Melatonin Differentially Modulated Autophagy And Apoptosis Through Sirt1 In Airway Inflammation Induced By Influenza A Virus

Background Influenza is an acute inflammatory disease caused by influenza virus,previous studies by our group have confirmed that influenza virus is one of the main pathogen causing acute exacerbation of chronic obstructive pulmonary disease in China,so it is necessary to study the pathogenesis of influenza virus-induced acute exacerbation of chronic airway inflammatory disease.Maintaining the balance of autophagy and apoptosis is of great significance to the severity and prognosis of the disease caused by viral infection.Autophagy plays an important role in viral infection,the double-membrane structure of the autophagosome protects the replication of the virus,on the other hand,autophagosomes degrade viruses after combining with lysosomes and present virus antigens to immune cells to activate immune response.In normal physiological processes,cells remove damaged or senescent organelles through apoptosis to maintain homeostasis.When invaded by pathogens,apoptosis activate the inflammatory response to generate immune response.Virus replicated in the autophagosome,the release of the virus is accomplished by apoptosis,then infect surrounding cells.Melatonin is an amine hormone secreted by the pineal gland of the hypothalamus,which play a role in improving sleep,anti-aging,and regulating immunity.This study aimed to explore the cellular and molecular mechanisms of autophagy and apoptosis in airway inflammation induced by IAV,and cross-talk between both pathway,whether melatonin has a protective effect in IAV induced airway inflammation.MethodsIn vitro,IAV co-cultured with DHBE cells,the expression of Sirt1 in DHBE cells were detected by western blotting;the expression of autophagy-related proteins Beclin1 and LC3B were detected by western blotting,electron microscope observation of intracellular autophagosomes,fluorescence confocal microscope observation of m RFP-GFP-LC3 adenovirus-labeled LC3 protein to track autophagy formation and changes in autophagy flux;the expression of apoptosis-related protein Bax and Bcl-2were detected by western blotting and flow cytometry were used to detect the level of apoptosis;the levels of IL-8 and TNF-? in the supernatant were detected by ELISA.The level of autophagy,apoptosis and related inflammatory indexes after pretreatment with melatonin for 4h.Lentivirus were used to knockdown or overexpress Sirt1 in DHBE cells,then pretreat with melatonin for 4h before IAV infection,after that detect the level of autophagy,apoptosis and related inflammatory indicators.In vivo,6-8 weeks,C57BL/6 male mice,were exposed to smoking for 6 months,in melatonin group,mice quantitative(20 mg/kg)and repetitive dose of melatonin in 28 days by peritoneal injection,and intratracheal administration of influenza virus suspension,then observe in the weight of the mice after influenza virus infection.One week later,the mice were sacrificed by exsanguination after anesthesia with sodium thiopental,and the serum,BALF were collected.The expression of Sirt1,Beclin1,LC3 B,Bax and Bcl-2 in lung tissue were detected by Western blotting,the expressions of Sirt1 and LC3 B were detected by immunofluorescence,and IL-8 and TNF-? in serum and BALF were detected by ELISA.ResultsResults shown that IAV infection induced suppressed expression of Sirt1,increased the level of autophagy and apoptosis.Detailed time course analyses showed that an early increase of autophagy in the first 24 h,a delayed reduction of autophagy after 48h;an early moderate increase developed within 24 h,preceding a substantial up-regulation of apoptosis in 48 h.In order to study the cross-talk between autophagy and apoptosis,the early autophagy inhibitor 3-MA,the late autophagy inhibitor CQ,and the apoptosis inhibitor Z-VAD-FMK were used before IAV infection.The results shown that pretreated with the autophagy inhibitor 3-MA,the level of apoptosis was reduced accordingly,combined with previous studies,it is considered that the occurrence of apoptosis is related to viral load.When inhibity the formation of autophagosomes,virus replication in cells was restricted,and viral load was reduced,then the level of apoptosis was decreased.CQ,a late stage inhibitor of autophagy,has been found to inhibit the combination of autophagosomes and lysosomes,result in the accumulation of autophagosomes,induce endoplasmic reticulum stress,then the level of apoptosis was increased.After pretreatment with the apoptosis inhibitor Z-VAD-FMK,the level of apoptosis was inhibited,and a large number of autophagosomes accumulated,which showed that the level of autophagy was increased.Pretreatment with melatonin for 4hours before IAV exposure alleviate the decrease of Sirt1,and inhibit the increase of apoptosis caused by IAV infection,further increase the level of autophagy.However,after knocking down Sirt1,the regulatory effect of melatonin on autophagy and apoptosis caused by IAV infection is significantly reduced.ConclusionOur study provides strong evidence that IAV infection simultaneously induced autophagy and apoptosis,autophagy was significantly earlier than apoptosis,melatonin administration before IAV infection significantly protects airway epithelial cells by promoting autophagy and inhibiting apoptosis.These findings implicate a new modulated mechanism of melatonin in airway inflammation caused by IAV.