The Role And Mechanism Of Adropin On Sepsis-induced Myocardial Injury

Section Ⅰ IntroductionSepsis is a life-threatening organ dysfunction caused by disordered response to infection,which is caused by a variety of pathogenic causes,accompanied by excessive activation of inflammatory cells,production of inflammatory factors and multiple organ dysfunction.Myocardial injury is one of the most serious and fatal complications of sepsis,also known as septic cardiomyopathy(sepsis-induced cardiomyopathy,SICM)or septic cardiac dysfunction(Sepsis-induced myocardial dysfunction,SIMD).It is characterized by myocardial systolic and diastolic dysfunction and decreased left ventricular output.Myocardial injury and cardiac dysfunction caused by sepsis further aggravate tissue microcirculation hypoperfusion and trigger a vicious circle,which is an important reason for the high mortality rate of sepsis.Therefore,in-depth interpretation of the molecular mechanism of septic myocardial injury to provide a theoretical basis for screening new treatment targets has important practical significance to improve the therapeutic effect and prolong the survival time of patients with septic myocardial injury.At present,it is believed that the pathogenesis of septic myocardial injury includes the imbalance between pro-inflammatory and anti-inflammatory factors caused by inflammatory cell infiltration,increased oxidative stress,mitochondrial dysfunction,endothelial dysfunction,calcium regulation disorder and so on.Adropin is a polypeptide encoded by energy homeostasis related gene(Energy homeostasis-related gene,Enho),which consists of 76 amino acids.The amino acid sequence is highly conserved among different species.Initial studies found that the function of adropin is to regulate energy metabolism and promote glucose and lipid metabolism.With the deepening of research,more and more scholars have found that adropin plays a unique protective role in the cardiovascular system.In particular,recent studies suggest that adropin is involved in anti-inflammatory and antioxidant stress.However,whether adropin plays a role in septic myocardial injury has not been fully clarified.Previous studies of our group have shown that adropin is involved in the protection of endothelial function in obese adolescents,and can play an anti-ventricular remodeling role through anti-inflammatory and anti-oxidative stress in the animal model of pathological myocardial hypertrophy.Therefore,we speculate that adropin may play an anti-inflammatory and antioxidant stress role in septic myocardial injury.The purpose of this study is to explore the role and mechanism of adropin in septic myocardial injury at molecular,cellular and animal levels.Section II Decreased expression of Adropin in septic myocardial injury model Objective:1)To investigate the expression of Adropin gene in heart tissue of patients with septic cardiomyopathy.2)To establish the model of septic cardiomyocyte injury induced by Lipopolys-accharide(LPS)and to explore the expression level of adropin.3)To establish an animal model of septic myocardial injury and to explore the changes of adropin expression.Methods:1)The GSE79962 data set of septic cardiomyopathy microarray was downloaded from GEO(Gene Expression Omnibus)database by bioinformatics method.The gene expression matrix was obtained by R and Perl software,and the expression value of Enho gene was extracted to analyze the difference of Enho gene expression between septic cardiomyopathy group and normal control group.2)The injury of H9C2 cardiomyocytes was induced by LPS in vitro.The levels of creatine kinase isoenzyme(Creatine Kinase-MB,CK-MB)and lactate dehydrog-enase(Lactate Dehydrogenase,LDH)in the supernatant of cardiomyocytes were detected by automatic biochemical analyzer,the m RNA levels of intracellular IL-6and TNF-α were detected by q RT-PCR,and the level of intracellular adropin protein was detected by Western-blot.3)In vivo,C57BL/6J mice were injected intraperitoneally with LPS to establish sepsis model.Echocardiography was used to evaluate cardiac function.Hematoxylin-eosin staining(Hematoxylin-Eosin staining,HE)and biochemical detection of serum myocardial injury markers(CK-MB and LDH)were used to evaluate the degree of cardiac injury.Western-blot and ELISA were used to detect the expression of adropin in heart tissue and serum.Result:1)Bioinformatics showed that the expression level of Enho gene in heart tissue of patients with septic cardiomyopathy was lower than that of normal controls(P <0.05).2)In vitro,the levels of myocardial injury markers CK-MB and LDH and the expression of IL-6 and TNF-α m RNA in the supernatant of H9C2 cells stimulated by LPS for 24 hours were increased,indicating that the model was established successfully.The expression of adropin protein in cardiomyocytes decreased in LPS model group(P < 0.05).3)In vivo experiment,24 hours after intraperitoneal injection of LPS,echocar-diography showed that the left ventricular ejection fraction was significantly decreased in LPS group(P < 0.05).HE staining and serum CK-MB and LDH detection showed myocardial injury.Serum ELISA showed that the levels of inflammatory factors IL-1β,IL-6 and TNF-α in LPS group were significantly increased,while the serum level of adropin and protein level in heart tissue were significantly decreased(P < 0.05).Conclusions:1)The expression level of Enho gene in cardiac tissue of patients with septic cardiomyopathy was decreased.2)Adropin decreased significantly in septic myocardial injury models in vivo and in vitro,suggesting that it may play an important protective role in septic myocardial injury.Section III Protective effect and mechanism of adropin on myocardial injury induced by lipopolysaccharideObjective:1)To study the role and mechanism of recombinant adropin in cardiomyocyte injury induced by lipopolysaccharide in vitro.2)Animal experiment in vivo to explore the role and mechanism of recombin-ant adropin in lipopolysaccharide-induced myocardial injury in mice.Methods:1)H9C2 cells were treated with bioactive recombinant adropin34-76 pept-ides.The cells were divided into four groups: blank control group(Control group),model group(LPS group),low dose LPS+Adropin group(LPS+Adropin-L),medium dose LPS+Adropin group(LPS+Adropin-M)and high dose LPS+Adropin group(LPS+Adropin-H).The effect of adropin on cell activity was detected by CCK-8 test after being pretreated with different concentrations of LPS+Adropin-M for 24 hours,and the optimum concentration of adropin intervention was determined.2)Cells were divided into four groups: Control group,Adropin group,LPS group and LPS+Adropin group.H9C2 cells were pretreated with Adropin and then induced with LPS(final concentration 10ug/ml)for 24 hours.The level of reactive oxygen species(Reactive Oxygen Species,ROS)in each group was detected by flow cytometry.3)Malondialdehyde(Malondialdehyde,MDA)and superoxide dismutase(Superoxide dismutase,SOD)detection kits were used to detect the levels of MDA and SOD in each group.4)The protein levels of intracellular inflammatory cytokines(IL-6 and TNF-α)and apoptosis related proteins(Bax,Bcl2 and cleaved-caspase3)were detected by Western-blot method.5)Animals were divided into four groups: Control group,Adropin group,LPS group and LPS+Adropin group.The septic myocardial injury model was established by LPS(10mg/kg).In the LPS+Adropin group,30 min was pretreated by tail vein injection of recombinant Adropin,followed by intraperitoneal injection of LPS(10mg/kg)for 24 hours.The cardiac function of mice in each group was evaluated by echocardiography,the levels of serum inflammatory factors were detected by ELISA method,and the levels of CK-MB and LDH in serum were detected by automatic biochemical instrument.6)The levels of MDA and SOD in heart tissue were detected,and the level of ROS in heart tissue was evaluated by Dihydroethidium(DHE)staining.7)Western-blot method was used to detect the protein levels of inflammatory cytokines(IL-1 β,IL-6 and TNF-α)and apoptosis related proteins(Bax,Bcl2 and Cleaved-caspase3)in each group.Result:1)The results of CCK-8 showed that in the LPS-induced H9C2 cell injury model,compared with Control group,LPS significantly decreased H9C2 cell activity,while after pretreatment with recombinant adropin34-76,compared with LPS group,LPS+Adropin-M(100ng/ml)group significantly increased H9C2 cell activity(P < 0.05).2)Flow cytometry showed that the intracellular ROS level in H9C2 group was significantly higher than that in Control group,while the intracellular ROS level in LPS+Adropin group was significantly lower than that in LPS+Adropin group after pretreatment with adropin(100ng/ml)(P < 0.05).3)The results of Western-blot detection showed that the protein levels of IL-6 and TNF-α in H9C2 cells induced by LPS group were significantly higher than those in Control group(P < 0.05),but after pretreatment with adropin(100ng/ml),the protein levels of IL-6 and TNF-α in LPS+Adropin group were significantly lower than those in LPS group(P < 0.05).The results of detection of apoptosis-related proteins(Bax,Bcl2 and Cleaved-caspase3)in each group showed that compared with Control group,the levels of Bax and Cleaved-caspase3 protein in LPS group were significantly higher,while Bcl2 protein was significantly lower(P < 0.05).However,after pretreatment with adropin(100ng/ml),the levels of Bax and Cleaved-caspase3 protein in LPS+Adropin group were significantly lower than those in LPS group,while the level of Bcl2 protein was significantly increased in LPS+Adropin group(P < 0.05).4)Echocardiography showed that left ventricular ejection fraction(LVEF)and left ventricular shortening(LVFS)in LPS group were significantly lower than those in Control group,while LVEF and LVFS in LPS+adropin group were significantly higher than those in LPS group after pretreatment with adropin(P < 0.05).5)The results of HE staining showed that compared with Control group,myocardial edema was obvious in LPS group,with myocardial rupture and a few inflammatory cell infiltration.After pretreatment with adropin,myocardial edema was improved,myocardial arrangement was more orderly,and inflammatory cell infiltration was reduced.6)The results of serum ELISA showed that the levels of IL-1 β,IL-6 and TNF-α in LPS group were significantly higher than those in Control group,but after pretreatment with adropin,the levels of serum inflammatory factors(IL-1 β,IL-6 and TNF-a)in LPS+adropin group were significantly lower than those in LPS group(P < 0.05).7)The results of DHE staining showed that the level of ROS in myocardial tissue in LPS group was significantly higher than that in Control group,but after pretreatment with adropin,the level of ROS in myocardial tissue in LPS+adropin group was significantly lower than that in LPS group(P < 0.05).8)The levels of MDA and SOD in myocardial tissue were measured.The results showed that after intraperitoneal injection of LPS,the content of MDA in myocardial tissue increased significantly,while the level of SOD decreased significantly(P < 0.05).After pretreatment with adropin,the level of MDA in heart tissue of LPS+Adropin group was significantly lower than that of LPS group,while the level of SOD was significantly higher than that of LPS+Adropin group(P < 0.05).9)The results of Western-blot detection of related proteins in mouse myocardial tissue showed that the protein levels of IL-1 β,IL-6 and TNF-α in myocardium were significantly increased after intraperitoneal injection of LPS,while pretreatment with adropin significantly decreased the protein levels of IL-1 β,IL-6 and TNF-α in myocardial tissue induced by LPS(P < 0.05).The results of apoptosis-related proteins(Bax,Bcl2 and Cleaved-caspase3)in cardiac tissue of each group showed that the protein levels of Bax and Cleaved-caspase3 in myocardial tissue of LPS group were significantly higher than those of Control group,while the level of Bcl2 protein was significantly lower than that of LPS group(P < 0.05).However,after pretreatment with adropin(0.2mg/kg),the protein levels of Bax and Cleaved-caspase3 in LPS+Adropin group were significantly lower than those in LPS group,while the protein level of Bcl2 in LPS+Adropin group was significantly higher than that in LPS+Adropin group(P < 0.05).Conclusions:1)At the cellular level in vitro,recombinant adropin34-76 peptide decreased the level of oxidative stress induced by LPS in H9C2 cells,alleviated intracellular inflammation and played an anti-apoptotic effect.2)In vivo animal experiments showed that recombinant adropin34-76 peptide could improve the cardiac function of mice induced by LPS,reduce the level of oxidative stress in heart tissue,reduce inflammatory reaction and reduce apoptosis in heart tissue.Section IV Adropin ameliorates myocardial injury induced by lipopolysaccharide through Nrf2/ARE signal pathwayObjective:1)To explore whether Nrf2/ARE signal pathway is involved in the mechanism of myocardial injury in sepsis.2)To prove that Adropin ameliorates septic myocardial injury by up-regulating Nrf2/ARE signal pathway.Methods:1)At the animal level,animals were divided into Control group,Adropin group,LPS group and LPS+Adropin group.The expressions of Nrf2,Gpx1 and Nqo1 related proteins in myocardial tissue of mice in each group were detected by Western-blot method.2)At the cellular level,cells were divided into Control group,Adropin group,LPS group and LPS+Adropin group.The expression of Nrf2,Gpx1 and Nqo1 protein in each group was detected by Western-blot method.3)The cells were pretreated with Nrf2 inhibitor ML385 for recovery experiment.The cells were divided into Control group,LPS group,LPS+Adropin group and LPS+Adropin+ML385 group.In LPS+Adropin+ML385 group,30 min of H9C2 cells was pretreated with adropin and ML385,and then LPS was added to treat cells for 24 hours.1 the levels of MDA and SOD in cells of each group were detected.2 the level of intracellular ROS in each group was detected by DHE method.3The m RNA expression of inflammatory cytokines(IL-6 and TNF-α)in each group was detected by q RT-PCR method.Western-blot method was used to detect the expression level of related proteins in each group.Result:1)In animal model,the protein levels of Nrf2,Nqo1 and Gpx1 in LPS group were lower than those in Control group,but after pretreatment with adropin,the protein levels of Nrf2,Nqo1 and Gpx1 in LPS+Adropin group were significantly higher than those in LPS group(P < 0.05).2)In the cell model,the protein levels of Nrf2,Nqo1 and Gpx1 in H9C2 cells induced by LPS were decreased,but after pretreatment with adropin,the levels of Nrf2,Nqo1 and Gpx1 in LPS+Adropin group were significantly higher than those in LPS group(P < 0.05).3)Compared with LPS+Adropin group,the intracellular ROS and MDA levels in LPS+Adropin+ML385 group were significantly higher,while the intracellular SOD level was significantly lower(P < 0.05).4)Compared with LPS+Adropin group,the m RNA and protein levels of inflammatory cytokines(IL-6 and TNF-α)in LPS+Adropin+ML385 group increased significantly after adding Nrf2 inhibitor ML385.5)Western-blot results showed that the expression of Nrf2/ARE pathway related proteins(Nrf2,Nqo1 and Gpx1)in LPS+Adropin+ML385 group was significantly lower than that in LPS+Adropin group after adding Nrf2 inhibitor ML385.6)Confocal results showed that the expression of Nrf2 in cytoplasm and nucleus of LPS group was lower than that of Control group.Pretreatment with Adropin could increase the expression of Nrf2 in cytoplasm and nucleus of cells.However,after administration of Nrf2 inhibitor ML385,the level of Nrf2 in cytoplasm and nucleus of LPS+Adropin+ML385 group was lower than that of LPS+Adropin group.Conclusions:1)Nrf2/ARE signal pathway is involved in the occurrence and development of myocardial injury in sepsis.2)Adropin may improve the oxidative stress of cardiomyocytes by up-regulating Nrf2/ARE signal pathway and play a protective role in cardiomyocyte injury induced by LPS.