The Effect And Mechanism Of Macrophage Migration Inhibitor Factor In The Early Warning And Prognosis For Myocardial Ischemia Injury

Objective: This study aims to investigate the effect and mechanism of macrophage migration inhibitor factor(MIF)in the early warning and prognosis for myocardial ischemia injury.Firstly,the study explored the value of MIF as a new marker for myocardial injury in early warning,in order to provide the basis for establishment the early risk assessment system for high-risk myocardial injury patients.Secondly,the predictive value of MIF as a biomarker for the clinical outcome of patients with myocardial ischemia injury was analyzed to provide support for the development of clinical intervention strategies.Finally,the effects of MIF on myocardial injury during severe ischemia reperfusion(I/R)were studied by gene interference in mice.Methods:(1)The case-control study consecutively recruited 498 adult patients with ST-elevation myocardial infraction(STEMI)attended the Department of Cardiology,the First Affiliated Hospital of Xinjiang Medical University during the period of January 2013 to June 2017 and underwent PCI within 12 h after onset of chest pain.137 healthy control and 40 stable angina pectoris(SAP)patients were selected as control group.At the same time,70 patients with non-ST-segment elevation myocardial infarction(NSTEMI)and 91 patients with unstable angina pectoris(UA)were selected as positive control.Plasma MIF levels at admission,4h and 24 h after PCI were measured using Quantikine MIF ELISA kits.Myocardial necrosis markers were collected,including peak and area under curve(AUC)of high-sensitivity troponin T(hs-TnT),creatine kinase MB(CK-MB),N-terminal pro-B-type natriuretic peptide(NT-pro BNP)and GRACE scores.The cut-off values of admission MIF level,peak of hs-TnT and NT-pro BNP,AUC of CK-MB,and GRACE score for predicting in-hospital mortality were determined by using the receiver operating characteristics(ROC)plot with the maximal corresponding values of Youden’s index(sensitivity+specificity-1).(2)A prospective cohort study was conducted to follow up 498 STEMI patients.Adverse cardiovascular events(MACE)and mortality were collected during hospitalization and long-term follow-up.The follow-up protocol included phone interview,outpatient visiting and in-hospital clinical records.A stepwise multivariable logistic regression was used to analyze all potential influencing factors associated with in-hospital mortality.Admission MIF level was also analyzed as a continuous variable using restricted cubic splines to assess for a non-linear relationship between admission MIF level and in-hospital MACE.To visualize the relationship between the cutoff value of these parameters and in-hospital mortality and MACE during the follow-up,Kaplan-Meier plots were generated,and the log-rank test was used to compare the resulting curves.Multivariate Cox proportional hazards regression analyses were applied to assess whether the admission MIF level was associated with time-to-mortality during follow-up.(3)Male wild-type(WT)and MIF knock-out(MIFKO)mice aged 12-16 weeks were selected and randomly divided into Sham and I/R group.I/R group were established with 60 min ischemia and 24 h reperfusion in vivo.Sham mice were only open chest without ligation of coronary artery.The in vivo effect of MIF was examined by measuring the infarct size,myocardial cell apoptosis,regional inflammatory response,cardiac function and chronic myocardial remodeling.The influence of MIF in RISK signaling pathway and energy metabolism were studied.The cardiac function of mice after 7 and 28 days of reperfusion was evaluated by echocardiography.Results:(1)(1)Admission MIF levels were elevated in 88.4% STEMI patients over the upper reference limit of healthy controls(56.0ng/mL)and it was 3-7 folds higher than that in SAP and control groups(122±61 vs.39±19 vs.17±8ng/mL,P<0.001).The MIF level of NSTEMI patients was higher,but still was lower than STEMI patients(104±46 vs.122±61ng/mL,P=0.018);(2)A serial plasma test demonstrated that MIF levels at 4h after PCI was slightly further increased(133±70ng/mL),and then declined back to the admission level at 24 h after PCI(120±64ng/mL).These changes did not reach statistical significance compared to admission MIF values(P=0.052);(3)MIF levels were correlated with admission hs-TnT(r=0.156,P=0.003),peak hs-TnT(r=0.154,P=0.003)and CK-MB(r=0.142,P=0.001),AUC of CK-MB(r=0.136,P=0.003)and Gensini score(r=0.125,P=0.005);(4)The MIF levels in deceased patients was significantly higher than that in survivors(211±82 vs.120±58ng/mL,P<0.001)with the highest level in the patient died of cardiac rupture(329.4ng/mL);(5)ROC of MIF for predicting in-hospital mortality in STEMI patients was used.The area under the ROC curve for MIF predicting mortality was 0.820.The recommended cut-off value for MIF based on the maximum of Youden’s index on the ROC curve was 127.8ng/mL and it had 85.7% sensitivity and 62.1% specifi city in predicting in-hospital mortality.The values of area under the ROC plot were similar among these parameters,including hs-TnT,CK-MB,NT-pro BNP and GRACE score(P=NS).Based on the MIF cut-off value,STEMI patients were further divided into the high-and low-MIF level groups.Patients with high MIF levels had higher levels of markers of myocardial necrosis and more severe coronary lesions;(2)(1)After discharged from hospital,484 STEMI patients were followed up with the median durati on of follow-up 39 months(range,12 to 67 months).During the follow-up period,12 patients lost contact and 137 patients(29.0%)developed MACE.Patients with MACE had higher admission MIF levels than those without MACE(137±67 vs.112±53ng/mL,P<0.001);(2)A further stepwise multivariable logistic regression analysis was performed to assess the association between in-hospital mortality and potential confounding factors The admission MIF levels either as a continuous variable(OR 1.6,95% CI: 1.29～2.06)or as a categorical variable(the cut off value of 127.8ng/mL,OR 9.1,95% CI: 1.75～47.25)was an independent predictor for in-hospital mortality.In spline regression models,higher admission MIF level was also associated with a greater risk of adverse outcomes.Kaplan-Meier curves demonstrated that in-hospital mortality was greater in STEMI patients with high-MIF levels than those with low-MIF levels,and the STEMI patients with higher cut-off value of peak hs-TnT,AUC of CK-MB,peak NT-pro BNP and GRACE score also had a greater in-hospital mortality;(3)Kaplan-Meier curves showed that the prevalence of MACE was significantly higher in patients with high-MIF levels than those with low-MIF levels during the 3.2-year follow-up period(P＜0.001)and the higher peak of hs-TnT and NT-pro BNP has the same predictive value for MACE during the 3.2-year follow-up.Multivariate analysis by stepwise Cox proportional hazards regression analysis confirmed that admission MIF levels(either as a continuous variable,natural square-transformed variable or in binary fashion according to the MIF cut-off value: 127.8ng/mL)remained as an independent predictor of MACCE(HR 1.2,95% CI:1.06-1.33 or HR 2.8,95% CI: 1.49-5.57,respectively);(3)(1)I/R(60min/24h)insult resulted in(45±4)% and(32±3)% of infarct size in WT and MIFKO mice,respectively(P=0.045);(2)The enzymetic test showed that LDH activity and plasma levels of Tn I of MIFKO mice after I/R(60min/24h)was lower than WT mice(1597.6±98.0 vs.1280.9±70.6U/L,P<0.05)and(17.2±2.0 vs.21.7±2.4ng/mL,P＜0.05);(3)The apoptotic rate in MIFKO mice was lower than WT mice after I/R injury [(16.5±1.6)% vs(22.4±1.4)%,P<0.05];(4)Immunofluorescence results showed that compared with WT mice,MIFKO could significantly reduce the infiltration of leukocytes and macrophages after I/R injury(P＜0.05);(5)Ecocardiography showed that MIFKO mice could significantly improve left venricular end diastolic volume and ejection fraction at 7 days and 28 days after I/R(P＜0.05);(6)Western blot results showed that MIFKO could significantly promote the phosphorylation of ERK1/2,p70S6 K and Akt protein after I/R(P＜0.05),and it also significantly promoted AMPK and ACC protein phosphorylation.Conclusion:(1)The plasma MIF level of STEMI patients increased rapidly in the early stage of injury,and was closely related to the degree of myocardial injury and coronary artery lesion.MIF and traditional markers of myocardial injury are equally effective in predicting hospital mortality;(2)A higher admission MIF level is an independent predictor for in-hospital mortality and long-term MACE in STEMI patients who underwent PCI.Compared with traditional myocardial necrosis markers,single measurement of MIF level at admission has predictive value for short-term and long-term prognosis,and has advantages of clinical application;(3)Knockout of MIF gene in mice has a protective effect on the heart.It may be related to the activation of RISK and AMPK pathways to promote cell survival and increase myocardial energy supply.Animal study has shown that MIF may mediate the proinflammatory response in myocardial ischemic injury,which results in adverse long-term prognosis.These results suggest that MIF is a potential therapeutic target for improving inflammation and hope to improve the prognosis of STEMI patients.