莱菔硫烷对ox⁃LDL诱导血小板活化的抑制作用
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R329.26

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国家自然科学基金(82003451);大理大学高层次人才科研启动基金项目(KYBS2021015)


The inhibitory effect and mechanism of sulforaphane on ox⁃LDL⁃induced platelet activation
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    摘要:

    目的:探讨莱菔硫烷(sulforaphane,SFN)对氧化低密度脂蛋白(oxidized low-density lipoprotein,ox-LDL)诱导血小板活化的影响及其可能的分子机制。方法:在体外实验中,将健康人纯化血小板与不同浓度的SFN(1.0、2.5、5.0 μmol/L)共同孵育40 min,然后用ox-LDL激活血小板20 min,并检测血小板活化的指标,包括CD62P的表达、胞内血小板因子4(platelet factor 4,PF4)和趋化因子配体5(chemokine ligand 5,CCL5)的释放水平。机制上,用Western blot蛋白免疫印迹法检测血小板肉瘤酪氨酸激酶(sarcoma tyrosine kinase,Src)及其下游的脾酪氨酸激酶(spleen tyrosine kinase,Syk)磷酸化水平;用活性氧(reactive ox- ygen species,ROS)检测试剂盒测定胞内总ROS水平。结果:ox-LDL诱导的血小板CD62P的表达以及PF4和CCL5的释放水平均可被 SFN 显著抑制(P < 0.05);SFN 显著下调 ox-LDL 诱导的血小板 Src 和 Syk 的磷酸化水平以及胞内总 ROS 水平(P < 0.05)。此外,ox-LDL诱导的血小板CD62P的表达、PF4和CCL5的释放可被Src家族激酶抑制剂PP2所抑制(P < 0.05),但PP2 与SFN联合使用时,无协同抑制效果(P > 0.05);Src家族激酶激活剂MLR-1023可逆转SFN对ox-LDL诱导的血小板活化的抑制作用(P < 0.05)。结论:SFN可显著抑制ox-LDL诱导的血小板活化,其机制可能是下调Src/Syk/ROS介导的信号通路。

    Abstract:

    Objective:The current study aims to determine the effects of sulforaphane(SFN)on oxidized low-density lipoprotein(ox- LDL)-induced platelet activation and its possible mechanism. Methods:Purified human platelets were treated with SFN(1.0,2.5,5.0 μmol/L)for 40 minutes in vitro,and then stimulated by ox-LDL for additional 20 minutes. The levels of platelet CD62P expression and intracellular PF4 and CCL5 release were measured to determine the effects of SFN on platelet activation. Moreover,the phosphorylation of sarcoma tyrosine kinase(Src)and its downstream spleen tyrosine kinase(Syk)were measured by Western blot. Reactive oxygen species(ROS)assay kit was used to measure the levels of total intracellular ROS generation. Results:The ox-LDL- increased platelet CD62P expression and PF4 and CCL5 release were significantly inhibited by SFN when compared with the control group(P < 0.05). SFN treatment greatly down-regulated Src and Syk phosphorylation and ROS generation stimulated by ox-LDL(P < 0.05). Furthermore,the ox-LDL-increased the expression of CD62P and release of PF4 and CCL5 were significantly abolished by PP2, a specific inhibitor of Src family kinases,which,nevertheless,showed no synergistic effects when combined with SFN(P > 0.05). In addition,the inhibitory effects of SFN on platelet activation induced by ox-LDL were reversed by an activator of Src family kinases MLR-1023. Conclusions:SFN attenuates platelet activation induced by ox-LDL possibly by down-regulating Src/Syk/ROS pathway.

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李玮琪,马永洁,黄新惠,周昕榆,伍春婷,牙甫礼.莱菔硫烷对ox⁃LDL诱导血小板活化的抑制作用[J].南京医科大学学报(自然科学版),2023,(5):684-690

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  • 收稿日期:2022-06-30
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  • 在线发布日期: 2023-05-18
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