The Cardio-Protective Role And Mechanism Of Hydrogen Sulfide In Sepsis-Induced Myocardial Dysfunction

Sepsis-induced myocardial dysfunction（SIMD）,which is a complication in sepsis,mainly characterized for myocardial systolic dysfunction,cardiac enlargement and decreased left ventricular ejection fraction（LVEF）,is the leading cause of death in patients with sepsis.Up to date,the mechanism for SIMD has not been fully elucidated.Inflammation is the cornerstone of sepsis,recent studies have shown that lipopolysaccharide（LPS）could induce endoplasmic reticulum stress（ERS）by activating Toll-like receptor 4（TLR4）and ERS plays an important role in multiple organ injury in sepsis,which becomes a focused filed in recent years.Hydrogen sulfide（H₂S）is the third gaseous mediator alongside carbon monoxide（CO）and nitric oxide（NO）.Studies have documented that H₂S,which is synthesized in mammalian tissues via endogenous enzymes,participates in many physiological systems process and plays an important role in the pathophysiology of some diseases.Until now,the role of H₂S in inflammation is still controversial.Could H₂S improve the cardiac function in SIMD by alleviating the inflammatory response and ERS through inhibiting TLR4?Up to now,there have been no reports of effect of H₂S on the pathogenesis of SIMD.Our study used echocardiography,hematoxylin-eosin staining（HE staining）,enzyme-linked immunosorbent assay（ELISA）and Western blotting（WB）technology,from the function,morphology,protein expression and other aspects,to estimate the effect of H₂S on cardiac function of SIMD and explore its mechanism.Then,we further evaluate the effect of exogenous H₂S on LPS induced myocardial dysfunction,to provide new ideas for the treatment of SIMD.This study is divided into five parts.Part one Clinical observation of the relationship between hydrogen sulfide and cardiac function in patients with sepsis-induced myocardial dysfunctionObjective:To observe the relationship between plasma H₂S level and cardiac function of SIMD patients.Methods:Ten SIMD patients in intensive care unit（ICU）of the fourth hospital of Hebei Medical University were selected as SIMD group,and ten healthy people for health examination in the same hospital were recruited as Control group.All patients gave their written informed consent.Demographics and clinical characteristics were recorded after allocating.Then collected blood was to determine white blood cells（WBC）,cardiac troponin I（cTnI）,creatine kinase（CK）,tumor necrosis factor-α（TNF-α）,interleukin-1beta（IL-1β）and H₂S concentrations in plasma.Cardiac ultrasound examination was done as soon as possible,and LVEF and left ventricular fractional shortening（LVFS）were measured for cardiac function index.Results:1.There was no difference in sex,age and weight between the two groups,all participants had no previous cardiac history.SIMD patients had obvious focus of infection,three patients were respiratory infection,four patients were abdominal infection,two patients were urinary tract infection,and one patient was enteric infection.Compared with the Control group,the body temperature,heart rate,respiratory speed,WBC were increased in the SIMD group,and LVEF is no more than 50%,which were consistent with the clinical diagnosis of SIMD.2.Compared with Control group,cardiac function changes in SIMD group were mainly manifested as left ventricular dilation,ventricular wall motion weakening,LVEF and LVFS decreased significantly,suggesting that SIMD patients mainly present with left ventricular systolic dysfunction.3.Compared with the Control group,plasma cTnI and CK levels were significantly increased in the SIMD group,indicating the existence of myocardial injury.4.Compared with the Control group,plasma TNF-αand IL-1βlevels were significantly increased in SIMD group.5.Compared with the Control group,plasma H₂S level was significantly decreased in SIMD group.6.There was a significant positive correlation between plasma H₂S level and LVEF as well as LVFS.Conclusions:SIMD patients mainly manifested as myocardial contractility dysfunction,accompanied by myocardial injury,increased inflammatory factor levels,and decreased endogenous H₂S level.In addition,endogenous H₂S levels were positively correlated with LVEF and LVFS.It is speculated that endogenous H₂S may be involved in the pathogenesis of SIMD and has a protective effect on the cardiac function of SIMD patients.Part two Reduction of endogenous hydrogen sulfide participated in the pathogenesis of LPS induced myocardial dysfunction of miceObjective:To observe the relationship between endogenous H₂S level and cardiac function of SIMD mice.Methods:C57BL/6J mice and CSE knock-out（CSE KO）mice were used in this part of the experiment.Replicating SIMD animal model by intraperitoneally inject of LPS（10mg/kg）.Six hours after intraperitoneal injection of LPS,observing the effect of LPS on the cardiac function of mice by echocardiography,the morphological changes of myocardial tissue of mice by HE staining,detecting cTnI,CK,TNF-αand IL-1βconcentrations in the plasma of mice,detecting H₂S concentrations in plasma and heart tissues.Furthermore,we also detected the three endogenous H₂S synthases activity and protein expressions in heart tissues of mice.Results:1.Compared with Control group,echocardiography showed that LVEF and LVFS were decreased significantly after 6h of intraperitoneal injection of LPS 10mg/kg in LPS group,and plasma cTnI,CK,TNF-αand IL-1βlevels were increased significantly.Inflammatory changes were also observed in heart tissues of LPS mice by HE staining.2.Compared with the Control group,H₂S levels of plasma and heart tissues were significantly dereased after 6h of intraperitoneal injection of LPS10mg/kg in LPS group.3.Compared with Control group,3-MST activity and CSE,CBS,3-MST protein expressions in heart tissues were significantly decreased after 6h of intraperitoneal injection of LPS 10mg/kg in LPS group.However,CSE&CBS activity in heart tissues of the two groups had no statistical difference.4.Compared with the WT+LPS group,LVEF and LVFS were decreased significantly in the CSE KO+LPS group,while plasma cTnI and CK levels were increased and the inflammatory changes in the myocardium were aggravated.Conclusions:Six hours after intraperitoneal injection of LPS 10mg/kg,we observed that cardiac ultrasound index was decreased,but inflammatory cytokines and myocardial enzymes were increased,inflammatory changes were observed in heart tissues of mice.The above results indicated that we successfully replicated the mouse model of SIMD.H₂S levels of plasma and heart tissues were decreased in SIMD mice,accompanied by reducing of3-MST activity and CSE,CBS and 3-MST protein expressions in heart tissues.Compared with WT mice,CSE knockout mice showed worsen cardiac function after giving LPS,which confirmed that endogenous H₂S participated in the pathophysiology of SIMD,and had cardioprotective effect.Part three Exogenous hydrogen sulfide could improve cardiac function f LPS induced myocardial dysfunction miceObjective:To observe the relationship between exogenous H₂S and cardiac function of SIMD mice.Methods:C57BL/6J mice and CSE knock-out（CSE KO）mice were used in this part of the experiment.Replicating SIMD animal model by intraperitoneally inject of LPS（10mg/kg）,NaHS was injected intraperitoneally after 3h of LPS injection.Six hours after LPS injection,echocardiography was used to observe the effect of NaHS on cardiac function in mice,and HE staining was used to observe the changes of myocardial histomorphology in mice.CK and cTnI levels of plasma,H₂S levels of plasma and heart tissues were detected.Results:1.Compared with LPS group,echocardiographys showed no obvious changes in LPS+10μmol/kg NaHS group,while LVEF and LVFS were significantly increased in LPS+50μmol/kg NaHS group and LPS+100μmol/kg NaHS group.However,there were no differences between LPS+100μmol/kg NaHS group and LPS+50μmol/kg NaHS group.Therefore,the dosage of50μmol/kg NaHS was selected for the subsequent experiment.2.Compared with Control group,echocardiographys showed that LVEF and LVFS were decreased significantly after 6h of intraperitoneal injection of LPS 10mg/kg in LPS group,plasma cTnI and CK levels were increased significantly,following with myocardial tissue swelling and inflammatory cell infiltration.Given 50 mol/kg NaHS could increase LVEF and LVFS in LPS+NaHS group and reduce plasma cTn I and CK levels,and ameliorate morphological changes in heart tissues.3.Compared with Control group,H₂S levels of plasma and heart tissues were significantly decreased after 6h of intraperitoneal injection of LPS10mg/kg in LPS group,while giving 50μmol/kg NaHS could increase H₂S levels in plasma and heart tissues.4.Compared with WT+LPS group,LVEF and LVFS were significantly decreased in CSE KO+LPS group,50μmol/kg NaHS could not but 100μmol/kg NaHS could advance LVEF and LVFS in CSE KO+LPS+NaHS group.Therefore,the dosage of 100μmol/kg NaHS was selected for the subsequent experiment.5.Compared with WT+LPS mice,echocardiographys showed that LVEF and LVFS were significantly decreased after 6h of intraperitoneal injection of LPS 10mg/kg in CSE KO+LPS group,plasma cTnI and CK levels were significantly increased,and inflammatory changes of myocardial tissue got worsen.Given 100μmol/kg NaHS could increase LVEF and LVFS in CSE KO+LPS+NaHS group,and reduce plasma cTnI,CK levels,and ameliorate morphological changes in heart tissues.Conclusions:We have found that exogenous supplementation of NaHS50μmol/kg could improve cardiac function in C57 SIMD mice.We observed a significant increase in LVEF,LVFS and H₂S levels of plasma and heart tissues,a decrease in plasma cTnI,CK levels and alleviated inflammatory changes in the heart.However,50μmol/kg NaHS could not ameliorate cardiac function in CSE KO SIMD mice,while 100μmol/kg NaHS treatment was shown to increase LVEF,LVFS and reduce plasma cTnI and CK levels and alleviate the inflammatory changes of myocardium in SIMD mice.It is proved that exogenous supplementation of H₂S had cardioprotective effect.Part four Hydrogen sulfide reduced LPS induced inflammation and ndoplasmic reticulum stress by inhibitiong TLR4Objective:to observe and discuss the mechanism of exogenous H₂S cardioprotective effect on SIMD.Methods:In this part,we detected plasma TNF-αand IL-1βlevels,and measured TLR4 and ERS-related markers protein expressions in heart tissues.Results:1.Compared with Control group,plasma TNF-αand IL-1βlevels were significantly increased after 6h of intraperitoneal injection of LPS 10mg/kg,giving 50μmol/kg NaHS could reduce plasma TNF-αand IL-1βlevels in LPS+NaHS group.2.Compared with Control group,TLR4 protein expression in heart tissues was increased significantly after 6h of intraperitoneal injection of LPS 10mg/kg in LPS group,giving 50μmol/kg NaHS could decrease TLR4protein expression in the heart tissues.3.Compared with Control group,the protein expressions of CHOP,Caspase-12,PERK,pPERK,IRE,pIRE,ATF6 were increased significantly after 6h of intraperitoneal injection of LPS 10mg/kg in LPS group,giving50μmol/kg NaHS could reduce the expression of the above indexes in heart tissues.There was no difference in GRP78 expression between the three groups.4.Compared with WT+LPS group,plasma TNF-αand IL-1βlevels were increased siginificantly after 6h of intraperitoneal injection of LPS10mg/kg in CSE KO+LPS group,giving 100μmol/kg NaHS could reduce the plasma levels of TNF-αand IL-1βin CSE KO+LPS+NaHS group.5.Compared with WT+LPS group,TLR4 protein expression in heart tissues was higher after 6h of intraperitoneal injection of LPS 10mg/kg in CSE KO+LPS group,giving 100μmol/kg NaHS could reduce the TLR4expression of heart tissues in CSE KO+LPS+NaHS group.6.Compared with WT+LPS group,the protein expressions of CHOP,Caspase-12,PERK,pPERK,IRE,pIRE,ATF6 were increased significantly after 6h of intraperitoneal injection of LPS 10mg/kg in CSE KO+LPS group,giving 100μmol/kg NaHS could reduce the expression of the above indexes in heart tissues.There was no difference in GRP78 expression between the three groups.Conclusions:LPS could upregulate the protein expressions of TLR4 and ERS markers such as CHOP,Caspase-12,PERK,p PERK,IRE,pIRE,and ATF6 of heart tissues in C57 mice,and increase the levels of TNF-αand IL-1βin plasma.Compared with WT mice,the above indexes increased more higher in CSE knockout mice.Giving 50μmol/kg and 100μmol/kg NaHS could respectively downregulate TLR4 and the ERS marker expression of myocardial tissues in WT mice and CSE KO mice.It is confirmed that exogenous H₂S could reduce LPS induced inflammation and ERS by inhibiting TLR4 and play cardioprotective role in SIMD.Part five Hydrogen sulfide alleviated renal damage in sepsis associatedacute kidney injuryObjective:To observe the effect of H₂S on renal function in sepsis associated acute kidney injuryMethods:In this part,ten sepsis associated acute kidney injury（SA-AKI）patients in ICU of the fourth Hospital of Hebei Medical University were selected as AKI group,and ten healthy people for health examination in the same hospital were recruited as Control group.All patients gave their written informed consent.Demographics and clinical characteristics were recorded after allocating.Then we collected patients’blood to determine the levels of TNF-α,IL-1β,serum creatinine（Cre）,blood urea nitrogen（BUN）and H₂S in plasma,and observed the relationship between Cre,BUN and H₂S.After that,C57BL/6J mice were given an intraperitoneal injection of 5mg/kg LPS to replicate the mouse model of AKI,and 50μmol/kg NaHS was given after 3h of LPS injection.Six hours after the injection of LPS,we detected the levels of TNF-α,IL-1β,Cre and BUN in plasma,and HE staining was used to observe the morphological changes in kidney tissues.We also detected the H₂S levels in plasma and kidney tissues,and activities and protein expressions of the three H₂S synthases in kidney tissues,inorder to observe the effect of LPS and NaHS on renal function in mice.Results:1.There was no difference in sex,age and weight between the two groups.All patients with AKI were infected with Gram-negative bacteria（covering 2 cases from Acinetobacter baumannii,2 cases from Pseudomonas aeruginosa,2 cases from Klebsiella,3 cases from Escherichia coli and 1 case from Clostridium difficile）,four patients were respiratory infection,three patients were abdominal infection,one patient were urinary tract infection,and two patients was enteric infection.All participants had no previous cardiac history,and renal injury caused by renal and post-renal factors has been excluded.Compared with the Control group,plasma Cre,BUN,TNF-α,IL-1βand WBC levels were increased in the AKI group,which were consistent with the clinical diagnosis of AKI.2.Compared with the Control group,the plasma H₂S level significantly decreased in AKI group,and H₂S level was negatively correlated with Cre and BUN levels.3.Compared with Control group,the levels of Cre,BUN,TNF-αand IL-1βin plasma were significantly elevated in AKI group after 6h of intraperitoneal injection of LPS 5mg/kg.Renal pathological changes such as glomerular atrophy,cell vacuolar degeneration,tubular dilation,tube formation and inflammatory cells infiltration were observed in AKI group,and kidney injury score was decreased.We have successfully replicated the mouse model of AKI.4.Compared with Control group,the H₂S levels in plasma and kidney tissues were significantly decreased in LPS group after 6h of intraperitoneal injection of LPS 5mg/kg,accompanied with reductions of 3-MST activity and protein expression in kidney tissue.5.Treatment of 50μmol/kg NaHS could increase the H₂S levels of plasma and kidney tissues in AKI group.6.Treatment of 50μmol/kg NaHS could reduce the plasma levels of Cre,BUN,TNF-αand IL-1βin AKI group,and improve the renal histom-orphology changes as well as renal injury score.Conclusions:In clinical trials,plasma levels of Cre,BUN,TNF-αand IL-1βwere increased in plasma,and H₂S levels of plasma and kidney tissues were decreased in AKI patients.H₂S levels were negatively correlated with Cre and BUN levels.In mice,intraperitoneal injection of LPS 5mg/kg could successfully complicate the mouse model of AKI.The H₂S levels of plasma and kidney tissues were decreased in AKI mice,companied with reduction of3-MST activity and protein expression in kidney tissues.It is confirmed that endogenous H₂S play an important role in the pathogenesis of AKI induced by LPS.Treatment of 50μmol/kg NaHS could increase H₂S levels of plasma and kidney tissues,and reduce the levels of TNF-α,IL-1β,Cre and BUN in plasma and ameliorate renal histomorphology.It is proved that H₂S could ameliorate the kidney injury induced by LPS through reducing inflammatory reaction.