Astragaloside Ⅳ Regulates Autophagy And Apoptosis In PM2.5-induced Acute Lung Injury Via AMPK/mTOR Pathway

BACKGROUNDSWith the rapid development of industrialization and urbanization in China,PM2.5has become one of the main risk factors for Chinese citizens to get sick.Long-term exposure of human body to PM2.5 will damage lung tissue,release a large number of inflammatory mediators to participate in airway inflammation and tissue damage,and eventually increase the incidence and mortality of respiratory diseases.In different stages of acute lung injury induced by PM2.5,autophagy and apoptosis play a dual role in immune response and regulation of pulmonary inflammation.On the one hand,the basic autophagy level of lung tissue cells is an important condition for the prognosis of many respiratory diseases.On the other hand,the abnormal autophagy activity induced by PM2.5 exposure can promote the pathological progress of lung injury by inducing cell death or initiating apoptosis procedure.Recent studies have shown that AMPK,which is an energy sensor of cells,is activated rapidly under the condition of energy deprivation,hypoxia and stress,and induces the up-regulation of autophage and also participates in the inflammatory response.In the study of environmental toxicology,AMPK inhibition mediated by PM2.5 exposure is highly correlated with the adverse effects of inflammation,insulin sensitivity,energy homeostasis and lipid metabolism.However,in the pathological process of lung injury induced by PM2.5,the mechanism by which cells maintain intracellular homeostasis through regulating AMPK/mTOR-autophagy,apoptosis and inflammation axis is not clear.In the early study on the prevention and treatment of respiratory tract infection and PM2.5 were studied by traditional Chinese medicine（TCM）.Our research group included PM2.5 as a new TCM etiology into the category of“environmental toxin”,and regarded qi deficiency and toxic sensation syndrome as an important pathogenesis of PM2.5,and discussed that qi detoxification is the main treatment method for PM2.5-induced acute lung injury.In the preliminary clinical trial of the project found that the representative of tonifying qi and detoxification,ganduqing（with Astragaloside IV as the quality control component）,has accurately therapeutic effect on treating respiratory tract infections related to PM2.5.Accordingly,we propose a scientific hypothesis:“the regulation of autophagy and apoptosis based on AMPK/mTOR pathway may be the molecular biological mechanism of Astragaloside IV’s protective effect on acute lung injury.”OBJECTIVEThis study focused on the relationship between PM2.5-induced acute lung injury and AMPK/mTOR signaling pathway,autophagy and apoptosis,as well as the protective mechanism of Astragaloside IV preadministration.This study mainly probes into the regulation of Astragaloside IV on the structural,functional and timing changes of autophagy in PM2.5-induced acute lung injury cells.Specific interference of pathway-related proteins was carried out in vivo to observe the protective effect of Astragaloside IV on acute lung injury.From the viewpoint of autophagy,apoptosis and inflammation,we are expected to explain the scientific connotation of Astragaloside IV in preventing and treating PM2.5-induced acute lung injury,and provide innovative idea and intervention target for drug research and development.METHODSPART 1:Subject investigated of the study is rat,which is to establish the animal model of acute lung injury by dripped PM2.5 suspension into the rats’airway.Eighty-four SD rats were given different doses of Astragaloside IV three days before modeling,and were killed at 6h,12h and 24h after modeling.According to execution time point,we set up the Normal saline group（NS）,PM2.5 model group（PM2.5）,Low dose group of Astragaloside IV(ASL,50mg·Kg^-1)and High dose group of Astragaloside IV(ASH,100mg·Kg^-1).First of all,after PM2.5 exposure,we measured the general situation,lung histomorphology,pulmonary edema index（dry-wet ratio,total lung water content）,HE staining,HE staining pathological score,alveolar lavage fluid cell count and classification,pulmonary ultrastructure,evaluated the establishment of acute lung injury model induced by PM2.5,and evaluated the safety and effectiveness of Astragaloside IV preadministration on acute lung injury induced by PM2.5.Secondly,we observed autophagy immunofluorescence,TUNEL fluorescence,apoptosis index,apoptosis pathway related proteins（Bax、Bcl 2、caspase 3、cleaved caspase 3、PARP1、cleaved PARP 1）,autophagy related proteins（LC3B、SQSTM 1、Beclin 1）,and pulmonary ultrastructure,determined the PM2.5-induced acute lung injury cell autophagy and apoptosis in the best monitoring time,and preliminarily discussed PM2.5-induced cells of the relationship between autophagy and apoptosis.PART2:Based on the optimal apoptosis observation time of PM2.5-induced acute lung injury,95 SD rats were divided into the following groups according to the experimental design:Normal saline group（NS）,Astragaloside IV in the control group(AS,100mg·Kg^-1),PM2.5 model group（PM2.5）,Low dose group of Astragaloside IV(ASL,50mg·Kg^-1)and High dose group of Astragaloside IV(ASH,100mg·Kg^-1),Rapamycin（RAPA）,Rapamycin+Astragaloside IV group（RAPA+AS）,Chloroquine group（CLQ）,Chloroquine+Astragaloside IV（CLQ+AS）,Compound C group（CC）and Compound C+Astragaloside IV group（CC+AS group）.Among them,NS group,AS group,PM2.5 group,ASH group,CC group and CC+AS group each had 10 rats（3rats in each group were randomly selected for AAV infection,and the remaining 7 rats were not injected）,and other groups each had 7 rats.First,we observed the TUNEL fluorescence staining,apoptosis index,apoptosis pathway related proteins（Bax,Bcl 2,caspase 3,cleaved caspase 3,PARP 1,cleaved PARP 1）,and inflammatory pathway related proteins（p-IκBα、p-p65）,and investigated the effect of Astragaloside IV on autophagy and inflammatory pathway of acute lung injury cells induced by PM2.5.Secondly,we observed pulmonary edema index（dry/wet ratio）,lung tissue HE staining,monitoring autophagy flow in AAV2/6-LC3-GFP-RFP carrier body,autophagy related proteins（LC3B,Beclin 1）and AMPK/mTOR pathway related proteins（AMPK,p-ampk,mTOR,p-mtor）,and investigated the effect of Astragaloside IV on apoptosis and AMPK/mTOR pathway of acute lung injury cells induced by PM2.5.In addition,after Compound C interfered with the expression of AMPK in lung tissue,the monitoring autophagy flow in AAV2/6-LC3-GFP-RFP carrier body was used to test AMPK protein,autophagin（LC3B,Beclin 1）,apoptosis pathway protein（Bax、Bcl 2、caspase 3、cleaved caspase 3、PARP 1、cleaved PARP 1）,inflammatory pathway protein（p-IκBα,p-p65）and ultrastructure of autophagy in lung tissue.Finally,chloroquine was used to inhibit autophagy,apoptosis pathway protein（Bax、Bcl 2、caspase 3、cleaved caspase 3、PARP 1、cleaved PARP 1）and inflammatory pathway protein（p-IκBα,p-p65）were observed.The study demonstrated that Astragaloside IV can regulate apoptosis and inflammation of acute lung injury induced by PM2.5 through autophagy.RESULTSPART1:（1）Pulmonary edema index（dry/wet ratio）,total lung water content,pathological score of HE staining and cell count of alveolar lavage fluid were increased in acute lung injury induced by PM2.5.the general condition of rats,pulmonary histomorphology,HE staining of lung tissue and ultrastructure of lung tissue,which all conform to the pathological process of acute lung injury.（2）During the course of acute lung injury induced by PM2.5,at the time point of 24h,the expression of autophagocyte-related proteins LC3 II,LC3 II/LC3 I and Beclin 1 was increased,the expression of SQSTM 1（p62）was decreased.It is suggested that the number of autophages in lung tissue increased after PM2.5 exposure.The results of immunofluorescence autophagy-lysosome co-localization showed that a large number of autophagy vesicles appeared in lung cells after acute exposure to PM2.5,and there was no fusion with autophagy lysosomes,and autophagosome showed degradation disorder.Transmission electron showed ultrastructure of early autophagy vesicle was increased.The main type of autophagy in acute lung injury induced by PM2.5 is alveolar macrophage.The above results suggested that cell autophagy activity was abnormal,autophagy flow was disordered,and leaded to autophagy degradation disorder in the course of PM2.5-induced acute lung injury.（3）At the time point of 24h in PM2.5-induced acute lung injury,the ratio of anti-apoptosis protein Bcl 2 and Bcl2/Bax decreased,the activation level of caspase 3 did not change significantly,but the activation level of PARP 1 increased.It suggests that PM2.5 induced apoptosis in lung tissue of acute lung injury.TUNEL fluorescence staining showed that there were a large number of positive apoptotic cells in lung tissue of acute lung injury induced by PM2.5,and the apoptosis index increased significantly.（4）The optimal time point for monitoring autophagy and apoptosis of acute lung injury cells induced by PM2.5 was24h after exposure.PART2:（1）Astragaloside IV inhibit Bcl2-Bax/caspase 3/caspase PARP 1 apoptosis pathway,decrease TUNEL fluorescence apoptosis index,and then inhibit downstream NF-κB pathway,reduce pulmonary inflammation,and play a protective role in protecting acute lung injury induced by PM2.5.（2）In the course of acute lung injury induced by PM2.5,the inhibition of AMPK activity can not only activate downstream mTOR 1 directly,but also break the interaction between ULK 1 and AMPK through Ser 757 of Phosphorylated ULK 1.The above two pathways can inhibit autophagy activity,block autophagy flow,lead to autophagy degradation disorder,and then promote apoptosis and induce acute pulmonary inflammation.（3）Astragaloside IV can activate autophagy through AMPK/mTOR/ULK 1 signal pathway,inhibit apoptosis and reduce pulmonary inflammation,so as to protect acute lung injury induced by PM2.5.Compound C,an inhibitor of AMPK,could block the effect of Astragaloside IV.This suggests that Astragaloside IV rely on AMPK targets to promote the activation of autophagy induced by PM2.5 in acute lung injury.（4）Through autophagy pathway,Astragaloside IV inhibit Bcl2-Bax/caspase 3/caspase PARP 1 apoptosis pathway,decrease TUNEL fluorescence apoptosis index,and then inhibit downstream NF-κB pathway,so as to play a protective role in acute lung injury induced by PM2.5.CONCLUSION（1）During the course of acute lung injury induced by PM2.5,the activity of AMPK was inhibited and downstream mTOR 1 was activated,and the Ser 757 of ULK1 was phosphorylation,which led to the obstruction of autophagy flow,which promoted apoptosis and induced acute pulmonary inflammation.（2）The preventive administration of Astragaloside IV can activate autophagy through AMPK/mTOR/ULK1 pathway,inhibit apoptosis and reduce pulmonary inflammation,so as to protect acute lung injury induced by PM2.5.This may be the potential mechanism of preventive administration of Astragaloside IV to protect acute lung injury induced by PM2.5.