Study On The Mechanism Of Abnormal Platelets Activation In Coronary Heart Disease With Qi Deficiency And Blood Stasis And The Regulatory Effect Of Buyang Huanwu Decoction

BackgroundAccording to the "2021 Report on Cardiovascular Health and Diseases in China",the incidence and mortality rates of cardiovascular diseases in China still rank first,and the prevention and control situation is severe.Coronary heart disease belongs to the category of chest tightness and heart pain in traditional Chinese medicine,and Qi deficiency and blood stasis syndrome is one of its main clinical types.Its pathogenesis is deficiency in essence and excess in manifestation,Qi deficiency and blood stagnation,resulting in poor blood circulation and stasis.Modern pharmacological research has found that the pathological changes of blood stasis syndrome include abnormal activation of platelets,abnormal regulation of inflammation,microcirculation disorders and other mechanisms.Platelets participate in the formation of thrombosis,causing myocardial infarction,and after activation,they enter the ischemic myocardium through microcirculation,accelerating heart failure.The previous 973 project of the team found that platelets are the biological basis of blood stasis,so regulating platelet function is an important part of the treatment of coronary heart disease with Qi deficiency and blood stasis syndrome.Platelets are rich in messenger RNA that participates in transcription,regulates translation,affects protein expression,and regulates platelet function.Further research on platelets is urgently needed.Buyang Huanwu decoction(BYHWD)was recorded in Wang Qingren’s "Medical Forest Correction Error" in the Qing Dynasty,which is a classic prescription for the treatment of qi deficiency and blood stasis,which was first used to treat stroke and is now widely used in the treatment of cardiovascular and cerebrovascular diseases.Studies have shown that BYHWD have pharmacological effects such as regulating angiogenesis after coronary heart disease,affecting blood rheology and blood cell function,anti-inflammatory,antioxidant,and regulating platelet aggregation,adhesion and secretion function,but how it affects the complex signals in platelets and regulates the gene transcription process is not clear.Therefore,firstly we carried out clinical research to analyze the clinical manifestations,biological characteristics,plateletrelated indexes in patients with coronary heart disease qi deficiency and blood stasis.On this basis,the model of qi deficiency and blood stasis in rats was constructed,the changes of platelet activation in the acute phase and convalescent phase were observed,and the regulatory effect of BYHWD on platelets was explored,in order to enrich the scientific connotation of qi deficiency blood stasis research and provide reference for clinical medication.ObjectiveTo explore the transcriptional characteristics of abnormal platelet activation in patients with coronary heart disease with qi deficiency and blood stasis,and to reveal the mechanism of BYHWD in regulating platelet activation in the acute and convalescent stages of myocardial infarction,and to provide experimental evidence for its clinical application.Part 1 Clinical researchStudy on platelet activation characteristics in patients with coronary heart disease with qi deficiency and blood stasisMethodsIn this study,51 healthy and 52 patients of coronary heart disease(CHD)with qi deficiency and blood stasis were included,and the enrollment and screening were based on the diagnostic criteria and exclusion criteria for coronary heart disease with qi deficiency and blood stasis.The ethical approval number was 2020XLA057.The detection indexes included TCM symptom score,vascular ultrasound,echocardiography,platelet aggregation(arachidonic acid,collagen,ADP induced),membrane glycoprotein expression(CD62p,PAC-1),four items of coagulation,transcriptomics and differential protein expression.Results1.1 Compared with healthy,the platelet aggregation rate of patients with coronary heart disease qi deficiency and blood stasis was increased,which was significantly positively correlated with blood stasis scores.Meanwhile in plasma,levels of blood inflammatory factors IL-6,IL-5,IL-10 and IL-17 elevated.1.2 Platelet abnormal activation,CD62p and PAC-1 expression were significantly increased,and the proportion of CD62p and PAC-1 co-expressed platelets decreased in CHD.Scanning electron microscopy showed that the outer membrane of platelets was rough,the mitochondrial structure was not clear,and the release of platelet particles was significantly increased under transmission electron microscopy.1.3 The expression of Rap1,Talin,and RIAM proteins in platelets with coronary heart disease qi deficiency and blood stasis was significantly upregulated,and the phosphorylation levels of Akt and PI3K proteins increased.Part 2 Experimental researchStudy on platelet activation characteristics in rats with myocardial infarction qi deficiency and blood stasis model and the regulatory effect of Buyang huanwu decoction2.1 Study of platelet activation mechanism in convalescent stage rats with myocardial infarction qi deficiency and blood stasisMethodsSD rats were divided into sham,model,positive,low-dose BYHWD and highdose BYHWD group by random number table method.By 6 weeks of sleep deprivation combined with left anterior descending branch ligation of coronary artery,a myocardial infarction model with qi deficiency and blood stasis was constructed.After successful modeling,0.1g/kg·d aspirin i.p.treatment for positive group,0.8 g/kg·d BYHWD i.p.and 1.6 g/kg·d BYHWD i.p.treatment for low-dose and high-dose groups for 28 days,rats in blank and model group were given the same volume of saline.The pulse amplitude,grip power,tongue image parameters,plantar blood flow,echocardiography,platelet aggregation,adhesion,extension function and blood clot retraction were detected.H&E staining was used to observe myocardial histopathology.Masson staining was used to observe the degree of myocardial fibrosis.Flow cytometry was used to detect the content of inflammatory factors(IL-1β,IL-6,IL-18,etc.)and platelet CD62p protein expression in serum.Telomerase expression in myocardial tissue was detected by Q-FISH fluorescence.Platelet morphology was observed under transmission electron microscopy and scanning electron microscopy.Platelet count was detected by hemocytometer,platelet mitochondrial function was detected by hippocampal respirator,and differential genes were screened by transcriptome sequencing,Western Blot and PCR detected the phosphorylation level and gene expression of platelet Rap1,PI3K,Akt,Src and other proteins.Results2.1.1 BYWHD significantly improved the left ventricular systolic function,increased cardiac ejection fraction and myocardial stroke volume.The contents of plasma IL-1 β,IL-6,IL-10,TNF-α,IFN-y and vascular endothelial growth factor in rats with myocardial infarction qi deficiency and blood stasis model were significantly increased,and BYHWD could significantly reduce the content of inflammatory factors.2.1.2 B YHWD significantly reduced the inflammatory infiltration of cardiac tissue and collagen volume fraction,reduced collagen volume fraction,regulated myocardial arrangement,and increased the expression of telomerase in cardiomyocytes.2.1.3 BYHWD significantly reduced the degree of platelet activation,CD62p expression and platelet aggregation,inhibited platelet adhesion to fibrinogen,and improved platelet mitochondrial basal respiration and maximum respiratory capacity.Meanwhile,BYHWD reduced blood viscosity and inhibited blood clot retraction.2.1.4 Under the field of view of transmission electron microscopy,it was seen that the cross-sectional area of platelets in the model group increased,and the release of platelet granules was increased by BYHWD.2.1.5 BYHWD regulated the expression of platelet Rap1 protein and mRNA in the convalescent period of myocardial infarction qi deficiency and blood stasis,and reduced the phosphorylation level of PI3K,Akt,Src and CDC42.2.2 Study on myocardial protective effect and regulation of platelet activation mechanism in acute stage rats with myocardial infarction qi deficiency and blood stasisMethodsSD rats were divided into sham,model,positive aspirin treatment group,and BYHWD treatment group by random number table method.A model of qi deficiency and blood stasis was constructed by sleep deprivation for 6 weeks.Saline was used in the blank group and model group,aspirin 0.1 g/kg·d in the positive drug group,1.6 g/kg·d in the BYHWD for 14 days.Left anterior descending coronary artery ligation was performed to construct an acute phase model of coronary heart disease qi deficiency and blood stasis.At 24 h after ligation,the left ventricular function of rats was evaluated by echocardiography,and the pulse amplitude,grip power,tongue image parameters,and plantar blood flow were detected.H&E staining were used for observing inflammatory cell infiltration,serum 6-keto-PGF1α and TXB2 levels were detected by ELISA,CD62p expression in rats by flow cytometry,coagulation was detected by biochemical method and the degree of internalization of platelets into myocardial tissue by immunofluorescence was detected.Platelet morphology was observed under transmission electron microscopy,transcriptome sequencing screened differential genes.PCR was used to detect mTOR,LC3,P62 and other gene expressions.Results2.2.1 BYHWD significantly reduced the area of myocardial infarction in the acute stage of myocardial infarction in rats with qi deficiency and blood stasis,reduced ECG elevation and the length of QRS period,and improved the inflammatory infiltration of myocardial tissue.2.2.2 BYHWD significantly reduced TXB2 content,while increased 6-ketoPGF1α content,decreased the average platelet aggregation rate and platelet CD62p expression.BYHWD decreased ITGB2 gene expression and inhibited platelet internalization.2.2.3 Transcriptome sequencing and PCR experiments found that BYHWD regulated platelet autophagy,affected mTOR/autophagy signaling pathway,and downregulated the gene expression of autophagy-related genes FUNDC1 and PINK meanwhile increased p62 and mTOR expression.2.3 Regulatory effect and mechanism of red peony on abnormal activation of plateletsMethodsThe drug target of red peony was predicted by compound structure fishing,and the molecular mechanism of active ingredient of red peony was predicted by network pharmacological methods.Platelet-rich plasma was divided into blank,model,aspirin,paeoniflorin,albiflorin and galloylpaeoniflorin group,except for the blank group,the remaining 5 groups were stimulated by hypoxia for 30 minutes.The concentration of paeoniflorin,albiflorin and galloylpaeoniflorin was 100 mg/mL,aspirin was 10 mg/mL,while the blank group and the model group were given the same amount of blank solution.Platelet aggregation was detected by agonist ADP stimulation,calcium ions and reactive oxygen species levels in platelets,platelet phosphatidylserine exposure and mitochondrial membrane potential changes were measured by ELISA method.Results2.3.1 KEGG pathway gene enrichment analysis found that the active ingredients in red peony regulated cytoskeleton,adhesion,cytokine interaction,as well as calcium ion signaling pathway,CAMP signaling pathway,Ras signaling pathway and PI3K-Akt signaling pathway.2.3.2 Paeoniflorin,albiflorin and galloylpaeoniflorin reduced ADP-induced platelet aggregation rate,decreaseded platelet reactive oxygen species release and intracellular calcium ion levels in hypoxic models.2.3.3 Paeoniflorin and albiflorin reduce hypoxia model platelet membrane phosphatidylserine exposure.Paeoniflorin improve mitochondrial membrane potential decline induced by hypoxic models.Conclusion1.In patients of coronary heart disease with qi deficiency and blood stasis,abnormal activation of platelets and ultrastructural changes were observed.Meanwhile CHD increased expression of platelet membrane glycoprotein CD62p and PAC-1,increased expression of Rap1,Talin protein,and phosphorylation of PI3K,Akt.2.Buyan Huanwu decoction regulated coronary heart disease with qi deficiency and blood stasis platelet transcription level,protected cardiac function via regulating Rapl protein and PI3K/Akt signaling pathway during the recovery period of myocardial infarction,and regulate mTOR/autophagy signaling pathway in the acute phase of myocardial infarction to play a therapeutic role.3.Red peony regulated abnormal activation of platelets in coronary heart disease qi deficiency and blood stasis,and paeoniflorin,albiflorin and galloylpaeoniflorin regulated reactive oxygen species and internal calcium content under hypoxic platelet stress,and paeoniflorin also regulated platelet phosphatidylserine exposure and reduced mitochondrial membrane potential decline.