| Coronary heart disease(CHD)is a disease caused by coronary atherosclerotic lesions causing vascular stenosis or obstruction,resulting in myocardial ischemia,hypoxia or necrosis,which seriously threatens human health.Traditional Chinese medicine has certain therapeutic effects and advantages in treating coronary heart disease.The combination of disease and syndrome animal model is the carrier of basic research in traditional Chinese medicine.The research team in the early stage believed that the basic pathogenesis of coronary heart disease is yang deficiency and phlegm stasis,with heart yang deficiency as the foundation and phlegm stasis as the standard.Furthermore,in-depth research was conducted on the mechanism of warming yang and benefiting the heart method and treating coronary heart disease with phlegm and blood stasis.This study aims to construct an animal model of coronary heart disease with heart yang deficiency and phlegm stasis syndrome that conforms to clinical characteristics based on previous research.Using metabolomics technology combined with drug target methods,the study aims to explore the mechanism of the treatment of coronary heart disease with heart yang deficiency and phlegm stasis syndrome with phlegm stasis syndrome using the same treatment formula for phlegm stasis.Objective:To construct a rat model of coronary heart disease with heart yang deficiency and phlegm stasis syndrome,and to study the therapeutic effect and mechanism of phlegm and blood stasis combination therapy on coronary heart disease with heart yang deficiency and phlegm stasis syndrome in rats using metabolomics technology combined with drug target methods.MethodExperiment 1:20 SPF grade male SD rats were randomly divided into a blank group and a coronary heart disease group with 10 rats in each group.The coronary heart disease group with the syndrome of heart yang deficiency,phlegm and blood stasis was fed with high-fat diet for 8 weeks,25 mg/kg of Propylthiouracil(PTU)was administered by gavage every day,600000 IU/kg,100000 IU/kg and 100000 IU/kg of vitamin D3 were injected intraperitoneally at the first,fourth and sixth weeks respectively,and the animal model was established by subcutaneous injection of 1 mg/kg isoproterenol every other day in the eighth week;The blank group received a normal diet,an equal amount of distilled water was administered by gavage,and an equal amount of physiological saline was injected intraperitoneally and subcutaneously.Observe the general state of rats,collect ear temperature,tongue picture,tail vein pulse wave,electrocardiogram,cardiac ultrasound,observe the pathological changes of aortic arch and myocardial tissue of rats with HE staining,detect hemorheology,and detect serum triglyceride(TG),total cholesterol(TC),low density lipoprotein cholesterol(LDL-C)High-density lipoprotein cholesterol(HDLC),inflammatory factor interleukin(IL)-6,tumor necrosis factor(TNF)-α、The expression levels of adenosine triphosphate(ATP)in T3,T4,and myocardial tissue were evaluated based on the integrated TCM syndrome integral table,animal signs,and biochemical indicators of heart yang deficiency phlegm stasis syndrome in coronary heart disease.Experiment 2:40 SPF grade male SD rats were randomly divided into a blank group,a model group,an atorvastatin group,and a phlegm and blood stasis treatment group,with 10 rats in each group.The modeling method is the same as experiment 1.From the fourth week of modeling,the phlegm and blood stasis Tongzhi decoction group was administered with 10g(crude drug)/kg/d of the water decoction of the phlegm and blood stasis Tongzhi decoction,the atorvastatin group was administered with 4.8mg/kg/d of the atorvastatin suspension,and the model group and the blank group were administered with the same amount of distilled water,continuously administered until the end of the eighth week of modeling.Observe the general state of rats,ear temperature,tongue picture,tail vein pulse wave,electrocardiogram,cardiac ultrasound changes,aortic arch and myocardial tissue pathological changes,detect blood rheology,and detect serum TG,TC,LDL-C,HDL-C,IL-6,TNF by ELISA-α、The expression levels of ATP in T3,T4,and myocardial tissue.Experiment 3:Collect rat serum from Experiment 2,use LC-MS non targeted metabolomics technology to detect serum samples,conduct database search on the obtained data,obtain qualitative and quantitative results of metabolites,and conduct multivariate statistical analysis to screen potential biomarkers for the treatment of heart yang deficiency and phlegm stasis syndrome in rats with coronary heart disease using the phlegm and blood stasis treatment formula.Through enrichment analysis of metabolites,reveal the biological significance of metabolites;Screening effective ingredients and drug targets for the simultaneous treatment of phlegm and blood stasis in the TCMSP database;Screening targets for the treatment of coronary heart disease in Gene Cards,TTD,OMIM,DisGeNET,and DrugBank databases,establishing a"phlegm stasis treatment formula active ingredients target coronary heart disease"network,conducting GO enrichment analysis and KEGG pathway analysis on common genes;Associate the potential biomarker enrichment metabolic pathway in metabolomics with the KEGG pathway obtained from network pharmacology research,establish a "drug component target metabolic pathway metabolite target"network,and analyze the mechanism of action of the phlegm and blood stasis combination therapy in treating rats with coronary heart disease with heart yang deficiency and phlegm stasis syndrome.ResultExperiment 1:Rats in the group of coronary heart disease with heart yang deficiency and phlegm stasis syndrome showed mental fatigue,curling up and little movement;In the open field experiment,the total distance and average speed of rat activity decreased(p<0.01),while the activity time decreased(p<0.01);Ear temperature decreased(p<0.01);The tongue texture is slightly dull,and the tongue image and plantar R,G,and B values increase(p<0.01);The amplitude of the tail static pulse wave decreases and the pulse wave widens;The ST segment of the electrocardiogram was significantly elevated(p<0.01);Left ventricular ejection fraction(EF),fraction shortening(FS),cardiac output(CO)and left ventricular mass(LV Mass)decreased(p<0.01);HE staining showed extensive necrosis and calcification of vascular smooth muscle necrosis in the tunica media of the aortic arch,and a large number of myocardial fibers under the endocardium were necrotic and dissolved,replaced by proliferative connective tissue;Whole blood viscosity and plasma viscosity increased(p<0.01);TC,LDL-C,IL-6,TNF in serum-α The content increases,T3 and T4 content decreases(p<0.01),and ATP content in myocardial tissue decreases(p<0.01);The levels of TG and HDL-C in serum decreased,but the difference was not statistically significant.Experiment 2:After the intervention of the phlegm and blood stasis treatment formula,the rats’ mental state improved and their activity increased;Compared with the model group,the total activity distance,average speed,and activity time of the rats in the phlegm and blood stasis treatment group increased(p<0.01);There is a downward trend in ear temperature;The tongue turned moist,and the R,G,and B values of the tongue image decreased(p<0.01),while the G and B values of the foot image decreased(p<0.05 or p<0.01);Pulse amplitude callback;The ST segment height of the electrocardiogram decreased(p<0.01);EF,FS,and left ventricular mass increased(p<0.05 or p<0.01);HE staining showed a small amount of necrosis and calcification of vascular smooth muscle necrosis in the middle membrane of the aortic arch,and a small amount of necrosis and dissolution of myocardial fibers were seen locally in the myocardial tissue;Reduced whole blood viscosity(p<0.05 or p<0.01);TC,LDL-C,IL-6,TNF in serum-α The content decreased(p<0.05 or p<0.01),the T3 content increased(p<0.05),and the TG and T4 content increased,but the difference was not statistically significant;The ATP content in myocardial tissue increased(p<0.05).Experiment 3:A total of 160 differential metabolites were identified between the model group and the blank group,mainly including glycerophosphate choline,amino acids,etc;The enrichment pathway of differential metabolites mainly includes amino acid biosynthesis,ABC transporter,unsaturated fat acid biosynthesis,pyrimidine metabolism,arachidonic acid metabolism,aminoacyl tRNA biosynthesis and other metabolic pathways;A total of 33 potential biomarkers were screened for the treatment of heart yang deficiency and phlegm stasis syndrome in coronary heart disease using the phlegm and blood stasis combination formula,mainly including phosphatidylcholine and fatty acids,including prostaglandin 12,taurine,thiamine monophosphate,etc;The differential metabolites were mainly enriched in the metabolic pathways of glycerol phospholipid,arachidonic acid,linoleic acid and thiamine;The metabolic pathway obtained from network pharmacology and the common signal pathway of Tanyu Tongzhi Recipe obtained from metabolomics in the treatment of coronary heart disease are arachidonic acid metabolism and linoleic acid metabolism,involving two metabolites of prostaglandin 12 and propylthiouracil.The effective components of Tanyu Tongzhi Recipe target PTGS2,CYP1A2,CYP3A4,AKR1C3,ALOX5,LTA4H,PLB1.ConclusionThe rat model of coronary heart disease with heart yang deficiency and phlegm blood stasis syndrome established by intragastric administration of propylthiouracil combined with high-fat diet,intraperitoneal injection of vitamin D3 and subcutaneous injection of isoproterenol is consistent with the clinical characteristics of coronary heart disease with heart yang deficiency and phlegm blood stasis syndrome.The combination of phlegm and blood stasis treatment formula can improve the mental state of rats with coronary heart disease syndrome of heart yang deficiency and phlegm stasis accumulation,improve heart function,increase energy metabolism,improve blood flow status,reduce blood lipids,alleviate inflammatory reactions,reduce arterial plaques,and exert an intervention effect on rats with coronary heart disease syndrome of heart yang deficiency and phlegm stasis accumulation.Phlegm Stasis Tongzhi Recipe may play a therapeutic role by regulating PTGS2,CYP1A2,CYP3A4,AKR1C3,ALOX5,LTA4H,PLB1 targets,and regulating linoleic acid metabolism and arachidonic acid metabolism. |
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