田蓟苷下调TLR4/Myd88/NF-κB信号通路抑制NLRP3炎症小体和炎症反应  

Tilianin downregulated TLR4/Myd88/NF-κB signaling pathway to inhibit NLRP3 inflammasome and inflammatory response

作　　者：张兴宇 徐磊 卡德尔业·卡德尔 王守宝[3] 邢建国 郑瑞芳 ZHANG Xing-yu;XU Lei;KADER Kaderyea;WANG Shou-bao;XING Jian-guo;ZHENG Rui-fang(College of Pharmacy,Xinjiang Medical University,Urumqi 830011,China;Xinjiang Institute of Pharmacy,Urumqi 830002,China;Institute of Materia Medica,Peking Union Medical College and Chinese Academy of Medical Sciences,Beijing 100050,China;School of Basic Medicine and Clinical Pharmacy,China Pharmaceutical University,Nanjing 211198,China)

机构地区：[1]新疆医科大学药学院,新疆乌鲁木齐830011 [2]新疆药物研究所,新疆乌鲁木齐830002 [3]中国医学科学院、北京协和医学院药物研究所,北京100050 [4]中国药科大学基础医学与临床药学学院,江苏南京211198

出　　处：《药学学报》2024年第7期2012-2019,共8页Acta Pharmaceutica Sinica

基　　金：新疆维吾尔自治区自然科学基金(2022D01D50,2023D01B46,2023D01E10);国家自然科学基金资助项目(82204767);中央引导地方科技发展专项(ZYYD2022A02);第二批天山英才培养计划青年托举人才项目(2023TSYCQNTJ0010);新疆维吾尔自治区科研业务费(ky2023091)。

摘　　要：本研究通过脂多糖(lipopolysaccharide,LPS)诱导RAW264.7细胞建立炎症反应模型,探讨田蓟苷(tilianin)对炎症反应的影响及其作用机制。通过CCK-8(cell counting kit-8)实验检测细胞活力,酶联免疫吸附实验(enzyme-linked immuno sorbent assay,ELISA)检测肿瘤坏死因子α(tumor necrosis factor-α,TNF-α)和白细胞介素6(interleukin6,IL-6)含量;Griess试剂法检测一氧化氮(nitricoxide,NO)含量;2',7'-二氯二氢荧光素二乙酸酯(2',7'-dichlorodihydrofluorescein diacetate,DCFH-DA)荧光探针检测细胞内活性氧(reactive oxygen radical,ROS)水平;Western blot检测Toll样受体4(Toll-like receptor 4,TLR4)、髓样分化因子88(myeloid differentiation primary response gene 88,Myd88)、核转录因子-κB p65(nuclear factor-κB p65,NF-κB p65)、磷酸化核转录因子-κB(phospho-nuclear factor-κB,p-NF-κB)、NF-κB抑制蛋白α(inhibitor of NF-κBα,IκBα)和磷酸化NF-κB抑制蛋白α(phospho-inhibitor of NF-κBα,p-IκBα)的蛋白表达水平;qRT-PCR法和Western blot检测Nod样受体蛋白3(Nod-like receptor protein 3,NLRP3)、IL-1β前体(pro-IL-1β)、IL-18前体(pro-IL-18)和半胱氨酸天冬氨酸蛋白酶-1前体(pro-caspase-1)的mRNA和蛋白水平。免疫荧光染色检测NF-κB p65核转位;使用TLR4抑制剂resatorvid(TAK242)进一步验证田蓟苷对TLR4/Myd88/NF-κB信号通路的影响。结果显示,田蓟苷显著降低了RAW264.7细胞上清中TNF-α、IL-6和NO水平以及细胞内ROS含量。田蓟苷降低了TLR4、Myd88、p-NF-κB p65和p-IκBα蛋白表达水平,抑制了NF-κB p65的核转位,同时显著降低了NLRP3、pro-IL-1β、pro-IL-18和pro-caspase-1的蛋白表达水平以及NLRP3和pro-IL-1β的mRNA水平。上述研究结果表明,田蓟苷能明显减轻LPS诱导RAW264.7细胞产生的炎症反应,其机制可能与下调TLR4/Myd88/NF-κB信号通路从而抑制NLRP3炎症小体有关。In this study,we investigated the anti-inflammatory effect and mechanism of tilianin in lipopolysaccharide(LPS)-induced RAW264.7 cells.The cell viability was detected by cell counting kit-8(CCK-8)assay.The content of tumor necrosis factorα(TNF-α)and interleukin-6(IL-6)were detected by enzyme-linked immuno sorbent assay(ELISA)kits.The content of nitric oxide(NO)was assayed by Griess reagent method.The level of intracellular reactive oxygen species(ROS)was detected by 2',7'-dichlorodihydrofluorescein diacetate(DCFH-DA)fluorescent probe.The protein levels of Toll like receptor 4(TLR4),myeloid differentiation primary response gene 88(Myd88),nuclear factorsκB p65(NF-κB p65),phosphorylated nuclear factorκB p65(p-NF-κB p65),nuclear factorκB inhibitory proteinα(IκBα)and phosphorylation nuclear factorκB inhibitory proteinα(p-IκBα)were detected by Western blot.The mRNA and protein levels of NLRP3,pro-IL-1β,pro-IL-18 and pro-caspase-1 were detected by qRT-PCR and Western blot.Immunofluorescence staining was used to detect the nuclear translocation of NF-κB p65.The effect of tilianin on the TLR4/Myd88/NF-κB signaling pathway was further validated by using the TLR4 signaling inhibitor restatorvid(TAK242).The results showed that tilianin significantly reduced the levels of TNF-α,IL-6 and NO in the supernatant of RAW264.7 cells and decreased the content of intracellular ROS.Tilianin reduced the levels of TLR4,Myd88,p-NF-κB p65 and p-IκBαprotein and nuclear translocation of NF-κB p65.Tilianin could reduce the protein levels of NLRP3,pro-IL-1β,pro-IL-18 and pro-caspase-1.Tilianin significantly inhibited the mRNA levels of NLRP3 and pro-IL-1β.The above research results indicated that tilianin could significantly alleviate the LPS-induced inflammatory response in RAW264.7 cells and its mechanism might be related to downregulate the TLR4/Myd88/NF-κB signaling pathway to inhibit NLRP3 inflammasome.

关 键 词：田蓟苷 脂多糖 RAW264.7细胞 TLR4/Myd88/NF-κB通路 NLRP3炎症小体 

分 类 号：R966[医药卫生—药理学]