| 1.BACKGROUNDAt present,more than 290 million people in China have suffered from cardiovascular diseases.Due to the high incidence of Acute coronary syndrome(ACS),the mortality rate of cardiovascular diseases is increasing and it is one of the main causes of death.Acute myocardial infarction(AMI)is an Acute interruption of coronary blood flow with persistent ischemia and anoxia,and is typical of ACS.Currently,despite significant advances in primary prevention and treatment of cardiovascular diseases,which have resulted in a decline in the 30-day mortality rate after AMI over the past 20 years,AMI is still the number one killer of diseases worldwide,often secondary to acute and subacute complications.Ventricular remodeling(VR)is an important pathologic basis in the progression of acute myocardial infarction to heart failure,and is also an important cause of increasing long-term cardiovascular end events.The concept of AMI is similar to "chest pain" or "thoracic obstruction".Many famous doctors in ancient China have rich theoretical knowledge and clinical experience in AMI treatment.At present,a large number of epidemiological studies focus on the characteristics and evolution of TCM syndromes of acute myocardial infarction.According to the existing research and the understanding of traditional Chinese medicine,Qi deficiency and blood stasis are the main pathogenesis of acute myocardial infarction.So,the method of "supplementing Qi and activating blood" is the main effective treatment.Wenxin Decoction is an effective treatment for AMI with Qi deficiency and blood stasis syndrome.As a member of the miR-126 family,miR-126-5p expression level was significantly reduced in patients with coronary heart disease(CHD),angina pectoris,multi-branch lesions,and high SYNTAX score,and was also overexpressed in myocardial cells with ischemia/reperfusion injury.High expression level of miR-126-5p can inhibit atherosclerosis(AS)and left ventricular hypertrophy.Bcl-2 gene is a proto-oncogene,which can inhibit apoptosis.Bcl-2 family protein is a key molecule regulating the balance between apoptosis and autophagy,and a key regulatory factor in mitochondrial pathway of apoptosis.Under normal circumstances,cysteine aspartic acid protease(Caspase)exists in living cells in the form of non-active proenzyme.Once stimulated by apoptosis-inducing signal,caspase is activated,triggering caspase cascade to mediate cell apoptosis.When the intracytoplasmic Bcl-2 of cardiomyocytes is stimulated by various regulatory stimuli,the over-opening of the mitochondrial permeability transformation pore is inhibited,thereby reducing the activity of caspase and inhibiting the apoptosis of cardiomyocytes.As a potential target for regulating cardiac apoptosis,miR-126-5p may be related to the targeted regulation of Bcl-2 expression.The research group previously used Wenxin Decoction to intervene in patients with unstable angina pectoris,and found that Wenxin Decoction could up-regulate the expression levels of miR-126-5p and Bcl-212(a member of the Bcl-2 family)in patients with unstable angina pectoris,which was negatively correlated with the expressions of caspase-9 and caspase-3.Therefore,this study developed a rat model of acute myocardial infarction to observe the effects of Wenxin Decoction on miR-126-5p/Bcl-2/Caspase-9/Caspase-3 regulatory network,and to explore the possible molecular mechanism of its inhibition of myocardial cell apoptosis.2.PURPOSETo observe the effects of Wenxin Decoction on cardiac function,pathological morphology of myocardial tissue,matrix metalloproteinase(MMP)and miR-126-5p/Bcl-2 pathway in rats with acute myocardial infarction,and to explore the mechanism of Wenxin Decoction in inhibiting the apoptosis of myocardial cells after myocardial infarction.Further clarifies the scientific connotation of the theory of supplementing Qi and activating blood.3.METHODS150 adult male Wistar rats underwent left anterior descending coronary artery(LAD)ligation to prepare the rat model of acute myocardial infarction.They were randomly divided into model group,trimetazidine hydrochloride group,Wenxin Decoction low-dose group,medium-dose group and high-dose group,and sham operation group,LAD only with suture without ligation.Each group was given continuous intragastric administration for 28 days.Left ventricular ejection fraction(LVEF)and left ventricular fraction shortening(LVFS)were measured by echocardiography,left ventricular internal dimension diastole(LVIDd),and left ventricular internal dimension systole(LVIDs)and the changes of cardiac function and cardiac structure were observed.The body mass(BM)and left ventricular mass(LVM)of the rats were measured and the left ventricular mass index(LVMI)was calculated.HE staining was used to observe the pathological changes of myocardial tissue.Collagen volume fraction(CVF)and Masson staining were used to detect the expression of collagen in rat myocardial tissue.Enzyme linked immunosorbent assay(ELISA)was used to detect the expression of tumor necrosis factor-α(TNF-α).The expression levels of miR-126-5p,Bcl-2,Caspase-9 and Caspase-3 were detected by real-time fluorescence quantitative PCR,and the expression levels of MMP-2,MMP-9,Bcl-2,Caspase-9 and Caspase-3 were detected by Western blot.4.RESULTS(1)Compared with the sham operation group,LVEF and LVFS of the model group were significantly decreased(P<0.05),while LVEDD and LVIDS were significantly increased(P<0.05);compared with model group,LVEF and LVFS in trimetazidine hydrochloride group and Wenxin Decoction group were significantly increased(P<0.05),while LVEDd and LVIDs were significantly decreased(P<0.05);compared with the three groups of Wenxin Decoction,LVEF in the medium-dose and high-dose groups of Wenxin Decoction was significantly increased,LVFS in the high-dose group of Wenxin Decoction was significantly increased,LVEDd in the high-dose group of Wenxin Decoction was significantly decreased,and LVIDs in the medium-dose and high-dose groups of Wenxin Decoction were significantly decreased(P<0.05).(2)Compared with sham operation group,LVMI in model group was significantly increased(P<0.05);compared with model group,LVMI in trimetazidine hydrochloride group and Wenxin Decoction group was significantly decreased(P<0.05);LVMI decreased in Wenxin Decoction low-dose and medium-dose groups,but the difference was not statistically significant(P>0.05).(3)Compared with the sham operation group,HE staining showed that the myocardial fibers in the model group were obviously broken,scattered and arranged,with different staining depth and widened cell space.The cells in the trimetazidine hydrochloride group and the Wenxin Decoction group were orderly and uniformly stained.Masson staining showed a large amount of collagen deposition in myocardial tissue after MI in model group.Compared with sham group,CVF was significantly increased(P<0.05).Collagen deposition in trimetazidine hydrochloride group and Wenxin Decoction group was significantly decreased,and CVF was decreased(P<0.05);compared with low-dose Wenxin Decoction group,CVF in medium-dose Wenxin Decoction group and high-dose Wenxin Decoction group was significantly decreased(P<0.05).(4)Compared with sham operation group,the expression level of TNF-α in serum of model group was increased(P<0.05);compared with model group,the expression level of TNF-α in serum of trimetazidine hydrochloride group and Wenxin Decoction group was significantly decreased(P<0.05);there was no significant difference among Wenxin Decoction low-dose,medium-dose and high-dose groups(P>0.05).(5)Compared with the sham operation group,the expression of miR-126-5p in myocardial tissue of model group rats after MI was significantly decreased(P<0.05),the expression of Bcl-2 gene was also decreased(P<0.05),and the expression of Caspase-9 and Caspase-3 genes were increased(P<0.05).The protein expression of Bcl-2 was decreased(P<0.05),while the protein expression of Caspase-9,Caspase-3,MMP-2 and MMP-9 were increased(P<0.05).Compared with model group,the expression of miR-126-5p in myocardial tissue after MI in trimetazidine hydrochloride group and Wenxin Decoction group was significantly increased(P<0.05),the expression of Bcl-2 gene showed an increasing trend(P<0.05),and the expression of Caspase-9 and Caspase-3 genes were decreased(P<0.05).The protein expression of Bcl-2 was increased(P<0.05),while the protein expression of Caspase-9,Caspase-3,MMP-2 and MMP-9 were decreased(P<0.05).Among them,compared with Wenxin Decoction low-dose group,medium-dose group and high-dose group,the gene expressions of miR-126-5p and Bcl-2 in Wenxin Decoction high-dose group were significantly up-regulated(P<0.05),while the gene expressions of Caspase-9 and Caspase-3 in Wenxin Decoction group were not significantly different(P>0.05).The protein expression of Bcl-2 in Wenxin Decoction high-dose group was significantly increased(P<0.05),the protein expression of Caspase-9 and Caspase-3 had no significant differences(P>0.05),and the protein expression of MMP-2 and MMP-9 in Wenxin Decoction high-dose group was significantly decreased(P<0.05).5.CONCLUSIONThe expression of miR-126-5p in myocardial tissue was significantly decreased after myocardial infarction.Wenxin Decoction with supplementing Qi and activating blood can improve cardiac function,inhibit myocardial fibrosis,and improve extracellular matrix metabolism in rats with acute myocardial infarction.The mechanism may be related to the up-regulation of miR-126-5p expression.It also regulates the expression of Bcl-2/Caspase-9/Caspase-3 gene and protein in the downstream apoptotic pathway. |
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