ALDH2 Alleviated Septic Myocardial Injury By Inhibiting Caspase-11 Mediated Noncanonical Pyroptosis

ObjectiveTo investigate the effect of aldehyde dehydrogenase 2（ALDH2）in cecal ligation and puncture（CLP）induced myocardial injury in mouse model and LPS-induced H9C2 cell injury focusing on caspase-11-mediated noncanonical pyroptosis.Methods Our experiments were divided into two parts:1.In vivo part:CLP induced sepsis model was established in C57BL/6 mice,and designed in 4groups: Sham group,CLP group,CLP + Alda-1（ALDH2 specific agonist）group and methyl sulfoxide（DMSO,the solvent of Alda-1）group.After 24 hours of intervention,the cardiac function including left ventricular ejection fraction（LVEF）,left ventricular fractional shortening（LVFS）,stroke volume（SV）and left ventricular end-systolic internal diameter（LVESD）were assessed by echocardiography,the changes of histological and ultrastructure injury were measured by H＆E staining and electron microscopy,the levels of tumor necrosis factor-α（TNF-α）was determined by enzymelinked immunosorbent assays（ELISA）,the levels of noncanonical pyroptosis associated proteins----caspase-11,gasdermin D（GSDMD）,high mobility group box 1（HMGB1）and the receptor for advanced glycation end products（RAGE）were determined by western blotting.In addition,whether ALDH2 interacted with noncanonical pyroptosis proteins was further explored by the co-immunoprecipitation（CO-IP）method.2.In vitro part:LPS-induced cardiomyocytes injury model was established and divided into 4groups: normal control group（NG）,LPS group（LPS）,LPS+ overexpression of ALDH2group（LPS+ ALDH2-GFP）,LPS+ GFP group.The cell viability by CCK-8 kit and lactate dehydrogenase（LDH）level were measured by spectrophotometer,and the protein levels of caspase-11,GSDMD,HMGB1,RAGE were determined by western blotting and immunofluorescence analysis.Results1.In vivo experiments:Compared with the Sham group,in the CLP group,left ventricular ejection fraction（LVEF）,left ventricular fractional shortening（LVFS）,and stroke volume（SV）were significantly decreased（P<0.01）,left ventricular end-systolic internal diameter（LVESD）was significantly increased（P<0.01）.H＆E staining showed the inflammatory infiltration was increased and the myocardial fibers were broken.Electron microscopy showed mitochondria were swelled and ruptured.The expression of TNF-α was significantly increased（P<0.05）.The expressions of caspase-11（P<0.01）,GSDMD（P<0.05）,HMGB1（P<0.01）,and RAGE（P<0.01）were significantly increased.Compared with the CLP group,in the CLP + Alda-1 group,LVEF,LVFS,and SV were significantly increased（P<0.01）,LVESD was significantly decreased（P<0.01）.H＆E staining and electron microscopy showed myocardial injury,inflammatory infiltration,and mitochondrial injury were alleviated.The expression of TNF-α was significantly decreased（P<0.05）.The expressions of caspase-11（P<0.01）,GSDMD（P<0.05）,HMGB1（P<0.01）,and RAGE（P<0.01）were significantly reduced.Furthermore,CO-IP showed that ALDH2 binds to HMGB1、RAGE and GSDMD in the total protein.2.In vitro experiments:Compared with the NG group,in the LPS group,the cell viability and LDH level were significantly increased（P<0.01）.The protein expressions of caspase-11（P<0.05）,GSDMD,HMGB1 and RAGE（P<0.01）were significantly increased.The fluorescence intensities of caspase-11,GSDMD,HMGB1,and RAGE（P<0.01）were significantly increased.Compared with the LPS group,in the LPS+ ALDH2-GFP group,the cell viability and LDH level were significantly decreased（P<0.01）.The protein expressions of caspase-11（P<0.05）,GSDMD（P<0.01）,HMGB1（P<0.05）,and RAGE（P<0.05）were significantly reduced.The fluorescence intensities of caspase-11（P<0.01）,GSDMD（P<0.01）,HMGB1（P<0.05）and RAGE（P<0.05）were significantly reduced.ConclusionOur findings revealed that ALDH2 protected mouse myocardium from septic injury through inhibiting caspase-11-mediated noncanonical pyroptosis.ALDH2 played the role through binding to GSDMD,HMGB1 and RAGE directly.