Study On The Protective Effect And Mechanism Of ANXA1sp Regulated SIRT3 On Sepsis-Induced Cardiomyopathy In Mice

Objective: To investigate the protective effect of Annexin-A1 bioactive peptide(ANXA1sp)on myocardial injury in sepsis and the effect of cardiac mitochondrial autophagy by establishing an animal model of the mice with sepsis myocardial injury by cecal ligature and puncture(CLP).Methods: 32 ad(?)lt wild-type male C57BL/6J mice with SPF grade were randomly divided into 4 groups(n=8).(1)Sham group;(2)CLP group(cecal ligation and perforation group);(3)ANX + CLP group(ANXA1sp group);(4)NS(normal saline)+ CLP group(NS group).Serum IL-6,TNF-α,CK-MB and CTN-Ⅰ were detected by Elisa to evaluate inflammatory markers and myocardial injury markers;Echocardiography was used to measure cardiac function and structure in mous to evaluate the model of septic myocardial injury.The expressions of Sirt3,AMPK,mTOR,LC3-Ⅰ,LC3-Ⅱ and Beclin-1 in myocardial tissue were detected by western blot.The (?)ltrastructure and autophagy of myocardial mitochondria were observed by electron microscope,ATP and ROS production in myocardial tissue were detected,mitochondria were extracted and membrane potential was detected.To investigate whether ANXA1 sp induces mitochondrial autophagy on myocardial protection by reg(?)lating Sirt3,32 ad(?)lt wild-type male C57BL/6J mice with SPF grade were randomly divided into 4 groups(n=8).(1)ANX+CLP(ANXA1sp group);(2)ANX+3TYP(Sirt3 inhibitor)+CLP(Sirt3 inhibitor group);(3)ANX+3MA(autophagy inhibitor)+ CLP(autophagy inhibitor group);(4)ANX+3TYP+3MA+CLP(Sirt3inhibitor group and group).The above methods were used to detect the myocardial related indicators,myocardial mitochondrial structure changes and related protein expression in sepsis.Res(?)lts: 1.After the establishment of CLP model,compared with the Sham group,the serum levels of IL-6,TNF-α,CK-MB and CTN-Ⅰ significantly increased(P<0.01).Left ventric(?)lar anterior systolic wall thickness(LVAWs),left ventric(?)lar anterior diastolic wall thickness(LVAWd),left ventric(?)lar posterior systolic wall thickness(LVPWs),left ventric(?)lar posterior diastolic wall thickness(LVPWd)significantly increased(P<0.05).Left ventric(?)lar diastolic diameter(LVIDd),left ventric(?)lar systolic diameter(LVIDs),left ventric(?)lar ejection fraction(LVEF)and left ventric(?)l-ar short axis shortening rate(LVFS)significantly reduced(P<0.05).The mice showed obviously slow movement,unsteady standing,drowsiness,increased nasal and mouth secretions,pyuria and watery stools.2.Compared with the CLP group and NS+CLP group,the myocardial mitochondrial membrane potential of mice in ANX+CLP group was significantly increased(P<0.01),ROS production was significantly decreased(P<0.0,1),ATP production was significantly increased(P<0.01),serum IL-6,TNF-α,CTN-Ⅰ,CK-MB levels were significantly decreased(P<0.01),LVAWs,LVAWd,LVPWs,LVPWd were significantly decreased(P<0.05),and LVIDd,LVIDs,LVEF,LVFS were significantly increased(P<0.05);Myocardial fiber rupture,mitochondrial edema,patchy destruction of inner and outer membrane,cavitation expansion,crest expansion with swelling,crest dissolution,electron density and mitochondrial number reduction in mice of CLP group and NS+CLP group were observed under electron microscope;In ANX+CLP group,only mild swelling of mitochondria,complete ridge structure and muscle fiber structure were observed.Compared with Sham group,the number of mitochondria was not significantly reduced and the number of mitochondrial autophagosomes increased.3.Western blot showed that Sirt3 expression in myocardial tissue of ANX+CLP group mice was significantly increased compared with that of CLP group and NS+CLP group(P<0.01).Sirt3 was confirmed to activate AMPK expression,and the expression of autophagy marker beclin-1,LC3Ⅱ/LC3 Ⅰ was increased through AMPK inhibitory effect on mTOR.However,after the addition of Sirt3 specific inhibitor(3TYP),the expression of the protein was reversed,and the protective effect of ANXA1 sp on myocardium and mitochondria was significantly decreased.4.After the addition of autophagy inhibitor(3MA),the protective effect of ANXA1 sp on myocardium and mitochondria decreased,and there was no significant difference between Sirt3 inhibition group,autophagy inhibition group and Sirt3 inhibition +autophagy inhibition group in evaluating the structure and function indexes of mitochondria and myocardium.Conclusion: ANXA1 sp can reduce the inflammatory response of sepsis,improve the mitochondrial injury and myocardial systolic dysfunction of myocardium caused by sepsis,and also activate the mitochondrial autophagy of myocardium,which may be induced by the activation of AMPK-MTOR pathway by reg(?)lating Sirt3.