Roles And Mechanisms Of Esketamine In Septic Myocardial Injury

Background: Sepsis has been widely focused for its high mortality.Heart is the main target organ vulnerable to sepsis and myocardial injury is one of the important indicators of poor prognosis of sepsis.As myocardial inhibitors,endotoxins and numerous inflammatory factors and reactive oxygen species(ROS)have been found to play an active role in septic myocardial injury,which could lead to myocardial dysfunction and even degeneration and necrosis through a variety of effects.Nuclear factor-erythroid 2-related factor 2 / heme oxygenase-1(Nrf2/HO-1)signal pathway is an important endogenous protective mechanism against oxidative stress.Several studies have shown that Nrf2/HO-1 is an effective target for improving septic myocardial injury as the cardio-protection.While other studies have shown that necroptosis as a novel mode of cell death may also be involved in septic myocardial injury.Ketamine could diminish sepsis-induced organ injury via anti-inflammatory and antioxidant stress in previous research.As the dextral enantiomers of ketamine,esketamine has similar pharmacological properties to ketamine with better analgesic effect and fewer side effects.Whether it has the same organ protection effect as ketamine remains to be fully elucidated.Therefore,this study aims to investigate the roles and mechanisms of esketamine in septic myocardial injury.The experiment was conducted from the following three parts.Part I Effects of esketamine on septic myocardial injuryObjective By establishing lipopolysaccharide-induced(LPS)septic myocardial injury model in rats,the effects of different doses of esketamine on septic myocardial injury were explored.Methods In total,60 SPF healthy adult male weighing 200-230 g Sprague-Dawley rats were randomized to 6 groups(n=10 each)using a random number table method: control group(C group),control plus 5 mg/kg esketamine group(CLK group),control plus 10 mg/kg esketamine group(CHK group),lipopolysaccharide group(L group),LPS plus 5 mg/kg esketamine group(LLK group)and LPS plus 10 mg/kg esketamine group(LHK group).Sepsis-induced myocardial injury was induced by injecting lipopolysaccharide 10 mg/kg intraperitoneally.In CLK group,CHK group,LLK group and LHK group,5 mg/kg or 10 mg/kg esketamine was intraperitoneally injected after 30 minutes saline or LPS injected,respectively.Twenty-four hours later,echocardiography was performed to evaluate the cardiac function of the rats.Then,blood samples and cardiac muscle tissue were prepared from the left ventricle for examination of the cardiac myocytes’ morphological changes(by hematoxylin-eosin staining and electron microscopy)and for determination of serum cardiac troponin I(c Tn I),lactate dehydrogenase(LDH),brain natriuretic peptide(BNP),creatine kinase-MB(CK-MB),tumor necrosis factor alpha(TNF-α),interleukin-1β(IL-1β),high mobility group box-1(HMGB1),and IL-6 level(by enzyme linked immunosorbent assay).Results 1.Several hours after intraperitoneal injection of LPS,the rats began to appear listless,shortness of breath,weakened response to the stimulus,and even died 12 hours later.The LPS-induced sepsis rat model was successfully established.Within 24 h of modeling,4 rats in the L group,2 in the LLK group and 1 in the LHK group died,and the surviving rats were included in the statistics.2.Compared with rats in the control group,the myocardial contractility,ventricular wall motion amplitude and left ventricular ejection fraction(LVEF)of rats in the sepsis groups were decreased,the pathological morphologic alterations of myocardial cell were apparent,and the serum concentrations of c Tn I,LDH,BNP,CKMB,TNF-α,IL-1β,HMGB1,and IL-6 were significantly increased.3.Compared with the L group rats,the LVEF of rats in LLK and LHK groups was significantly increased,the cardiac function and myocardial pathological morphology and structure were significantly improved,and the contents of serum myocardial injury markers c Tn I,BNP,LDH and CK-MB and inflammatory factors TNF-α,HMGB1,IL-6,and IL-1β were significantly decreased.In addition,the improvement of septic myocardial injury in rats in high dose of esketamine group was more significant.Conclusion Esketamine can reduce the septic myocardial injury in rats with anti-inflammatory actions,and high dose esketamine has a more significant protective effect.Part II Esketamine reduces septic myocardial injury by regulating the Nrf2/HO-1 signal pathwayObjective To observe the effects of esketamine on myocardial oxidative stress and Nrf2/HO-1 signal pathway in septic myocardial injury rats.Methods Forty SPF healthy adult male SD rats were randomized to equipartition 4 groups(n=10 each): control group(C group),lipopolysaccharide group(L group),LPS plus 5 mg/kg esketamine group(LLK group)and LPS plus 10 mg/kg esketamine group(LHK group).Same as the first part,LPS-induced septic myocardial injury was induced by injecting lipopolysaccharide 10 mg/kg intraperitoneally.In LLK group and LHK group,5 mg/kg or 10 mg/kg esketamine was intraperitoneally injected after 30 minutes LPS injected,respectively.Twenty-four hours later,the serum concentrations of c Tn I,BNP,LDH,CK-MB and myocardial malondialdehyde(MDA)and superoxide dismutase(SOD)levels of myocardium in rats of each group were measured to determine the degree of myocardial oxidative stress.The expression levels of Nrf2,HO-1 and bric-a’-brac,tramtrack and broad complex and cap’n’collar homology 1(Bach 1,BTB and CNC Homology 1)were detected by immunohistochemistry and Western blotting.Results 1.In this part of the experiment,3 rats in the L group,1 in the LLK group and 1 in the LHK group died within 24 h of modeling,and the surviving rats were included in the statistics.Compared with rats in the control group,the myocardial concentrations of SOD in the sepsis groups were decreased,the serum contents of myocardial injury markers and the myocardial concents of MDA were increased.Compared with rats in the L group,the myocardial concentrations of SOD in the LLK and LHK group were increased,the contents of myocardial injury markers and MDA were decreased.2.Compared with rats in the control group,the myocardial expressions of Nrf2 and HO-1 in the sepsis groups were down-regulated,while the Bach1 were up-regulated.Compared with rats in the L group,the myocardial expressions of Nrf2 and HO-1 in the LLK and LHK group were up-regulated,while the Bach1 were down-regulated.Moreover,the changes of these indicators in high dose esketamine group were more significant.Conclusion Esketamine can effectively reduce the level of oxidative stress in myocardial cells of septic rats,and this effect may be related to the activation of Nrf2/HO-1 endogenous protective antioxidant signal pathway.Part III The roles of necroptosis in esketamine reduction of septic myocardial injuryObjective With the purpose of investigating the roles and possible mechanisms of necroptosis in septic myocardial injury and observing the effects of esketamine on necroptosis in this part.Methods A total of 60 SPF healthy adult male SD rats were randomly assigned to equipartition 6 groups(n=10 each): control group(C group),control plus 10 mg/kg esketamine group(CHK group),lipopolysaccharide group(L group),LPS plus 10 mg/kg esketamine group(LHK group),LPS plus 1 mg/kg receptor interaction protein 1(RIP1)inhibitor(Necrostatin-1)group(LN group),and LPS plus 10 mg/kg esketamine and 1 mg/kg Necrostatin-1 group(LNK group).Same as the first part,LPS-induced septic myocardial injury was induced by injecting lipopolysaccharide 10 mg/kg intraperitoneally.In CHK group and LHK group,10 mg/kg esketamine was intraperitoneally injected after 30 minutes saline or LPS injected,respectively.In LN group and LNK group 1 mg/kg Necrostatin-1 was intraperitoneally injected after 30 minutes LPS or esketamine injected,respectively.Twenty-four hours later,echocardiography was performed to evaluate the cardiac function of the rats.Then,blood samples and cardiac muscle tissue were prepared from the left ventricle for examination of the cardiac myocytes’ morphological changes(by hematoxylin-eosin staining and electron microscopy)and for determination of serum c Tn I,LDH,BNP,CK-MB,TNF-α,IL-1β,and IL-6 level(by enzyme linked immunosorbent assay),and the apoptosis rate of cardiomyocytes(by TUNEL staining),and expression of RIP1、RIP3、P65 and P38(by Western blot).Results 1.In this part of the experiment,3 rats in the L group,1 in the LHK group and 1 in the LN group died within 24 h of modeling,and the surviving rats were included in the statistics.Compared with rats in the control group,the myocardial contractility,ventricular wall motion amplitude and LVEF of rats in the sepsis groups were decreased,the pathological morphologic alterations of myocardial cell were apparent,and the serum concentrations of c Tn I,LDH,BNP,CK-MB,TNF-α,IL-1β,and IL-6 were significantly increased.While compared with the L group rats,the LVEF of rats in LHK,LN and LNK group was significantly increased,the cardiac function and myocardial pathological morphology and structure were significantly improved,and the contents of serum myocardial injury markers and inflammatory factors were significantly decreased.In addition,the improvement of septic myocardial injury in rats in LNK group was more significant.2.The myocardial cell apoptosis was increased in the sepsis groups,while esketamine or Necrostatin-1 did not reduce the apoptosis rate.Compared with rats in the control group,the myocardial expressions of RIP1、RIP3、P65 and P38 in the sepsis groups were up-regulated.While compared with rats in the L group,the expressions of RIP1 、 RIP3 、 P65 and P38 in LHK,LN and LNK group were down-regulated.Moreover,the changes of these indicators in LNK group were more significant.Conclusion Esketamine may play a protective role on septic myocardial injury in rats by inhibiting the trigger of necroptosis and associated inflammatory pathways.