin in vivo and inhibited autophagy.