Study On The Mechanism Of Qishen Granules In Treating Cardiac Insufficiency With Qi Deficiency And Blood Stasis Syndrome

Aims:Based on the thought of network pharmacology,using transcriptome RNA-Seq technology and methodology NMR metabolomics combined from the perspective of gene regulation expression levels of metabolites,research on the effective drug targets and pathways may exist and may can be the biomarker of the heart dysfunction with QDBS,and the effector mechanisms permit and further explore traditional Chinese medicine treatment of coronary heart disease check against possible pharmacological mechanism for the Chinese drug development,and also providing solid foundation to find effective multi-target drug therapy.Methods:1.We used the Ameroid constriction ring placed on the left anterior descending coronary to copy miniature pig coronary artery disease and chronic heart failure QDBS animal models.Using the sham group as a control group,and after three weeks the evaluation was given to all the model animals.The fit model animals were took administration with traditional Chinese medicine high dose of Qishen particles,medium and low dose group,western positive drug dogxin which were already on the market,and TCM positive drug Qishenyiqi drop pills for treatment.2.Using metabolomics NMR techniques,the model group,sham operation group,Qishen particles high dose group after 8 weeks of endogenous serum samples of small molecule metabolites comprehensive identification and comparative analysis of the difference,and trying to find Qishen particle therapy of coronary heart disease heart failure pathway metabolic pathways may exist,or pharmacodynamic mechanism of action.3.The differentily gene sequencings were deceted by transcriptome RNA-Seq technology,and using the model group,sham operation group,the high-dose group Qishen particles ischemic myocardial tissue which was on the marginal zone.From the perspective of gene regulation,expression patterns of clustering,GO functional classification,Pathway,network-level interaction,the Qishen effective drug targets and pathways effector mechanisms were be studied which may be the important the factors of cardiac dysfunction in coronary artery disease,and further explore traditional Chinese medicine treatment of coronary heart disease check against possible the pharmacological and molecular biological methods for their verification prompt node key molecules and effector mechanisms.Results:1.After 3 weeks,the animals showed no difference between the groups,2,4,8 weeks after administration,miniature swine in model group activity had been restored,but unresponsive to external stimuli,struggling weakness,fur gradually lose their luster,messy hair,reducing the number wag,food intake decreased water intake;symptoms were improved in other groups.2.Before administered an animal model of heart failure QDBS integration increased significantly,there is a comparison with the sham group which was significant differenctly(P<0.05 or P<0.01).After administration,the animals in each treatment group symptom scores decreased compared exist eight weeks,compared with the model group it was a significant difference.(P<0.05 or P<0.01)3.Before administration,compared with the presence of model group,the sham group tongue existed significant difference(P<0.05 or P<0.01).After administration Qishenyiqi pills and Qishen particles different dose group were varying degrees of improvement(P<0.05 or P<0.01).4.Hemorheology showed that before administration,whole blood viscosity at different shear rates,reduced viscosity and blood cell aggregation index varying degrees increased in each group(P<0.05 or P<0.01);8 weeks after administration each drug group permissions under different shear rate and plasma viscosity were improved(P<0.05 or P<0.01).5.Echocardiography showed that before administration,in model group,LVSV increased significantly(P<0.05),LVEF significantly decreased(P<0.01),FS decreased significantly(P<0.05);LVSV,LVDV increased significantly(P<0.05 or P<0.01).2 weeks after administration,digoxin group and QGGH,LVSV and LVDV increased significantly(P<0.05 or P<0.01),LVEF significantly decreased(P<0.01).4 weeks after administration,digoxin group,Qishenyiqi drop pills group,QGGH LVSV increased significantly(P<0.05),LVEF signilicantly decreased(P<0.01).8 weeks after administration of digoxin group,LVSV increased significantly(P<0.05),LVHF significantly decreased(P<0.01);Qishenyiqi dripping pill group and QGGH LVEF decreased significantly(P<0.05 or P<0.01).With the progression of the model group,the different levels of indicators fell.6.After 8weeks,pigs in the the Qishen granules group and the positive control group were more active than pigs in the model group.Compared with pigs in the sham-operated group,systolic blood pressure(SBP),diastolic blood pressure(DBP),and mean arterial pressure(MAP)of the model group decreased signifccantly(P<0.05);pigs in the Qishen granules group and the positive control group showed significant improvement on SBP,SBP and MAP(P<0.05).In vertricular hemodynamic,left vertricular systolic pressure(LVSP),maximal rate of left vertrcular systolic pressure(LV+dP/dt max)and maximal rate of left ventricular diastolic blood pressure(LV-dP/dt max)of the model group were significantly down-regulated when compared with the sham-operated group(p<0.05);the Qishen granules group and sodium digoxin tablet increased the LVSP,LV+dP/dt max and LV-dP/dt max(p<0.05),and thus improved ventricular hemodynamic in pigs with heart failure.7.Heart weight ratio,the heart of BMI in model group was significantly higher than the sham group;each group after adminstration was improved cardiac mass index,especially in Qishen group,obvious improvement in the high-dose group;but the improvement in the positive control group is not obvious in cardiac remodeling.8.8 weeks after administration,cTNT and BNP in model group were increased,and BNP was significantly higher than the sham group;Qishen particles in each dose group and digoxin group can significantly reduce BNP levels(P<0.05).9.After HE staining,model group miniature swine cells were disarray,a large number of myocardial necrosis,loss of normal structure,the occurrence of muscle fibers dissolve;digoxin group of cells were disorganized,the cell gap was increased with fuzzy boundaries;In Qishenyiqi dripping pill group,the cells were disarray,some cells showed nuclear condensation and other performance;Qishen particles high dose group was more neatly arranged,the cells maintained their basic form,some new cells appeard;Qishen particle middle dose group was sparsed arrangement,the more muscle fibers and tidy;Qishen particles low dose group cells lost cardiomyocytes morphology,cell shape changed,some nuclei aggregation.10.After Masson dyeing visible,in model group myocardial cell was collagen fibrous instead with diffuse sexual and infiltration sexual distribution.In diogxin group there were much collagen deposition in the myocardial cell.Qishenyiqi drops pill group were visible large collagen deposition.QGGH existed few collagen organization distribution,and the collagen deposition was less than positive drug group and control group;Qishen particle middle dose group were visible collagen deposition,more clustered around the vascular area,Qishen particles with low dose group was a large number of collagen tissu,showing distribution of characteristics such as diffuse and infiltrating.11.Model group and QGGH,sham group with 1H-NMR found total 25 differences metabolism material,including histidine?acetoacetate?acetate?ethanol?valine?aspartate?glycoproteins?proline?citrate?urea?hippurate?leucine?lysine?creatinine?myo-inositol?succinate?glycerol?dimethylglycine?dimethylamine?methionine?choline?phenylalanine?b-Hydroxybutyrate?b-Glucose?1-Methylhistidine.After 8 weeks adminstration,compared with the sham-operated group,glycoprotein,creatinine,dimethyl glycine,beta-hydroxybutyrate were increased significantly in model group;acetoacetic acid,acetic acid,valine,proline,urea,dimethylamine were reduced.8 weeks after adminstration,compared with the model group,acetoacetic acid,proline,urea,acetic acid,ethanol,1-Methylhistidine werer significantly increased,dimethyl glycine was significantly reduced.12.RNA-Seq results showed that when Model VS Control,90 genes were up-regulated,4 genes were down-regulate;when Treatment VS Model,12 genes were up-regulated,14 genes were down-regulate.When Model VS Control and Treatment VS Model,the differences genes were 10.When Model VS Control,then KEGG pathway maintained p53 signaling pathway,leukocyte transendothelial migration,primary immunodeficiency,cell adhesion molecules.When Treatment VS Model,the KEGG pathways mainly were fatty acid biosynthesis,complement and coagulation cascade,rennin-angiotensin system,and finally the differenced genes were found with their GO classification,clustering pattern and network interaction.13.Molecular biology authentication prompted that compared with the sham group,the model group and each group with administration on JAK2,STAT3,HIF-1-?,NOX2,NOX4 gene expression levels increased in varying degrees,and AC02,MDH,IDH,with varying degrees of expression of PPAR-y were in the lower level.Conclusion1.An herbal formula Qishen particles can effectively improve Ameriod cardiac dysfunction caused by constriction ring miniature pigs QDBS macroscopic signs of improvement in cardiac function as well as physical and chemical parameters.2.1H-NMR metabolomics results suggested the differenced metabolites were concentrated on the major metabolite of amino acid metabolism,glucose metabolism and energy metabolism.There is certain contribution in pharmacodynamic mechanism for clinical treatment to find new drugs target.3.RNA-Seq transcriptome results suggested the important pharmacological pathway maybe closed related to energy metabolism pathway and oxidative stress in Qishen of particles which can treat coronary heart failure with QDBS.