作 者：
申栋帅;杨亚军;刘希望;孔晓军;秦哲;李世宏;焦增华;李剑勇
关键词：
阿司匹林丁香酚酯;血小板聚集;二磷酸腺苷(ADP);蛋白质印迹法
摘 要：
将空白血小板样品分为8组,包括溶剂对照组以及模型组、阿司匹林组、丁香酚组、联合给药组、阿司匹林丁香酚酯(aspirin eugenol ester,AEE)低剂量组、AEE中剂量组和AEE高剂量组这7个处理组。各组加入对应的药物,并在37℃孵育3min,而后各处理组加入血小板激活剂二磷酸腺苷(ADP),并于37℃孵育5min,从而激活血小板。最后,通过ELISA检测试剂盒分别对血小板胞内cAMP含量变化以及血小板ATP释放量进行测定;利用荧光分光光度法以及钙离子特异结合的荧光探针Fura-2/AM对血小板胞内钙离子浓度([Ca~(2+)]i)进行测定;通过蛋白质印迹法(Western blot)对血小板胞内Akt、ERK、JNK以及P38蛋白磷酸化程度进行测定。结果显示,AEE各组及其前药对照组均能显著降低胞内钙离子浓度以及ERK蛋白磷酸化(P<0.05),但对胞内cAMP含量变化以及JNK、P38和Akt蛋白的磷酸化过程无显著性影响(P>0.05)。此外,不同浓度AEE均可显著降低血小板ATP释放量(P<0.01),而阿司匹林、丁香酚以及两者联用则均无显著性影响(P>0.05)。在终浓度为25μmol/L时,AEE的降钙作用以及抑制ERK2蛋白磷酸化作用效果均优于其前药对照组,且该浓度下AEE能够显著降低血小板ATP释放量(P<0.01)。由此可见,AEE通过降低血小板胞内钙离子含量和ATP释放量,以及抑制ERK2磷酸化从而抑制ADP诱导的血小板聚集,且相比于其前药增加了降低血小板ATP释放量这一新的作用途径。
译 名：
Mechanism of platelet aggregation inhibition of aspirin eugenol ester
作 者：
SHEN Dong-shuai;YANG Ya-jun;LIU Xi-wang;KONG Xiao-jun;QIN Zhe;LI Shi-hong;JIAO Zeng-hua;LI Jian-yong;Key Lab of New Animal Drug Project of Gansu Province/Key Lab of Veterinary Pharmaceutical Development,Ministry of Agriculture/Lanzhou Institute of Husbandry and Pharmaceutical Sciences,CAAS;
单 位：
SHEN Dong-shuai%YANG Ya-jun%LIU Xi-wang%KONG Xiao-jun%QIN Zhe%LI Shi-hong%JIAO Zeng-hua%LI Jian-yong%Key Lab of New Animal Drug Project of Gansu Province/Key Lab of Veterinary Pharmaceutical Development,Ministry of Agriculture/Lanzhou Institute of Husbandry and Pharmaceutical Sciences,CAAS
关键词：
aspirin eugenol ester(AEE);;platelet aggregation;;ADP;;Western blot
摘 要：
Aspirin eugenol ester is a new compound synthesized by using the theory of piecing the structure of compounds together,and previous researches demonstrated that AEE has the effect of inhibiting the platelet aggregation.This study aims to research the relevant mechanism of platelet aggregation inhibition of aspirin eugenol ester while making a distinction between AEE and its prodrugs.The blank platelets samples were divided into eight groups,covering solvent group and seven treatment groups including model group,aspirin group,eugenol group,integration group,low concentration of AEE group,middle concentration of AEE group,high concentration of AEE group.0.5% DMSO were add to solvent group and model group,the other groups were respectively added with 25μmol/L aspirin,25μmol/L eugenol,25μmol/L aspirin and 25μmol/L eugenol,12.5μmol/L AEE,25μmol/L AEE,50μmol/L AEE,with the same volume of 0.5% DMSO.After incubating for 3 min at 37℃,the treatment groups were added with 10μmol/L ADP while adding isometric DMSO to solvent group followed by incubation for 5 min at 37℃.Finally,cAMP and ATP kits were used to respectively measure the cAMP concentration and the release quantity of ATP of platelets;fluorescence anisotropy assay and Fura-2/AM were used to determine[Ca~(2+)]i;Western blotting for detecting the extent of phosphorylation of Akt,ERK,JNK and P38.The results demonstrated that all groups of AEE and its prodrugs groups can significantly reduced[Ca~(2+)]iand the phosphorylation of ERK2(P<0.05),but there was not markedly effect of AEE or its prodrugs on the concentration intracellular cAMP and the phosphorylation of JNK,P38 and Akt of platelets(P>0.05).Besides,the amount of ATP release of platelets was significantly reduced by all of different concentrations AEE groups(P<0.01),but not aspirin,eugenol or integration group(P>0.05).At the final concentration of 25μmol/L,the effect of reducing[Ca~(2+)]i,and suppressing the phosphorylation of ERK2 of AEE has an advantage over the prodrugs groups of AEE(P<0.05),furthermore,AEE can signally lower the amount of ATP release of platelets at 25μmol/L(P<0.01).Thus it can be seen that AEE inhibit platelet aggregation induced by ADP via reducing[Ca~(2+)]iand the amount of ATP release and suppressing the phosphorylation of ERK2 in which includs a new operating pathway that reduces the more amount of ATP release of platelets than its prodrugs.
