| Platelet dysfunction is the main pathological basis for the development of cerebral infarction(CI),and antiplatelet therapy has been an important initiative in the prevention and treatment of CI.P2Y12 receptors play multiple roles in CI,and activation of P2Y12 triggers platelet activation and aggregation,further causing thrombotic inflammation,expanding the infarct size and aggravating the pathological damage of CI.Therefore,platelet P2Y12 receptors have long been the main target of antiplatelet drugs,and platelet P2Y12 antagonists such as clopidogrel are widely used.However,P2Y12 antagonists have side effects such as bleeding,post-infarction recurrence,and drug resistance in clinical applications,and the combination of safety and efficacy has become a major problem in the prevention and treatment of thrombotic diseases.Because of their multi-target,multi-effect and multi-pathway synergistic effects,traditional drugs that activate blood circulation and resolve blood stasis are increasingly becoming the focus of Chinese medicine research with great prospects.Blood Stasis is a symptom caused by poor blood flow in the body,obstruction of the meridians and blood stagnation,which is the most common among cardiovascular diseases in TCM.Buyang Huanwu decoction(BYHW)is a classic formula for the treatment of Qi Deficiency and Blood Stasis in CI,which is based on the cubic formula of "the glory of Qi and blood,and the stagnation of meridians".Previous studies have shown that BYHW has the effect of inhibiting platelet activation and anti-inflammatory factor expression.The specific alterations of platelets and the existence of differentially expressed genes in patients with CI Qi Deficiency and Blood Stasis,as well as whether BYHW,a herbal compound with clear clinical efficacy in activating blood circulation and resolving blood stasis,can modulate P2Y12 receptors and related signaling pathways,thus playing an anti-platelet activation and aggregation role,are still unclear and need to be further explored in combination with clinical and basic experiments.Part 1 Domestic and international bibliometric analysis of platelets in the field of cerebral infarction research based on CiteSpace and VOSviewerObjective:By visualizing and analyzing the platelet-related literature research in CI field in the past 10 years at home and abroad,we can macroscopically explore the popular research and frontier directions in this field.Methods:The Chinese and English literature data sources were obtained by searching the CNKI and WOS Chinese and English databases for the last 10 years,respectively.CiteSpace was used to obtain maps of network visualization(research institutions,authors,references),cluster analysis and timeline visualization analysis(keywords)and citation burst(references,keywords),including double mapping maps(dual-map)of journals;VOSviewer was used to obtain maps of network visualization(country/region,keywords),year overlay visualization(country/region,keyword)map,and density visualization(country/region,keyword)map.Results:(1)A total of 1,974 publications were screened for inclusion based on the search strategy.The number of literatures of platelet research in CI showed an overall trend of steady climbing increase.(2)These Chinese and English publications are mainly from China and the United States,with the largest contribution from Chinese scholars in particular.(3)The main research institutions were Jilin University,Hebei Medical University,Capital Med University,and Wurzburg University.(4)Chinese Journal of Practical Neurological Disorders,Chinese Medical Guide,CIRCULATION,and NEW ENGL J MED were the most studied and co-cited journals.(5)Among the study authors,Wang Congjun from Beijing Tiantan Hospital,Capital Medical University,Yi Xingyang from Deyang People’s Hospital,Bernhard Nieswandt and Guido Stoll from Würzburg University,ranked high in terms of number of publications.(6)Among the keywords,the strongest cited occurrences in Chinese and English literature were clopidogrel,prevention,adverse effects,mechanical clot retrieval and intravenous thrombolysis,thrombectomy,neutrophil extracellular traps,etc.;cluster analysis formed#0 clopidogrel,#1 stroke,#2 cerebral infarction,#3 atrial fibrillation,#4 platelets,#5 antiplatelets,#6 secondary prevention and#0 ischemic cerebral infarction,#1 Clopidogrel,#2 acute ischemic stroke,#3 platelet activation#4 atrial fibrillation,#5 eicosanoic acids;the latest clustering keywords on the time axis are neurological factors,neurological recovery,inflammatory indicators,fibre-lowering therapy,secondary prevention,cerebral blood flow,immune function.predictive value,and early management,dual anti-platelet therapy,cell death,cerebral venous thrombosis,intracerebral hemorrhage,predicts,etc.Conclusion:In the past 10 years,domestic and international research on platelets in CI has focused on antiplatelet agents,platelet function,thrombus extraction,early management and secondary prevention,prediction,neuronal cell repair,immune inflammation,biomarkers and biomics technologies as popular and cutting-edge research directions in this field.Part 2 Clinical study of platelet function and morphological changes in patients with cerebral infarction with Qi Deficiency and Blood Stasis evidenceObjective:Based on the clinical study,we observed the biological indexes and micromorphological changes related to platelets in patients with CI Qi Deficiency and Blood Stasis evidence.Methods:A clinical cross-sectional study was used to include subjects who met the criteria of CI Qi Deficiency and Blood Stasis evidence study group and healthy control group,and to compare the maximum aggregation rate,coagulation function,basal parameters and other clinical biological indexes of platelets in the two groups;flow cytometry was used to detect platelet activation indexes CD62P,PAC-1 and 12 inflammatory cytokines to observe the degree of platelet activation and inflammatory response;transmission electron microscopy was used to observe the microscopic morphological changes of platelet precipitates.Results:(1)The mean score of Qi Deficiency and Blood Stasis symptoms in the study group was 34.55±3.95;the mean NIHSS score in the study group was 10.87±3.86;the TG(t=3.17,P<0.01),GGT(t=2.39,P=0.0187),GLU(t=3.74,P<0.01),UA(t=2.77,P<0.01)were significantly higher than those in the control group.(2)Among the platelet aggregation rate indexes,PAg-AA,PAg-ADP,and PAg-COL were significantly higher in the study group than in the control group(tPAg-AA=3.213,P<0.01;tPAg-ADP=7.682.P<0.01;tPAg-COL=2.766,P<0.01);among the coagulation function indexes,APTT,TT were significantly lower than those of the control group(tAPTT=2.22,P<0.05;tTT=2.57.P<0.05),and the FIB of the study group was significantly higher than that of the control group(tFIB=3.15,P<0.01);among the platelet parameters,PLT and PCT of the study group were significantly lower than those of the control group(tPLT=2.27,P<0.05;tPCT=3.10,P<0.01).(3)The flow cytometry results showed that the expression levels of platelet activation indexes CD62P and PAC-1 were significantly increased in the study group compared with the control group(P<0.01).(4)Compared with the control group,the expression levels of inflammatory factors IL-4 and IL-8 were decreased in the study group(P<0.01,P<0.01);while the expression levels of IL-5.IL-10 and IFN-γ were increased in the study group(P<0.05,P<0.01,P<0.05).(5)Transmission electron microscopy showed that the platelets in the control group were oval in shape,with clear cell membrane boundaries,clearly visible intracellular granules and intact cell structure;the platelets in the CI Qi Deficiency and Blood Stasis study group had broken cell membranes,large vacuoles,high intracellular granule destruction,mitochondrial cristae fracture and disorganized cell structure,as well as a tendency of pseudopod protrusion.Conclusion:Patients with CI Qi Deficiency and Blood Stasis evidence have markedly broken platelet structure,high platelet activation aggregation and coagulation dysfunction,along with an immune inflammatory response.Part 3 Correlation and predictive value of the severity of Qi Deficiency and Blood Stasis evidence in CI with platelet biological indicatorsObjective:To explore the correlation between the severity of symptoms and platelet biological indexes in patients with CI with Qi Deficiency and Blood Stasis,and to explore the clinical predictors of the occurrence of CI with Qi Deficiency and Blood Stasis.Methods:A general linear bivariate Pearson linear correlation was applied to test the correlation between clinical platelet biology-related indexes,such as platelet aggregation rate,coagulation function,platelet parameters,and Qi Deficiency and Blood Stasis symptoms(QDBSS score)and neurological deficits(NIHSS score);binary logistic regression(Enter method)was used to analyze the independent factors influencing the severity of Qi Deficiency and Blood Stasis symptoms and neurological deficits;the clinical value of each index level to predict Qi Deficiency and Blood Stasis symptoms and severe neurological deficits was tested by survival curve(ROC).Results:(1)There was a linear positive correlation between QDBSS score and NIHSS score(r=0.786,P<0.01),and its linear regression equation was Y=19.29+1.43X.(2)The severity of Qi Deficiency and Blood Stasis evidence was positively correlated with PAg-AA(r=0.448,P=0.013)and FIB(r=0.362,P=0.008),and negatively correlated with APTT(r=-0.331,P=0.015)was negatively correlated;the degree of neurological deficits was positively correlated with PAg-AA(r=0.420,P=0.021),PAg-ADP(r=0.528,P=0.005),FIB(r=0.276.P=0.047)and negatively correlated with APTT(r=-0.338,P=0.013)was negatively correlated(P<0.05).(3)PAg-ADP(OR=1.306,95%CI 1.014-1.684,P=0.039).FIB(OR=17.749,95%CI 1.834-17.178,P=0.013),PLT(OR=0.760,95%CI 0.617-0.936,P=0.010),PCT(OR=5.920,95%CI 4.027～8.703,P=0.015),PDW(OR=12.645,95%CI 1.322～12.092,P=0.028)indexes were independent influencing factors for the severity of CI Qi Deficiency and Blood Stasis evidence.(4)PAg-ADP(OR=1.589,95%CI 1.076～2.348,P=0.020),TT(OR=8.71 1,95%CI 1.204～63.010,P=0.032),FIB(OR=7.216,95%CI 1.135～45.870.P=0.036),PLT(OR=0.739,95%CI 0.581-0.940,P=0.014),and PCT(OR=5.832,95%CI 8.631-3.940,P=0.020)indicators were independent influencing factors on the severity of neurological deficits in CI Qi Deficiency and Blood Stasis evidence.(5)PAg-ADP,FIB,PLT,and PCT were used to predict the AUC of CI Qi Deficiency and Blood Stasis evidence of 0.9310,0.7196,0.6273,and 0.6608,respectively,all of which had some predictive value,with PAg-ADP and FIB having the most significant predictive value.Conclusion:CI Qi Deficiency and Blood Stasis evidence exist platelet aggregation and coagulation abnormalities,PAg-ADP and FIB levels are common independent risk factors affecting the severity of the disease,with prominent clinical relevance,and long-term monitoring has important significance and assessment value for the prognosis and regression of the disease.Part 4 Transcriptomic analysis of platelets in patients with CI with Qi Deficiency and Blood Stasis evidenceObjective:To search for differentially expressed genes between CI Qi Deficiency and Blood Stasis evidence and healthy population,to reveal the molecular mechanism leading to the occurrence of CI Qi Deficiency and Blood Stasis evidence,and to provide a chain of evidence for clinical research and basic experiments.Methods:Platelets were collected from elbow venous blood and isolated from the study and control groups,total RNA was extracted,total RNA from platelet precipitates was detected,a transcriptomic library was established,sequenced using an Illumina HiSeqTM 2500 sequencer,and the differentially expressed genes and related signaling pathways in platelets of patients with CI Qi Deficiency and Blood Stasis were mined by GO enrichment,KEGG enrichment and protein interaction network analysis,and validated by RT-qPCR.Results:(1)There were 2253 differentially expressed genes between the two groups,1731 genes were up-regulated and 522 genes were down-regulated.(2)The total number of GO enrichment entries between the two groups was 5492,including 3790 in the BP category,656 in the CC category and 1046 in the MF category.The differentially expressed genes were mainly enriched in extracellular exosome,neutrophil degranulation,platelet degranulation,blood coagulation,and inflammatory response.inflammatory response,nucleosome assembly,platelet alpha granule lumen.focal adhesion,tertiary granule lumen,platelet alpha granule lumen,platelet alpha granule lumen,platelet alpha granule lumen,platelet alpha granule lumen,platelet alpha granule lumen,platelet alpha granule lumen.granule lumen.platelet aggregation,platelet activation,etc.(3)The total number of KEGG enrichment entries between the two groups was 319,including 91 in HD category,82 in OS category,21 in CP category,32 in EIP category,19 in GIP category,and 74 in M category.The main pathways of differentially expressed gene enrichment were Gap junction,Focal adhesion,Necroptosis,Rap1 signaling pathway,PI3K-Akt signaling pathway(PI3K-Akt signaling pathway),IL-17 signaling pathway,NF-kappa B signaling pathway,Toll-like receptor signaling pathway,NOD receptor signaling pathway,NOD-like receptor signaling pathway,Fluid shear stress and atherosclerosis,Platelet activation,Vascular smooth muscle contraction Vascular smooth muscle contraction,Human cytomegalovirus infection,etc.(4)Validation by RT-qPCR revealed that the key hub mRNAs(GP1BA,GP1BB,VWF,IGF2,ITGA2B)and differentially expressed P2Y receptor family mRNAs(P2RY1,P2RY2,P2RY10,P2RY12,P2RY13,P2RY14)in platelets from patients with CI Qi Deficiency and Blood Stasis evidence were found to be significantly different compared with the control group.P2RY14)were consistent with the RNA-seq results,indicating that the sequencing results of RNA-seq were accurate and reliable.Conclusion:Abnormally expressed mRNA expression profiles exist in platelets of patients with CI Qi Deficiency and Blood Stasis evidence.mRNAs expressed by the P2Y receptor family may be involved in the development of platelet pathological changes in CI Qi Deficiency and Blood Stasis evidence.Part 5 Pathological changes in a combined model of CI with Qi Deficiency and Blood Stasis and the effect of BYHW in ratsObjective:To observe the pathological changes in rats in a combined model of CI Qi Deficiency and Blood Stasis evidence,and the effects of the intervention by BYHW.Methods:Ninety SD rats were fed ad libitum for 3 days with sleep deprivation for 2 weeks first,and the multiple cerebral infarction model was induced by microspheres,combined with the incomplete sleep deprivation method on the horizontal table to establish the evidence of Qi Deficiency and Blood Stasis,and together they constructed an animal model combining CI Qi Deficiency and Blood Stasis disease,and the specimens were collected after 1 month of corresponding drug treatment.The rats were divided into sham operation group(Sham),model group(Model),clopidogrel positive drug group(CL,6.75g/kg/d),ADP agonist group(ADP,0.2mg/kg ADPβS diluted in 0.5ml saline,tail vein injection),tonic Yang Returning Five Soup high dose group(BYHW-H,1.8g/kg/d),tonic Yang(BYHW-L,0.9g/kg/d),15 rats in each group.The rats were monitored daily for general phenotypes,and neurological deficits were scored at 6h and 24h post-modeling,and the maximum grip force was recorded at four time points before,7 days,14 days and 28 days post-modeling using a rat grip force meter;the rats were monitored by PeriCam PSI HR laser scattered blood flow video for perfusion in the caudal peripheral circulation;the pathological changes in the hippocampal region of brain tissue were observed by HE staining.The pathological changes in the hippocampal region of brain tissue were observed by HE staining.Results:(1)The rats in the Model group were thinner than those in the Sham group,with hemiplegic limbs,slow response,and yellowish and lusterless hair;the rats in the BYHW group were in a slightly better condition,with relatively normal physical development.(2)There was no significant difference in body weight change between Model group and Sham group from before modeling to day 7;on day 14,the body weight of rats in Model group showed a decreasing trend compared with Sham group(P<0.01);on day 28,the body weight of rats in Model group also decreased compared with Sham group(P<0.01),compared with Model group,the body weight trend of CL and BYHW-H group was relatively higher.(2)The body weight trend was relatively higher in the CL and BYHW-H groups compared with the Model group and lowest in the ADP group during the same period.(3)The maximum grip force values of rats in each group before modeling were not significantly different;on day 7 and 14,the Model group showed a significant reduction in grip force compared with the Sham group(P<0.01),and the maximum grip force values of rats in the BYHW-H group showed an increasing trend compared with the Model group(P<0.05);on day 28,the Model group showed a decrease in grip force compared with the Sham group(P<0.01),and the other groups showed a decrease in grip force compared with the Model group(P<0.01).Compared with the Model group,all the other dosing groups showed a relative increase,and the highest grip force values were found in the CL and BYHW-H groups,while the BYHW-L and ADP groups were relatively lower during this period.(4)The red pixels on the tail surface of the rats were dense and highly chromogenic,suggesting good blood circulation;the red pixels in the Model group were sparse and less chromogenic than those in the Sham group,suggesting poor blood circulation;compared with the Model group.CL perfusion was the highest,BYHW-H was the second highest,and ADP group was relatively lower,suggesting that the tail microfluidic perfusion in the BYHW-H group improved after drug administration.(5)The neurological deficit scores of rats in the Model group were higher than those in the Sham at 6h and 24h after modeling(P<0.01);compared with the Model group,the neurological deficit scores of rats were lower than those in the Model group at 24h postoperatively between all dosing groups(P<0.05).(6)Microscopically,the structural damage was obvious in the Model group compared with the Sham group,which showed sparse and scattered neuronal arrangement in the CA1 region of the hippocampus,edema and degeneration of neurons,significantly reduced cellular hierarchy,and the presence of inflammatory cell lymphocyte clusters,and vacuolar degeneration with nuclear consolidation and even necrosis in some cells;compared with the Model group,the structural changes in the BYHW-H group were relatively mild,and the pathological changes were less than those in the other dosing groups and close to those in the Model group.Compared with the Model group,the BYHW-H group had less structural changes and fewer pathological changes than the other dosing groups and was closer to the Sham group.Conclusion:1.Rats with CI Qi Deficiency and Blood Stasis had symptoms of weakness and peripheral microcirculation disorders,combined with neurological deficits and pathological structural damage in the hippocampus.2.BYHW can improve the severity of Qi Deficiency and Blood Stasis symptoms and reduce the neurological deficits and pathological damage in CI rats with Qi Deficiency and Blood Stasis.Part 6 Regulation mechanism of platelet and neuroinflammation in CI Qi Deficiency and Blood Stasis evidence by BYHW mediated by P2Y12 receptor based on AC/PKA signaling pathwayObjective:To explore the effects of P2Y12 receptor-mediated effects on platelet function and neuropathy in CI Qi Deficiency and Blood Stasis evidence,and to explore where the mechanisms of BYHW modulation lie.Methods:Sixty SD rats were divided into sham-operated group(Sham),model group(Model),clopidogrel positive drug group(CL,6.75g/kg/d),ADP agonist group(ADP,0.2mg/kg ADPβS diluted in 0.5ml saline,tail vein injection),and tonic yang hui wu tang group(BYHW,1.8g/kg/d)by random number method.15 animals in each group.Immunofluorescence double-labeling was used to co-localize P2Y12 with microglia Iba-1;Micro PET/CT scan was used to assess the inflammatory expression of TSPO in rat brain;flow cytometry was used to detect the expression levels of platelet surface membrane glycoproteins CD62P and CD61;PL-12 automatic platelet aggregation meter was used to detect the platelet aggregation rate;hemagglutination meter was used to detect blood coagulation function;glycine silver Western-Blot assay for P2Y12,AC,PKA,p-VASPser157 protein expression.Results:(1)The results of immunofluorescence double-labeling showed that P2Y12 receptor was co-expressed with microglia Iba-1 and specifically expressed on microglia;compared with Sham group,P2Y12 expression was significantly increased in Model group and microglia activation was significant;compared with Model group,P2Y12 expression was the lowest in CL group and microglia activation was the lowest in BYHW group.This indicates that the Chinese herbal compound BYHW can synergistically play the advantage of reducing the inflammatory response and brain damage.(2)The expression of TSPO in the Model group was significantly higher than that in the Sham group,reaching a peak on day 7,then gradually decreasing slightly and leveling off after day 14;compared with the Model group,the expression in the BYHW group was similar on day 1 and also peaked on day 7,but its expression was still lower than that in the Model group,then decreasing significantly and leveling off after day 14.(3)The expression levels of CD62P and CD61 in rat platelets were significantly higher in the Model group compared with the Sham group(P<0.01);the expression levels of CD62P in rat platelets in the CL and BYHW groups were lower than those in the Model group(P<0.01).It showed that both BYHW had inhibitory effect on platelet activation.(4)Compared with the Sham group,the maximum and mean aggregation rates(MAR and AAR)of platelets in the Model group induced by ADP inducer were significantly higher than those in the Sham group(P<0.05);compared with the Model group,the BYHW group could significantly reduce the MAR(P<0.05)and AAR of platelets in rats with CI Qi Deficiency and Blood Stasis evidence induced by ADP(P<0.05).(5)Compared with the Sham group,PLT was significantly decreased(P<0.05)and PT,PTA and Fbg were significantly increased(P<0.05,P<0.01,P<0.05)in the Model group rats.Compared with the Model group,PLT was increased in the BYHW group(P<0.01)and Fbg showed a decrease(P<0.05).It was suggested that BYHW could improve platelet activation aggregation and increase PLT level,while decreasing Fbg expression in rats with CI Qi Deficiency and Blood Stasis evidence.(6)Compared with the Sham group,there was a decrease in cAMP level in the brain tissue of rats in the Model group(P<0.01)and an increase in cGMP level(P<0.01);compared with the Model group,there was an increase in cAMP level in the brain tissue of rats in the CL and BYHW groups(P<0.05)and a decrease in cGMP level in the brain tissue of rats in the CL group(P<0.05).It is suggested that BYHW promotes neural regeneration by increasing the expression level of cAMP in the brain tissue of rats after ischemia.(7)Compared with the Sham group,the rats in the Model group showed a significant increase in the total action trajectory distance(P<0.05),a significantly longer escape latency period(P<0.01),a decreasing trend in the mean speed(P<0.01),and a decrease in the number of platform crossings(P<0.01);the rats in the BYHW group showed a decrease in the total swimming distance and escape latency duration compared with the Model group(P<0.05),while the number of platform crossing was increased(P<0.05).It showed that BYHW could improve the spatial learning memory ability of rats.(8)Glycine silver staining(silver plating)staining was observed microscopically in the hippocampal region of the rats in the Model group compared with the Sham group,and a large number of black streaks were visible microscopically,a large number of wrinkled neurons were visible and stained dark brown.and the neuronal axons were black and thicker in shape,especially in the CA3 region;the distribution of black streaks in the hippocampal region of the rats in the BYHW group was relatively lighter,and the black The distribution of neuronal axons was reduced.(9)Transmission electron microscopy of rat platelets showed that the platelets in the Model group were more agglomerated than those in the Sham group,and the cell structure was severely damaged,with a tendency of pseudopods protruding;the microstructure of platelets in the BYHW group improved significantly,and the edges of the cell membrane were more intact,without obvious pseudopods.Ultrastructural observation of microglia in brain tissue revealed that microglia in the Model group were more broken than those in the Sham group,with irregularly shaped nuclei and obvious local depression;the degree of structural destruction of microglia in the CL and BYHW groups was less than that in the Model group;the destruction in the ADP group was obvious.It is suggested that CI Qi Deficiency and Blood Stasis evidence will show increased inflammatory expression of microglia,and BYHW can reduce the degree of microglia damage after ischemia.(10)WB results showed that compared with the Sham group,platelet P2Y12 protein expression level was significantly increased(P<0.01)and AC,PKA,p-VASPser157 protein expression was decreased(P<0.05,P<0.01,P<0.01)in the Model group,while AC,PKA.p-VASPser157 protein expression level in the ADP group decreased(P<0.01,P<0.05);compared with the Model group,the platelet P2Y12 protein expression levels of rats in CL and BYHW groups were decreased(P<0.05),while AC,PKA,p-VASPser157 protein levels were increased to different degrees(P<0.05).This indicates that BYHW can increase the expression levels of AC and PKA proteins in platelets with CI Qi Deficiency and Blood Stasis evidence,promote the dephosphorylation of phosphorylated VASP(p-VASPser157),and exert anti-platelet activation effects.Conclusion:1.Brain tissues of rats with CI Qi Deficiency and Blood Stasis evidence showed co-expression of P2Y12 and microglia Iba-1,significantly increased platelet activation and aggregation levels,impaired coagulation,broken and deformed electron microscopic microstructures,as well as high expression of inflammatory response levels in brain tissues,and significantly reduced spatial learning and cognitive memory ability.2.BYHW can inhibited the changes of platelet function mediated by P2Yi2 receptor,and enhance the expression of p-VASPSer157 protein by regulating AC/PKA signaling pathway to inhibit platelet activation and aggregation in CI syndrome of Qi deficiency and blood stasis.Compared with the western medicine P2Y12 receptor antagonist clopidogrel,BYHW can also play a synergistic role in reducing neuroinflammatory response and improving nerve function injury. |
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