The Effect Of Autophagy On Lung Injury Induced By H9N2 Influenza Virus

Influenza A virus(IAV)often causes serious economic loss to breeding farms and affects human health.H9N2 influenza virus can infect poultry,pigs and humans,so the study of H9N2 influenza virus has important public health significance.The major pathogenic factor of influenza A virus is the direct damage caused by the effective replication of the virus,the release of inflammatory factors induced by H9N2 influenza A virus and the oxidative stress triggered by virus,however,the exact mechanism needs to be further studied.It was reported that autophagy was induced by the influenza virus,however,the mechanism still needs to be further explained.Therefore,this study explores the role of autophagy in the pathogenesis of influenza virus.The main findings are as follows:1.Autophagy was induced by H9N2 influenza virus in A549 cellsA549 cells were infected with H9N2 influenza virus,the autophagolysosomes with single-layer film was observed by transmission electron microscopy and increased LC3-II level was found by western blot.In addition,this study established A549 cell line with stable expression of GFP-LC3,named A549/GFP-LC3.The laser confocal test revealed an increase in the GFP-LC3 puncta in the A549/GFP-LC3infected with H9N2 virus,indicating that autophagy was induced by H9N2 influenza virus in A549.2.Autophagy and the replication of H9N2 influenza virusTo study whether autophagy affects virus replication,3-MA,LY294002 and Atg5siRNA were used in this study to inhibit autophagy and autophagy inducer rapamycin was used to induce autophagy,then the mRNA and protein level of NP were detected by real-time PCR and western blot and the titer of virus was detected.The results showed that LY294002 and Atg5 siRNA could significantly reduce the mRNA and the expression levels of NP,and the virus titer.3-MA also reduced the NP mRNA levels and the virus titer.Moreover,the autophagy inducer rapamycin could increase the NP mRNA level and the virus titer,indicating that inhibition of autophagy could decrease the replication of influenza virus.3.Autophagy was involved in the inflammatory responses in A549 cells infected by H9N2 influenza virusA549 cells were treated with 3-MA or Atg5siRNA,the mRNA levels of TNF-?,IL-1?,IL-8 and CCL5 were detected by real-time PCR.The results showed that inhibition of autophagy significantly reduced the increase in the mRNA levels of inflammatory factors induced by H9N2 influenza virus.Inhibition of autophagy can reduce the significant differences in elevated cytokine and chemokines levels induced by H9N2 influenza virus with different MOI to the same level.So the results showed that the decrease of inflammatory factors induced by inhibition of autophagy was not due to the decrease of virus titer caused by inhibition of autophagy.In addition,Akt-mTOR regulated autophagy induced by H9N2 influenza virus,and autophagy was involved in the release of inflammatory factors induced by H9N2 influenza virus by regulating p38 MAPK,JNK,ERK1/2 and NF-?B signals.4.Autophagy was involved in the oxidative stress induced by H9N2 influenza virusAutophagy is inhibited or induced,then the levels of SOD1,ROS and MDA were detected to investigate the relationship between autophagy and oxidative stress.The results showed that inhibition of autophagy by autophagy inhibitors 3-MA,LY294002and Atg5 siRNA significantly reduced the increase in oxidative stress induced by H9N2 influenza virus.Inhibition of autophagy can reduce the significant differences in elevated oxidative stress levels induced by H9N2 influenza virus with different MOI to the same level.Autophagy induced by rapamycin could further enhance the oxidative stress in A549 cells.So the results showed that the decrease of oxidative stress induced by inhibition of autophagy was not due to the decrease of virus titer caused by inhibition of autophagy.In addition,Simultaneous treatment with NAC and Atg5siRNA could further reduce oxidative stress levels,NP mRNA levels and viral titers,while H₂O₂ had the opposite effect.So,autophagy was involved in the virus replication by regulating the oxidative stress induced by influenza virus,which was regulated by the signaling pathway Akt/TSC2/mTOR.5.Autophagy was involved in acute lung injury induced by H9N2 influenza virus in miceIn this study,we investigated whether autophagy was involved in acute lung injury induced by H9N2 influenza virus in mice through evaluation of the relationship between autophagy and viral replication,virus-induced release of inflammatory factors,and virus-induced oxidative stress injury.The results showed that inhibition of autophagy by 3-MA and Atg5siRNA could reduce the mortality of mice,the pulmonary edema,the pathological changes.Inhibition of autophagy could reduce the replication of influenza virus,reduce the release of inflammatory factors in mice,and also reduce the oxidative stress levels.In addition,Akt-mTOR can regulate autophagy induced by H9N2 influenza virus,and autophagy is involved in the release of inflammatory factors in H9N2 influenza virus-induced acute lung injury mice by regulating p38 MAPK,JNK,ERK1/2,and NF-?B signals.CONCLUSIONS:(1)Inhibition of autophagy can inhibit H9N2 influenza virus replication.(2)Inhibition of autophagy can reduce the release of virus-induced inflammatory factors in A549 cells.Akt-mTOR regulates autophagy,and autophagy mediates the release of inflammatory factors through p38MAPK,JNK,ERK1/2 and NF-?B pathways.(3)Inhibition of autophagy reduced oxidative stress in A549 cells induced by H9N2 influenza virus.Autophagy?mediated oxidative stress influences the replication of H9N2 influenza virus,which is regulated by the Akt/TSC2/mTOR signal.(4)Autophagy is involved in acute lung injury induced by H9N2 influenza virus in mice by regulating viral replication,the release of inflammatory factors and oxidative stress.Inhibition of autophagy can alleviate acute lung injury in mice induced by H9N2 influenza virus.Therefore,autophagy is one of the important pathogenic mechanisms of H9N2 influenza virus.