摘要
目的旨在通过体外实验探讨姜黄素的主要代谢活性产物四氢姜黄素(tetrahydrocurcumin,THC)对高糖(high glucose,HG)诱导人血小板聚集和活化的作用及机制。方法将溶剂对照(0.05%DMSO)或THC(0.5、1、10μmol/L)与健康人来源的纯化血小板在体外共同孵育40 min,随后加入Normal glucose(NG,5 mmol/L)或HG(25 mmol/L)溶液继续作用90 min后,使用血小板聚集仪测定血小板的最大聚集率,并利用流式细胞术评估血小板经典活化标志物CD62P的表达情况以及胞内总活性氧(Reactive oxygen species,ROS)生成水平。同时,通过Western blot蛋白免疫印迹实验检测血小板p53蛋白的磷酸化水平。结果与NG组相比,HG干预可显著增加血小板聚集(P<0.05)和CD62P的表达(P<0.001),且可被不同浓度的THC显著抑制(P<0.05)。机制上,与溶剂对照组相比,THC显著降低HG诱导的血小板内总ROS生成水平(P<0.001),显著下调p53蛋白磷酸化水平(P<0.05);此外,通过加入ROS清除剂N-乙酰半胱氨酸(N-acetylcysteine,NAC),观察到HG诱导的血小板内总ROS生成(P<0.001)以及p53蛋白的磷酸化水平(P<0.05)均显著降低,进一步将其与THC(10μmol/L)联合使用时并未观察到协同抑制效果(P>0.05)。HG诱导的血小板聚集和活化均可被NAC和p53特异性抑制剂PFT-μ显著抑制(P<0.05),THC(10μmol/L)与NAC联合干预时对HG诱导血小板聚集和活化无协同抑制作用(P>0.05),与PFT-μ联合干预时对血小板聚集具有协同抑制作用(P<0.05),但对血小板活化无协同抑制作用(P>0.05)。结论在体外实验中,THC对HG诱导的健康人血小板聚集与活化具有显著的抑制作用,其作用机制可能是下调ROS/p53介导的信号通路,本研究为THC改善糖尿病及其相关慢性代谢性疾病中血栓形成提供参考。
Objective This study aims to explore the efficacy of tetrahydrocurcumin(THC),the major active metabolite of curcumin,on high glucose(HG)-induced human platelet aggregation and activation as well as to clarify the underlying mechanisms in vitro.Methods Purified platelets prepared from healthy subjects were pre-incubated with various concentrations of THC(0.5μmol/L,1μmol/L or 10μmol/L)or vehicle control(0.05%DMSO)for 40 min at 37°C,followed by the stimulation of normal glucose(NG,5 mmol/L)or HG(25 mmol/L)for additional 90 min.The maximal aggregation rate was determined by an aggregometer.Flow cytometry was used to measure platelet surface expression of CD62P(a typical marker of platelet activation)and generation of total intraplatelet reactive oxygen species(ROS).Meanwhile,the phosphorylation level of platelet p53 was detected by Western blot assay.Results Compared with NG group,HG intervention significantly increased platelet aggregation(P<0.05)and CD62P expression(P<0.001),which were greatly inhibited by different concentrations of THC(P<0.05).Mechanistically,when compared with solvent control,THC significantly decreased the level of total ROS production(P<0.001)and p53 phosphorylation(P<0.05).In addition,HG-induced total intraplatelet ROS generation(P<0.001)and p53 phosphorylation(P<0.05)were greatly attenuated by adding a ROS scavenger N-acetyl-L-cysteine(NAC).The combination of NAC with THC(10μmol/L)showed no additive inhibitory effects(P>0.05).Moreover,platelet aggregation and activation induced by HG were greatly decreased by NAC and a p53 specific inhibitor PFT-μ(P<0.05).The combination of THC(10μmol/L)and NAC resulted no additive inhibitory effects on HG-increased platelet aggregation and activation(P>0.05).THC(10μmol/L)exhibited additive inhibitory effects on platelet aggregation(P<0.05)but no additive inhibitory effects on platelet activation when combined with PFT-μ(P>0.05).Conclusions THC exerts a protective effect on HG-induced platelet aggregation and activation possibly through down-regulating ROS/p53 signaling pathway in human platelets in vitro.The current study may provide potential value for THC to improve thrombosis in diabetes mellitus and the related chronic metabolic diseases.
作者
胡锦秋
毕晓艳
马军羽
李梦瑶
李荣
牙甫礼
张春梅
HU Jinqiu;BI Xiaoyan;MA Junyu;LI Mengyao;LI Rong;YA Fuli;ZHANG Chunmei(Department of Nutrition,School of Public Health,Dali University,Dali 671000,Yunnan,China;不详)
出处
《实用医学杂志》
北大核心
2025年第3期305-312,共8页
The Journal of Practical Medicine
基金
国家自然科学基金项目(编号:82260638)
云南省基础研究专项面上项目(编号:202401AT070081)。
关键词
四氢姜黄素
血小板聚集
血小板活化
高糖
活性氧
tetrahydrocurcumin
platelet aggregation
platelet activation
high glucose
reactive oxygen species
作者简介
通信作者:张春梅,E-mail:ZhangCM2023@yeah.net。
