Clinical Study On Association Between IMAR And The Severity Of Coronary Artery Stenosis And Experimental Study On Relationship Of Hepcidin And Diabetic Cardiomyopathy

BackgroundAcute coronary syndrome(ACS)is a very serious type of coronary heart disease,which is a clinical syndrome that caused by acute myocardial ischemia.That the rupture or erosion of coronary unstable plaque causes thrombosis is the pathology of ACS.Assessing the severity of coronary artery disease is the key step to make a clinical decision which is vital for the treatment of ACS.Coronary angiography is the gold standard for finding the location and assessing the extent of coronary stenosis,but it is invasive,risky,and costly,limited by the conditions of the medical institution and the level of the operator.Therefore,it is of great clinical significance to find a non-invasive and early detectable biomarker for assessing the severity of coronary artery stenosis.Ischemia modified albumin(IMA)is a marker of myocardial ischemia discovered in recent years,which is transformed from human serum albumin(ALB)that flows through ischemic tissue.The amino terminus of serum albumin contains a unique amino acid sequence which is a binding site for transition metals such as copper,nickel and cobalt.When myocardial ischemia or ischemia-reperfusion occurs,the production of oxygen free radicals in ischemic tissue increases.Oxygen free radicals cause a structural change in the amino terminus of serum albumin,resulting in disability of albumin to bind to cobalt,copper,nickel and other transition metal ions.The albumin that underwent these structural changes is called ischemia-modified albumin.IMA rapidly increases in a few minutes after myocardial ischemia,and is highly sensitive in early diagnosis of myocardial ischemia.However,the predictive value of IMA in the extent of coronary artery stenosis remains controversial.The method of IMA is to use the albumin cobalt binding(ACB)test,which is affected by serum albumin levels.Albumin has a certain antioxidant effect and can bind to free radicals,and low albumin concentration is correlated with the occurrence of atherosclerosis.Therefore,studies have used the ischemic modified albumin/serum albumin ratio(IMA/ALB ratio,IMAR)as an indicator of IMA to assess oxidative stress levels and disease severity.There are no studies to investigate whether IMAR is associated with the severity of coronary lesions.Therefore,we designed this case-control study to explore the clinical value of IMAR in the assessment of severity of coronary artery stenosis.Purposes(1)To investigate the serum IMA and IMAR levels in patients of ACS;(2)To study the association between IMAR and the severity of coronary artery stenosis;(3)Compare the value of IMA and IMAR in predicting the severity of coronary artery stenosis.Materials and MethodsAccording to the inclusion criteria and exclusion criteria,219 hospitalized patients with acute coronary syndrome diagnosed by Qilu Hospital of Shandong University,including 167 patients with unstable angina pectoris and 52 patients with acute myocardial infarction,enrolled the trial.At the same time,56 patients with no coronary stenosis or stenosis<50%showed by coronary angiography are involved in the control group.We collected medical records information of patients during hospitalization.,including general conditions,medical history,laboratory indexes and medication history before coronary angiography,as well as coronary angiography results.The Gensini score was calculated based on the angiographic results,and IMAR was calculated based on serum IMA and ALB levels.The data were analyzed by IBM SPSS Statistics 24 software.P<0.05 was considered statistically significant.Result(1)There was no significant difference in gender and age between the control group,UA group and AMI group.Compared with the control group,SBP was significantly increased in the UA group(P<0.01)and the AMI group(P<0.05),and the heart rate was significantly lower in the AMI group(P<0.05).Compared with the control group,the ratio of AMI patients with hypertension(P<0.05)increased,while the ratio of UA and AMI patients with diabetes(P<0.001)was significantly increased.In terms of laboratory indexes,HDL-c(P<0.01)was significantly lower in the UA group than in the control group,and there was no statistically significant difference in other laboratory indexes between the two groups.Compared with the control group,FBG(P<0.05),CK-MB(P<0.05),cTnI(P<0.001),AST(P<0.001),WBC(P<0.001),NEU(P<0.001)in the AMI group,NEU%(P<0.01)were significantly increased,while HDL-c(P<0.001)was significantly lower.There was no significant difference in other laboratory indexes between the two groups.Compared with the UA group,the FBG(P<0.05),CK-MB(P<0.01),cTnI(P<0.001),AST(P<0.001),WBC(P<0.001),and NEU(P<0.001),NEU%(P<0.01)in AMI group was significantly increased,while HDL-c(P<0.01)and ALB(P<0.01)were significantly decreased.About the history of medication,patients who has used aspirin(P<0.001),clopidogrel/ticagrelor(P<0.001),? blockers(P<0.001),ACEI/ARB(P<0.01),diuretics(P<0.001),nitrates(P<0.05),statin(P<0.001)significantly increased,while AMI group used aspirin(P<0.001),clopidogrel or Tigrelor(P<0.001),beta blockers(P<0.001),ACEI/ARB(P<0.01),diuretics(P<0.001),nitrates(P<0.05),statins The ratio of drug(P<0.001)and LMWH(P<0.001)in the UA group was more the control group.Compared with the UA group,the ratio of patients who has used ACEI/ARB(P<0.01)and LMWH(P<0.001)was significantly increased in the AMI group.(2)IMA was positively correlated with age(r=0.161,P=0.009),with TC(r=-0.140,P=0.023),TG(r=-0.167,P=0.007),HDL-c(r=-0.244,P<0.001),ALT(r =-0.176,P=0.004),RBC(r =-0.289,P<0.001),and Hb(r =-0.328,P<0.001)showed a negative correlation.HDL-c(?=-0.242,P<0.001),TG(?=-0.126,P=0.034),Hb(?=-0.347,P<0.001),NEU%(?=-0.163,P=0.007),clopidogrel/ticagrelor(?=-0.143,P =0.015)were independent factors affecting IMA.IMAR was associated with age(r=0.186,P=0.002),with HDL-c(r=-0.234,P<0.001),ALT(r=-0.178,P=0.004),RBC(r=-0.304,P<0.001)and Hb(r =-0.367,P<0.001)showed a negative correlation.HDL-c(?=-0.257,P<0.001),Hb(p=-0.347,P<0.001),PLT(p=-0.163,P=0.005),ALT(p=-0.263,P=0.003),AST(?=0.312,P<0.001),clopidogrel/ticagrelor(P=-0.143,P=0.013)were independent factors affecting IMAR.(3)Compared with the control group,there was no significant difference in IMA and IMAR between the UA group,and the IMA(P<0.01)and IMAR(P<0.001)increased in the AMI group.There were no significant differences in IMA and IMAR between the UA and AMI group.Besides,after controlling the confounding factors,the result is the same.(4)There was no significant correlation between IMA and Gensini scores in the ACS group.There was a positive correlation between IMAR and Gensini scores(r=0.215,P=0.009).IMAR(?=0.180,P=0.009)and age(?= 0.180,P=0.009)were independent influence factors for the Gensini score.Conclusion(1)The levels of IMA and IMAR in patients with acute myocardial infarction were higher than those in patients with unstable angina and without coronary heart disease.(2)There was no significant difference in IMA and IMAR between patients with unstable angina and control subjects.(3)There was no significant correlation between IMA and severity of coronary artery stenosis,and IMAR was positively correlated with the severity of coronary artery stenosis,which could reflect the severity of coronary artery lesions.BackgroundDiabetes mellitus is a chronic metabolic disease that gravely threatens human health globally,which is estimated to affect more than 592 million people worldwide by 2035.Diabetic Cardiomyopathy(DCM)is one of the severe cardiovascular complications of diabetes that have contributed tremendously to high morbidity of heart failure and high mortality among diabetic patients.Therefore,prevention and treatment of DCM has prominently clinical significance.DCM is characterized by diastolic dysfunction,the main pathological changes of which include cardiomyocyte apoptosis and myocardial fibrosis.The mechanism of the occurrence and regulation of these changes is complicated and yet not fully clarified.Available evidence suggests that oxidative stress plays an important role in both cardiomyocyte apoptosis and myocardial fibrosis in DCM.Iron is an essential metal element for nearly every living organism,participates in biological processes.However,as redox active metal,free iron generate a large amount of reactive oxygen species(ROS)through Haber-Weiss reaction and Fenton reaction to induce oxidative stress.Iron overload cardiomyopathy(IOC)is reported to be the main cause of death in patients with thalassemia and sickle-cell anemia that are dependent on frequent blood transfusions.In IOC animal models,the enhancement of oxidative injury caused by iron overload in heart results in pathological changes including cardiomyocyte hypertrophy,apoptosis,and fibrosis,and eventually leads to cardiac dysfunction.Furthermore,excessive iron deposition in myocardium has been observed in animal models of diabetes.Therefore,iron metabolism is regarded as a new breakthrough point for in-depth study of DCM.Hepcidin(Hep)is an important systemic iron homeostasis regulatory polypeptide.Previous studies demonstrate the level of Hep in patients with T2DM and gestational diabetes mellitus(GDM)is higher than that in their respective control groups.The effect of Hep on cellular iron metabolism is dependent on Ferroportinl(Fpnl),the only recognized mammalian iron export transporter on the cell surface.After binding to Hep,Fpnl internalizes and degrades in lysosomes,thereby effectively preventing efflux of iron in cells.Hep causes abnormal distribution of iron inside and outside the cell via Fpnl,and then affects the function of the target organ,which is the pathophysiological basis of Hep involved in iron-related disorders.Liver is the main site of the synthesis of Hep,and the cardiomyocytes also express Hep.A recent research revealed that iron content of the cardiomyocyte fraction was significantly lower in mice with a cardiomyocyte-specific deletion of the Hamp gene that encodes Hep than in control group,which indicates Hep plays an essential role in cardiac iron homeostasis.However,the effect of Hep as a key regulator of iron metabolism in cardiomyocytes on the structure and function of DCM remains unclarified.In this study,we used gene-silencing technique to explore the role and potential mechanism of Hep in DCM in T2DM mice.Purposes(1)to explore the relationship between myocardial iron metabolism and diabetic cardiomyopathy;(2)To explore the effect of Hep gene silencing on cardiac structure and function of diabetic cardiomyopathy;(3)To explore the mechanism of Hep gene silencing to improve diabetic cardiomyopathy.Materials and Methods(1)There were 36 male C57BLKS db/db mice at 7 weeks.After 3 week of acclimatization,the mice were randomly assigned into DM group,DM + vehicle group and DM + Hep-shRNA group.The mice of last group were injected via tail vein with type 9 recombinant adeno-associated virus(rAAV9)that harboring short hairpin RNA against Hep gene(Hep-shRNA),and the mice of DM + vehicle group were injected with a control empty virus by the tail vein.The mice were sacrificed and serum and heart specimens were collected after 16 weeks of feeding.(2)At the end of experiment,the left ventricular geometric pattern and cardiac function of mice were measured echocardiogram.The M-mode echocardiogram imaging were captured at the long-axis,The mitral-valve pulsed Doppler and tissue Doppler imaging were obtained from the apical four-chamber view.(3)The serum extracted from the venous blood samples of experimental animals were used for analyses of fasting blood glucose(FBG),total cholesterol(TC),triglycerides(TG),high density lipoprotein cholesterol(HDL-c),low density lipoprotein cholesterol(LDL-c),free fatty acid(FFA),and fasting insulin(FINS).Then we calculated insulin sensitivity index(ISI).(4)The myocardial tissue were stained with hematoxylin-eosin(HE),Masson-Trichrome,Picrosirius red,and Oil Red O.Perivascular area/luminal area(PVCA/LA)ratio,collagen volume fraction(CVF),and myocardial lipid content were obtained by quantitative morphometry with automated image analysis.(5)Immunohistochemical staining for observation of collagen ?,collagen ?,Hep,Fpnl,H-ferritin and L-ferritin im myocardial tissue.(6)Western blot analysis for collagen ?,and collagen ?,Hep,Fpnl,H-ferritin,and L-ferritin.(7)The supernatant of myocardial tissue was analysed for total superoxide dismutase(SOD)activity and glutathione peroxidase(GSH-Px)activity with respective assay kits.(8)TUNEL assay were used in detection of apoptotic cells in t myocardial tissues.Result(1)At the end of the experiment,there was no significant difference in body weight,FBG,FINS,ISI,TC,HDL-c,LDL-c,TG and FFA.(2)Compared with DM + vehicle group,echocardiogram showed E'/A' were elevated(0.98±1.34 vs.1.3 6±0.28,P<0.05)and E/E' were decreased(30.26±5.07 vs.23.05±3.97,P<0.05)in DM + Hep-shRNA group.There was no significant difference in E/A,LEVDd,IVSd,LVPWd,EF and FS.(3)HE staining showed that the myocardial cells in DM and DM + vehicle group were twisted,fractured and disorganized.While the myocardial cells in DM + Hep-shRNA group were less twisted and fractured,the arrangement of myocardial cells was relatively orderly.the areas of the myocardial interstitial and perivascular fibrosis were significantly reduced in the DM + Hep-shRNA group.Compared with DM + vehicle groups,PVCA/LA ratio in the DM + Hep-shRNA group was significantly decreased(0.54± 0.34 vs.0.30±0.13,P<0.05),as well as CVF(9.61±4.14%vs.5.12±1.80%,P<0.05).Oil red O staining showed that there was no significant difference in lipid deposition among three groups.(4)Immunohistochemistry and western blot showed that the content of collagen I and the collagen I/III ratio were significantly decreased in DM + Hep-shRNA group than in the DM + vehicle group.(5)Compared with DM + vehicle group,the expression levels of Hep,H-ferritin and L-ferritin were increased in myocardial tissue in DM + Hep-shRNA group,while the level of Fpnl was significantly elevated(6)The SOD activity level of unit protein was significantly increased in the DM +Hep-shRNA group compared with the DM+ vehicle group(92.81± 7.47 U/mgprot vs.79.95± 9.62 U/mgprot,P<0.05),and the activity of GSH-Px was also significantly increased(887.11±59.11 U/mgprot vs.801.43±3 1.02 U/mgprot,P<0.05)(7)TUNEL assay analysis demonstrated that in the DM + Hep-shRNA group,apoptosis cell count was significantly lower than that in the DM + vehicle groupConclusion(1)Oxidative stress damage caused by iron deposition is an important mechanism in diabetic cardiomyopathy,and iron deposition is a potential therapeutic target for diabetic cardiomyopathy.(2)Hep gene silencing can reverse left ventricular pathological remodeling,improve diastolic function,and delay the progression of diabetic cardiomyopathy by attenuating iron-mediated oxidative stress injury.