| BackgroundYiqihuoxue druges Huangqi,ginseng,angelica,Chuanxiong and Panax notoginseng are the clinical experience medications used by professor to treat myocardial infarction.Our previous study of network pharmacology found that Calmodulin-dependent protein kinase II(CaMKII)was one of the main targets for the treatment of myocardial ischemic with Yiqihuoxue druges.After administration of Yiqihuoxue druges to myocardial infarction rats.It was speculated that CaMKII may be an important target for the treatment of myocardial ischemic diseases with Yiqihuoxue druges.Several studies have suggested that CaMKII signal transduction pathway plays a key role in the development of cardiac hypertrophy.Therefore,CaMKII may be a key regulatory target during the regulation of myocardial hypertrophy in rats with myocardial infarction.PurposeThis study was designed to observe the effect of Yiqihuoxue druges on cardiomyocyte hypertrophy and CaMKII molecular target in myocardial infarction rats and to explore whether the effect of Yiqihuoxue druges on myocardial.hypertrophy in myocardial infarction rats is related to CaMKII-HDAC4-MEF2C pathway..MethodA rat model of myocardial infarction was constructed by ligation of the left anterior descending coronary artery and divided into model group,Yiqihuoxue group,perindopril group,and sham operation group.The sham group only had threading and no ligation.Each group was intragastrically administered on the second day after the operation.The drug group was given the corresponding drug(clinical equivalent dose).The sham operation group and the model group were given sterile distilled water.The changes of indexes were observed at 7 days and 28 days after surgery.And detect changes in related indicators.Small animal ultrasound was used to detect the changes of myocardial structure and function in infarcted rats at each time point.HE staining and optical microscope were used to observe the changes of myocardial tissue and myocardial cell hypertrophy in each group.Western Blotting was used to detect the expression of CaMKⅡ,p-CaMKⅡ,CaMKIIS,CaMKIIS,HDAC4 and p-HDAC4 in the marginal region of myocardial infarction.Immunofluorescence was used to detect the localization of HDAC4.The expression of MEF2C mRNA was detected by real-time fluorescence quantitative RT-PCR.ResultExperiment one,the effect of Qi-enhancing and blood-activating drugs on heart function in infarcted rats.Small animal ultrasound detection of cardiac function in each group of rats:at 7 days and 28 days after operation,the left ventricular ejection fraction(LVEF)and left ventricular short axis contraction rate in the model group were compared with the sham operation group.left ventricular fraction shortening(LVFS)was significantly decreased(P<0.01,P<0.05).The left ventricular end-systolic volume(LVESV),left ventricular end-diastolic volume(LVESV),left ventricular end-systolic diameter(LVIDs),and left ventricular end-diastolic dimension(LVIDd)were significantly higher in the model group than in the sham group(P<0.01).).At 7 days after operation,LVEF and LVFS were significantly increased in all groups compared with the model group(P<0.05);LVESV in the perindopril group was lower than that in the model group(P<0.05).At 28 days after operation,LVEF and LVFS were significantly higher(P<0.01)and LVEDV and LVESV were significantly lower(P<0.01)in each drug-administered group compared with the model group;LVIDs and LVIDd were significantly lower in the Yiqihuoxue group than in the model group.(P<0.01);LVIDs in perindopril group were lower than those in model group(P<0.05).The results showed that left ventricular dilatation and systolic function were decreased and heart function was significantly reduced in rats with myocardial infarction.The medication group can significantly reduce the left ventricular dilatation state in infarcted rats and improve heart function in infarcted rats.Experiment 2,the effect of Yiqihuoxue drugs on myocardial tissue morphology and myocardial cell hypertrophy in infarct marginal zone of myocardial infarction rats.HE staining was used to observe the histomorphological changes of myocardial cells in each group by light microscopy.Myocardial cells in the infarct margin of infarcted rats were swollen and disorganized,and the infiltration of inflammatory cells increased and the myocardium fibers were disrupted.Compared with the model group,the cardiomyocyte swelling of the drug group was reduced,the arrangement was more orderly,and the infiltration of inflammatory cells was significantly reduced.Cross-sectional morphological parameters of myocardial cells were measured.Results:The cross-sectional area,perimeter,and mean diameter of rat myocardial cells in the model group were higher than those in the sham operation group(P<0.05).At 7 days after operation,compared with the model group,the cross-sectional area,perimeter,and mean diameter of cardiomyocytes in the Yiqihuoxue group decreased(P<0.01,P<0.05);the cross-sectional area and perimeter of the perindopril group were similar to those of the model group.Compared with the model group(P<0.05).At 28 days after surgery,the area,perimeter,and mean diameter of each drug group decreased compared with the model group(P<0.01,P<0.05).The results showed that myocardial tissue in the infarct margin of myocardial infarction rats was severely damaged and myocardial cells were hypertrophied.Yiqihuoxue drugs and perindopril can significantly reduce myocardial structural damage in myocardial infarction rats,and improve myocardial cell hypertrophy.Experiment 3,the effect of Yihuoxue drugs on CaMKⅡ molecular target.The expression of CaMKⅡ,p-CaMKⅡ,CaMKⅡδ in cytoplasm and nucleus were detected.CaMKⅡ protein expression:At the 7th day after operation,CaMKⅡ protein expression was increased in the model group compared with the sham group(P<0.01),and CaMKⅡ protein expression was decreased in each group compared with the model group(P<0.01,P<0.05).There was no statistically significant difference between the groups at 28 days after surgery.Expression of p-CaMKⅡ protein:The expression of p-CaMKⅡ protein in the model group was significantly higher than that in the sham group(P<0.05).Seven days after operation,Yiqihuoxue group was lower than the model group(P<0.01),but there was no significant difference between the perindopril group and the model group.At 28 days after surgery,the medication group was lower than the model group(P<0.01).The protein expression of CaMKⅡδ in cytoplasm and nucleus:The expression of CaMKⅡδ in the nucleus of the model group was significantly higher than that of the sham group(P<0.01)at the 7th day after operation,and the medication group was significantly reduced compared with the model group.(P<0.01),There was no statistically difference in CaMKⅡδ cytoplasmic expression between the groups.At the 28 days after surgery,compared with the sham group,CaMKⅡδ protein expression in the cytoplasm and nucleus was increased in the model group(P<0.01),and the medication group were significantly lower than the model group.The results showed that the expression of CaMKII protein and activation of phosphorylation in the infarcted rats increased,especially the level of phosphorylation increased more significantly.The expression of CaMKⅡδ in CaMKⅡ,the main isoform of CaMKII,increased in both nucleus and cytoplasm of infarcted rats,and increased mainly in nucleus expression in early stage.Yiqihuoxue druges can inhibit its abnormal expression.CaMKII is highly expressed and active in infarcted rats.The Yiqihuoxue drugs and perindopril had a certain inhibitory effect on their expression and activity.Experiment 4:Effect of Yiqihuoxue druges on the expression of HDAC4 and MEF2C in infarct marginal zone in rats with myocardial infarction.HDAC4 and MEF2C are downstream factors of CaMKⅡ and myocardial cell hypertrophy.HDAC4 protein expression:On the 7th and 28th day,the expression of HDAC4 in the model group was higher than that in the sham group(P<0.01),The drug group was lower than the model group but the difference was not statistically significant.Expression of p-HDAC4 protein:At 7 days,the expression of p-HDAC4 protein in the model group was significantly higher than that in the sham group(P<0.01),and the expression level of the medication groups was significantly lower than that of the model group(P<0.01).At 28 days,the expression of p-HDAC4 was significantly higher in the model group,the Yiqihuoxue group,and the perindopril group than in the sham operation group(P<0.01).The Yiqihuoxue group was reduced compared with the model group(P<0.01),and the perindopril group was lower than the model group but the difference was not statistically significant.HDAC4 immunofluorescence assay:HDAC4 in the sham-operated group was basically located in the nucleus,whereas the model group was mainly expressed in the cytoplasm.The drug groups were somewhere in between and there is a certain amount of expression in the nucleus cytoplasm.MEF2C mRNA expression:The MEF2C mRNA in the model group rats at 7 and 28 days was significantly higher than that in the sham operation group(P<0.01).Compared with the model group,MEF2C mRNA expression was significantly reduced in the drug groups and the difference was statistically significant(P<0.01 or P<0.05).This experimental study showed that HDAC4 and its phosphorylation activation levels were significantly increased in infarcted rats,and their phosphorylation levels increased so that they moved from the nucleus into the cytoplasm.The inhibition of MEF2C was weakened,and the expression of MEF2C mRNA was significantly increased.Yiqihuoxue druges significantly inhibited the phosphorylation of HDAC4,which in turn led to decrease in the expression level of MEFF2C gene and eventually inhibited the expression of cardiac hypertrophy genes.In conclusion1.Yiqihuoxue druges can effectively improve the heart structure and function of infarcted rats.2.Yiqihuoxue druges can inhibit the abnormal expression of CaMKⅡ molecular target and its phosphorylation activation level in infarcted rats.3.Yiqihuoxue drugs may improve myocardial hypertrophy in infarcted rats by inhibiting abnormal pathological signal transduction of CaMKⅡ-HDAC4-MEF2C pathway. |
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