Study On The Mechanism Of PM2.5 Inducing Ferroptosis Leading To Acute Lung Injury By Regulating AMPK-beclin1 Signaling Pathway

Objective:PM2.5 is an important source of global air pollution.Although it occupies a relatively low proportion in the earth’s atmospheric components,it can increase the morbidity and mortality of respiratory diseases such as acute lung injury(ALI)and acute exacerbation of chronic obstructive pulmonary disease(AECOPD)due to its characteristics such as multiple toxic components,long retention time in the air and long transport distance.At the cellular level,the mechanism of PM2.5 causing ALI is very complex,involving changes in various cell death patterns and signaling pathways.Recent studies have reported that PM2.5 can lead to ferroptosis in endothelial cells by inducing oxidative stress and iron overload.However,whether PM2.5 can lead to ALI by inducing ferroptosis and its molecular mechanism remains unclear,and further studies are needed.Methods:This experiment mainly includes two parts.The first part mainly constructs PM2.5induced ALI models in vivo and in vitro,and establishes the pathogenic role of ferroptosis in PM2.5 induced ALI.In vitro,we selected bronchial epithelial cells(BEAS-2B cells)as the research objects,prepared different concentrations of PM2.5organic extraction solutions,and co-cultured with the cells to detect cell viability,lipid peroxidation,iron levels,and ferroptosis characteristic proteins.Then,we constructed the mice ALI model with PM2.5 intranasal infusion,and further verified the toxic effect of ferroptosis on PM2.5 induced ALI in vivo.The second part explores the specific molecular mechanism of ferroptosis on the basis of the experiment in the first part,and uses molecular biology(knockdown,overexpression,q PCR),immunology(Western Blot,co-immunoprecipitation)and other methods to explore the main role of AMPKBeclin1 signaling pathway in the occurrence of ferroptosis.The molecular mechanism of PM2.5 promoting ferroptosis by activating AMPK-Beclin1 signaling pathway was elucidated.In addition,PM2.5 nasal drops were used to induce ALI model in mice,and a new mechanism of ferroptosis in mice induced by PM2.5 activation of AMPKBeclin1 signaling pathway was confirmed in vivo.Results:1.In vitro,PM2.5 decreased cell viability,enhanced lipid peroxidation,and disrupted expression of ferroptosis-related proteins in a dose-dependent manner.The expression of iron storage proteins(FTH-1,FTL)and iron inward transporter(TFR)was enhanced,the expression of iron outward transporter(FPN)was weakened,the expression of lipid peroxides(COX-2,NOX-4)was enhanced,and the expression of ferroptosis characteristic proteins(amino acid transporter x CT/SLC7A11)was weakened.2.In the process of ferroptosis triggered by PM2.5,AMPK-Beclin1 signaling pathway is activated and SLC7A11 expression is inhibited.After Beclin1 knockdown,the ferroptosis caused by PM2.5 was partially alleviated,manifested as the reduction of lipid peroxidation and iron overload.Compared with the control group,the cell viability in vitro was significantly increased,and the degree of ALI and inflammation was relieved in vivo.Beclin1 overexpression showed the opposite result to knockdown,lipid peroxidation level and iron overload degree were aggravated compared with the control group,cell viability was decreased,and the degree of lung injury was aggravated.The results of co-immunoprecipitation showed that Beclin1 inhibited System XC-activity by directly binding to SLC7A11,resulting in reduced cystine uptake and GSH synthesis,triggering the occurrence of ferroptosis.Similarly,administration of AMPK inhibitor Compound C and activator AICAR to regulate Beclin1 expression by affecting AMPK activity showed similar experimental results as those of Beclin1 knockdown and overexpression.These results indicate that AMPKBeclin1 signaling pathway plays an important role in PM2.5 induced ferroptosis.The results of real-time PCR confirmed that the transcription of SLC7A11 was activated by low dose PM2.5 and slightly inhibited by high dose PM2.5,which confirmed that the inhibition of System XC-activity mainly occurred at the protein level rather than the transcription level.3.In vivo,PM2.5 caused obvious ALI,manifested as alveolar fluid exudation,edema and infiltration of inflammatory cells,significantly enhanced iron storage,mitochondrial shrinkage and rounding,membrane thickening,and cristae destruction.Giemsa staining showed obvious infiltration of neutrophils and lymphocytes in the lavage fluid,and ELISA showed that the levels of inflammatory cytokine(IL-1β,IL-6,TNF-α)were significantly increased.The expression of iron storage protein(FTH-1)was enhanced,and the protein characteristic of ferroptosis(SLC7A11)was similarly inhibited.4.Beclin1 is closely related to autophagy,and the autophagy inhibitors hydroxychloroquine(HCQ)and Bafilomycin A1 only slightly reversed the decrease in cell viability caused by PM2.5,while lipid peroxidation and iron overload were not significantly alleviated.PM2.5 can activate autophagy,and autophagy inhibitors block autophagy flux,but no significant inhibition of ferroptosis was observed,suggesting that Beclin1 activated by PM2.5 is mainly induced by ferroptosis,rather than autophagy process,leading to ALI.Conclusion:In summary,this study explored the role of ferroptosis mediated by AMPKBeclin1 signaling pathway in PM2.5-induced ALI and its molecular mechanism,providing new ideas for understanding the toxicological effects of PM2.5 on respiratory diseases and a new target for the treatment of lung injury.