药学学报, 2021, 56(5): 1343-1351
引用本文:
王丹姝, 燕柳艳, 孙姝婵, 姜瑜, 阴苏月, 王守宝*, 方莲花*, 杜冠华. 葛根素通过TLR4/Myd88/NF-κB抑制NLRP3炎症小体抗大鼠心肌缺血再灌注损伤[J]. 药学学报, 2021, 56(5): 1343-1351.
WANG Dan-shu, YAN Liu-yan, SUN Shu-chan, JIANG Yu, YIN Su-yue, WANG Shou-bao*, FANG Lian-hua*, DU Guan-hua. Puerarin protects against myocardial ischemia/reperfusion injury by suppressing NLRP3 inflammasome activation via TLR4/Myd88/NF-κB pathway in rats[J]. Acta Pharmaceutica Sinica, 2021, 56(5): 1343-1351.

葛根素通过TLR4/Myd88/NF-κB抑制NLRP3炎症小体抗大鼠心肌缺血再灌注损伤
王丹姝, 燕柳艳, 孙姝婵, 姜瑜, 阴苏月, 王守宝*, 方莲花*, 杜冠华
中国医学科学院、北京协和医学院药物研究所, 天然药物活性物质与功能国家重点实验室, 药物靶点研究与新药筛选北京市重点实验室, 北京 100050
摘要:
本研究旨在探究葛根素对大鼠心肌缺血/再灌注(myocardial ischemia/reperfusion,MI/R)损伤的治疗作用及其机制。采用冠状动脉左前降支结扎60 min再灌注24 h制备大鼠MI/R损伤模型,于再灌注前20 min灌胃给予葛根素(10、30及100 mg·kg-1)。考察葛根素对心功能、心肌梗死范围、心肌损伤标志物、炎症因子及细胞凋亡的影响,并应用Western blot法检测Nod样受体蛋白3(Nod-like receptor protein 3,NLRP3)炎症小体和Toll样受体4/髓样分化因子88/激活核转录因子-κB(TLR4/Myd88/NF-κB)通路相关蛋白。所有动物实验均遵循中国医学科学院药物研究所伦理委员会的规定。结果显示,葛根素能够显著改善MI/R损伤大鼠的心功能,减小心肌梗死范围,降低血清中乳酸脱氢酶(lactic dehydrogenase,LDH)、天冬氨酸转氨酶(aspartate transaminase,AST)、肌酸激酶MB同工酶(creatine kinase-MB,CK-MB)和心肌肌钙蛋白T(cardiac troponin T,cTnT)的含量,抑制心肌细胞凋亡。此外,葛根素可以显著降低心肌炎症因子白细胞介素-1β(interleukin-1β,IL-1β)、白细胞介素-6(IL-6)和肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)的水平。Western blot分析结果显示,葛根素能够明显下调MI/R损伤大鼠心肌TLR4、Myd88及NLRP3、凋亡相关斑点样蛋白(apoptosis-associated speck-like protein containing a CARD,ASC)、切割型半胱氨酸天冬氨酸蛋白酶-1(cleaved-caspase 1)、切割型gasdermin-D(cleaved-GSDMD)、IL-1β、IL-18蛋白水平,同时减少NF-κB抑制蛋白α(inhibitor of NF-κB α,IκBα)、IκB激酶β(IκB kinase β,IKKβ)和NF-κB蛋白磷酸化。上述研究结果表明,葛根素对大鼠MI/R损伤具有明显的改善作用,其机制可能为通过下调TLR4/Myd88/NF-κB通路抑制NLRP3炎症小体激活,从而减轻炎症反应。
关键词:    葛根素      心肌缺血再灌注损伤      Nod样受体蛋白3炎症小体      TLR4/Myd88/NF-κB通路      炎症反应     
Puerarin protects against myocardial ischemia/reperfusion injury by suppressing NLRP3 inflammasome activation via TLR4/Myd88/NF-κB pathway in rats
WANG Dan-shu, YAN Liu-yan, SUN Shu-chan, JIANG Yu, YIN Su-yue, WANG Shou-bao*, FANG Lian-hua*, DU Guan-hua
State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Beijing Key Laboratory of Drug Target Identification and Drug Screening, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, China
Abstract:
This study was to investigate the protective effects of puerarin on myocardial ischemia/reperfusion (MI/R) injury and the underlying mechanism. The MI/R-model was established by ligating the left anterior descending artery (LAD) for 60 min followed by 24 h reperfusion, puerarin (10, 30, and 100 mg·kg-1) was orally administered 20 min before reperfusion. Cardiac function, myocardial infarct index, cardiac damage markers, inflammatory cytokines, and apoptosis index were measured to evaluate the protective effects of puerarin on MI/R injury. The activation of Nod-like receptor protein 3 (NLRP3) inflammasome and Toll like receptor 4 (TLR4)/myeloid differentiation factor 88 (Myd88)/nuclear factor kappa B (NF-κB) pathway were determined by Western blot. All animal experimental procedures were approved by the ethics committee of the Institute of Materia Medica, Peking Union Medical College, Chinese Academy of Medical Sciences. The results showed that puerarin could significantly improve cardiac function, reduce myocardial infarct size, decease the levels of lactic dehydrogenase (LDH), aspartate transaminase (AST), creatine kinase-MB (CK-MB), and cardiac troponin T (cTnT) and suppress cardiomyocyte apoptosis. Meanwhile, puerarin could notably decrease the levels of inflammatory cytokines such as interleukin-1β (IL-1β), IL-6, and tumor necrosis factor-α (TNF-α). Western blot analysis revealed that puerarin could downregulate the expression of TLR4, Myd88, NLRP3, apoptosis-associated speck-like protein containing a CARD (ASC), cleaved-caspase 1, cleaved-gasdermin-D (GSDMD), IL-1β, and IL-18, as well as the phosphorylation levels of inhibitor of NF-κB α (IκBα), IκB kinase β (IKKβ), and NF-κB. These findings demonstrated that puerarin could alleviate MI/R injury by suppressing NLRP3 inflammasome activation, possibly via TLR4/Myd88/NF-κB pathway.
Key words:    puerarin    myocardial ischemia/reperfusion injury    Nod-like receptor protein 3 inflammasome    TLR4/Myd88/NF-κB pathway    inflammation   
收稿日期: 2021-01-21
DOI: 10.16438/j.0513-4870.2021-0124
基金项目: 国家自然科学基金资助项目(81773935,81673422);北京市自然科学基金资助项目(7192131);中国医学科学院医学与健康科技创新工程创新团队(2016-I2M-3-007);“重大新药创制”国家科技重大专项(2018ZX09711001).
通讯作者: 王守宝,Tel:86-10-63165313,E-mail:shoubaowang@imm.ac.cn;方莲花,E-mail:fanglh@imm.ac.cn
Email: shoubaowang@imm.ac.cn;fanglh@imm.ac.cn
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