| Coronary atherosclerotic heart disease is a kind of heart disease caused by coronary artery atherosclerosis,which leads to stenosis or obstruction of vascular cavity,resulting in myocardial ischemia,hypoxia or necrosis,which is often called "coronary heart disease".At present,the disease ranks the highest in the incidence of all kinds of heart diseases in China,and still shows an upward trend.The treatment of traditional Chinese medicine obeys the thought of syndrome differentiation,such as Zhang Zhongjing putting forward "chest arthralgia and pain" in synopsis of the Golden Chamber,and as recorded in Huangdi’s Internal Classic,"if the qi of the heart meridian loses,the pulse will be blocked,and if the pulse is blocked,the blood will not flow.",indicating that qi deficiency and blood stasis syndrome is the basic syndrome type of coronary heart disease.In this experiment,multi-factor combined disease and syndrome model were used.The rat model of syndrome of qi deficiency and blood stasis was established by sleep deprivation for 6 weeks,and then the combined model of syndrome of qi deficiency and blood stasis of coronary heart disease was established by ligating the left anterior descending coronary artery of rats.On this basis,the effects of coronary heart disease syndrome of qi deficiency and blood stasis on platelet function and morphology in rats were studied.In addition,different doses of Buyang Huanwu decoction were given to study the effect of Buyang Huanwu decoction on protecting rats with coronary heart disease qi deficiency and blood stasis syndrome by improving platelet function.The possible target and related mechanism of Buyang Huanwu decoction treating platelet disease were predicted by network pharmacologic methods.The suggested GPVI protein and its downstream pathway were verified and studied,which provided clinical application basis for the treatment of coronary heart disease with syndrome of qi deficiency and blood stasis and also a new research idea on antiplatelet drug targets.This study is divided into five parts:1.Research on Changes of Platelet Function in Rat Model with Qi Deficiency and Blood Stasis Syndrome of Coronary Heart Disease.Aims To study the effect of coronary heart disease model with qi deficiency and blood stasis syndrome on platelet function in rats.Methods SD rats were randomly divided into normal group,model group and aspirin group.After 6 weeks of irregular sleep deprivation in rats with syndrome of deficiency of qi and blood stasis formed,the coronary heart disease model was established by ligation of left anterior descending coronary artery in rats.The expression of CD62p was measured by flow cytometry assay,the platelet aggregation rate induced by ADP and COL,the contents of 5-HT and TXA2 in serum,the content of COX-1 and ATP in platelets were measured by resistance assay.Results 1.Compared with the normal group,the activated platelet ratio and the median expression of CD62p in the model group were significantly lifted(P<0.01).The levels of MAR and AAR,5-HT and TXA2 in serum,COX-1 and ATP in platelets were significantly increased(P<0.01).2.Compared with the model group,the aspirin group significantly reduced the ratio of activated platelets and the median expression of CD62p(P<0.01 or P<0.05),The levels of 5-HT,TXA2,COX-1 and ATP released in rat platelets were significantly decreased(P<0.01 or P<0.05),and the levels of MAR and AAR induced by ADP and COL were significantly decreased(P<0.01).Summary The syndrome of deficiency of Qi and blood stasis of coronary heart disease can lead to the excessive activation and aggregation of platelets and the significant increase of CD62p in rats.The platelet aggregation rate increased significantly under the condition of ADP and COL,indicating that the platelet function was disordered and thrombosis was accelerated.Platelet degranulation in rats was abnormal,the level of COX-1 in platelets increased,the ATP release increased,and the contents of TXA2 and 5-HT in serum increased significantly.The degranulation phenomenon will further expand the activation ratio of platelets,and more platelets will adhere to the injury site,aggregate and form thrombotic clots.2.Morphological Study on Platelet Function in Coronary Heart Disease Rats with Qi Deficiency and Blood Stasis SyndromeAims To explore the effect of Qi deficiency and blood stasis syndrome in coronary heart disease(CHD)on the morphology of platelet related function in rats.Methods SD rats were randomly divided into normal group,model group and aspirin group.The method of modeling was the same as before.After 48 hours,the rats were killed.The ultra microstructure of platelet and the thrombosis of coronary artery were observed by scanning electron microscope.The intracellular Ca2+concentration of platelets was detected by fluorescence micrograph.The fluorescence intensity of Ca2+expression in platelets of rats was observed by fluorescence microscope.HE staining and Masson trichrome staining were used to view the myocardial tissue morphology of rats.Results 1.Compared with the normal group,the ultra microstructure of platelets in the model group showed that pseudopodia protruded more frequently,platelets were connected with each other,and there were more than one thrombus mass.There were many sticky "gels" in the inner wall of the rats’ coronary artery,and more platelets and red blood cells attached to the inner walls of the coronary artery.The concentration of fluorescence calcium ion in platelets increased significantly(P<0.01).HE staining showed more scar tissue,disappearance of myocytes and increase of inflammatory cells.Masson staining showed that the area of myocardial fibrosis was larger,and cardiac myocytes were divided and replaced by fibrous tissue.2.Compared with the model group,aspirin group can effectively reduce the aggregation of platelets in rats,but the improvement of pseudopodia protrusion is not obvious.Improving the inner wall of coronary artery,few thrombus and occasionally red blood cell adhesion were seen;The concentration of fluorescence calcium ion in platelets was obviously decreased(P<0.05).HE staining showed that the necrotic area of cardiac myocytes was reduced,the cardiac myocyte arrangement was more complete,the inflammatory cells were reduced;Masson staining showed that myocardium fibrosis area was reduced obviously,lateral cardiac myocytes arranged orderly,and myocardium tissue was thick.Summary The syndrome of deficiency of Qi and blood stasis in coronary heart disease leads to the formation of atherosclerotic plaque in the inner wall of coronary artery in rats,leading to the increase of calcium ion concentration in platelets,and the ultra microstructure of platelets shows that they are over-activated and aggregated,promoting the dysfunction of platelets,and accelerate thrombosis.At the same time,the model can also lead to severe myocardial tissue damage,cardiac function damage,severe fibrosis,and promote the occurrence of heart failure.3.Effects of Buyanghuanwu Decoction on Syndrome and Related Indexes in Rats with Coronary Heart Disease of Qi-Deficiency and Blood Stasis.Aims To explore the pharmacodynamic effect of Buyanghuanwu Decoction on coronary heart disease with qi deficiency and blood stasis syndrome by improving platelet function.Methods SD rats were randomly divided into normal group,model group,aspirin group,Buyanghuanwu Tang 6 g/kg group and Buyanghuanwu 13 g/kg group.The method of modeling was the same as before,and the dosage of 1 mL/100 g was given by intragastric administration for 14 days after operation.To detect the index of TCM syndrome:tongue,pulse,grip;Medical testing(coagulation function,cardiac function index):coagulation Function(APTT,PT,TT,FIB),hemorheology,echocardiography,myocardial ischemia area,myocardial damage markers(CK,CK-MB,LDH)and myocardial pathological staining(HE,Masson);Platelet function evaluation index:CD62p expression,platelet aggregation rate(ADP,COL),calcium ion expression,scanning electron microscope observation of platelet ultra microstructure and coronary artery ultra microstructure and other indicators.Results 1.Compared with the normal group,the pulse amplitude of rats in the model group was significantly reduced(P<0.01),the R and G values of the lingual surface and floor were significantly reduced(P<0.01),the maximum grip value was significantly reduced(P<0.01),the viscosity of whole blood was significantly increased under the action of low-cut(5s-1),medium-cut(60s-1)and high-cut(150s-1)shear stress(P<0.01),plasma viscosity(100s-1)also increased significantly(P<0.01),activated partial thrombin time(APTT)was significantly prolonged(P<0.01),prothrombin time(PT)was shortened(P<0.01),fibrinogen(FIB)value was significantly increased(P<0.01),echocardiography showed that LVAWs,LVAWd,CO,EF,and FS were significantly reduced(P<0.01),and LVIDSs,LVIDd,LVVs and LVVd were significantly increased(P<0.01).The myocardial ischemia area of rats increased significantly(P<0.01),and serum CK,LDH and CK-MB activities were significantly increased(P<0.01).The results of HE staining showed that the rats had a large area of necrosis area,increased inflammatory cells,and the necrotic area was mainly scar tissue,and the results of Masson staining showed that myocardial fibrosis was serious and myocardial cells were arranged disordered.The platelet aggregation rate induced by ADP and COL increased significantly(P<0.01),the activated platelet ratio and the median CD62p expression increased significantly(P<0.01),calcium ion expression increased(P<0.01),and the platelet ultra microstructure showed that the platelet pseudopodia of the model group were intertwined,the aggregation was obvious,the inner wall of the coronary artery was uneven,and there were more thrombotic masses attached.2.Compared with the model group,the pulse amplitude of the rats in Buyanghuanwu decoction group was significantly higher than that in the model group,and the pulse mobility was better,and the values of R,G and R on the lingual surface were effectively improved in Buyanghuanwu decoction 6 g/kg group,and the R and B values in lingual surface were significantly improved in Buyanghuanwu decoction 13 g/kg group(P<0.01).The maximum grip rate of rats was significantly improved in each dose group(P<0.01 or P<0.05).The whole blood viscosity decreased significantly under the action of low shear,middle shear and high shear stress,and the plasma viscosity of each dose group also decreased significantly(P<0.01 or P<0.05).The APTT of Buyanghuanwu decoction 6 g/kg group was prolonged(P<0.01).In Buyanghuanwu decoction 13 g/kg group,APTT was significantly prolonged and FIB was significantly decreased(P<0.01).Echocardiography results showed that LVAWs,LVAWd,LVPWs,CO,EF,FS increased significantly(P<0.01 or P<0.05),LVIDSs,LVIDd,LVVs and LVVd decreased significantly(P<0.01),Buyanghuanwu decoction 13 g/kg group could also increase LVPWd(P<0.01):Each dosage group could effectively reduce the area of myocardial infarction(P<0.05),and the activities of serum CK,LDH and CK-MB in rats were significantly reduced(P<0.01 or P<0.05).The results of HE showed that each dosage group of Buyanghuanwu decoction could reduce the area of myocardial necrosis,reduce the infiltration of inflammatory cells,thicken the lateral myocardium and protect the cardiac function.The results of Masson staining showed that the fibrosis region of myocardium was obviously reduced,the lateral myocardium was arranged orderly and compactly;The platelet aggregation induced by ADP and COL was significantly decreased(P<0.01 or P<0.05),the median expression of CD62p and the activated platelet ratio were significantly decreased(P<0.01 or P<0.05).The platelet ultra microstructure showed that each dose of Buyanghuanwu decoction could reduce the pseudopodia protrusion and aggregation of platelets in rats,and the adhesion of platelets to the inner wall of coronary artery was reduced and the inner wall of coronary artery was smooth.Summary In this part,the animal model of coronary heart disease with syndrome of qi deficiency and blood stasis was established by ligating the left anterior descending branch of coronary artery in rats with sleep deprivation.The indexes of TCM syndrome were as follows:tongue,pulse,grip,and the indexes of blood and heart function in western medicine:blood coagulation,hemorheology,echocardiography,myocardial ischemic area,myocardial injury markers and myocardial pathological staining.The evaluation indexes of platelet function:CD62p expression,platelet aggregation rate,calcium ion expression,platelet ultra microstructure,coronary artery ultra microstructure and so on,to explore the pharmacological protective effect of Buyanghuanwu decoction by improving platelet related function in the treatment of coronary heart disease rats with qi deficiency and blood stasis syndrome.It could increase pulse amplitude and grip,improve tongue image,prolong APTT and PT,decrease FIB content,decrease whole blood and plasma viscosity,increase EF,FS,CO to improve cardiac function,increase LVAWs,LVAWd,LVPWs,LVPWd,decrease LVIDs,LVIDd,LVVs,LVVd to inhibit ventricular remodeling,reduce myocardial infarction area,reduce calcium ion expression and decrease platelet membrane glycoprotein CD62p expression and decrease platelet aggregation rate induced by ADP and COL.According to the comparison of various indexes,it was found that Buyanghuanwu decoction 13 g/kg had a better protective effect on the model of qi deficiency and blood stasis syndrome in rats with coronary heart disease.4.Study on the Pathway Prediction of Buyanghuanwu Decoction in Treating Platelet Diseases Based on Network Pharmacology.Aims The potential target and mechanism of Buyanghuanwu Decoction in treating platelet diseases were explored through network pharmacology method.Methods LC-MS/MS technique were used to obtain the information of 13 ingredients of Buyanghuanwu decoction.The drug target information of Buyanghuanwu decoction was obtained by PharmMapper,TCMSP,ETCM,BATMAN and Pubchem.Platelet disease targets were acquired by CTD library and Gene Cards library.By Bioinformatics&Evolutionary Genomics,GENEMANIA,STRING,KEGG and other platforms to predict the effect of Buyanhuanwu decoction in the treatment of platelet diseases target.Results Buyanghuanwu Decoction may play a role in treating platelet diseases through protein binding,signal receptor binding,heme binding,same protein binding,enzyme binding and other biological processes.The mechanism may be related to fluid shear stress,atherosclerosis,AGE-RAGE,IL-17,HIF,platelet activation,PI3K/AKT in diabetes mellitus.Summary Buyanghuanwu Decoction treats platelet diseases through multi-component,multi-target and multi-pathway synergy.Platelet activation signaling pathway,fluid shear stress,atherosclerosis pathway and expression of membrane protein receptor GPVI may be one of the key mechanisms,which will be further verified and discussed in subsequent experiments.5.Study on the change of platelets in rats with coronary heart disease syndrome of blood stasis and the effect of Buyanghuanwu Decoction.Aims To explore the mechanism of Buyanghuanwu decoction regulating platelet GPVI and reducing platelet activation to protect the rats with syndrome of blood stasis and qi deficiency of coronary heart disease.Methods SD rats were randomly divided into normal group,model group,aspirin group,Buyanghuanwu 6 g/kg group and Buyanghuanwu 13 g/kg group.The levels of 5-HT,TXA2 and sGPVI in serum,COX-1 content and ATP release in platelets were detected by Elisa,the expression of GPVI,Syk,IP3 and CALDAG-GEFI protein were detected by Western Blot,and the phosphorylation of PLCy2 was detected.Results 1.Compared with the normal group,the levels of serum TXA2 and 5-HT in the model group were significantly higher(P<0.01),and the level of COX-1 in platelets was significantly higher in the control group(P<0.01).The expression of GPVI was significantly increased(P<0.01),the content of sGPVI in serum was significantly increased(P<0.01),the expression of Syk,IP3 and CALDAG-GEFI were significantly increased(P<0.01),and the phosphorylation of PLCγ2 was significantly increased(P<0.01).2.Compared with the model group,the levels of TXA2 and 5-HT in serum and COX-1 in platelets of rats were significantly decreased in each dose group(P<0.01 or P<0.05).The expression of platelet membrane glycoprotein GPVI(P<0.01 or P<0.05)and the content of sGPVI in the serum of the rats were significantly reduced(P<0.01 or P<0.05).The expression of platelet protein Syk and CALDAG-GEFI in rats was significantly decreased(P<0.01).The expression of IP3 and the phosphorylation of platelet protein PLCγ2 were significantly decreased(P<0.01)in 13 g/kg group.Summary Based on the prediction results of pharmacology and network pharmacology in the early stage,this part further discusses the mechanism of Buyanghuanwu decoction improving platelet function to protect coronary heart disease with qi deficiency and blood stasis syndrome(Fig 1).The results showed that Buyanghuanwu decoction could not only regulate and decrease the expression of platelet membrane glycoprotein GPVI and the content of sGPVI in serum,but also decrease the expression of Syk,IP3,CalDAG-GEFI and PLCγ2 phosphorylation in the downstream pathway of the protein,and effectively inhibit the degree of platelet activation mediated by collagen.It can also reduce the content of 5-TH and TXA2 in serum,the content of COX-1 and ATP in rat platelets,effectively inhibit the platelet degranulation reaction,and reduce the occurrence of further platelet activation reaction.These mechanisms are in good agreement with the prediction results of the fourth part of the network pharmacology,which indicates that Buyanghuanwu decoction,a traditional Chinese medicine for invigorating qi and activating blood circulation,may play an important role in protecting rats with coronary heart disease with qi deficiency and blood stasis syndrome by regulating platelet membrane glycoprotein Ⅵ. |
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