| BackgroudEpidemiological studies have shown that the global incidence of cardiovascular disease continues to increase year by year,with the prevalence and mortality of coronary heart disease remaining high in China,and the total number of patients has exceeded 11 million.Stable coronary artery disease(SCAD)is the main type of coronary heart disease that the occurrence of cardiovascular events can be controlled.Active anti-platelet therapy is the key to delaying atherosclerosis(AS)and thus controlling SCAD.Western anti-platelet drugs can effectively inhibit platelet activation and significantly improve the prognosis of SCAD patients,but there are still insurmountable side effects such as dose-dependent bleeding,drug resistance,and gastrointestinal adverse reactions.Herein,how to give full play to the advantages of traditional Chinese medicine and deeply explore the anti-platelet activation herbal medicine has become the focus of medical practitioners and researchers.Yuxintong Capsule(YXTC)is a representative prescription for supplementing qi and promoting blood circulation developed by academician ChenKeji’s team for treating coronary heart disease.YXTC is composed of Red ginseng,Panax notoginseng and Rhizoma corydus,with the efficacy of tonifying qi and promoting blood circulation to relieve pain.A large number of clinical and basic studies carried out in the early stage have confirmed that YXTC could effectively improve angina symptoms,myocardial ischemia-reperfusion injury,and blood rheology abnormalities,etc.in patients with SCAD.However,whether YXTC could inhibit platelet activation and its mechanisms are not yet clear.Recently,an increasing number of scholars have focused on the effect of blood flow shear stress on the formation of AS.The blood flow shear stress can affect the functional state of vascular endothelial cells and platelets,and then influence the formation of AS.Based on the above studies,this study will focus on platelet activation and evaluate the effects of YXTC on platelet aggregation rate and thromboelastogram through clinical studies,and then explore whether the effects of YXTC on AS after platelet activation is related to the effect of blood flow shear stress through basic studies.Objective1.A multicenter,randomized,double-blind,placebo-controlled clinical trial was conducted to clarify the efficacy and safety of YXTC in improving platelet function in patients with SCAD with Qi deficiency and blood stasis,providing evidence-based basis for the promotion and application of YXTC.2.To clarify whether the effects of YXTC on the development of AS induced by platelet-endothelial adhesion is related to the influence of blood flow shear stress.3.To confirm whether the effects of YXTC on platelet-endothelial adhesion induced by low shear stress(LSS)is related to its regulation of MMP-2/PAR-1/p38-MAPK signaling pathway.Methods and Results1.Clinical study on improving platelet function of patients with SCAD with Qi deficiency and blood stasis syndromeMethods:A multi-center,randomized,double-blind,placebo-controlled clinical study design method was adopted.Eighty patients with SCAD and syndrome differentiation of Qi deficiency and blood stasis who met the test criteria and were admitted to Beijing Anzhen Hospital Affiliated to Capital Medical University,Dongzhimen Hospital of Beijing University of Chinese Medicine and the Third Affiliated Hospital of Guangzhou Medical University from March 2021 to November 2021 were included as case enrollment.A statistician who was not involved in the statistical analysis of this trial used SAS statistical software to generate a random number table according to grouping ratio.The patients were randomly divided into the experimental group and the control group at a ratio of 1:1,with 40 cases in each group.The experimental group was treated with YXTC on the basis of conventional western medicine,and the control group was treated with YXTC simulator on the basis of conventional western medicine.The intervention methods of the two groups were 4 pills each time,3 times a day for 12 weeks.Visits were conducted at weeks 0,8 and 12.The main efficacy indicators were platelet aggregation rate induced by AA and ADP,conventional thromboelastography,AA-induced thromboelastography and ADP-induced thromboelastography.The secondary efficacy index was Qi deficiency and blood stasis syndrome scores.The safety indicators included blood routine,urine routine,liver function,renal function,coagulation routine,vital signs,and electrocardiogram.Results:(1)Comparison of baseline data between the two groups:80 subjects were included in this trial,40 in the experimental group and 40 in the control group.A total of 8 cases dropped out after the study,including 3 cases in the experimental group and 5 cases in the control group.Finally,a total of 72 cases met the full analysis set(FAS),including 37 cases in the experimental group and 35 cases in the control group.The average age was 56.68±10.34 years in the experimental group and 55.14±10.25 years in the control group.There were no significant differences in baseline characteristics between the two groups(P>0.05).(2)Comparison of the main efficacy indicators between the two groups:the platelet aggregation rate(AA)of the experimental group was 28.76±30.96 before treatment and 16.37±19.67 at the end of 12 weeks of treatment,which was significantly lower than that before treatment(P=0.033<0.05);The platelet aggregation rate(ADP)of the experimental group was 49.29±20.96 before treatment and 38.23±18.27 at the end of 12 weeks of treatment,which was significantly lower than that before treatment(P<0.001).In terms of thromboelastography(AA inhibitory effect),the value of coagulation time(R)in the experimental group before treatment was 7.24±1.67,and 8.46±1.13 at the end of 12 weeks of treatment,which was significantly longer than that before treatment(P=0.009<0.01);The clot formation time(K)value before treatment in the experimental group was 1.91±0.47 and at the end of 12 weeks of treatment was 2.23±0.53,which was significantly longer than that before treatment(P=0.030<0.05);The function of fibrin(Angle)was 63.77±6.59 in the experimental group and 62.01±7.56 in the control group at the end of 12 weeks of treatment,and there was no significant difference between the two groups(P=0.429>0.05);The maximum elastic force of thrombus(MA)value before treatment was 62.34±2.34 in the experimental group,and 55.73±7.68 at the end of 12 weeks of treatment,with a statistically significant difference in MA values before and after treatment(P=0.001<0.01),and the MA value at the end of treatment 12 in the control group was 61.61±5.49,with a statistically significant difference between the experimental and control groups after treatment(P=0.008<0.01);There were no significant differences in LY30,EPL and TPI between the experimental and control groups at the end of 12 weeks of treatment(P>0.05);The AA inhibition rate(IR)value before treatment was 77.78±22.75 in the experimental group,and 87.85±9.52 at the end of 12 weeks of treatment,which was significantly higher than that before treatment(P=0.034<0.05)In terms of thrombelastogram(ADP inhibitory effect),the value of coagulation time(R)in the experimental group before treatment was 0.60±0.17,and 0.82±0.11 at the end of 12 weeks of treatment,which was significantly longer than that before treatment(P<0.001);The clot formation time(K)value before treatment in the experimental group was 3.00±1.21 and at the end of 12 weeks of treatment was 4.52±2.19,which was significantly increased after treatment(P<0.001);At the end of 12 weeks of treatment,the function of fibrin(Angle)was 5 7.06±17.26 in the experimental group and 54.9±10.44 in the control group,and there was no significant difference between the two groups(P=0.139>0.05);The maximum elastic force of thrombus(MA)value before treatment was 43.7±10.19 in the experimental group,and 37.96±8.49 at the end of 12 weeks of treatment,which was significantly lower than that before treatment(P=0.001<0.01);There were no significant differences in LY30,EPL and TPI between the two groups at the end of 12 weeks of treatment(P>0.05);The ADP inhibition rate(IR)value of the experimental group was 33.95±17.02 before treatment and 61.90±27.12 at the end of 12 weeks of treatment,which was significantly higher than that before treatment(P<0.001).The IR value of the experimental group was significantly higher than that of the control group after treatment(t=3.237,P=0.002<0.01).In terms of thromboelastography(conventional coagulation function),the coagulation index(CI)value was-0.75±2.02 in the experimental group and-1.24±3.54 in the control group at the end of 12 weeks of treatment,with no statistical difference between the two groups(P=0.52>0.05).(3)Comparison of the secondary efficacy indicators between the two groups:the score of Qi deficiency and blood stasis syndrome in the experimental group was 8.27±1.26 before treatment,4.76±0.93 at the end of 8 weeks of treatment,and 2.41±1.21 at the end of 12 weeks,which were significantly lower than those before treatment(P<0.001).The scores of the experimental group were significantly lower than those of the control group at the end of 8 weeks and 12 weeks of treatment(P<0.001).(4)Safety evaluation:there were no obvious abnormalities in blood,urine routine,electrocardiogram and four vital signs in the two groups.2.Pharmacodynamic evaluation of YXTC on low shear stress model ratsMethods:(1)Experimental grouping:SD rats were randomly divided into 6 groups:sham-operated group(Sham),model group(Model),low-dose group of YXTC(YXTC-L),medium-dose group of YXTC(YXTC-M),high-dose group of YXTC(YXTC-H),and aspirin group(Aspirin).(2)Establishment of animal model:The low shear stress(LSS)rat model was established by partial ligation of the left carotid artery in Model,YXTC-L,YXTC-M,YXTC-H and Aspirin groups.The Sham group was treated with the same method without ligation of the artery.(3)Animal administration:rats in YXTC-L,YXTC-M and YXTC-H groups were treated with 0.18g/kg,0.36g/kg and 0.72g/kg by gavage,respectively.The dose of Aspirin was 4.5mg/kg.The rats in Sham group and Model group were given the same amount of sterile distilled water by gavage every day.The drug was administered once daily for 8 weeks.(4)The left common carotid artery(LCCA)was detected by ultrasound,and the left ventricular end diastolic diameter(Dr)and peak systolic flow velocity(Vm)of LCCA were measured,and the average value of three cardiac cycles was taken.(5)Whole blood viscosity(η)was measured by automatic hemorheology analyzer.(6)The flow shear stress of the LCCA was calculated according to the formula:τm＝4×η×Vm/Dr.(7)HE staining was used to observe the pathological changes of the LCCA,and the intima-media thickness and lumen cross-sectional area of the LCCA were calculated.(8)Sirius red staining was performed to observe the changes of collagen fibers in the LCCA,and the area and proportion of collagen fibers were calculated.Results:(1)In terms of peak systolic flow velocity(Vm),compared with the Sham group,the Vm of the LCCA in the Model group was significantly decreased(P<0.01),and the YXTC-M(P<0.05)and YXTC-H(P<0.01)and Aspirin(P<0.01)were significantly higher compared with the Model group.(2)Regarding the end-diastolic diameter of the LCCA(Dr),compared with the Sham group,the Dr in the Model group was significantly reduced(P<0.01).Compared with the Model group,the Dr of YXTC-M,YXTC-H and Aspirin group was significantly increased(P<0.01).(3)In terms of whole blood viscosity(η),compared with the Sham group,there was no significant change in η of the Model group(P>0.05);compared with the Model group,the η of the YXTC-L group was significantly decreased(P<0.05),while η of YXTC-M,YXTC-H and Aspirin groups did not change significantly(P>0.05).(4)In terms of shear stress,compared with the Sham group,the blood flow shear stress of the LCCA in the Model group decreased significantly(P<0.01);compared with the Model group,the blood flow shear stress in the YXTC-M,YXTC-H and Aspirin group was significantly increased(P<0.01).(5)HE staining showed that the vascular endothelial cells at the low shear stress(LSS)area of the LCCA were arranged disorderly and inflammatory infiltration was obvious in the Model group compared with the Sham group.Compared with the Model group,the endothelial inflammatory injury in the YXTC-L and YXTC-M groups was not significantly alleviated,but the morphological structure was slightly improved.The inflammatory infiltration was significantly attenuated in the YXTC-H and Aspirin groups.With regard to the intima-media thickness of the LCCA,the Model group was significantly thicker compared with the Sham group(P<0.01).Compared with the Model group,the YXTC-L,YXTC-H and Aspirin groups had a tendency to decrease,but there was no statistical significance(P>0.05).Regarding the luminal cross-sectional area of the LCCA,there was a tendency to increase the luminal cross-sectional area in the Model group compared with the Sham group,but the difference was not statistically significant(P>0.05),and the luminal cross-sectional area in the YXTC-L,YXTC-M,YXTC-H,and Aspirin groups were all decreased to different degrees compared with the Model group,but none of the differences were statistically significant(P>0.05).(6)Sirius red staining showed that the collagen fiber content in the Model group was significantly increased compared with the Sham group(P<0.01).Compared with the Model group,the content of collagen fibers in YXTC-L,YXTC-H and Aspirin groups were significantly decreased(P<0.01),and YXTC-M also decreased compared with the model group(P<0.05).3.Pharmacological study on the inhibition of YXTC to platelet-endothelial adhesion induced by LSSMethods:(1)Immunohistochemical staining(IHC)was performed to detect the expression of GPIba in the vascular tissue of LCCA.(2)ELISA was used to determine the levels of vWF and GPIbα in serum.(3)Cell grouping and culture:RVECs were randomly divided into 4 groups:Control group,Model group,YXTC group and Asprin group,then 1×107 platelets were added to RVECs of each group,and cultured for 24 hours in 37℃ and 5%CO2 incubator.The Control group was perfused with DMEM medium containing blank serum and loaded with normal shear stress(NSS)of 15 dyne/cm2.The Model group was perfused with DMEM medium containing blank serum and loaded with LSS of 4 dyne/cm2.In YXTC group,DMEM medium containing YXTC drug serum was perfused and loaded with LSS of 4 dyne/cm2.The Aspirin group was perfused with DMEM medium containing blank serum and aspirin solution,and loaded with LSS of 4 dyne/cm2.(4)Fluorescence microscopy was performed to observe the adhesion of platelets to endothelial cells.(5)ELISA was conducted to detect the expression of vWF and E-selectin in the cell supernatant.Results:(1)The results of immunohistochemical staining showed that compared with the Sham group,the expression of positive particles was significantly increased,and the number of positive cells and integral optical density were significantly increased in the Model group(P<0.01).Compared with the Model group,the positive particles in the vascular endothelium of the YXTC-L,YXTC-M,YXTC-H and Aspirin groups had different degrees of reduction,and the number of positive cells in the YXTC-L and YXTC-H and Aspirin groups was significantly lower(P<0.01),the integral optical density of YXTC-L and YXTC-H was significantly lower(P<0.01),the number of positive cells in YXTC-M was also significantly lower(P<0.05),and the integral optical density was slightly reduced compared with the model group,but the differences were not statistically significant(P>0.05).(2)ELISA results of serum showed that the levels of GPIba and vWF protein were significantly higher in the Model group compared with the Sham group,and the differences were statistically significant(P<0.01).Compared with the Model group,the levels of GPIba and vWF protein in the YXTC-L group had a tendency to decrease,but the differences were not statistically significant(P>0.05).The expression of vWF protein was significantly lower in the YXTC-M group(P<0.05),but the expression of GPIba was not significantly changed(P>0.05);the GPIba and vWF protein contents were dramatically lower in the YXTC-H and Aspirin groups compared with the Model group(P<0.01).(3)The results of fluorescence staining showed that the number of RVECs and platelet adhesions were significantly increased in the Model group compared with the Control group(P<0.01);the number of RVECs and platelet adhesions were significantly decreased in the YXTC and Asprin groups compared with the Model group(P<0.01).(4)ELISA results of cell supernatant showed that the protein expression of E-selectin and vWF were significantly increased in the Model group compared with the Control group(P<0.01);the protein expression of E-selectin and vWF were significantly down-regulated in the YXTC and Aspirin groups compared with the Model group(P<0.01).4.Pharmacological mechanism of inhibition of YXTC to platelet-endothelial adhesion induced by LSSMethods:(1)Immunofluorescence double staining was used to observe the co-localization of MMP-2 and PAR-1 in the LCCA of rats.(2)Real-time fluorescent quantitative PCR(RT-qPCR)was performed to detect the mRNA expression of MMP-2,PAR-1,p38-MAPK,ICAM1 and VCAM1 in the LCCA of rats.(3)Western-blot assay to detect the protein expression levels of MMP-2,PAR-1,p38-MAPK,ICAM1 and VCAM1 in the LCCA of rats.(4)ELISA to determine the expression of MMP-2 and Fibronectin protein in serum.(5)Cell grouping and treatment:RVECs were randomly divided into 7 groups:normal control group(NSS+Vehiche),PAR-1 inhibitor group(NSS+RWJ56110),model group(LSS+Vehiche),YXTC group(LSS+YXTC),LSS+PAR-1 inhibitor group(LSS+RWJ56110),YXCT+PAR-1 inhibitor group(LSS+YXTC+RWJ56110)and aspirin group(LSS+Aspirin),and then 1×107 platelets were added to the RVECs of each group,and were co-cultured for 24 hours in a 5%CO2 incubator at 37℃.NSS+Vehiche group was perfused with DMEM medium containing blank serum and loaded with NSS of 15 dyne/cm2.In NSS+RWJ56110 group,DMEM medium containing blank serum and RWJ56110 solution was perfused,and NSS of 15 dyne/cm2 was loaded.In LSS+Vehiche group,DMEM medium containing blank serum was perfused and loaded with LSS of 4 dyne/cm2.LSS+YXTC group was perfused with DMEM medium containing YXTC drug serum and loaded with LSS of 4 dyne/cm2.In LSS+RWJ56110 group,DMEM medium containing blank serum and RWJ56110 solution was perfused and loaded with LSS of 4 dyne/cm2.In LSS+Aspirin group,DMEM medium containing blank serum and aspirin solution was perfused and loaded with LSS of 4 dyne/cm2.(6)RT-qPCR was applied to detect the mRNA levels of MMP-2,PAR-1,p38-MAPK,VCAM1,ICAM1 and GPIba in the supernatant of each group.(7)Western-blot assay was performed to detect the expressions of MMP-2,PAR-1,p38-MAPK,VCAM1,ICAM1 and GPIbα in the supernatant of each group of cells.Results:(1)The results of LCCA tissue immunofluorescence double staining showed that the fluorescence intensity of MMP-2 and PAR-1 in the Model group was significantly higher than that in the Sham group,and the co-localization expression of MMP-2 and PAR-1 was significantly increased(P<0.01).Compared with the Model group,the fluorescence intensity of MMP-2 and PAR-1 in the YXTC-L group did not change significantly,and the co-localization expression of MMP-2 and PAR-1 did not decrease significantly(P>0.05).The co-localization expression of MMP-2 and PAR-1 in YXTC-M,YXTC-H and Aspirin groups was significantly decreased(P<0.01).(2)The results of RT-qPCR of LCCA tissues showed that the mRNA levels of MMP-2,PAR-1,vWF,p38-MAPK,ICAM1 and VC AM1 in the Model group were significantly higher than those in the Sham group(P<0.01).Compared with the Model group,the levels of vWF,ICAM1 and VCAM1 mRNA in the YXTC-L group decreased,and the differences were statistically significant(P<0.01).The mRNA of MMP-2,PAR-1 and p38-MAPK in the YXTC-L group decreased,but the difference was not statistically significant(P>0.05).The mRNA levels of MMP-2,PAR-1,vWF,p38-MAPK,ICAM1 and VCAM1 in YXTC-M,YXTC-H and Aspirin groups were significantly decreased(P<0.05).(3)Western-blot results showed that compared with the Sham group,the expression levels of MMP-2,PAR-1,p38-MAPK,vWF,VCAM and ICAM1 proteins in the Model group were significantly increased(P<0.01).Compared with the Model group,the protein levels of MMP-2,PAR-1,P38-MAPK,VCAM1,ICAM1 and vWF in the YXTC-L group were decreased,but the differences were not statistically significant(P>0.05).The expression of VCAM1 and vWF protein in the YXTC-M group was significantly lower than that in the Model group(P<0.05).The expression levels of PAR-1,vWF,VCAM and ICAM1 proteins in the YXTC-H group were significantly lower than those in the control group(P<0.05).The protein expression of PAR-1,P38-MAPK,VCAM1,ICAM1 and vWF in the Asprin group was significantly lower than that in the model group(P<0.05).(4)ELISA results of serum showed that compared with the Sham group,the levels of MMP-2 and fibronectin protein in the Model group were significantly increased(P<0.01).Compared with the Model group,the levels of MMP-2 and fibronectin protein in the serum of the YXTC-L group were decreased,but the differences were not statistically significant(P>0.05).The contents of MMP-2 and fibronectin protein in YXTC-M group were decreased(P<0.05),while thier contents in both YXTC-H and Asprin groups were significantly lower than those in the Model group(P<0.01).(5)RT-qPCR results of cell supernatant showed that compared with the NSS+Vehiche group,the mRNA levels of MMP-2,PAR-1,p38-MAPK,VCAM1,ICAM1 and GPIba in the NSS+RWJ56110 group did not change significantly(P>0.05),while the mRNA levels of MMP-2,PAR-1,p38-MAPK,VCAM1,ICAM1 and GPIba were significantly increased in the LSS+Vehiche group(P<0.01).Compared with the LSS+Vehiche group,the mRNA levels of MMP-2,PAR-1,p38-MAPK,ICAM1 and VCAM1 in the LSS+YXTC and LSS+RWJ56110 groups were significantly decreased(P<0.01).Consistent with the changes in LSS+YXTC group,the mRNA expression of MMP-2,PAR-1,p38-MAPK,ICAM1 and VCAM1 in LSS+Asprin group was significantly down-regulated(P<0.01).Compared with LSS+YXTC group and LSS+RWJ56110 group,the mRNA levels of MMP-2,PAR-1,ICAM1 and VCAM1 in LSS+YXTC+RWJ56110 group were significantly decreased(P<0.01);However,the mRNA levels of p38-MAPK did not change significantly(P>0.05).(6)Western-blot results of cell supernatant showed that compared with the NSS+Vehiche group,the expressions of MMP-2,PAR-1,p38-MAPK,VCAM1,ICAM1 and GPIba in the NSS+RWJ56110 group did not change significantly(P>0.05).The protein expression levels of MMP-2,PAR-1,p38-MAPK,VCAM1,ICAM1 and GPIba in the LSS+Vehiche group were significantly increased(P<0.01);Compared with the LSS+Vehiche group,the expression of MMP-2,PAR-1 and ICAM1 was significantly down-regulated in the LSS+YXTC and LSS+RWJ56110 groups(P<0.01),and p38-MAPK expression was also significantly decreased(P<0.05),while the expression of VCAM1 and GPIba did not change significantly(P>0.05),and ICAM1 expression was significantly down-regulated in LSS+Asprin group(P<0.01),but the expression of MMP-2,PAR-1,p38-MAPK,VCAM1 and GPIba did not change significantly(P>0.05);Compared with LSS+YXTC group or LSS+RWJ56110 group,the expression levels of MMP-2,PAR-1,p38-MAPK,VCAM1 and GPIba in LSS+YXTC+RWJ56110 group were significantly decreased(P<0.01).In addition,ICAM1 expression was significantly down-regulated in the LSS+YXTC+RWJ56110 group compared with the YXTC group(P<0.01),but there was no significant difference compared with RWJ56110 group(P>0.05).5.Study on chemical composition and constituents migrating to blood of YXTCMethods:Ultra-high performance liquid chromatograph(ACQUITY UPLC H-Class)and four-pole time-of-flight high resolution mass spectrometer(Xevo G2-S QTOF)were used to detect the chemical composition and constituents migrating to blood of YXTC.Results:(1)There were 20 chemical components identified in YXTC,including six alkaloids,namely protopine,glaucine,tetrahydropalmatine,canadine,tetrahydrocoptisine and corydaline,respectively,and 14 ginsenosides:notoginsenoside R1,ginsenoside Rg1,ginsenoside Re,ginsenoside Rf,notoginsenoside R2,ginsenoside Rb1,ginsenoside Rg2,ginsenoside Rc,ginsenoside Rb2,ginsenoside Rb3,ginsenoside Rd,ginsenoside Rd isomer,ginsenoside Ro,ginsenoside Rg3.(2)Ginsenoside Rbl was detected in the serum of rats after administration of YXTC.Conclusions1.YXTC could significantly improve the fibrinolytic activity and enhance the inhibitory effect of antiplatelet drugs on ADP channels in SCAD patients.However,there was no significant improvement in platelet aggregation rate,platelet coagulation function,and the total score of Qi deficiency and blood stasis syndrome.2.YXTC inhibits platelet-endothelial adhesion mediated AS development by affecting blood flow shear stress.3.YXTC inhibits platelet-endothelial adhesion induced by LSS through inhibiting the activation of MMP-2/PAR-1/p38-MAPK signaling pathway. |
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