| Background:China Cardiovascular Disease Report 2018 shows that the number of cardiovascular diseases in China was 290 million and the mortality rate was the highest.Acute myocardial infarction(AMI)is one of the main causes of cardiovascular death.By 2030,patients of myocardial infarction in China is expected to increase to 22.6 million which has directly caused serious economic and social burdens.It has become a major public health problem in China.Therefore,active research,prevention and treatment of myocardial infarction are needed to reduce its morbidity and mortality,improve the quality of life and prognosis of myocardial infarction,which has very important social significance and economic value.Velvet antler(VA),sweet and salty in taste,warm in temperament,is endowed with the quality of pure Yang and contains the Qi of generating hair.It has the functions of tonifying kidney and strengthening yang,promoting essence and blood circulation,reinforcing marrow and bone as well as activating blood circulation and removing blood stasis.Our research group has devoted itself to the application of VA in cardiovascular diseases for many years,and has got some conclusion:For clinical trials,VA can improve the heart function of heart failure patients with heart-kidney-yang deficiency,improve the clinical symptoms and life quality with traditional Chinese medicine;As for animal experiments,VA can improve the cardiac function index of rats with myocardial infarction;Furthermore,in vitro cell experiments,VA protein can reduce cardiomyocyte apoptosis induced by ischemia and hypoxia and alleviates cardiomyocyte injury.Many studies have shown that apoptosis is closely related to the prognosis of AMI.Anti-apoptotic therapy can effectively protect myocardial by reducing myocardial injury,decreasing infarct size and improving cardiac function.Cardiomyocyte autophagy plays an important role in maintaining the stability of cardiomyocyte structure and function both of which are of great significance to the pathophysiological changes of myocardial cells and the improvement of cardiac function after myocardial infarction.However,apoptosis and autophagy play different roles in different stages of the disease,protective effects on the body or the possibility of damage.The specific regulatory mechanisms and roles of apoptosis and autophagy after myocardial infarction are still controversial.Therefore,a thorough study of the inducing factors and transmission pathways of apoptosis and autophagy after myocardial infarction,and a clear target of VA to selectively activate or inhibit apoptosis and autophagy.All of these can provide a new idea for the comprehensive prevention and treatment of myocardial infarction.Based on this,we put forward a hypothesis that VA can improve acute myocardial infarction injury by regulating apoptosis and autophagy of myocardial cells and carry out relevant experiments to verify it.Experiment 1 Study on cardioprotective effect of VA on rat model of acute myocardial infarctionObject:To observe the cardioprotective effect of VA on rat model of acute myocardial infarction.Methods:Rat models of AMI were established by ligation of left anterior descending branch of coronary artery(sham-operated group only threaded,without ligation).They were randomly divided into model group,low-dose VA group[100mg/(kg.d)],medium-dose VA group[200mg/(kg.d)],high-dose VA group[400mg/(kg.d)]and enalapril maleate group[abbreviated as Pril group,1mg/(kg.d)].At the same time,sham-operated group was established with 10 rats in each group.Each treatment group was given corresponding drugs by gavage,sham operation group and model group were given 0.5%sodium carboxymethyl cellulose by gavage every day.The changes of electrocardiogram of rats in each group were detected after operation.24 hours after operation,the changes of cardiac troponin Ⅰ(cTnI)were detected by enzyme linked immunosorbent assay(Elisa).At the end of the seventh day after operation,the indexes of cardiac function were detected by echocardiography,the pathological changes of myocardium were observed by hematoxylin-eosin staining(HE),and the changes of tumor necrosis factor-α(TNF-α)in serum were detected by Elisa.Results:24 hours after operation:Compared with the sham operation group,the level of cTnI in the model group increased significantly(P<0.01).Compared with the model group,the serum cTnI levels of each dose of VA group and enalapril maleate group decreased,but there was no statistical difference(P>0.05).At the 7th day after operation:(1)Compared with the sham-operated group,the left ventricular short axis shortening(FS)and ejection fractions(EF)were significantly decreased,left ventricular end-diastolic dimension(LVEDD),left ventricular end-systolic dimension(LVESD),left ventricular end systolic volume(ESV)and left ventricular end diastolic volume(EDV)increased significantly in the model group(P<0.01).Compared with model group,EF increased,EDV and ESV decreased in low-dose VA group(P<0.05);EF and FS increased,LVESD decreased(P<0.05),LVEDD,EDV and ESV decreased significantly in medium-dose VA group(P<0.01);LVEDD,LVESD,EDV and ESV decreased significantly(P<0.01);EF,FS increased significantly in high-dose VA group(P<0.01);LVEDD,LVESD,EDV and ESV decreased significantly(P<0.01),EF,FS increased significantly in Pril group(P<0.01).(2)HE staining showed that the myocardium of model group had obvious histomorphological changes,including the destruction of myocardial cell structure in infarcted area,disordered arrangement,blurred fascicular space,infiltration of inflammatory cells,dissolution,rupture or necrosis of myofibrils.Compared with the model group,the range of myocardial lesion in each dose of VA group was reduced,the number of viable myocardium was more,the cell arrangement was more orderly,the degree of fibrosis was reduced,and a less inflammatory cells infiltrated.(3)Elisa showed that the content of TNF-α in model group was significantly higher than that in sham-operated group(P<0.01);compared with model group,the content of TNF-α in high dose VA group and pril group was lower(P<0.05).Conclusions:(1)VA can improve cardiac function and myocardial infarction injury in AMI rats,and has significant cardioprotective effect.(2)Different doses of VA can alleviate myocardial pathological changes,inhibit inflammation,improve myocardial injury and protect myocardial cells in a dose-dependent manner.The effect of high dose VA[400mg/(kg.d)]was the most obvious.Experiment 2 Study on the mechanism of VA in improving myocardial injury of AMI rats.Object:To observe the effects of VA on apoptosis and autophagy of myocardial cells in the marginal zone of myocardial infarction in rats with AMI.Methods:The sham operation group,model group and optimal dose antler group(high dose VA group)rats were selected in experiment 1.Apoptotic rate in the marginal zone of myocardial infarction was detected by TdT-mediated dUTP Nick-End Labeling(TUNEL);changes of myocardial actin microfilament(F-actin)staining were observed by immunofluorescence;and apoptotic protein B lymphoma-2 gene(B-cell lymphoma-2,Bcl-2)was detected by Western blot(WB)in the marginal zone of myocardial infarction as well as Bcl-2 associated X protein(Bax),cysteinyl aspartate specific proteinase-9(Caspase-9),cysteinyl aspartate specific proteinase-3(Caspase-3),and autophagy associated protein Beclin-1 and light chain marker of microtubule-associated protein 3(LC3).The expression levels of Bcl-2,Bax and Beclin-1 in the marginal zone of myocardial infarction were detected by immunohistochemistry.Results:(1)TUNEL results showed that the apoptotic rate of myocardial cells in the marginal zone of myocardial infarction in model group was significantly higher than that in sham-operated group(P<0.01);compared with model group,the apoptotic rate in VA antler group was significantly lower(P<0.05).(2)Immunofluorescence showed that compared with sham-operated group,F-actin of cardiac myocytes in model group was disordered and dissolved,and F-actin of cardiac myocytes in VA group was effectively repaired and improved.(3)WB results showed that the expression level of Bcl-2 protein in model group was lower than that in sham operation group(P<0.05),while the expression levels of Bax,Caspase 9 and Caspase 3 protein were higher(P<0.01);compared with model group,the expression level of Bcl-2 protein in VA group was higher(P<0.05),while the expression levels of Bax,Caspase-9 and Caspase-3 protein were lower(P<0.05,P<0.01).(4)Immunohistochemistry showed that the expression of Bax and Bcl-2 in model group increased significantly and decreased significantly when compared with sham-operated group(P<0.01,P<0.05).Compared with model group,the expression of Bax and Bcl-2 in VA group decreased and increased(P<0.01).(5)WB showed that the expression of Beclin-1 and LC3-Ⅱincreased in model group when compared with sham operation group(P<0.05,P<0.01),while the expression of Beclin-1 and LC3-Ⅱ decreased in VA group compared with model group(P<0.05,P<0.01).(6)Immunohistochemistry showed that the expression of Beclin-1 protein in the model group was significantly higher than that in the sham-operated group(P<0.01),while the expression of Beclin-1 protein in the VA group was significantly lower than that in the model group(P<0.01).Conclusions:(1)VA can significantly inhibit apoptosis of myocardial cells in the marginal zone of myocardial infarction in AMI rats,and its mechanism may be closely related to the regulation of mitochondrial apoptosis pathway represented by Bcl-2/Bax-Caspase 9-Caspase 3.(2)VA can inhibit the excessive autophagy of myocardial cells in the marginal zone of myocardial infarction in AMI rats,which is closely related to the decrease of Beclin-1 and LC3-Ⅱ expression.(3)VA may inhibit cardiomyocyte apoptosis and excessive autophagy at the same time.Its mechanism is through regulating the expression level of Beclin-1、Bcl-2、Bax at the upstream junction of apoptosis and autophagy. |
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