Hydrogen Sulfide （H₂S） Inhibits BECN1/SLC7A11 Pathway Mediated Ferroptosis Pathway To Reduce Myocardial Damage In Sepsis

Objective:To investigate whether exogenous H₂S donor Na HS can improve myocardial injury in sepsis by inhibiting BECN1/SLC7A11 pathway mediated ferroptosis pathway.Methods:Cell experiments:Different concentrations of LPS（1,3,5,10,20μg/m L）to stimulate H9c2 rat cardiomyocytes to induce sepsis myocardial injury cell model.Before LPS stimulation,cells were pretreated with different concentrations of Na HS（20,50,100,150,200 mmol/L）for 1 hour to screen the optimal concentration of Na HS for administration.H9c2 cells were divided into three groups:Control group,LPS group and LPS+Na HS group.CCK-8 method was used to detect cell viability,the kit was used to detect the level of Fe²⁺,LDH,CK-MB,GSH,MDA in myocardial cells,the fluorescent probe was used to detect the changes of ROS and mitochondrial membrane potential JC-1 in myocardial cells,immunofluorescence was used to detect the expression location of BECN1 and SLC7A11 in myocardial cells,small interfering RNA（si RNA）was used to reduce the expression of BECN1 in H9c2 cells,and western blot method was used to detect the ferroptosis regulatory proteins BECN1,p-BECN1,SLC7A11,Ferritin,GPX4 expression level.Animal experiment:In order to further verify the in vivo results,45 SD rats（8-10 weeks）were random Ly divided into sham operation（Sham）group,sepsis（CLP）group,sepsis+Na HS（CLP+Na HS）group with 15 rats in each group,using the in vivo model of sepsis myocardial injury induced by cecal ligation and puncture（CLP）Echocardiography was used to observe the cardiac systolic and diastolic functions of rats in each group,HE staining was used to observe the pathological changes of myocardial tissue of rats in each group,transmission electron microscopy was used to observe the structural changes of myocardial mitochondria,and western blot method was used to detect the expression levels of ferroptosis regulatory proteins BECN1,p-BECN1,SLC7A11,Ferritin,GPX4.Paired student’s t test was used to compare the means of two samples;≥3 samples were compared using one-way ANOVA with complete randomization and post hoc Bonferroni’s correction.P<0.05 was considered to indicate a statistically significant difference.The correlation between Fe²⁺and cell activity,myocardial enzyme,cell oxidative stress,lipid peroxidation,iron death marker protein and other indicators was analyzed.The correlation between rat cardiac function indicators and iron death marker protein was analyzed.Pearson correlation coefficient|r|>0.8 indicates a high correlation,and P<0.05 indicates a significant linear correlation.Results:（1）In the sepsis induced myocardial injury cell model of H9c2 rat cardiomyocytes stimulated by LPS at different concentrations,compared with Control,the activity of H9c2 cells decreased,the concentration of Fe²⁺increased,the levels of MDA and ROS increased,the monomer of mitochondrial JC-1increased,the protein expression levels of p-BECN1 were increased,and the protein expression levels of Ferritin,GPX4 and SLC7A11 were down-regulated,and the myocardial cell injury increased after LPS stimulation（p<0.05）.（2）Compared with LPS group,Na HS intervention can reduce LPS induced H9c2 cell damage and increase of Fe²⁺concentration,reduce LPS induced H9c2 cell oxidative stress and iron metabolism disorder,down-regulated the expression level of p-BECN1 protein,up-regulated the expression levels of Ferritin,GPX4 and SLC7A11 proteins,and alleviated myocardial cell injury（p<0.05）.（3）After the intervention of BECN1 si RNA,inhibition of BECN1 expression can enhance the antioxidant capacity of cells and reduce the sensitivity of LPS induced ferroptosis of H9c2 cells.The expression levels of ferroptosis regulatory proteins SLC7A11,Ferritin and GPX4 increased after combined Na HS administration（p<0.05）.（4）Compared with Sham group,the systolic and diastolic functions of the heart of rats in CLP group were weakened.HE staining showed that the arrangement of myocardial tissue was disordered,and the infiltration of inflammatory cells was obvious.Transmission electron microscopy showed that the mitochondria became smaller,the crista decreased,the membrane broke,and the expression level of ferroptosis regulatory proteins SLC7A11,Ferritin,GPX4 decreased,The expression level of p-BECN1 was significantly higher（p<0.05）.（5）Compared with CLP group,cardiac dysfunction was relieved after Na HS administration,cardiac histopathology was improved,mitochondrial damage was alleviated,the expression of SLC7A11,Ferritin,GPX4 in myocardial tissue was increased,and the expression level of p-BECN1 was decreased（p<0.05）.Conclusion:Exogenous H₂S donor Na HS ameliorates septic myocardial injury by inhibiting BECN1/SLC7A11 pathway mediated ferroptosis pathway.