The Effect Of The Medicine For Replenishing Qi And Activating Blood On The Expression Of MiR-133a And Left Ventricular Remodeling In Rats With Myocardial Infarction

Myocardial infarction（MI）is one of the cardiovascular diseases with a high mortality rate.Ventricular remodeling after MI is the key pathological change,which leads to decreased cardiac compliance,decreased cardiac function,and even heart failure.Therefore,it’s of great importance to relieve or inhibit ventricular remodeling for the prognosis of patients with MI_With the continuous development of research,people gradually recognize the important regulatory roles of non-coding small RNAs（miRNAs）in life activities.It has been confirmed that miRNAs are widely involved in the pathophysiological processes of human body.In particular,the change of myocardial miR₁33a in cardiac diseases such as cardiac hypertrophy,MI,and heart failure is attracting much attention,and miR-133a is considered as potential targets for intervention in ventricular remodeling of these cardiovascular diseases.Traditional Chinese medicine has a long history and characteristic theory in the prevention and treatment of cardiovascular diseases such as MI.The methods of benefiting Qi and activating blood circulation are the basic treatment of MI.Whether the efficacy and pharmacological mechanism of Yiqihuoxue medicines in the prevention and treatment of MI are related to the regulation of miR-133a and improvement of left ventricular remodeling is a scientific problem worthy of study.Therefore,firstly,the rat model of MI was established by left coronary artery ligation in this study.In the first experiment,the expression changes of miR-133a and the left ventricular remodeling indictors such as fibrosis and apoptosis were observed.Subsequently,on the basis of experiment one,the intervention and the possible signaling mechanism of Yiqihuoxue medicines including the patent prescription Qidanlixin Pill and the listed medicine Qiliqiangxin Capsule on expression of miR-133a and left ventricular remodeling in rats with MI were further studied through experiment two and experiment three.Through these experiments,we hope to preliminarily explain the molecular pharmacological mechanisms of Yiqihuoxue medicines for its prevention and treatment of MI from miRNA level and related downstream pathway.Objective:1.To study the pathomorphological features of left ventricular remodeling and the expression of miR-133a in rats with MI induced by ligation of the left coronary artery.2.To investigate the interventions of Qidanlixin Pill and Qiliqiangxin Capsule on expression of miR-133a,and the possible molecular pharmacological mechanism related to the left ventricular remodeling in rats with MI.Methods:1.To establish a rat model of MI and observe the pathomorphological manifestations of left ventricular remodeling:the rat model of MI was established by ligation of the left anterior descending coronary artery,and the control group with parallel operation but no ligation（namely sham group）.The changes of ST segment and Q wave were recorded before and after surgery by electrocardiogram.The left ventricular mass（LVM）and body weight（BM）were obtained to calculate the left ventricular mass index（LVMI）.Comparing heart structure and morphology by photos of the heart.The histopathological changes of rat heart were observed by hematoxylin-eosin staining（HE staining）.The expression change of collagen was detected by Masson staining.The apoptosis of cardiomyocytes was detected and observed by TUNEL fluorescent staining.2.The cardiac hemodynamics,miR-133a expression and left ventricular remodeling related indexes and signaling pathway changes were observed in experimental rats:Cardiac hemodynamic indexes including left ventricular systolic pressure（LVSP）,left ventricular end-diastolic pressure（LVEDP）,the maximal rate of increase of left ventricular pressure（+dp/dt max）and the maximal rate of decrease of left ventricular pressure（-dp/dt max）were used to evaluate changes in cardiac function.Detection of miR-133a expression in left ventricular myocardium by RT PCR.The pathological changes of left ventricular remodeling were reflected by LVMI,HE staining,collagen volume fraction（CVF）and apoptotic index.RT-PCR was used to detect the changes of genes related to fibrosis like transforming growth factor-β1（TGF-β1）,connective tissue growth factor（CTGF）,Smad2,Smad3,and genes related to apoptosis like inositol requiring enzyme 1（IRE1）,glucose-regulated protein 78（GRP78）,X-box binding protein-1（XBP-1）,activated transcription factor 4（ATF-4）,activated transcription factor 6（ATF-6）,cysteinyl aspartate specific proteinase 12（Caspasel2）,C/EBP homologous protein（CHOP）,cysteinyl aspartate specific proteinase 9（Caspase9）and cysteinyl aspartate specific proteinase 3（Caspase3）.The protein expressions of TGF-β1 CTGF,Smad2,Smad3,Caspase9,Caspase3 and cleaved-Caspase3 were detected by Western blot.3.Pharmacological study of the Yiqihuoxue medicines in prevent and treatment of left ventricular remodeling induced by MI in rats:experimental rats were randomly divided into model group,sham group（control group）,captopril group（positive control group）and Qidanlixin Pill group（experiment 2）Or Qiliqiangxin Capsule group（experiment 3）.All rats were treated through the intragastric administration once a day for 4 weeks by corresponding drug treatment.After treatment,detected the pathological morphology,cardiac hemodynamics,miR-133a expression and indexes related to left ventricular remodeling in each group.And exploring the gene or protein changes of related signal pathway to partially explain the pharmacodynamic effects and molecular pharmacology mechanisms of the Yiqihuoxue medicines.Results:1.In the rat model of MI,the I lead and the V2-V6 lead of ECG showed obviously raised ST segment after surgery,and the pathological Q wave appeared on the ECG at 24 hours after ligation operation,while the sham group did not show these typical changes on the ECG At2 and 4weeks after coronary artery ligation,compared with the sham group,the left ventricular anterior wall and apex in the model group showed obvious infarction.Specifically,the infarcted area was paler than the non-infarcted area,the ventricular wall became thinner and partially collapsed,and the heart volume enlarged obviously.In the model group,HE staining showed obvious myocardial necrosis in the infarct area of left ventricular,and the pathological manifestations including broken myocardial fiber,widened gap of myocardial fiber,uneven cytoplasmic staining,nuclear pyknosis and deep staining in some nucleus.Masson staining showed much collagen located in the infarct margin of the model group,which was the typical pathological feature of myocardial fibrosis.Moreover,increased apoptotic nuclei were observed in MI rats by TUNEL fluorescent staining.At 4 weeks after coronary artery ligation,LVM and LVMI increased,miR-133a expression significantly decreased in the model group when compared with the sham group（P<0.01）.2.The results of Qidanlixin Pill on miR-133a expression and left ventricular remodeling in rats with MI:compared with the sham group,LVSP decreased,LVEDP increased,±dp/dt max decreased,LVM and LVMI increased,CVF increased,apoptotic index increased,and miR-133a expression decreased in the model group（P<0.05 or P<0.01）.The gene expressions of TGF-β1,CTGF,Smad2,Smad3,Caspase9 and Caspase3 increased in the marginal zone of MI（P<0.05 or P<0.01）.Furthermore,the protein expressions of TGF-β1,Smad2,Caspase9 and Caspase3 increased（P<0.05 or P<0.01）,while the protein expressions of CTGF and Smad3showed no difference（P>0.05）.Compared with the model group,LVSP increased,LVEDP decreased,±dp/dt max increased,LVM showed no significant change（P>0.05）,LVMI decreased,CVF decreased,and apoptotic index decreased,miR-133a increased in the Qidanlixin Pill group（P<0.05 or P<0.01）.The gene expressions of TGF-β1,CTGF,Smad2,Smad3 and Caspase9 were all decreased in the marginal zone of myocardial infarction（P<0.05 or P<0.01）,while there was no difference in the gene expression of Caspase3（P>0.05）.The protein expressions of TGF-β1 Smad2 and Caspase3 were decreased.while the protein expressions of CTGF,Smad3 and Caspase9 showed no difference（P>0.05）.3.The results of Qiliqiangxin Capsule on miR-133a expression and left ventricular remodeling in rats with MI:compared with the sham group,LVSP decreased,LVEDP increased,±dp/dt max decreased,CVF increased significantly,apoptotic index increased,and miR-133a expression decreased in the model group（P<0.05 or P<0.01）.The gene and protein expressions of TGF-β1 increased,and the gene expressions of CTGF,GRP78,ATF-4,CHOP,Caspasel2,Caspase9 and Caspase3 increased（P<0.05 or P<0.01）.There was no difference in the gene expression of IRE1,ATF-6,XBP-1 and in the protein expression of CTGF（P>0.05）.The protein expressions of Caspase9,Caspase3 and cleaved-Caspase3 increased significantly（P<0.05 or P<0.01）.Compared with the model group,LVSP showed no difference,LVEDP decreased,±dp/dt max increased,CVF decreased,apoptotic index decreased,and miR-133a expression increased in the Qiliqiangxin Capsule group（P<0.05 or P<0.01）.The gene and protein expressions of TGF-β1 decreased,while the gene and protein expression of CTGF showed no difference（P>0.05）.The gene expressions of IRE1,GRP78,XBP-1,ATF-4,ATF-6,Caspase12,CHOP,Caspase9 and Caspase3 decreased（P<0.05 or P<0.01）.Moreover,the protein expressions of Caspase9,Caspase3 and cleaved-Caspase3 decreased significantly（P<0.05 or P<0.01）.Conclusion:1.Typical left ventricular remodeling appeared in the MI rats induced by the coronary artery ligation,which showed obvious pathomorphological manifestations including increased myocardial fibrosis and apoptosis.2.The expression of miR-133a in myocardial tissue after MI changed obviously,which showed a significant decreased.3.The patent prescription Qidanlixin Pill and the listed medicine Qiliqiangxin Capsule,which were all developed by the theory of traditional Chinses medicine for benefiting Qi and activating blood circulation,could improve cardiac hemodynamics and partially inhibit left ventricular remodeling after MI.The underlying mechanisms might be related to increasing the expression of miR-133a,and regulating downstream gene and protein involved in the fibrosis and apoptosis signaling pathway.