| ObjectiveBased on preliminary research,Qishen Granule(QSG)has beneficial effects on heart failure in rats after myocardial infarction and chronic myocardial ischemia in Chinese miniswine.On this basis,our study will observe the effects of QSG on myocardial protection in mice with transverse aortic constriction,especially in the mechanism of inhibiting myocardial hypertrophy.Materials and Methods1.Six-week-old C57BL/6 male mice were equally and randomly divided into the sham operation,model,QSG and Fosinopril group.The mice in the model and QSG group were subjected to TAC.The sham operation group was threaded without constriction.After the operation,the QSG group mice intervene with QSG14.66g/kg(Clinical equivalent dose)and the Fosinopril group mice intervenes with Fosinopril 1.83mg/kg(Clinical equivalent dose).All drugs are dissolved by water.The daily gavage volume was 1.0ml/100g and last 4 weeks.The other groups were gavaged an equal volume of normal saline.2.A Vevo 2100 small animal ultrasonic apparatus was used to test the blood flow rate in the coarctation of the aorta and parameters of cardiac function.3.Fixed tissue of the heart,using paraffin-embedded technology,was made into paraffin sections.After taking paraffin sections,hematoxylin-eosin staining(H&E)and Mallory staining were used to analyze the pathological variations of heart tissue.4.The heart tissue was tested with Gene Expression Profile Chips(Affymetrix MTA1.0).5.Real-time fluorescence quantitative PCR was used to examine the expression of the p21,p53,Fas,caspase8 and cleaved-caspase3 genes in the heart;6.The expression of the p21,p53,Fas,caspase8 and cleaved-caspase3 proteins was assessed using western blotting.Results(1)As shown in ultrasonic cardiograms,the blood flow rate of the model group was>2,400 mm/s in the coarctation of the aorta.The result meets the requirements of TAC model mice.(2)Compared with the sham group,the left ventricular internal diameter end systole(LVIDs),left ventricular internal diameter end diastole(LVIDd),left ventricular posterior wall thickness end systole(LVPWs),left ventricular posterior wall thickness end diastole(LVPWd),left ventricular anterior wall thickness end diastole(LVAWd),left ventricular anterior wall thickness end systole(LVAWs),left ventricular end-diastolic volume(LVEDV),left ventricular end-systolic volume(LVESV)and left ventricular mass(LVM)were significantly increased in model group mice(P<0.05).The ejection fraction(EF)and fractional shortening(FS)were significantly decreased(P<0.05).Compared with the model group,the left ventricular internal diameter end systole(LVIDs),left ventricular posterior wall thickness end systole(LVPWs),left ventricular posterior wall thickness end diastole(LVPWd),left ventricular anterior wall thickness end diastole(LVAWd),left ventricular anterior wall thickness end systole(LVAWs),left ventricular end-systolic volume(LVESV)and left ventricular mass(LVM)were significantly decreased in QSG group mice(P<0.05).The ejection fraction(EF)and fractional shortening(FS)were significantly increased(P<0.05).(3)Compared with the sham group,the amount of collagenous tissue between myocardial cells was increased,and the cell arrangement was obviously disordered in model group mice.Compared with the model group,the amount of collagenous tissue between myocardial cells was reduced,and the cell arrangement was obviously improved in QSG group mice.(4)Compared with the sham group,the number of differentially expressed genes in the model group was 171 and compared with the model group,the number of differentially expressed genes was 54.Among them,44 genes are present in both comparisons according to the GeneChip analysis.The results of the Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway analysis suggested that QSG had a significant effect on the p53 and apoptosis pathway in the QSG group compared with the effect of the model group(P<0.05).(5)Based on the Real-time fluorescence quantitative PCR results,p21 expression was obviously decreased in the model group compared with the sham group(P<0.05)and obviously increased in the QSG group compared with the model group(P<0.05).P53 expression was decreased in the model group compared with the sham group and increased in the QSG group compared with the model group,but no significant difference(P>0.05).Fas and caspase8 expression was increased in the model group compared with the sham group and decreased in the QSG group compared with the model group,but no significant difference(P>0.05).(6)Western blotting showed an obvious decrease in the expression of the p21 and p53 proteins and increase in the expression of the Fas,caspase8 and cleaved-caspase3 proteins in the model group compared with the sham group(P<0.05).An obvious increase in the expression of the p21 and p53 proteins and decrease in the expression of the Fas,caspase8 and cleaved-caspase3 proteins in the QSG group compared with the model group(P<0.05).Conclusions1.We confirmed the effects of QSG intervention by small animal heart ultrasound and heart histopathology test.The ventricular hypertrophy wall is thinner,enlarged ventricular volume increased,heart weight obviously lighten and heart function recovery significantly.Histopathological results also showed that there are significant effects of QSG in preventing myocardial cell proliferation,myocardial fibrosis and collagen myocardial cell hypertrophy;2.Based on the signal transduction pathway of the cell cycle,the expression of p21 gene in myocardial tissue of mice was significantly increased at the gene level.Although there was no statistical difference in p53 gene,there was a significant trend.At the protein level,the results of the experiment are very good to verify the results of the chips.QSG significantly increased the expression of p53 and p21 protein in myocardial tissue of mice.The results indicated that QSG could significantly regulate the expression of p21 and p53,and have a positive effect on cell cycle.It can be seen that QSG may regulate the cell cycle and has a positive effect on myocardial hypertrophy;3.Based on the apoptosis signaling pathway,the expression of Fas and caspase8 gene in myocardial tissue of mice was decreased at the gene level,but there was no statistical difference.At the protein level,the expression of Fas,caspase8 and cleaved-caspase3 protein in the myocardial tissue of mice was significantly decreased.The results indicated that QSG could significantly regulate the expression of Fas,caspase8 and cleaved-caspase3,and had a positive effect on cell apoptosis.It can be seen that QSG can regulate the apoptosis of myocardial hypertrophy.In conclusion,this study explored the mechanism of QSG in cell cycle and Fas signaling pathway.The study revealed that QSG has important intervention effects in myocardial cell cycle and Fas signaling pathway.All of data were preliminarily explained the effects and mechanisms of QSG on cardiac hypertrophy,and provided exact scientific basis of a new drug for prevention and treatment of cardiovascular disease. |
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