| Objective:The present study was designed to explore the regulatory effect of mitophagy to alveolar macrophage(AM)pyroptosis and its molecular mechanism during sepsis-associated acute lung injury(ALI).Methods:1.miRNAs targeting NLRP3 were predicted by bioinformatics analysis and miR-138-5p was screened.The targeted binding relationship between miR-138-5p and NLRP3 mRNA 3’-Un-Translated Region(3’-UTR)was verified by double luciferase reporter gene assay.Bioinformatics analysis was used to predict the distribution of Cytosine-phosphate-guanine(Cp G)islands in the promoter region of miR-138-5p.2.AM was stimulated with Lipopolysaccharide(LPS)to construct an in vitro model of sepsis-associated lung injury.AM was treated with PBS,LPS,LPS + sh-NC,LPS + sh-NLRP3,LPS + mimic-NC,LPS + miR-138-5p-mimic,LPS + DMSO,LPS+ 5-Aza,LPS + 5-Aza + inhibitor-NC,LPS + 5-Aza + miR-138-5p-inhibitor,LPS +CCCP or LPS + CCCP + mtDNA.Electron microscopy was used to observe the formation of cell membrane pore.Fluorescence microscope analysis of FAM-FLICA caspase was used to detect caspase activity and cell membrane permeability.Western Blot was used to examine the expression levels of pyroptosis and inflammation-related proteins.The levels of IL-1β and IL-18 in culture supernatant were determined by ELISA.The expression level of miR-138-5p was detected by qRT-PCR.The methylation level of miR-138-5p promoter was determined by methylmion-specific PCR(MSP)and chromatin immunoprecipitation(CHIP).3.In vivo model of sepsis-associated lung injury was established by performing caecal ligation and puncture(CLP)surgery.Wild type(WT)mice were treated with sham operation,CLP,CLP + AgomiR-NC,CLP + AgomiR-138-5p,CLP + 5-Aza,CLP + 5-Aza + AntagomiR-NC,CLP + 5-Aza + AntagomiR-138-5p,CLP + CCCP or CLP + CCCP + mtDNA;NLRP3 knockout mice were treated with sham surgery or CLP.HE staining was used to observe lung injury level.Immunohistochemistry was applied to examine the expression levels of pyroptosis and inflammation-related proteins in lung tissues.Transmission electron microscopy was applied to observe the formation of mitophagosome(MP)and mitolysosome(ML).Western Blot was used to determine the expression level of autophagy-related protein LC3 B.The expression level of miR-138-5p in lung tissues was detected by qRT-PCR.MSP was used to detect the methylation level of miR-138-5p promoter in lung tissues.Results:1.LPS-treated AM showed cell membrane pore formation and inflammatory injury.Transfection of NLRP3 lentivirus inhibited LPS-induced AM pyroptosis and inflammation.NLRP3 gene knockout alleviated sepsis-associated lung injury of CLP mice.2.Double luciferase reporter gene assay confirmed the direct targeting relationship between miR-138-5p and NLRP3 mRNA 3’-UTR,and overexpression of miR-138-5p could reduce AM pyroptosis and inflammation to weaken sepsis-induced lung injury by inhibiting the expression level of NLRP3.3.In sepsis-associated ALI,the expression of miR-138-5p in AM decreased in a methylation-dependent manner;4.Pretreatment with 5-Aza,a DNA methylation inhibitor,alleviated sepsis-induced lung injury in vitro and in vivo,while functional deletion of miR-138-5p reversed the effect of 5-Aza,suggesting that 5-Aza alleviated lung inflammation by inducing demethylation of miR-138-5p promoter.5.Compared with sham group,MP was observed in lung tissues of CLP group,and LC3BⅡ/Ⅰ ratio was increased.CCCP pretreatment could further enhance mitophagy.In addition,enhanced mitophagy induced demethylation of miR-138-5p promoter by decreasing cytoplasmic mtDNA level.6.CCCP-enhanced mitophagy inhibited AM pyroptosis and inflammation,which may alleviate septic lung injury.Conclusions:Mitophagy induced the demethylation of the miR-138-5p promoter by reducing the cytoplasmic mtDNA level,thus increasing expression level of miR-138-5p,which may subsequently inhibit NLRP3 inflammasome activation to alleviate AM pyroptosis and inflammation in sepsis-associated lung injury. |
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