The Mechanism Of IL-37 Ameliorates Sepsis-induced Myocardial Dysfunction

BackgroundSepsis,a complex organ dysfunction syndrome induced by infection,is a high mortality disease that occupied public health concern in global.Sepsis-induced myocardial dysfunction(SIMD)is one of the most severe complications of sepsis.Currently,the therapy of SIMD is still limited to conventional managements.With the in-depth research and understanding of the pathogenesis of sepsis-induced myocardial dysfunction,potential drugs or therapies for SIMD are being proposed,but they are still in studies.Therefore,finding interventions targeting to SIMD pathogenesis is still the point of researches.Immune dysfunction has been shown to be the core pathogenesis of sepsis and SIMD.In the early stage of sepsis,pathogenic substances such as lipopolysaccharide(LPS)were recognized by Toll-like receptors(TLR)of macrophages,making numerous secretions of pro-inflammatory factors and chemokines,which aggravates the inflammatory response in the body.Excessive inflammatory response destroys the metabolism of cells to a certain extent,induces apoptosis,aggravates tissue ischemia and ultimately aggravates tissue damage.However,the role and specific mechanisms of macrophages in SIMD are still unclear,and further exploration is needed.Interleukin-37(IL-37),a new number of IL-1 family,plays a protective role in a variety of inflammatory diseases,autoimmune diseases and tumors by inhibiting immunity.Extracellularly,IL-37 binds to receptors IL-18Ra and IL-1R8 to form a complex that transduces anti-inflammatory signals though inhibiting NF-κB and MAPK pathway,thereby inhibiting NLRP3 and AIM inflammasomes,ultimately reducing tissue damage.However,the role and specific mechanism of IL-37 in SIMD are still unclear.Therefore,we put forward hypothesis:IL-37 can reduce the myocardial injury induced by sepsis though NF-κB pathway and NLRP3 inflammasome,playing a protective role in sepsis-induced myocardial dysfunction.In vivo,we took C57BL/6J mice as the research object,established sepsis models by intraperitoneal injection of LPS with intervention by intraperitoneal injection of IL-37.At the same time,In vitro,we took RAW264.7 macrophage cell line and H9C2 cardiomyocyte cell line as the research objects,stimulated RAW264.7 cells with LPS and IL-37.and cultured.H9C2 cells were intervened with IL-37 and incubated with the conditioned medium which from the supernatant of RAW264.7 cells stimulated with LPS.Aim to explore the role and mechanism of IL-37 on myocardial injury in SIMD.Objective1.Intraperitoneal injection of LPS to establish sepsis model,with intraperitoneal injection of IL-37 to evaluate the systemic inflammatory response though survival rate and the inflammatory cytokines expression levels in serum.2.Evaluate the cardiac function and myocardial inflammatory injury though echocardiography and cardiac histopathology,aiming to estimate the effect of IL-37 on SIMD.3.Explore the effect of IL-37 on NF-κB pathway and NLRP3 inflammasome by in vitro experiments,aiming to estimate the effect of IL-37 on myocardial inflammatory injury.Materials and methods1.General condition and survival rate analysisA total of 60 male C57BL/6J mice,4-6 weeks old,were randomly divided into 4 groups:control group,IL-37 group,LPS group,LPS+IL-37 group.Mice in the LPS group were intraperitoneally injected with LPS(20mg/kg).Mice in the IL-37 group were intraperitoneally injected with IL-37(1mg/kg).Mice in the IL-37 group were intraperitoneally injected with LPS and IL-37 simultaneously.Mice in the control group received the same dose of normal saline.Feeding for 7 days,observed and recorded the activity,hair,diet and other general conditions and the survival of the mice to construct the survival curve.2.Animal experimentsAnother 48 mice were randomly divided into 4 groups according to step 1,and were anesthetized and sacrificed after 24 hours for further research.Mice were anesthetized by isoflurane inhalation,and cardiac hemodynamic parameters were measured by echocardiography,to evaluate the cardiac function.Routine anesthesia and made pathological sections of myocardial tissue.HE staining and immunohistochemical staining were performed to evaluate the level of myocardial tissue damage,inflammatory responses and infiltration of macrophages.Serum samples were collected for ELISA to detect the expression levels of inflammatory cytokine.Western blot was used to detect the expression levels of NLRP3,Caspase1 and ASC in total protein of cardiac tissue to evaluate the expression levels of NLRP3 inflammasome in myocardial tissue.3.Cell experiments3.1 RAW264.7 cells were stimulated with LPS(1 μg/ml)at different time gradients.Total protein was collected to detect the phosphorylation of NF-κB by western blot.RAW264.7 cells were treated with different concentration gradients of IL-37.Western blot detected the phosphorylation of NF-κB to evaluate the effect of IL-37 on the phosphorylation of NF-κB.3.2 RAW264.7 cells were divided into 4 groups:control group,IL-37 group,LPS group and LPS+IL-37 group,was treated with LPS and IL-37.Nuclear protein and plasma protein were collected respectively,to detect the nuclear translocation of NFκB by western blot.Total protein was collected to detect the expression level of upstream protein of NF-κB pathway.Total RNA was collected for RT-PCR to detect the expression levels of IL-1β IL-6 and TNF-α mRNA.3.3 Conditional medium was mixed by RAW264.7 cell supernatant which were collected from step 3.2,and 10%DMEM at a ratio of 1:1.H9C2 cells were incubated by conditional medium and treated with IL-37 to explore the influence of macrophages on myocardial cell.Total protein was collected to detect the expression levels of NLRP3 inflammasome relating proteins by western blot.Results1.IL-37 improves the general condition and survival of sepsis miceThe mice in the control group and IL-37 group were in good condition:active activity,smooth and soft hair,good diet,sensitive reaction capacity and good mental state.The mice in LPS group were in poor condition:weak condition,dim and rough hair,less diet,sluggish reaction capacity and poor mental state.This poor condition was particularly remarkable within 3-4 days after receiving LPS stimulation and then gradually recovered.The general condition of mice in LPS+IL-37 group was between the above two,remarkable within 2-3 day and gradually recovered at the forth day.The survival rate of mice in control group and IL-37 group was up to 100%.2.IL-37 reduced the expression levels of inflammatory cytokines in serum of sepsis miceComparing with the expression levels of IL-1β,IL-6 and TNF-α in serum from control group,these in serum from LPS group were increased.While the expression levels of IL-1β,IL-6 and TNF-α in serum from LPS+IL-37 group were all decreased.3.I1-37 alleviated cardiac dysfunction and attenuated the myocardial injury of sepsis miceCompared with the cardiac function of mice in control mice,LVEF and FS of mice in LPS group were decreased and LVID(s)was increased.However,LVEF and FS were increased and LVID(s)decreased in LPS+IL-37 group.Further,the HE staining of myocardial tissues showed,in control group,the cells were arranged regularly,with no necrosis and edema in tissue.As for LPS group,the results showed that cells were arranged in disorder,and the sarcolemma was ruptured,accompanying by necrosis interstitial hyperemia,edema and infiltration of inflammatory cells in tissue.These pathological damages were alleviated in LPS+IL-37 group.4.IL-37 attenuated inflammation and macrophage infiltration in myocardial tissue of sepsis miceCompared with control group,the expression levels of IL-1β,IL-6 and TNF-αwere increased in LPS group,and the number of F4/80-positive cells was increased.However,the expression levels of 1L-1β,IL-6,TNF-α and the F4/80-positive cells were decreased in LPS+IL-37 treatment group.5.LPS promoted NF-κB phosphorylation in time-dependent while IL-37 inhibited NF-κB phosphorylation in concentration-dependent in RAW264.7 cellsLPS increased the phosphorylation of NF-κB in RAW264.7 cell line in a timedependent manner,with the most significant effect was at 60 min.IL-37 decreased LPSinduced phosphorylation of NF-κB in a concentration-dependent manner.The effect was most obvious in LPS+IL-37(1 μg/ml)group.Therefore,60min was selected for LPS stimulation,and 1μg/ml was the selected for the concentration of IL-37 to be used in following experiments.6.IL-37 inhibited LPS-induced the activation of NF-κB pathway and expression of inflammatory cytokines in RAW264.7 cellsCompared with control group,the nuclear translocation of NF-κB and phosphorylation levels of IκBα and IKKα/β were increased in LPS group.While the nuclear translocation of NF-κB and phosphorylation levels of IκBα and IKKα/β were decreased in LPS+IL-37 group.Furthermore,the results of RT-PCT showed,compared with control group,the expression levels of IL-1β,IL-6 and TNF-α mRNA in LPS group were up-regulated.The expression levels of IL-1β and IL-6 mRNA in LPS+IL37 group were down-regulated.However,the expression levels of TNF-α mRNA in the LPS+IL-37 group and LPS group has no statistically different.7.IL-37 reduced the expression level of NLRP3 inflammasome in H9C2 cells cultured in conditional mediumCompared with those in control group,the expression levels of NLRP3,Caspase1 and ASC were increased in LPS group.Further,the expression levels of NLRP3,Caspase1 and ASC in LPS-CM group were higher than those in LPS group.In addition,the expression levels of NLRP3 and ASC in IL-37+LPS-CM group were decreased compared with those in LPS-CM group.The expression level of ASC in LPS+IL-37 group was also decreased compared with LPS group.But there was no significant difference in NLRP3 expression between LPS+IL-37 group and LPS group.There was no statistically difference in the expression of Caspase1 between IL-37+LPS-CM group and LPS-CM group,nor between LPS+IL-37 group and LPS group.8.IL-37 inhibited the expression level of NLRP3 inflammasome in myocardial tissue of sepsis miceImmunohistochemical staining of myocardial tissue showed that,compared withcontrol group,the expression level of NLRP3 in LPS group was significantly increased.However,the expression level of NLRP3 in LPS+IL-37 group was decreased.Furthermore,western blot of myocardial tissue showed that the expression levels of NLRP3,Caspase1 and ASC in LPS group were increased,compared with control group.The expression levels of NLRP3,Caspase1 and ASC in LPS+IL-37 group were all decreased.Conclusions1.IL-37 reduces systemic inflammation and mortality in sepsis mice2.IL-37 ameliorates myocardial dysfunction,reduces the infiltration of macrophages and alleviates myocardial inflammatory injury in sepsis mice.3.IL-37 reduces LPS-induced macrophages inflammatory responses and sepsisinduced myocardial inflammatory injury though inhibiting NF-κB pathway and NLRP3 inflammasome.