| Objective:1.The health status of patients with acute myocardial infarction before onset,clinical characteristics at onset,treatment strategies and programs,lifestyle changes after treatment,medication compliance and other factors,which will have different nature and degree of influence on long term prognosis.This is a prospective cohort study to describe and compare the basic characteristics,coronary lesion characteristics,coronary atherosclerotic burden of patients with acute myocardial infarction,as well as the one-year and five-year end events of patients and their influencing factors,to explore new prognostic indicators,to predict the prognosis of patients with acute myocardial infarction,and to provide guidance optimization provide objective basis for diagnosis and treatment strategy.2.According to the findings of the first part of the study,to explore the long term prognosis and clinical significance of patients with acute myocardial infarction after PCI residual coronary artery atherosclerosis burden.In view of the fact that atherosclerotic burden leads to coronary heart disease patients persistent presence varying degrees of myocardial hypoxia,the residual Gensini score was used to quantify atherosclerotic burden to reflect the degree of myocardial hypoxia,and the effect of persistent different degrees of myocardial hypoxia on long term prognosis was explored.3.HIF1α(Hypoxia inducible factor 1),are the main transcription factors and core regulators for cells to adapt to hypoxia.They are involved in the regulation of energy supply,autophagy,apoptosis and other hypoxia adaptive responses,and have short term and long term effects on organ structure,function and metabolism.According to the results of the first two parts,the relationship and significance between the levels of HIF1α,autophagy related protein beclin1and apoptosis related protein bcl2in serum of patients with acute myocardial infarction and the long term prognosis of patients with acute myocardial infarction were analyzed clinical significance.4.Through the H9C2 rat cardiomyocyte experiment,simulate different clinical conditions,explore the intrinsic mechanism of HIF1αregulating cardiomyocyte autophagy and apoptosis under different hypoxia conditions.Methods:1.The first part is a prospective cohort study of patients with acute myocardial infarction,and a one-year and five-year follow up results and prognosis study.Consecutive patients with acute myocardial infarction from December 2012 to June2014 were enrolled.Study approved by ethics committee informed consent was signed by all patients.Baseline enrollment was completed records were collected demographic data,past status,clinical features,laboratory parameters,and treatment.All patients were followed up for1month,1year and 5year after discharge including life status,lifestyle changes,exercise,medication status,medication compliance and mace events(all cause death,non fatal myocardial infarction,unplanned coronary revascularization,heart failure,angina pectoris and stroke etc.).Univariate analysis,Kaplan-Meier method,Log-rank test were used to compare the survival curves.Univariate Cox regression analysis was used to screen out potential prognostic factors according to the level ofα=0.10,and all the selected factors were included in Cox regression model for multivariate analysis.The statistical test level was set atα=0.05.All the data were analyzed by IBM SPSS 25.0 statistical software.2.According to the first part of the study,rGS can quantify the burden of residual coronary atherosclerosis,is an independent predictor of acute myocardial infarction PCI five-years mace after treatment.The second part grouped by rGS median,compared the high and low rGS baseline characteristics,mortality and mace incidence between the two groups.Kaplan-Meier method was used to draw the survival curve,and Log-rank test was used to compare the differences between one-year and five-year mortality and mace.Univariate Cox regression analysis was used to screen out potential prognostic factors according to the level ofα=0.10,and all the selected factors were included in Cox regression model for multivariate analysis.The statistical test level was set atα=0.05.All the data were analyzed by IBM SPSS 25.0statistical software.3.The levels of HIF-1α,Beclin-1and Bcl-2 were detected by ELISA.The HIF-1α,Beclin-1and Bcl-2 were used as biomarkers,and the median levels were divided into high and low concentration groups.Baseline characteristics,mortality and mace incidence were compared between the high and low concentration groups.Objective to explore their relationship and predictive value with AMI patients and long term prognosis.Kaplan-Meier method was used to describe the survival curve,and Log-rank method was used to test the difference.Univariate Cox regression analysis was used to screen out potential prognostic factors according to the level ofα=0.10,and all the selected factors were included in Cox regression model for multivariate analysis.The statistical test level was set atα=0.05.All the data were analyzed by IBM SPSS 25.0statistical software.4.Application H9C2 rat cardiomyocytes,through plasmid construction and transfection to establish the over expression of HIF-1αcardiomyocyte cell model.The cell model of HIF-1αknock down was established by RNA silencing technique.The expression of HIF-1α,autophagy related proteins(Beclin-1,LC3,p62)and apoptosis related proteins(Bcl-2,Caspase3,Bax,BNIP3)were detected by Western blot for comparative analysis.Result:1.A total of 249 patients with acute myocardial infarction enrolled,one month followup death 2 person(0.8%),mace event occurrence 2 person(0.8%);one year followup death 7 person(2.8%),mace event occurrence 55 times(22.1%);five year followup death 27person(10.8%),mace events 97 people(38.9%).The endpoint was one year mace.Univariate Cox regression analysis showed that age(P=0.001),EF(P=0.017),rGS(P=0.001),delta GS(P=0.005),complete revascularization(P=0.01),number of coronary artery lesions(P=0.039),chronic renal insufficiency(P=0.002),possible one-year mace independent influencing factors.The screening factors were included in Cox multivariate regression model,age(HR=1.031,95%CI:1.004-1.058,P=0.024),EF(HR=0.969,95%CI:0.940-0.999,P=0.040),delta GS(HR=0.984,95%CI:0.972-0.997,P=0.013),complete revascularization(HR=0.381,95%CI:0.162-0.898,P=0.027)was an independent influencing factor of one-year mace.The endpoint events were five-year mace,univariate Cox regression analysis showed that age(P=0.001),EF(P=0.001),rGS(P=0.001),complete revascularization(P=0.003),history of stroke(P=0.012),chronic renal insufficiency(P=0.002),and BMI(P=0.043),possible five-years mace independent influencing factors.The screening factors were included in the Cox multivariate regression model,and EF(HR=0.972,95%CI:0.950-0.995,P=0.015),rGS(HR=1.009,95%CI:1.001-1.018,P=0.025)were the independent influencing factors of five year mace incidence.3.Patient were divided into two groups according to rGS median number.Compared with the low R rGS group rGS≤11),the high rGS group(rGS>11)was older(62.0 vs.59.0).The rates of combined chronic renal insufficiency and stroke were high(16.0%vs.6.2%,P=0.013;18.5%vs.8.5%,P=0.020);the rates of single vessel disease were low(14.3%vs.67.7%,P=0.001),the rates of complete revascularization were low(15.1%vs.81.5%,P=0.001).The proportion of staging PCI treatment was low(7.6%vs.19.6%,P=0.025).The incidence of mace in high rGS group was higher than that in low rGS group.The difference of one-year mace,five-year mace and five-year mortality between the two groups was statistically significant.The one-year mortality in high rGS group was higher than that in low rGS group,and the difference was not statistically significant.The Kaplan-Meier survival curve was drawn,the high rGS group one-year cumulative mace incidence was higher than the low rGS group,(28.6%vs.16.2%,χ~2=6.114,P=0.013);the high rGS group five-year cumulative mace incidence was higher than the low rGS group,(47.9%vs.30.8%,χ~2=8.609,P=0.003);high rGS groupfive yearscumulative death ratehigher than low rGS group,(15.1%vs.6.9%,χ~2=4.497,P=0.034).Univariate Cox regression analysis,rGS,delta GS,for one-year mace event independent influencing factor;rGS for five years mace and death independent influencing factor.Multivariate Cox regression analysis showed that age(HR=1.031,95%CI:1.0041.058,P=0.024),EF(HR=0.969,95%CI:0.940-0.999,P=0.040),delta GS(HR=0.984,95%CI:0.972-0.997,P=0.013),complete revascularization(HR=0.381,95%CI:0.162-0.898,P=0.027)is the independent influencing factor of one-year mace;EF(HR=0.972,95%CI:0.950-0.995,P=0.015),rGS(HR=1.009,95%CI:1.001-1.018,P=0.025)is the independent influencing factor of five-year mace;age(HR 1.044 95%CI:1.005-1.084,P=0.027)and EF(HR0.975,95%CI:0.921-0.994,P=0.027)were the independent influencing factors of five-year death.3.The serum concentrations of HIF-1αand Bcl-2 in 1-year mace group were lower than those in 1-year non-mace group,Beclin-1was higher,(P<0.001).In the 5-year mace group,the serum concentrations of HIF-1αand Bcl-2 were low,Beclin-1 high,(P<0.001).In the5-year death group,the serum concentrations of Bcl-2 were lower than those in the control group,(P<0.001);HIF-1αand Beclin-1were lower than those in the control group.Kaplan-Meier survival curve was drawn,high HIF-1αgroup 1-year cumulative mace incidence low,(5.6%vs.38.4%,χ~2=38.032,P<0.001);high Beclin-1group 1-year cumulative mace incidence high,and Bcl-2 group 1-year cumulative mace incidence low,(12.9%vs.31.2%,χ~2=12.089,P=0.001).High HIF-1αgroup 5-year cumulative mace incidence low,(7.3%vs.70.4%,χ~2=104.272,P<0.001);high Beclin-1 group 5-year cumulative mace incidence high,(50.8%vs.27.2%,χ~2=16.227,P<0.001);high Beclin-1group 5-year cumulative mace incidence high,(50.8%vs.27.2%,χ2=16.227,P<0.001);High Bcl-2 group 1-year cumulative mace incidence low,(18.5%vs.59.2%,χ~2=41.315,P<0.001).High HIF-1αgroup 5-year cumulative low mortality,(3.2%vs.18.4%,χ~2=14.596 P<0.001);high Bcl-2 group 5-year cumulative low mortality,(4.0%vs.17.6%,χ~2=11.5,P=0.001).In univariate Cox regression analysis,HIF-1α,Bcl-2 and Beclin-1were the independent influencing factors of mace;HIF-1αand Bcl-2 were the independent influencing factors of 5-year death when the endpoint event was 5-year death.Multivariate Cox regression analysis,age(HR=1.031,95%CI:1.004-1.058,P=0.022),rGS(HR=1.036,95%CI:1.019-1.054,P<0.001),HIF-1α(HR=0.019,95%CI:0.005-0.074,P<0.001),Bcl-2(HR=0.738,95%CI:0.615-0.085,P=0.001)is an independent influencing factor of 1-year mace;age(HR=1.029,95%CI:1.008-1.050,P=0.007),rGS(HR=1.039,95%CI:1.027-1.052,P<0.001),complete revascularization(HR=0.533,95%CI:0.307-0.927,P=0.026),HIF-1α(HR=0.010,95%CI:0.004-0.028,P<0.001),Bcl-2(HR=0.658,95%CI:0.579-0.747,P<0.001),Beclin-1(HR=1.376,95%CI:1.045-1.790,P=0.023)are the independent impact factors of 5-year mace.They are:rGS(HR=1.026,95%CI:1.009-1.043,P=0.003),chronic renal insufficiency(HR=3.513,95%CI:1.434-8.609,P=0.006),HIF-1α(HR=0.177,95%CI:0.046-0.673,P=0.011),Bcl-2(HR=0.667,95%CI:0.509-0.875,P=0.003)is 5-year death independent influencing factor.4.The over expression of HIF-1αand the knockdown expression of HIF-1αin the normoxic group,hypoxic group,hypoxic and hypoxic hypoxic groups were significantly different,(P<0.05).The expression level of HIF-1αin hypoxic group,hypoxic reoxygenation group and hypoxic and hypoxic reoxygenation group was higher than that in normoxic group,(P<0.05),the difference was significant.The expression level of HIF-1αin hypoxic and hypoxic reoxygenation groups was higher than that in hypoxic and hypoxic reoxygenation groups,(P<0.05),the difference was significant.There was no significant difference in the expression of HIF-1αbetween the hypoxic and hypoxic groups.There was no difference in the expression of BNIP3 in normoxic group,normoxic over expression HIF-1αgroup and normoxic knockdown HIF-1αgroup.The expression level of BNIP3 in hypoxia and hyporexia knockdown HIF-1αgroup was lower than that in hypoxia and over expression HIF-1αgroup,hypoxia and over oxygenation HIF-1αgroup,and hypoxia and hypore oxygenation and over expression HIF-1αgroup,(P<0.05),showing significant difference.There was no difference in the expression of BNIP3 in HIF-1αover expression under hypoxia,hypoxia and hypoxia and hypoxia and hypoxia conditions,(P<0.05).The expression level of Beclin-1 in the normoxic group was not different from that in the hypoxic HIF-1αgroup,hypoxic HIF-1αgroup,and hypoxic HIF-1αgroup.There was no difference in the expression degree between them and hypoxic and hypoxic groups.The expression level of Beclin-1in the normoxic group was lower than that in the hypoxic and over expressed HIF-1αgroup,the hypoxic and over oxygenated HIF-1αgroup,and the hypoxic and over oxygenated HIF-1αgroup,with significant difference(P<0.05).There was no difference in LC3 expression in the normoxic group,the normoxic over expression HIF-1αgroup,and the normoxic knockdown HIF-1αgroup.The expression degree of hypoxic group,hypoxic and hypoxic group,hypoxic and hypoxic group,hypoxic and hypoxic over expression of HIF-1αgroup,hypoxic and hypoxic over expression of HIF-1αgroup,hypoxic and hypoxic and hypoxic over expression of HIF-1αgroup,hypoxic and hypoxic and hypoxic over expression of HIF-1αgroup,hypoxic and hypoxic and hypoxic over expression of HIF-1αgroup,hypoxic and hypoxic and hypoxic over expression of HIF-1αgroup,hypoxic and hypoxic and hypoxic and hypoxic over expression of HIF-1αgroup,and hypoxic and hypoxic and hypoxic over expression of HIF-1αgroup were all higher than that of normal oxygen group,(P<0.05),with significant difference.There was no difference in the expression of P62 in the normoxic group,hypoxic group,hypoxic reoxygenated group and hypoxic reoxygenated group.The expression degree of P62 in the normoxic group was higher than that in the normoxic over expression HIF-1αgroup,hypoxic over expression HIF-1αgroup,hypoxic reoxygenated over expression HIF-1αgroup,and hypoxic and hyporeoxygenated over expression HIF-1αgroup,with significant differences(P<0.05).The expression degree of P62 in normoxic group was higher than that in hypoxic HIF-1αgroup,hypoxic and hypoxic HIF-1αgroup,and hypoxic and hypoxic HIF-1αgroup,with significant difference(P<0.05).The expression level of Bcl-2 in the normoxic group was higher than that in the hypoxic and over expressed HIF-1αgroup,the hypoxic and reoxygenated HIF-1αgroup,and the hypoxic and hypooxy genated hypoxic and under oxygenated HIF-1αgroup,and the difference was significant(P<0.05).There was no difference between the two groups.The expression level of Bax in normal oxygen group was significantly lower than that in hypoxia and hyporeoxygenation group(P=0.0134),hypoxia and hyporeoxygenation group(P<0.0001)and HIF-1αknockdown group(P<0.0001).There was no difference between the two groups.The expression level of Bax in normal-oxygen-overexpressed HIF-1αgroup was higher than that in hypoxic and reoxygenated HIF-1αgroup(P=0.0115),and lower than that in hypoxic and reoxygenated HIF-1αgroup(P=0.0057)and knockdown HIF-1αgroup(P=0.0185),with significant differences.There was no difference between hypoxia and hyporeoxygenation group(P=0.9568).The expression level of caspase-3 in the normoxic group was lower than that in the hypoxic group,the hypoxic and hypoxic groups,the hypoxic and hypoxic groups,the hypoxic and hypoxic groups,the hypoxic and hypoxic groups,and the hypoxic and hypoxic groups,and the HIF-1αknockdown groups,with significant differences(P<0.05).There was no difference between the two groups.By Pearson test,HIF-1αexpression was correlated with BNIP3,Beclin-1,LC3 and p62(P<0.05).The expression of BNIP3 was correlated with HIF-1α,Beclin-1,LC3 and p62,(P<0.05);Beclin-1 expression was correlated with HIF-1α,BNIP3 and p62,(P<0.05);LC3 expression was correlated with BNIP3,Bax and Caspase-3,(P<0.05);The expression of p62 was correlated with HIF-1α,BNIP3 and Beclin-1,(P<0.05);The expression of Bax was correlated with Caspase-3 and LC3,(P<0.05);Caspase-3 expression was correlated with LC3 and Bax(P<0.05).Bcl-2expression was not correlated with the others.Conclusion:1.Age,EF value,whether combined chronic renal insufficiency,whether coronary artery three vessel disease and other commonly used clinical indicators,in this study one-year and five-year mace events have predictive value,can play evaluation and guidance role for clinical decision making.Complete revascularization and rGS evaluation AMI patients PCI residual atherosclerotic burden after treatment,one-year and five-years mace have good predictive value.It is the first time to quantify the severity of acute myocardial infarction after 5-years of PCI.2.Aiming at a therosclerotic burden coronary heart disease patients persistent existence varying degrees of myocardial hypoxia,rGS as a new tool for measuring residual atherosclerotic burden in patients with AMI,reflecting the degree of persistent myocardial hypoxia,is an independent factor for predicting 5-year mace after acute myocardial infarction,for long term.The clinical outcome has predictive value.3.HIF-1α,Bcl-2,Beclin-1 as biomarkers have an intrinsic relationship with the long term prognosis of patients with acute myocardial infarction,which has the potential to become a new independent predictor of five-year mace and death in patients with acute myocardial infarction.Hypoxia HIF-1αhas an intrinsic effect on autophagy and apoptosis,which can balance the response state and compensatory ability of cardiomyocytes under hypoxia and determine whether cardiomyocytes can survive or not.Under acute hypoxia,myocardium may survive by increasing autophagy and inhibiting apoptosis.4.HIF-1α,BNIP3,LC3,Bax,Caspase-3were sensitive to hypoxia,while Beclin-1,p62,Bcl-2 were not sensitive to hypoxia.The expression of HIF-1αwas correlated with BNIP3and autophagy proteins Beclin-1,LC3,p62.The expression of Bcl-2 was not correlated with others.Under hypoxia,HIF-1αfurther regulates autophagy by regulating BNIP3;under hypoxia,autophagy and apoptosis are cross linked and interact with each other,which depends on the duration and degree of hypoxia.Long and severe hypoxia leads to greater apoptosis increment and decreased cell viability;short and mild hypoxia leads to greater autophagy increment and decreased cell viability... |
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