Protective Effect And Mechanism Of β1 Blocker Combined With Levosimendan On Myocardial Injury Induced By Lipopolysaccharide In Rats

Objective:The purpose of this study was to investigate the effect of esmolol（ES）.β1-blocker,and levosimendan（LE）on lipopolysaccharide（LPS）-induced cardiac injury and the possible mechanism.Methods:170 adult male SD rats were selected.According to the random number table method,the animals were divided into 7 groups:sham group（Sham）,sepsis model group（LPS）.esmolol intervention group（LPS+ES）.levosimendan intervention group（LPS+LE）,esmolol combined with levosimendan intervention group（LPS+ES+LE）,3-methyladenine group（LPS+3-MA）,rapamycin group（LPS+RAP）.Sham group was divided into 5 subgroups（0h,3h,6h,12h,24h）;LPS was divided into 4 subgroups（3h,6h.12h,24h）;LPS+ES.LPS+3-MA and LPS+RAP groups were divided into two subgroups（12h,24h）;LPS+LE and LPS+ES+LE groups（24h）;there were 10 rats in each subgroup.All groups underwent right internal jugular vein catheterization,and LPS（10mg/kg）was injected intraperitoneally to establish sepsis models.Depending on the time point,in the LPS+ES,LPS+LE and LPS+ES+LE groups,esmolol injection(15mg·kg^-1·h^-1).levosimendan injection（4mg·kg-1·d-1）or esmolol injection(15mg·kg^-1·h^-1)combined with levosimendan injection（4mg·kg-1·d-1）were continuously pumped through the right internal jugular vein,respectively;in the Sham.LPS.LPS+3-MA（15mg/kg）and LPS+RAP（4mg/kg）groups,the same volume of normal saline（0.5ml/h）was continuously pumped through the right internal jugular vein:except for the Sham group,LPS was injected intraperitoneally 30min after administration in the other groups.The degree of myocardial injury was analyzed by hematoxylin-eosin（HE）staining,and the myocardial injury score was calculated.The concentration of cardiac troponin I（cTnI）was measured by enzyme-linked immunosorbent assay（ELISA）.Western blotting（WB）was used to detect the expression of autophagy-related proteins Beclin-1,LC3-II.p-AMPK.p-ULK1 and p-mTOR in myocardial tissue.Autophagosome formation and mitochondrial ultrastructure damage were observed by transmission electron microscopy.Results:①Survival and death of rats in each group at 24 h:after drug intervention for 24 h,the rats in the LPS and LPS+3-MA groups appeared drowsily,with wilting hair color,curled up in the corner of the rat cage,with little activity,and the water and food intake decreased sharply.In the Sham group,the hair color was white and shiny,the activity was good,and the diet was normal.The rats in the LPS+ES,LPS+LE,LPS+ES+LE and LPS+RAP groups showed decreased activity,listlessness,upright hair and shipping.The overall state of the rats in the LPS+ES,LPS+LE,LPS+ES+LE and LPS+RAP groups was better than those of the LPS and LPS+3-MA groups,but worse than the Sham group.In the Sham group,LPS group,LPS+ES group,LPS+LE group,LPS+ES+LE group,LPS+RAP group,LPS+3-MA group,the mortality was 0.0%,40.0%,20.0%,0.0%,10.0%,10.0%,40.0%,respectively.There was no significant difference in survival rate between groups（P>0.05）.② Pathological changes of myocardium induced by LPS:under light microscope,the myocardium of the Sham group showed normal structure;in the LPS group,myocardial fibers were disordered,myocardial cells were obviously dissolved and necrotic,and vacuolization and degeneration were observed.The injury in the LPS group was time-dependent.The myocardial injury score was as follows:12h-LPS group>6h-LPS/24h-LPS group>3h-LPS group>Sham group（P<0.05）.③Pathological changes of myocardium in each group at 12h and 24h:at 12 h and 24h,the myocardial fibers in the Sham group were arranged neatly and re gularly,the transverse lines were clear,the intercellular space was normal,and th ere was no obvious edema,congestion,degeneration and necrosis;the myocardial tissue injury of the LPS and LPS+3-MA groups was significantly aggravated at 12h and 24h;at 12h and 24h,the LPS+ES group and LPS+RAP group had sig nificantly reduced myocardial fiber disorder,myocardial cell lysis,necrosis,myoc ardial interstitial laxity and inflammation;at 24h,the LPS+LE and LPS+ES+LE groups had alleviated myocardial injury,as shown by reduced degree of myocard ial cell destruction,regular arrangement of myocardial fibers,and reduced degree of myocardial cell edema.The myocardial injury score was as follows:12h:LP S>LPS+3-MA>LPS+ES/LPS+RAP>Sham（P<0.05）.24h:LPS/LPS+3-MA>LPS+ES/LPS+LE/LPS+ES+LE/LPS+RAP>Sham（P<0.05）.④ Myocardial enzyme changes at 24h:serum cTnI concentrations in the LPS and LPS+3-MA groups were significantly higher than those in the Sham group,and cTnI in the LPS+ES,LPS+RAP,LPS+LE and LPS+ES+LE groups were significantly lower than those in the LPS and LPS+3-MA groups（P<0.05）.⑤ Beclin-1 and LC3-II protein expression in myocardial tissue of LPS group:the autophagy induced by LPS was time-dependent.Compared with the Sham group,the expression of LC3-II and Beclin-1 in the 3h-LPS group increased（P<0.05）.The expression levels of LC3-II and Beclin-1 in the 6h-LPS.12h-LPS and 24h-LPS groups were significantly lower than those in the 3h-LPS or Sham group（P<0.05）.⑥Beclin-1.LC3-II,p-AMPK.p-ULK1 and p-mTOR protein expression in myocardial tissue of each group after LPS induction for 12h and 24h:at 12h and 24h.compared with the Sham group,the expressions of Beclin-1,LC3-II,p-AMPK and p-ULK1 proteins in the LPS group were significantly increased,and the expression of p-mTOR protein was significantly decreased（P<0.05）.Compared with the LPS group at the same time point.the expression of Beclin-1,LC3-II,p-AMPK and p-ULK1 proteins was increased,while the expression of p-mTORwas decreased in the LPS+ES group（P<0.05）.Compared with the LPS group at the same time point,the LPS+RAP group had significantly increased protein expression of Beclin-1 and LC3-II and significantly decreased protein expression of p-mTOR.and significantly increased protein expression of p-ULK1 at 12h（P<0.05）.There was no significant difference between the LPS+3MA group and the LPS group at the same time point（P>0.05）.Compared with the 24h-LPS group,the expression levels of Beclin-1,LC3-II,p-AMPK and p-ULK1 were increased,and the expression of p-mTOR was decreased in the LPS+LE and LPS+LE+ES groups（P<0.05）.⑦ Myocardial mitochondria and autophagosomes in each group at 24 h:a normal mitochondrial structure and a small number of autophagosomes were observed in the Sham group,and complete mitochondrial membrane and cristae were shown.Compared with the Sham group,the number of autophagosomes in myocardial tissue of the LPS group was significantly reduced,the mitochondria were obviously swollen,the cristae was blurred,and the vacuolization was increased.Compared with the LPS group,the mitochondrial damage was partially reversed and a larger number of autophagosomes were observed in the LPS+ES,LPS+LE,LPS+ES+LE and LPS+RAP groups（P<0.05）.There was no significant difference between the LPS+3-MA group and the LPS group（P>0.05）.Conclusion:ES,LE or ES+LE alleviates LPS-induced myocardial damage through regulating autophagy through AMPK/m TOR/ULK1 signaling pathway.