首页|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

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

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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(interleukin 6，IL-6)含量;Griess试剂法检测一氧化氮(nitric oxide，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 ofNF-κB α，IKBα)和磷酸化NF-κB抑制蛋白 α(phospho-inhibitor of NF-κB α，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-1 β、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。
Tilianin downregulated TLR4/Myd88/NF-κB signaling pathway to inhibit NLRP3 inflammasome and inflammatory response
In this study,we investigated the anti-inflammatory effect and mechanism of tilianin in lipopolysac-charide(LPS)-induced RAW264.7 cells.The cell viability was detected by cell counting kit-8(CCK-8)assay.The content of tumor necrosis factor α(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 factors κB p65(NF-κB p65),phosphorylated nuclear factor κB p65(p-NF-κB p65),nuclear factor κB inhibitory protein α(IκBα)and phosphorylation nuclear factor κB inhibitory protein α(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-IκBα protein 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 signifi-cantly 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.

tilianinlipopolysaccharideRAW264.7 cellTLR4/Myd88/NF-κB pathwayNLRP3 inflammasome

张兴宇、徐磊、卡德尔业·卡德尔、王守宝、邢建国、郑瑞芳

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新疆医科大学药学院,新疆乌鲁木齐 830011

新疆药物研究所,新疆乌鲁木齐 830002

中国医学科学院、北京协和医学院药物研究所,北京 100050

中国药科大学基础医学与临床药学学院,江苏南京 211198

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

新疆维吾尔自治区自然科学基金新疆维吾尔自治区自然科学基金新疆维吾尔自治区自然科学基金国家自然科学基金资助项目中央引导地方科技发展专项第二批天山英才培养计划青年托举人才项目新疆维吾尔自治区科研业务费

2022D01D502023D01B462023D01E1082204767ZYYD2022A022023TSYCQNTJ0010ky2023091

2024

10.16438/j.0513-4870.2024-0228

药学学报
中国药学会 中国医学科学院药物研究所

药学学报

CSTPCD北大核心
影响因子：1.274
ISSN：0513-4870
年,卷(期)：2024.59(7)
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