Role Of MicroRNA-125b In Sepsis Induced Cardiac Dysfunction

BackgroundSepsis is defined as Systemic Inflammatory Response Syndrome(SIRS)which caused by definite or suspected infection.Sepsis will lead to severe sepsis,septic shock and multiple organ dysfunctions(MODS)with futher development.Heart is one of the easily vulnerable organs in sepsis,about 50%of sepsis patients exhibit inhibition of myocardial function in the early stage of sepsis.Cardiac dysfunction is is one of the serious complications of sepsis.Sepsis induced cardiac dysfunction has the characteristics of high incidence and high mortality,the mortality of patients who have cardiac dysfunction will be increased from 20%to 70%-90%.The research on sepsis induced cardiac dysfunction is developing,however the pathological and physiological basis of its occurrence and development is unclear.Pathogenesis of sepsis induced cardiac dysfunction mainly include cascade amplification of the inflammatory response,apoptosis of myocardial cells,endothelial dysfunction,energy metabolism disorder of cardiomyocytes and so on.We are also lack of effective prevention and treatment of sepsis induced cardiac dysfunction nowdays.MicroRNAs(miRNA)have become a research hotspot in recent years,and they are non-coding single stranded RNAs.MiRNAs play roles in negative regulating gene expression through the post transcriptional level.MiR-125b is a homologous gene of Lin-4 which is the first discovered miRNA.MiR-125b plays an important role in cell proliferation,apoptosis,differentiation and inflammatory response.It has been shown that miR-125b inhibits cell apoptosis through direct interaction with the target gene p53 and Bak.Researches have also confirmed that activation of NF-?B(Nuclear factor-Kappa B)inhibits the expression of miR-125b.In addition,the expression of miR-125b decreased after TLR4 ligand lipopolysaccharide(LPS)stimulates Raw264.7 cells,while overexpression of miR-125b can inhibite tumor necrosis factor-?(TNF-?)expression.Our groups have found that miR-125b protects against myocardial ischaemia/reperfusion injury via targeting TRAF6 and preventing NF-?B binding activity.On the other hand,miR-125b inhibites apoptosis of myocardial cells by targeting p53 mediated apoptotic signalling.The inflammatory response mediated by TRAF6/NF-?B signaling pathway and apoptosis of myocardial cells play important roles in sepsis induced cardiac dysfunction.However,little is known about the role and mechanism of miR-125b in sepsis induced cardiac dysfunction,and further study will be helpful to provide new strategies for prevention and treatment of sepsis.ObjectiveThis study will be divided into three parts.Firsly we will determine the protective or destructive role of miR-125b in sepsis induced cardiac dysfunction(Part ?).And then we will further explore the mechanism of the role of miR-125b played in sepsis induced cardiac dysfunction by animal study(Part ?)and cell study(Part ?).We will focus on weather inflammatory response and apoptosis will play an important role in it.Part ? Effect of microRNA-125b on sepsis induced cardiac dysfunctionMethod1.Examination of miR-125b level in LPS induced J774a.1 and H9c2 cellsJ774a.1 macrophages and H9c2 rat cardiomyoblasts were divided into untreated group(Con)and lipopolysaccharide treated group(LPS),cells were stimulated with 100ng/ml LPS for 12 hours.The level of miR-125b in these two groups were evaluated by quantitative real-time PCR(qRT-PCR).2.Examination of miR-125b level in CLP induced mice heart and serumCecal ligation and puncture(CLP)was used to establish the sepsis in vivo model.Mice were divided into sham operation group(Sham)and surgery group(CLP).The serum and cardiac tissue of mice were collected 6 hours after operation.The changes of miR-125b expression in these two groups were evaluated by qRT-PCR.3.Mice treatment and experimental groupsTransfection of lentivirus miR-125b(LmiR-125b)and negative control lentivirus miR-Con(LmiR-Con)into mouse hearts:Mice were transfected with one hundred microliters(about 1x 108 IU)of LmiR-125b or LmiR-Con through the right carotid artery into heart.We select C57BL/6J mice aged 10-12 weeks,and they were randomly assigned to six groups:injected no lentivirus but had sham operation group(Untransfected+Sham),injected no lentivirus but had CLP operation group(Untransfected+CLP),injected LmiR-Con and 7 days later had sham operation group(LmiR-Con+Sham),injected LmiR-Con and 7 days later had CLP operation group(LmiR-Con+CLP),injected LmiR-125b and 7 days later had sham operation group(LmiR-125b+Sham),injected LmiR-125b and 7 days later had CLP operation group(LmiR-125b+CLP)4.Detection of efficiency of infection in mice heart with LmiR-125b and LmiR-ConExpression of miR-125b in mouse myocardium was detected by immunofluorescence of green fluorescent protein(GFP)and qRT-PCR.5.Detection of mice cardiac functionMice cardiac function was assessed by echocardiography 6 hours after CLP operation.6.Comparison of mice survival rateMice in Untransfected+CLP,LmiR-Con+CLP and LmiR-125b+CLP 3 groups were compared with 7 days survival rate,using log-rank test for statistical analysis.ResultJ774a.1 macrophages and H9c2 rat cardiomyoblasts were stimulated by LPS for 6h,the expression of miR-125b was decreased in both macrophages and H9c2 cells.The expression of miR-125b was also decreased in mice myocardium and serum after CLP operation.These indicate that miR-125b participate in sepsis development.CLP operation was used to establish sepsis in vivo model,results of echocardiography showed that CLP operation induced cardiac dysfunction and significantly decreased ejection fraction(EF%)by 30.6%,fractional shortening(FS%)by 38.8%,stroke volume(SV)by 63.8%,and cardiac output(CO)by 72.0%compared with sham operation group.In contrast,transfection of LmiR-125b into the myocardium significantly attenuated CLP induced cardiac dysfunction.Compared with untransfected+CLP group,LmiR-125b+CLP group increased EF%by 32.6%,FS%by 43%,SV by 71.4%and CO by 75.9%respectively.Overexpression of miR-125b significantly improved the survival rate of mice following CLP.Transfection of LmiR-Con did not alter CLP induced cardiac dysfunction and mortality outcome.Conclusions1.Polymicrobial sepsis significantly decreases the levels miR-125b in mice myocardium and in circulation.2.Overexpression of miR-125b attenuates mice cardiac dysfunction following CLP sepsis.3.Overexpression of miR-125b improves mice survival following CLP sepsis.Part? Mechanisms of microRNA-125b in attenuating sepsis induced cardiac dysfunction(In vivo study)Method1.Detection of inflammatory cytokines in mice circulationLevels of TNF-? and IL-1? in mice serum were detected by enzyme linked immunosorbent assay(Elisa)2.Detection of macrophages and neutrophils infiltration and adhesion molecules in myocardiumImmunohistochemisty were used to detect the infiltration of macrophages and neutrophils into mice myocardium.Western blot was used to detect the levels of intercellular cell adhesion molecule-1(ICAM-1)and vascular cell adhesion molecule-1(VCAM-1)in myocardium.3.Detection of NF-?B binding activity and TRAF6 in myocardiumThe binding activity of NF-?B and the expression of TRAF6 in mice myocardium were detected by electrophoretic mobility shift assay(EMSA)and western blot respectively.4.Detection of myocardial apoptosisTunel assay was used to count the apoptosis of myocardial cells in mice.Activity of Caspase-3/7 and Caspase-8 in myocardium was detected by Caspase-Glo assay kit.5.Detection of p53,Bax and Bak expression in mice myocardiumWestern Blot was used to detect the expression of p53,Bax and Bak in mice myocardium.ResultsCLP operation was used to establish sepsis in vivo model.Six hours after CLP,the levels of TNF-? and IL-1? markedly increased in mice serum compared with sham group.In contrast,transfection of LmiR-125b significantly suppressed TNF-?and IL-1? production in serum by 55%? 29.7%(P<0.01).Overexpression of miR-125b also significantly reduced the expression of ICAM-1 and VCAM-1 in mice myocardium following CLP.CLP significantly increased the infiltration of macrophages and neutrophils in myocardium compared with sham group.However,overexpressing of miR-125b markedly attenuated CLP induced infiltration of macrophages and neutrophils into the myocardium.In addition,CLP significantly increased the binding activity of NF-?B in septic mice myocardium,while transfection of LmiR-125b into the myocardium decreased CLP induced NF-?B binding activity by 27.1%(P<0.01)compared with LmiR-Con group.Overexpression of miR-125b decreased the expression of TRAF6 in presence or absence of CLP.The expression of TRAF6 in LmiR-125b+CLP group decreased by 35.5%compared with LmiR-Con+CLP group(P<0.01).In addition,The expression of TRAF6 in LmiR-125b+sham group decreased by 39.5%compared with LmiR-Con+Sham group(P<0.01).CLP markedly increased myocardial apoptosis by 5.7 fold compared with sham control(P<0.01),while overexpression of miR-125b significantly reduced myocardial apoptosis following CLP.In addition,the results showed that 6h after CLP,Caspase-3/7 and Caspase-8 activity significantly increased in myocardium compared with sham group.However transfection of LmiR-125b attenuated sepsis induced Caspase-3/7 and Caspase-8 activity by 26.1%(P<0.05)? 23.2%(P<0.01).Overexpression of miR-125b significantly inhibited the CLP induced expression of p53,Bak and Bax in myocardium.Conclutions1.MiR-125b attenuates sepsis induced cardiac dysfunction via inhibiting the expression of TRAF6 and reducing the NF-?B binding activity to effectively inhibit excessive inflammatory response in myocardium.2.MiR-125b attenuates sepsis induced cardiac dysfunction via targeting p53 mediated myocardial apoptotic signalling.Part? Mechanisms of microRNA-125b in attenuating sepsis induced cardiac dysfunction(In vitro study)Method1.Endothelial cells transfected with miR-125b mimicsMiR-125b mimics were transfected into human vein endothelial cells(HUVECs)so that miR-125b overexpressed in endothelial cells.2.HUVECs treatment and experimental groupsHUVECs stimulated with 100ng/ml LPS to constructed sepsis in vitro model.Cells were devided into 6 groups:did't transfected with mimics either stimulated with LPS(Untransfected+Con),did't transfected with mimics but stimulated with LPS(Untransfected+LPS),transfected with miR-Con mimics for 48 hours but did't stimulated with LPS(miR-Con+Con),transfected with miR-Con mimics for 48 hours and then stimulated with LPS(miR-Con+LPS),transfected with miR-125b mimics for 48 hours but did't stimulated with LPS(miR-125b+Con),transfected with miR-125b mimics for 48 hours and then stimulated with LPS(miR-125b+LPS).3.Detection of inflammatory cytokines released by endothelial cellsTNF-? and IL-1? in endothelial cell culture supernatant was determined by enzyme linked immunosorbent assay(Elisa).4.Detection of adhesion molecules in endothelial cells.The expression of intercellular adhesion molecule-1(ICAM-1)and vascular cell adhesion molecule-1(VC AM-1)in endothelial cells was detected by Western Blot.5.Detection of NF-?B binding activity and TRAF6 expression in endothelial cellsThe binding activity of NF-?B and expression of TRAF6 in endothelial cells were detected by Electrophoretic Mobility Shift Assay(EMSA)and Western Blot respectively.ResultsHUVECs were transfected with miR-125b and miR-Con mimics for 48 hours and then stimulated with LPS for 12h.The contents of TNF-?,IL-1?and IL-6 in culture medium were significantly increased after stimulated by LPS in HUVECs.Overexpression of miR-125b reduces TNF-? and IL-6 release from the LPS-stimulated HUVECs by 44.8%and 13%(P<0.01)compared with miR-con group.Overexpression of miR-125b didn't change LPS induced increased levels of IL-1? in culture medium.In addition,LPS increased protein expression of ICAM-1 and VCAM-1 in HUVECs,while miR-125b overexpression inhibited such induction by 21.3%(P<0.05)and 27.4%(P<0.01)respectively.Overexpressing miR-125b also significantly inhibited LPS induced NF-?B binding activity in HUVECs.In addition,overexpressing miR-125b reduced the protein expression of TRAF6 in HUVECs in the presence or absence of LPS.Compared with untransfected+Con group,the protein expression of TRAF6 in miR-125b+Con group decreased by 22%(P<0.05).Additionally,expression of TRAF6 in miR-125b+LPS group decreased by 23.9%(P<0.05)when compared with untransfected+LPS groupConclusion1.Overexpression of miR-125b inhibits LPS induced inflammatory response in HUVECs.2.Another important mechanism of miR-125b prevent sepsis induced cardiac dysfunction is reduced inflammatory response to protect against endothelial cells dysfunction,at least in part via targeting TRAF6 and inhibiting NF-kB binding activity.