Protective Role And Mechanisms Of Recombinant Human Angiotensin-converting Enzyme 2 In Sepsis-induced Cardiac Dysfunction

Sepsis is defined as lethal organ dysfunction induced by dysregulated host’s response to confirmed or suspected infection,which can cause disa-bility even death.Evidences accumulated over the past decades show a high incidence of sepsis,especially in the intensive care unit（ICU）all over the world.Though the understanding and management of sepsis have been impro-ving during the past years due to advances in medical science,the reported mortality remains as high as 40%.Sepsis-induced cardiac dysfunction（SICD）is a common complication of sepsis,which is characterized by reversible systolic and/or diastolic left and/or right ventricular dysfunction which is inconsistent with the coronary area.In patients with SICD,the mortality is increased 2-3 times,which is reported as approximately 70-90%.Precise me-chanism and effective therapy in patients with SICD remain unclear.Depre-ssive myocardial inhibitors,dysregulated Ca²⁺,adrenergic disorder,imbalan-ced metabolic alteration and mitochondrial dysfunction derived from and contributed back to the dysregulated inflammation and disturbed redox cycle were all demonstrated to participate in the development of myocardial depres-sion,injury or necrosis which lead to cardiac dysfunction finally.However,interventions based the available mechanisms showed limited therapeutic value in clinical settings.Further exploration is needed to improve its manage-ment and outcomes.The renin-angiotensin-aldosterone system（RAAS）includes classic path-way in which angiotensin II（Ang II）is the main effector and non-classic RAAS pathway in which angiotensin 1-7（Ang 1-7）plays the key role.Both pathways are expressed in various cells in heart including cardiomyocytes,fibroblasts,endothelia,and immune cells.Ang II is known to promote heart failure through inducing cardiac hypertrophy,fibrosis,and ventricular remo-deling depending on its pro-inflammatory effects.On the contrary,Ang 1-7 in heart exerts anti-inflammatory effect and cardiac protective role opposite to Ang II.Angiotensin-converting enzyme 2（ACE2）,which can inactivate Ang II by catalyzing it to Ang 1-7 shows cardiac beneficial effects.Evidences indicated that Ang II is enhanced in sepsis and involved in the pathophy-siology of organ injury.During sepsis,Ang II leads to an up-regulation of pro-inflammatory cytokines such as tumor necrosis factor-α（TNF-α）and interleu-kin-1（IL-1）,participates activation of nucleotide-binding domain,leucine-rich-repeat containing family,pyrin domain-containing 3（NLRP3）inflam-masome and induces macrophage infiltration,microvascular ischemia,and organ damege finally including cardiac dysfunction.Treatment targeted to balance the disturbed RAAS in septic animal models show anti-inflammatory effects and are beneficial to heart function and survival.Angiotensin-conver-ting enzyme 2（ACE2）,which is a homolog of angiotensin-converting enzyme（ACE）,is a negative regulator of RAAS by converting Ang II to Ang 1-7.A recombinant human ACE2（rh ACE2）has been found to have protective effect on cardiac dysfunction and injury in both inflammatory and non-inflam-matory cardiac injury models and to be safe,with no negative hemodynamic effects in healthy volunteers and in patients with acute respiratory distress syndrome（ARDS）.The NLRP3 inflammasome,which consists of 3 members,namely apoptosis-associated speck-like protein containing a CARD（ASC）,NLRP3,and pro-caspase-1,is a vital mediator pathway of the innate immune system and has been shown to be involved in development of SICD.And Ang II is verified to activate the NLRP3 inflammasome through nuclear factor-κB（NF-κB）pathway.Therefore,we aimed to investigate the association between Ang II plasma concentration and development of SICD in patients with sepsis as well as their prognosis.To explore the role of Ang II in the development of SICD,we established a mouse model of LPS-induced cardiac dysfunction by LPS intraperitoneal injection.On detecting the changes of Ang II and NLRP3inflammasome activation as well as the followed effects including inflam-mation,cell pyroptosis and systolic function of left ventricular after treatment with rh ACE2 for LPS-induced cardiac dysfunction,we objected to clarify the role of rh ACE2 on SICD and the probable signal pathways of it,which is expected to give new idea to the understanding and treatment to SICD.Part One Correlation analysis between level of angiotensin II and SICD in patients with sepsisObjective:To investigate the correlation between the plasma concen-tration of Ang II and development of SICD on evaluating the differences of Ang II level in plasma among non-septic patients,patients with sepsis and patients with SICD.Methods:Patients admitted to ICU of Cangzhou Central Hospital from February 1,2022 to January 31,2023 who met the criteria of Sepsis 3.0 were enrolled.The septic patients were divided into sepsis group and SICD group depending on occurrence of SICD or not.Patients with stroke but without sepsis were enrolled as control group.Basic clinical data were collected.Plasma concentrations of Ang II and cytokines including IL-1βand TNF-αwere detected.Heart function parameters of patients including LVEF,LVFS,and E/A were detected through bedside ultrasound.Independent t test,rank and column chi-square test,one-way ANOVA or non-parametric test,pearson or spearman correlation analysis,and binary logistic regression was used to analyze the data.Results:1.A total of 204 patients with sepsis admitted to the three departments were enrolled continuously and 90 patients were included to complete all the analysis according to the exclusion.Depending on occurance of SICD or not56（60%）cases and 34（40%）cases were distributed into sepsis group and SICD group respectively.Accoring to the same exclusion,20 patients with stroke admitted to EICU of neurology branch were enrolled as control.2.Compared with the control group,patients in the sepsis group and the SICD group all had infection and showed higher PCT,WBC,APACHE II and SOFA score,heart rate,lactate and kidney function paremeters（P<0.05）.The blood pressure and oxygenation index of the the patients in the sepsis group and SICD group were lower than those in the control group（P<0.05）.ICU stay of patients in the sepsis group and the SICD group were longer than those in the control group（P<0.05）.Compared with the control group,plasma concen-trations of Ang II,IL-1βand TNF-αin patients of the sepsis group and SICD group increased significantly（P<0.001）.3.Distribution of infection sources,PCT,WBC,oxygenation index,kid-ney function paremeters and ICU stay in the sepsis group and SICD group were comparable（P>0.05）.Compared with the sepsis group,the patients in the SICD group showed higher APACHE II and SOFA scores,lower blood pressure and higher lactate（P<0.001）.4.Compared with the sepsis group,plasma levels of Ang II,IL-1βand TNF-αincreased significantly（P<0.001 or P<0.05）.Ang II level was positi-vely correlated with BNP,c Tn I and IL-1βlevels in plasma（r_s=0.5198,P<0.001;r=0.579,P<0.001;r_s=0.562,P<0.001,respectively）according to the correlation analysis.5.The 28-day-mortality in SICD group was significantly higher than that in the sepsis group（P<0.05）.According to the binary logistic regression analysis,APACHE II score,plasma concentrations of BNP,TNF-αand Ang II,and occurance of SICD were the the independent risk factors of 28-day-mortality of patients with sepsis.Summary:1.Plasma concentrations of Ang II were significantly higher in patients with sepsis than those without sepsis.2.The situations of patients with SICD were more critical with longer ICU stay;In patients with SICD,plasma concentrations of c Tn I,BNP,Ang II and IL-1βwere significantly higher than those in patients with sepsis only;Ang II plasma level was positively correlated with BNP,c Tn I and IL-1βplasma levels.3.The short-term outcomes of patients with SICD were poorer indicated by higher 28-day-mortality.APACHE II score,plasma concentrations of Ang II and TNF-α,and occurance of SICD were the independent risk factors of28-day-mortality of patients with sepsis.Part Two Change of angiotensin II in plasma in mice with SICDObjective:To explore the changes of plasma concentration of Ang II in SICD by establishing mouse model of SICD.Methods:We built mice with SICD with healthy,pathogen-free,and adult-male C57BL/6N mice by LPS 10mg/Kg intraperitoneal injection.Left ventricular cardiac function including LVEF and left ventricuar fraction shortening（LVFS）were evaluated at 3 hours and 6 hours after LPS admini-stration.At 6 hours after LPS or saline administration,all mice were sacrificed for blood and myocardial tissue collection when cardiac function re-evalu-ation was finished.Cardiac troponin I（c Tn I）,TNF-α,IL-1βand Ang II in plasma were measured with commercial enzyme-linked immunosorbent assay（ELISA）kits.Myocardium injury was investigated by hematoxylin and eosin（HE）stained heart tissue sections and scored with a semi-quantitative method.Paired and independent t test were used to compare the changes or differences among groups of the parameters.Results:1.LPS intraparitoneal injection at dose of 10mg/kg modulated the clinical manifestations of sepsis.Three hours after LPS injection,the mice had mental depression,loose and disordered hair,reduced activity,reduced eating and drinking water,weakened responsiveness to stimulation,accelerated heart rate and respiratory rate,chills,viscous secretions on eyelids,diarrhea.2.LVEF and LVFS decreased significantly at 3 hours after LPS injec-tion（P<0.0001）,and decreased further at 6 hours after LPS injection.Com-pared with those in the control group,LVEF and LVFS of mice in the LPS group at 3 hours and at 6 hours after LPS injection were all lower（P=0.001;P<0.0001,respectively）.3.Plasma concentration of c Tn I in the LPS group was significantly higher than those in the control group（P<0.0001）.Mice in the LPS group showed obvious myocardial tissue damage and myocardial injury scores were significantly higher than those in the control group（P<0.0001）.4.The plasma concentrations of TNF-α,IL-1βand Ang II in the LPS group were significantly higher than those in the control group（P<0.0001）.Summary:1.LPS intraparitoneal injection at dose of 10mg/kg can establish mice model of SICD successfully.Mice with SICD showed significantly decrease of LVEF and LVFS accompanied by significantly myocardial injury.2.TNF-α,IL-1βand Ang II in plasma increased significantly in mice with SICD induced by LPS injection.Part Three Effects of recombinant human angiotensin-converting enzy-me 2 on myocardial injury and cardiac function by modula-ting levels of angiotensin and inflammationin mice with SICDObjective:On supplementing rh ACE2 through intraperitoneal injection,we aimed to explore the effects of rh ACE2 on myocardial injury and contrac-tility in mice model of SICD.Methods:In the preliminary part,two different doses of rh ACE2（100ug/kg and 200ug/kg respectively）were administrated to mice with SICD to determine the appropriate dose.Results showed that rh ACE2 at dose of 200ug/kg improved cardiac dysfunction in mice with SICD and was adopted in the following experiments.In the following experiment,adult male C57BL/6N mice were chose and allocated to the control group,LPS group and LPS+rh ACE2 group（n=6）randomly.At 3 hours after LPS injection,the mice in the three groups received ultrasound investigation and then 0.9%saline,0.9%saline and rh ACE2 200ug/kg injection respectively.After Re-evaluation by ultrasound at 6 hours after LPS injection,all mice were sacrificed.And samples of blood and heart tissue were collected and stored at appropriate temperature.Plasma concentrations of c Tn I,Ang II,Ang 1-7,TNF-α,and IL-1βwere measured with commercial ELISA kits.Myocardium injury was investigated by HE stained heart tissue sections and scored with a semi-quantitative method.Results:1.Accoring to the preliminary experiment,dose of 200ug/kg intraperi-toneal injection improved cardiac function and septic manifestation in mice with SICD（P=0.002）,which was adopted in the following experiments.2.Mice in the LPS group showed signs of sepsis,which were improved sinificanlty in LPS+rh ACE2 group.3.LVEF and LVFS in the LPS group were significantly lower than those in the control group（P<0.0001）.And LVEF and LVFS in the LPS+rh ACE2group were significantly improved（P=0.001）.4.Plasma concentration of c Tn I in the LPS group was significantly higher than those in the control group（P=0.001）.And plasma concentration of c Tn I in the LPS+rh ACE2 group were significantly decreased（P=0.027）.Mice in the LPS group showed obvious myocardium injury,myocardial injury score was significantly higher than those in the control group（P<0.0001）.Com-pared with those in the LPS group,changes of myocardial tissue morphology and myocardial injury score in the LPS+rh ACE2 group improved significantly（P=0.003）.5.Plasma concentrations of Ang II（P<0.001）and Ang 1-7（P=0.001）in LPS group were significantly higher than those in the control group.Plasma concentration of Ang II in LPS+rh ACE2 group was significantly inhibited（P=0.001）,while plasma concentration of Ang 1-7 in LPS+rh ACE2 group was further increased（P=0.007）.6.Plasma concentrations of TNF-αand IL-1βin the LPS group were significantly higher than those in the control group（P<0.0001）.And plasma concentrations of TNF-α（P=0.011）and IL-1β（P=0.038）in the LPS+rh ACE2group were significantly inhibited.Summary:1.Rh ACE2 administration ameliorated myocardial injury and improved left ventricular systolic function in mice with SICD.2.Rh ACE2 administration decreased Ang II level and further increased Ang 1-7 level in plasma in mice with SICD.3.Rh ACE2 administration reduced TNF-αand IL-1βlevel in plasma in mice with SICD.Part Four Effects of recombinant human angiotensin-converting enzy-me 2 on activation of NLRP3 inflammasome and related in-flammatory cytokines and cell pyroptosis in mice with SICDObjective:On administrating rh ACE2 to mice with SICD,we objected to investigate the effects of rh ACE2 on activation of NLRP3 inflammasome in mice with SICD and the probable signaling pathways involved in.Methods:Mice were grouped and treated in the same way to the third part of the experiment.Blood and heart tissue were collected in the same way to the third part of the experiment.IL-1βand IL-18 in plasma were detected by ELISA kits.Protein expression of NLRP3,Caspase-1 and GSDMD activa-tion,and related signaling pathways involved in NLRP3 inflammasome acti-vation（including NF-κB,p38 MAPK and AMPK-α1 pathways）in heart tissue were investigated by western blots（WB）.Cell pyroptosis rates in heart tissue were examined with terminal deoxynucleotidyl transferase（Td T）d UTP nick-end labeling（TUNEL）staining.One-way ANOVA was used to analyzed the differences of the paremeters among three groups followed by a post hoc analysis between two groups by least significance difference（LSD）test or Tamhane’s T2 test.Correlation between plasma concentration of Ang II and expression in heart tissue of NLRP3 was analyzed by pearson analysis.Results:1.Protein expression of NLRP3,activation of Caspase-1 and GSDMD in heart tissue of mice in the LPS group were significantly higher than those in control group（P<0.0001）;and protein expression of NLRP3（P=0.025）,activ-ation of Caspase-1（P=0.037）and GSDMD（P=0.006）in LPS+rh ACE2 group were significantly inhibited compared with those in LPS group.2.Plasma concentrations of IL-1βand IL-18,the inflammatory factors matured through NLRP3 inflammasome activation,increased significantly in the LPS group compared with those in control group（P<0.0001）,and Plasma concentrations of IL-1β（P=0.038）and IL-18（P=0.011）were significantly inhibited in the LPS+rh ACE2 group compared with those in LPS group.3.TUNEL staining of heart tissue sections showed that pyroptosis cell rates in the LPS group increased significantly compared with those in control group（P<0.001）;and compared with those in LPS group,pyroptosis cell rates decreased significantly in LPS+rh ACE2 group（P=0.001）.4.Significant positive correlation between Ang II in plasma and NLRP3expression in heart tissue was revealed by correlation analysis（r=0.884,P<0.0001）.5.The levels of activated NF-κB and p38 MAPK in heart tissue in the LPS group were significantly higher than those in control group（P<0.0001）.And the evels of activated NF-κB（P=0.033）and p38 MAPK（P=0.001）in heart tissue in the LPS+rh ACE2 group were significantly lower than that in the LPS group.On the contrary,levels of activated AMPK-α1 decreased in the LPS group（P<0.0001）recovered partly in the LPS+rh ACE2 group（P=0.014）.Summary:1.Rh ACE2 decreased NLRP3 inflammasome related infulammatory factors（IL-1βand IL-18）in plasma and inhibited activation of NLRP3inflammasome through suppression of NF-κB and p38 MAPK pathways and enhancement of AMPK-α1 pathway in mice with SICD.2.NLRP3 expression in heart tissue were positively correlated with the plasma concentration of Ang II,indicating the inhibitive effects of rh ACE2 on NLRP3 inflammasome activation may be related to the decrease of Ang II.Part Five Effects of angiotensin 1-7 blocker on the role of rh ACE2 on NLRP3 suppression in heart tissue and cardiac protection in mice with SICDObjective:On administrating A779,the specific Mas receptor（Mas R）blocker of Ang 1-7,to mice with SICD who accepted rh ACE2 treatment,we aimed to investigate the influence of A779 on cardiac protective role of rh-ACE2 on SICD and to explore whether change of Ang 1-7 in plasma is invol-ved in the effects of rh ACE2 on SICD.Methods:Mice were randomly allocated to the LPS group,LPS+rh-ACE2 group and LPS+rh ACE2+A779 group（n=6）.At 3 hour after LPS injec-tion,after echocardiographic examination all mice in the four groups were treated with saline,saline,rh ACE2 and A779+rh ACE2 respectively.After Re-evaluation of the LVEF and LVFS at 6 hours after LPS injection,the mice were sacrificed for plasma and myocardial tissues collection.Plasma concen-tration of c Tn I was detected with ELISA kit.Myocardium injury was investi-gated through HE stained heart tissue sections and scored with a semi-quanti-tative method.Cell pyroptosis rates in heart tissue were examined with ter-minal deoxynucleotidyl transferase（Td T）d UTP nick-end labeling（TU-NEL）staining and calculated with Image J system.NLRP3 expression in heart tissue was investigated by western blot（WB）.One-way ANOVA was used to analyz the differences of the paremeters four groups followed by a post hoc analysis between two groups by least significance difference（LSD）test or Tamhane’s T2 test.Results:1.The LVEF and LVFS in LPS group were significantly lower than those in control group（P=0.003）and were significantly improved in the LPS+rh ACE2 group（P=0.016）.While compared with those in the LPS+rh ACE2 group,the LVEF and LVFS in the LPS+rh ACE2+A779 group deter-iorated significantly（P=0.028）.2.Plasma concentration of c Tn I in the LPS group was significantly higher than that in the control group（P=0.002）and was significantly decrea-sed in the LPS+rh ACE2 group（P=0.033）.While compared with that in LPS+rh ACE2 group,plasma concentration of c Tn I in the LPS+rh ACE2+A779group increased significantly（P=0.04）.HE stained heart tissue sections in the LPS group showed obvious myocardium injury and injury score was signifi-cantly higher than that in the control group（P<0.0001）.Compared those in the LPS group,the changes of myocardial tissue morphology,myocardial injury score in the LPS+rh ACE2 group improved significantly（P=0.008）.While in the LPS+rh ACE2+A779 group,myocardial injury and injury score significant higher myocardial injury score deteriorated significantly compared with those in the LPS+rh ACE2 group（P=0.014）.3.Cell pyroptosis rates in heart tissue of mice in LPS group increased significantly than those in the control group（P<0.0001）and decreased signifi-cantly in the LPS+rh ACE2 group（P=0.021）.While in the LPS+rh ACE2+A779 group,cell pyroptosis rates in heart tissue of mice increased signifi-cantly compared with those in the LPS+rh ACE2 group（P=0.031）.4.NLRP3 expression in heart tissue in the LPS group was significantly higher than those in the control group（P<0.0001）,and was significantly lower than those in the LPS+rh ACE2 group（P=0.03）.While compared with those in the LPS+rh ACE2 group,NLRP3 expression in the LPS+rh ACE2+A779group increased again（P=0.04）.Summary:Not only A779 attenuated the inhibitive effects of rh ACE2on NLRP3 expression and cell pyroptosis enhancement in mice with LPS-induced cardiac dysfunction,but also it mitigated the protective role of rh ACE2 on cardiac injury and cardiac systolic function,which indicated that increase of Ang 1-7 due to rh ACE2 treatment may account for the cardiac protective role of rh ACE2 in mice with SICD.Conclusions:1.In patients with SICD,plasma concentrations of Ang II,IL-1βand TNF-αwere significantly higher than patients in sepsis group.Their situations were more critical,ICU stay and 28-day mortality were significantly increa-sed.Ang II and inflammatory factors in plasma were significantly higher and were related to occurance of SICD and increase of 28-day mortality.2.The way of LPS intraperitoneal injection into C57BL/6N mice simu-lated the manifestation of sepsis and replicated the conditions of SICD.The characteristics of mice with SICD included decline of LVEF and LVFS,myo-cardium injury indicated in HE stained heart tissue section,increase of myo-cardial injury indicator and inflammatory cytokines in plasma,and increase of levels of Ang II and Ang1-7 in plasma,which were consistent with what were observed in patients with sepsis in clinical part of the study.3.The activation of NLRP3 inflammasome and cell pyroptosis in heart tissue increased significantly in mice with SICD.Rh ACE2 inhibited the activ-ation of NLRP3 inflammasome by suppressing NF-κB and p38 MAPK path-ways and enhancing AMPK-α1 pathway in mice with SICD,through which rh ACE2 mitigated the inflammatory response and cell pyroptosis in heart,relieved myocardial injury and improved cardiac function.4.Rh ACE2 decreased the level of Ang II in plasma in mice with SICD,and correlation analysis showed that the NLRP3 expression in heart tissue correlated with the level of Ang II in plasma positively,which indicated that the protective effects of rh ACE2 may partly derived from its catalyzing action of Ang II.5.Not only A779,the specific Mas R blocker of Ang 1-7,attenuated the inhibitive effects of rh ACE2 on NLRP3 expression and cell pyroptosis enhan-cement in mice with SICD,but also it mitigated the protective role of rh ACE2on cardiac injury and systolic function,which indicated that the increase of Ang 1-7 due to rh ACE2 treatment may account for the cardiac protective role of rh ACE2 in mice with SICD.In summary,rh ACE2 plays a protective role in SICD,ameliorating cardiac injury and dysfunction through inihibiting NF-κB and p38 MAPK signaling pathways and enhancing the AMPK-α1 signaling pathway,thus inhibiting NLRP3 inflammasome ativation depending on converting Ang II to Ang 1-7.