Cigarette Smoking Promotes Senescence Of Alveolar Epithelial Type 2 Cells To Induce Pulmonary Fibrosis

Background:IPF is an aging-related and interstitial lung disease.Its median survival time from diagnosis is 2-4 years.Currently,the effective therapeutic strategy for IPF is still lacking.With increasing global aging and COVID-19 spread,IPF incidence is likely to be higher in future,which will make larger impacts on patients and societies,thus there is a pressing need to seek effective therapeutic targets.Cigarette smoking(CS)is a strong risk factor for IPF and can lead to pulmonary fibrosis in rats,with unclear mechanism.Cellular senescence of alveolar epithelial type 2 cell(AT2 cells)is a key player in IPF development.And autophagy is a hot issue in cellular senescence and IPF.Here we aim to explore whether CS induced AT2 cells senescence and lung fibrosis in an autophagy dependent pathway,how CS regulated autophagy and how autophagy dysregulation led to AT2 cells senescence.Methods:In vivo,mice were exposed to CS(5 cigarettes/time,30min/time,2 times/day)for 4 weeks.And then autophagy and cellular senescence of AT2 cells were evaluated.The effects of SIRT1 activator SRT1720 and mitochondrial-targeted reactive oxygen species(mtROS)antioxidant mitoquinone(MitoQ)on AT2 cells senescence and pulmonary fibrosis were examined.In vitro,AT2 cells strain MLE12 were stimulated with cigarette smoke extract(CSE),and then cellular senescence,autophagy,mitophagy and mtROS were detected.Then,we examined the effect of autophagy inducer rapamycin(RAPA)and inhibitor 3MA on mitophagy,mtROS and cellular senescence and the impact of MitoQ on cellular senescence.Next,how CSE regulate SIRT1 expression and activity was determined,and the effects of SRT1720 and SIRT1 inhibitor Ex527 on autophagy and cellular senescence were evaluated.In addition,the ratio of NAD+to NADH was detect,and the effects of NAD+precursorβ-nicotinamide mononucleotide(NMN)and PARP1 inhibitor Olaparib(OLA)on SIRT1 activity,autophagy and cellular senescence were assessed.Results:In vivo,smoking mice exhibited increased senescence and decreased autophagy of AT2 cells.SRT1720 and MitoQ elevated autophagy and inhibited senescence of AT2 cells and protect against CS induced pulmonary fibrosis.In vitro,0.5%CSE stimulation for 36h promoted cellular senescence,declined autophagy and mitophagy and increased mtROS level.MitoQ suppressed CSE-induced senescence.RAPA pretreatment prevented CSE-related senescence and declined mtROS,while 3MA alone showed similar effects with CSE.Then,we found CSE inactivated SRT1720.SRT1720 reversed CSE-induced senescence and rescued autophagy and these effects were eliminated by 3MA.Next,we found CSE led to DNA damage and downregulated the ratio of NAD+to NADH,while did not change NAD+ content.Both NMN and OLA activated SIRT1,restored autophagy and inhibited senescence.The above effects of NMN and OLA were reversed by Ex527.Additionally,RAPA and MitoQ decreased NAD damage and increased SIRT1 activity.Conclusion:Cigarette smoking-induced DNA damage activated PARP1 and subsequential NAD+consumption to inactivate SIRT1,which induce AT2 cells senescence by promoting mitochondrial oxidative stress in an autophagy and mitophagy dependent pathway.Autophagy reduction return inactivated SIRT1 through increasing mitochondrial oxidative stress,which resulted in DNA damage,PARP1 activation and NAD+consumption.Therefore,a positive feedback loop between SIRT1 and autophagy was formed in CS-treated AT2 cells.Targeting SIRT1 and mtROS prevented CS-induced pulmonary fibrosis and AT2 cells senescence in mice.Consequently,SIRT1 inactivation contributed to CS-induced AT2 cells senescence and pulmonary fibrosis by promoting mitochondrial oxidative stress in an autophagy dependent pathway.