| Platelet P2Y12 antagonist is an important part of clinical treatment strategy for patients with acute coronary syndrome,ischemic stroke and other arterial thrombotic diseases.However,recurrent ischemic events,such as myocardial infarction and stent thrombosis,still occurred in some patients after taking P2Y12 antagonist.This is related to the high on-treatment platelet reactivity caused by drug resistance,among which clopidogrel resistance was the most common.How to improve drug resistance and reduce the risk of recurrent ischemic events is a difficult and hot topic in the field of cardiovascular and cerebrovascular research.Blood-activating and stasis-resolving herbs has the advantage of multi-target and multi-approach synergistic effect,which can be widely used in the prevention and treatment of cardiovascular and cerebrovascular diseases.In recent years,studies have focused on the effect of blood-activating and stasis-resolving herbs on clopidogrel resistance,but there has been no systematic evaluation.Tetramethylpyrazine is one of the active ingredients of chuanxiong.Previous studies have shown that tetramethylpyrazine inhibits platelet activation by regulating thromboxaneA 2-prostacyclin system,reducing the concentration of calcium and affecting the metabolism of phosphatidylinositol.P2Y12 receptors and related signaling pathways are the important pathways that affecting drug reactivity and the potential targets for improving drug resistance.Studies have shown that the P2Y12 receptor and the related pathways are one of the targets of blood-activating and stasis-resolving herbs to regulate platelet activation and antithrombogenesis.It is unclear that whether tetramethylpyrazine can regulate P2Y12 receptor and its related signaling pathways.Based on the above background,we carried out the following researches:(1)a meta analysis of the effect of blood-activating and stasis-resolving herbs on improving clopidogrel resistance;(2)the effect of tetramethylpyrazine on platelet activation;(3)the associated molecular mechanism of tetramethylpyrazine on inhibiting platelet activation.Part 1 A meta analysis of the effect of blood-activating and stasis-resolving herbs on improving clopidogrel resistanceObjective:To systematically evaluate the efficacy and safety of improving clopidogrel resistance by activating blood circulation and removing blood stasisMethods:All randomized controlled trials(RCTs)listed in the CNKI,WFDP,VIP,CBM,PubMed,EMBASE,Cochrane Library that were published in English or Chinese were searched,and the retrieval time range was from database inception to November 31,2019.The search terms include "clopidogrel resistance" "low response to clopidogrel" "clopidogrel" "high residual platelet reactivity","Traditional Chinese Medicine" "activating blood circulation".The quality of the evidence reported in the results of Meta-analysis was analyzed with the Cochrane Handbook for Systematic Review of Interventions,and a comprehensive meta-analysis of the effect and safety of blood-activating and stasis-resolving herbs on improving clopidogrel resistance of all publications was performed with RevMan 5.3 software.Results:A total of 977 documents were retrieved and six trials with 602 participants were included in this Meta-analysis.Compared with the using conventional treatment alone,according to the statistical analysis,the combination of blood-activating and stasis-resolving herbs increase the inhibition rate of platelet aggregation[MD=6.08,95%CI(3.63,8.53),P<0.00001]and reduce the incidence of adverse events of cardia and cerebrovascular disorders[RR=0.48,95%CI(0.34,0.69),P<0.0001]more effectively.Conclusion:The meta analysis indicates that the combination of blood-activating and stasis-resolving herbs with the conventional treatment was superior to conventional treatment alone in increasing the inhibition rate of platelet aggregation and reducing the incidence of cardia and cerebrovascular adverse events.Part 2 Effect of tetramethylpyrazine on platelet activationObjective:To observe the effect of tetramethylpyrazine on P2Y12 receptor mediated platelet activationMethods:Platelet suspension was prepared,and the P2Y1 receptor was inactivated by MRS2179,and then inducer was given to establish the P2Y12 receptor-mediated platelet activation model.Six groups were set up in the following experiments,including the resting group,the control group,model group,positive control group(ticagrelor)and tetramethylpyrazine groups of different doses(lmmol/L,2mmol/L,3mmol/L).The platelet aggregation rate was measured by light turbidimetry,the cell toxicity of tetramethylpyrazine was determined by lactate dehydrogenase(LDH)leakage rate,flow cytometry was used to detect the expression of the platelet receptor CD62p and PAC-1,and the effect of tetramethylpyrazine on clot retraction was observed.Results:Tetramethylpyrazine(1-3mmol/L)did not damage the integrity of platelet membrane.Compared with the resting group,the levels of platelet aggregetion and platelet receptor CD62p and PAC-1 were increased in the control group(P<0.001).Compared with the control group,the levels of platelet aggregation decreased in the model group(P<0.001),while the levels of platelet receptor CD62p and PAC-1 were increased(P<0.05).Compared with the model group,tetramethylpyrazine(1-3mmol/L)decreased the levels of platelet aggregetion(P<0.001)and platelet receptor CD62p and PAC-1(P<0.05)significantly.Compared with the model group,tetramethylpyrazine(2-3mmol/L)decreased the clot contraction area(P<0.05).Compared with the positive control group,tetramethylpyrazine(3mmol/L)reduced the levels of platelet aggregation(P<0.001)and platelet receptor CD62p and PAC-1(P<0.05)and decreased the clot contraction area(P<0.05).Conclusion:By inhibiting P2Y12 receptor-mediated platelet aggregation,secretion and adhesion,tetramethylpyrazine can inhibit platelet activation.Part 3 The mechanism of tetramethylpyrazine on inhibiting platelet activation.Objective:To investigate the mechanism of inhibition of P2Y12 receptor mediated platelet activation by tetramethylpyrazine.Methods:Platelet suspension was prepared,and the P2Y1 receptor was inactivated by MRS2179,and then inducer was given to establish the P2Y12 receptor-mediated platelet activation model.Eight groups were set up in the following experiments,including the resting group,the control group,model group,positive control group(ticagrelor),tetramethylpyrazine group,AC inhibitor group(SQ22536),tetramethylpyrazine+AC inhibitor group,PI3K inhibitor group(LY294002),and tetramethylpyrazine+PI3K inhibitor group.The expression of the phosphorylation of VASpser157(p-VASpser157),Akt,the phosphorylation of Aktser473 and AktThr308(p-Aktser473 and p-AktThr308)were analyzed by western blot.The levels of cAMP,IL-1 beta,and sCD40L were determined by elisa.The platelet aggregation rate was measured by light turbidimetry.Flow cytometry was used to detect the expression of the platelet receptor CD62p and PAC-1.Results:Compared with control group,the levels of platelet aggregation(P<0.001)were decreased,the levels of CD62p and PAC-1,IL-1 beta and sCD40L(P<0.05)was increased in model group.Besides,the levels of cAMP and p-VASPser157(P<0.05)were decreased,whlie the levels of p-Aktser473 and p-AktThr308(P<0.05)were increased in model group.Compared with model group,the levels of platelet aggregation(P<0.001),CD62p and PAC-1(P<0.05),IL-1 beta and sCD40L(P<0.05)were decreased,the levels of cAMP(P<0.05)and p-VASPser157 were increased,and the levels of p-Aktser473 and p-AktThr308(P<0.05)were decreased in tetramethylpyrazine group.After the addition of SQ22536,the levels of platelet aggregation(P<0.001),CD62p and PAC-1,IL-1 betas and sCD40L(P<0.05)were increased and the levels of cAMP and p-VASPser157(P<0.05)were decreased in the tetramethylpyrazine+AC inhibitor droup when compared with the tetramethylpyrazine group.After the addition of LY294002,the levels of platelet aggregation(P<0.001),CD62p and PAC-1,IL-1 beta and sCD40L(P<0.05),p-Aktser473 and p-AktThr308(P<0.05)were decreased in the tetramethylpyrazine+PI3K inhibitor group when compared with the tetramethylpyrazine group.Conclusion:The mechanism of tetramethylpyrazine inhibiting platelet activation mediated by P2Y12 receptor may be related to the upregulating of AC/cAMP signaling pathway and the inhibition of p-Akt. |
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