| BackgroudVentricular remodeling is a series of changes resulted from myocardial injury or overload,it includes the changes of size,shape and structure of heart,ventricular wall thickness.It is compensated and repaired procedure of heart injury,moreover,it is pathological.As a result,damaged myocardium is hypertrophy and replaced with fibrotic tissue consequently leading to contractile dysfunction and ultimately heart failure.It is one of the most important pathological mechanism of heart failure resulted from myocardial infraction.The mechanisms of ventricular remodeling include inflammatory,fibrotic,and neovascularization responses that involve regulated cell recruitment and function.Aldehyde dehydrogenase 2(ALDH2)is the key enzyme of the metabolism of aldehydes.It is confirmed that ALDH2 is cardiac-protective,by accelerating clearance of reactive aldehydes,or any other mechanisms.However,in the past and present,the studies on the effects of ALDH2 on cardiovascular diseases were more concentrated on acute myocardial infarction(AMI)or heart failure(HF)stage,there was no report about the effects of ALDH2 in the long-term development of ventricular remodeling after myocardial infarction.BYHWD can protect heart from VR after MI remarkably.In this study,we used HE staining,Mallory staining,immunohistochemistry,and TUNEL staining,ALDH2 activity detection,Caspase-3 activity detection,Western-Blot technology,to observe changes of ALDH2 expression and enzyme activity after MI,to explore the effects of ALDH2 on VR after AMI,and explore whether BYHWD inhibits VR by regulation of ALDH2,to find out more mechanisms,therapeutic targets and more effective ways of prevention of VR,and enriched the mechanisms of traditional Chinese medicine.ObjectiveTo explore the effects of ALDH2 on long-term ventricular remodeling after MI and the under mechanisms,and explore the effects of BYHWD on long-term ventricular remodeling after MI,whether or not it was related to regulation of ALDH2,and explore the under mechanisms.MethodsThe research is divided into two parts.In the first part,ventricular remodeling animal model was made by ligation of the anterior descending branch of coronary artery of rat.1 week after myocardial infarction surgery,all successfully ligated rats were checked with echocardiography,and randomly assigned into three experimental groups,including model group,Alda-1 treated group,daidzin treated group,and sham-operated rats served as sham group.Each groups was 18 rats,all animals were through gavage administration,for 20 weeks.Alda-1 group rats were treated with Alda-1(16mg/kg per day),and daidzin group rats were treated with daidzin(5mg/kg per day),the sham group and model group were given normal saline of the same quantity.Noninvasive transthoracic echocardiography was checked once a month during the treatment.After the sacrifice,hearts were removed,and weight of each hearts was measured.Left ventricles were separated,freeze-clamped and were stored at-80? or fixed in 10%formalin.In order to detect the extent of fibrotic areas,histologic samples were stained with HE staining and Mallory staining,type I and type III collagen were measured by immunohistochemistry.The protein expression of ALDH2 was measured by western blot,and ALDH2 enzyme activity was also checked.Content of toxic aldehydes(4-HNE)was measured by immunohistochemistry.The cell apoptosis was monitored by P53,Bax,Bcl-2 protein expression,Caspase-3 activity and TUNEL staining,to explore the effects of ALDH2 on long-term ventricular remodeling after MI and the under mechanisms.In the second part of the study,ventricular remodeling animal model was made by ligation of the anterior descending branch of coronary artery of rat.1 week after myocardial infarction surgery,all successfully ligated rats were checked with echocardiography,and randomly assigned into four experimental groups,including model group,BYHWD group,Alda-1 treated group,perindopril treated group,and sham-operated rats served as sham group.Each groups was 18 rats,all animals were through gavage administration,for 20 weeks.The BYHWD group rats were treated with Bu Yang Huan Wu Decoction(lOg/kg.d),Alda-1 group rats were treated with Alda-1(16mg/kg per day),and Perindopril group rats were treated with(0.41 mg/kg per day),the sham group and model group were given normal saline of the same quantity.Noninvasive transthoracic echocardiography was checked once a month during the treatment.After the sacrifice,hearts were removed,and weight of each hearts was measured.Left ventricles were separated,freeze-clamped and were stored at-80? or fixed in 10%formalin.In order to detect the extent of fibrotic areas,histologic samples were stained with HE staining and Mallory staining,the protein expression of ALDH2 was measured by western blot,and ALDH2 enzyme activity was also checked.Content of toxic aldehydes(4-HNE)was measured by immunohistochemistry.The cell apoptosis was monitored by TUNEL staining,to explore the effects of BYHWD on long-term ventricular remodeling after myocardial infarction,whether or not it was related to regulation of ALDH2,and explore the under mechanisms.ResultsPart1.Effects of ALDH2 on ventricular remodeling1.1 The effects of Alda-1 and daidzin on the survival of VR rats.The effects of Alda-1 and daidzin on the survival rate were assessed for 20 weeks after AMI.The mortality of different groups was great different.The mortality rates of the model group was greatly decreased,compared with sham group(5.6%vs44%,P<0.05).The mortality rates of the Alda-1 group was 16.7%,it was significantly increased,compared with model group(16.7%vs 44.4%,P<0.05).And daidzin group(28%)tended to lower than the model group(44.4%),but there was no difference between them(P=0.3765).1.2 Effects of Alda-1 and daidzin on HW/BW,histological,and cardiac functionThe HW/BW ratio in different groups were significantly different(F=171.17,P<0.001).Compared with the sham group,the HW/BW ratio of model group was significantly increased(P<0.001).HE staining showed that the cardiomyocyte hypertrophy and myocardial fibrosis increased,the number of myocardial cells at 400x magnification decreased(P<0.001),and the cardiac function of model group deteriorated.The left ventricular dimension at end diastole(LVIDd,P<0.001)and left ventricular dimension at end systole(LVIDs,P<0.001,)significantly increased.Meanwhile,the model group showed lower left ventricular ejection fraction(LVEF,P<0.001)and left ventricular fractional shortening(LVFS,P<0.001)compared with the sham group.These result indicated that the ventricular remodeling model was successfully established,as previously reported.The Alda-1 group showed significantly lower HW/BW ratio(P<0.001),higher myocardial cell number(P<0.001),and improved cardiomyocyte hypertrophy and cardiac function,compared with the model group.The treatment also significantly reduced LVIDd(P<0.001)and LVIDs(P<0.001),as well as increased LVEF(P<0.001)and LVFS(P<0.001).However,no difference was found between the model and daidzin groups in the following parameters:HW/BW ratio(P = 0.107),myocardial cell number(P = 0.139),LVIDd(P = 0.680),LVIDs(P = 0.529),LVEF(P = 0.970)and LVFS(P = 0.471).1.3.Effects of Alda-1 and daidzin on collagen formationThe results of collagen volume fraction(CVF)in different groups were significantly different(F=125.458,P<0.001).The collagen formation increased and CVF of model group were significantly increased(P<0.001),compared with the sham group(P<0.001).Immunohistochemistry illustrated that the expression of collagen Types I and III of the model group significantly increased,compared with sham group(P<0.001,P<0.001,respectively).However,the increase of former type exceeded that of the latter,and the collagen Type I/III ratio increased(P<0.001).Mallory staining showed that Alda-1 treatment reduced collagen fiber amount,collagen volume(P<0.001),collagen Type I and Type III expression,and collagen Type I/III ratio(P<0.001).No significantly differences in collagen volume(P = 0.327)and collagen Type I/III ratio were detected between the model and daidzin groups.However,naked eye inspection of Mallory staining revealed that the collagen volume,collagen Type I/III ratio and collagen fiber amount were slightly lower in the daidzin group than in the model group.1.4.Effects of Alda-1 and daidzin on cell apoptosisCell apoptosis was detected through TUNEL assay,evaluating P53,Bax,and Bcl-2 expression and caspase-3 activity.The apoptosis index of different groups were significantly different(F=326.88,P<0.001).The number of TUNEL-positive cells and apoptotic index(P<0.001)significantly increased in the model group.The expression of P53,and Bax and the activity of Casapse-3 showed the same trend.However,the expression of anti-apoptosis gene Bcl-2decreased,compared with the sham group.Alda-1 treatment significantly down regulated cell apoptosis,including TUNEL positive cell number,apoptotic index(P<0.001),p53 and Bax protein expression,and casapse-3 activity(P<0.001)and increased Bcl-2 expression.No differences in the protein expression levels of p53,Bax and Bcl-2 were observed between the model group and daidzin group.However,daidzin treatment significantly increased the apoptotic index(P= 0.021)and caspase-3 activity(P<0.001).This result indicated that daidzin increased apoptosis might probably by interacting with the caspase family.1.5.Effects of Alda-1 and daidzin on ALDH2 enzymatic activity and ALDH2 expressionThe ALDH2 enzymatic activity assay showed that ALDH2 activity results were significantly different(F=322.304,P<0.001).The ALDH2 activity and expression were suppressed in the model group(P<0.05),compared with sham group.Alda-1 treatment significantly upregulated ALDH2 enzymatic activity(P<0.001),but did not affect ALDH2 expression compared with the model group.Similar to Alda-1,daidzin significantly decreased ALDH2 enzymatic activity(P<0.001),but did not affect ALDH2 expression compared with the model group.No difference in ALDH2 expression was observed among the model group,Alda-1 and daidzin groups.1.6 Effects of Alda-1 and daidzin on 4-HNE expressionThe 4-HNE stain positive rats in different groups were significantly different(F=158.046,P<0.001).Immunohistochemistry illustrated that 4-HNE was accumulated in the model group,and that 4-HNE stain-positive rate significantly increased(P<0.001),compared with the sham group.By contrast,the results of immunohistochemistry showed that Alda-1 treatment reduced 4-HNE expression(P<0.001),compared with the model group.Daidzin treatment increased 4-HNE accumulation,as indicated by the increased 4-HNE stain positive rate(P<0.001),compared with the model group.Part2.The effect of BYHWD on the regulation of ALDH2 in ventricular remodeling2.1 The effects of BYHWD on survival of VR rats.The mortality rates of the model group was greatly decreased,compared with sham group(5.6%vs44.4%,P<0.05).The mortality rates of the Alda-1 group was 16.7%,it was significantly increased,compared with model group(16.7%vs 44.4%,P<0.05).The mortality rates of the BYHWD group was 22.2%,and the And The mortality rates of the Perindopril group was 16.7%.No difference was found among the Alda-1 group,BYHWD group and Perindopril group(P=0.453).The cause of death was mostly because of heart failure.2.2 Effects of BYHWD on HW/BW,histological,and cardiac function.The HW/BW ratio in different groups were significantly different(F=15.729,P<0.001).Compared with the sham group,the HW/BW ratio of model group was significantly increased(P<0.001).HE staining showed that the cardiomyocyte hypertrophy and myocardial fibrosis increased,the number of myocardial cells at 400x magnification decreased(P<0.001),and the cardiac function of model group deteriorated.The left ventricular dimension at end diastole(LVIDd,P<0.001)and left ventricular dimension at end systole(LVIDs,P<0.001)significantly increased.Meanwhile,the model group showed lower left ventricular ejection fraction(LVEF,P<0.001)and left ventricular fractional shortening(LVFS,P<0.05)compared with the sham group.These result indicated that the ventricular remodeling model was successfully established,as previously reported.The Alda-1 group showed significantly lower HW/BW ratio(P<0.001),higher myocardial cell number(P<0.001),and improved cardiomyocyte hypertrophy and cardiac function,compared with the model group.The BYHWD treatment also significantly reduced LVIDd(P<0.001)and LVIDs(P<0.001),as well as increased LVEF(P<0.001)and LVFS(P<0.001).Compared with model,Perindopril significantly reduced LVIDd(P<0.001)and LVIDs(P<0.001),as well as increased LVEF(P<0.001)and LVFS(P<0.001).However,no difference was found among the Alda-1,BYHWD group and Perindopril group in the following parameters:HW/BW ratio,myocardial cell number,LVIDd,LVIDs,LVEF and LVFS.2.3 Effects of BYHWD on collagen fibrous formationThe results of Mallory staining showed that lots of fibrous tissue was founded in infarct area and non-infract area of rats in the model group,and the fiber was arranged disorderly.The collagen volume fraction(CVF)in different groups was significantly different(F=161.820,P<0.001).The CVF of model group was significantly increased(P<0.001),compared with the sham group.The amount of fiber were significantly reduced in BYHWD group,Alda-1 group and Perindopril group(P<0.001,P<0.O001 P<0.001,respectively),compared with the model group.There was no difference among the above three groups(P=0.838,P=0.923,respectively).2.4 Effects of BYHWD on cell apoptosisThe number of TUNEL-positive cells and apoptotic index was greatly different in different groups(F=236.799;P<0.001).The number of TUNEL-positive cells and apoptosis index(P<0.001)were significantly increased in the model group,compared with sham group.BYHWD and Alda-1 treatment significantly down regulated cell apoptosis,reduced apoptotic index(P<0.001,P<0.001,respectively),and the apoptotic index of Perindopril group was also significantly decreased(P<0.001).No differences was found,when BYHWD group compared with Perindopril group(P=0.688),and Alda-1 group compared with Perindopril group(P=0.10).2.5 Effects of BYHWD on 4-HNE expressionImmunohistochemistry illustrated that 4-HNE stain positive rates were significantly different in different groups(F=234.69,P<0.001).It was significantly increased in the model group(P<0.001),compared with sham group.By contrast,the BYHWD treatment,Alda-1 treatment,and Perindopril group reduced 4-HNE expression,significantly decreased 4-HNE stain positive rate(P<0.001,P<0.001,P<0.001,respectively),compared with the model group.2.6 Effecs of BYHWD on ALDH2 expression and ALDH2 enzymatic activityAccording to the Western-blot result,the expression of ALDH2 of different groups was significantly different(F=72.71,P<0.001).The expression of ALDH2 was suppressed in the model group,compared with sham group(P<0.001).The treatment ofAlda-1 and Perindopril did not have affects on ALDH2 expression,compared with the model group(P=0.857,P=0.829).Increased ALDH2 expression was observed in the BYHWD group,compared with the model group,The ALDH2 enzymatic activity assay showed that ALDH2 activity was greatly different in different groups(F=317.809,P<0.001).The ALDH2 enzymatic activity was significantly decreased in the model group(P<0.001),as compared with sham group.But it was significantly increased in the BYHWD group(P<0.001)and Alda-1 group(P<0.001),compared with model group.There was no differernce between BYHWD group and Alda-1 group(P=0.421).There was also no difference between model group and Perindopril group(P=0.092).Conclusion1.Alda-1 treatment can improve the survival rate and cardiac function,prevent heart from ventricular remodeling after MI.2.The expression and enzyme activity of ALDH2 were decreased significantly in ventricular modeling after myocardial infarction.It suggests that ALDH2 expression and enzyme activity play important roles in the VR,which may be one of the mechanisms of VR after MI.In addition,aldehydes overload may aggravate ventricular remodeling,and ALDH2 agonist prevents heart from ventricular remodeling by reducing aldehydes overload.3.BYHWD significantly prevents heart from ventricular remodeling,reduces the accumulation of 4-HNE,increases ALDH2 expression and enzyme activity,the regulation of ALDH2 may be one of the mechanisms of its function of prevention of ventricular remodeling. |
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