| 廖玉娇,陈志伟.七叶苷通过下调 Toll样受体 4/核因子 κB信号通路抑制脂多糖诱导的 RAW264.7巨噬细胞炎症[J].安徽医药,2025,29(1):34-39. |
| 七叶苷通过下调 Toll样受体 4/核因子 κB信号通路抑制脂多糖诱导的 RAW264.7巨噬细胞炎症 |
| Esculin Inhibits Lipopolysaccharide-induced Inflammation in RAW264.7 Macrophages by Toll-like receptor 4/ nuclear factor κB Signaling Pathway |
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| DOI：10.3969/j.issn.1009-6469.2025.01.006 |
| 中文关键词: 七叶苷 RAW264.7 脂多糖 炎症 核因子 κB |
| 英文关键词: Esculin RAW264.7 Lipopolysaccharide Inflammation Nuclear factor κB |
| 基金项目:温州市基础性科研项目( Y2020043) |
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| 中文摘要: |
| 目的探讨七叶苷通过调控 Toll样受体 4/核因子 κB(TLR4/NF-κB)途径抑制脂多糖( LPS)诱导的 RAW264.7巨噬细胞炎症。方法 2022年 10月至 2023年 6月，培养 RAW264.7巨噬细胞，用脂多糖诱导炎性损伤，分为正常组、 LPS诱导的模型组，七叶苷低、中、高浓度组(七叶苷的浓度分别为 25，100和 200 μmol/L)。 RAW264.7巨噬细胞先用上述浓度七叶苷处理 12 h，后用脂多糖诱导 12 h。用噻唑蓝溴化四氮唑( MTT)法检测各组细胞活性，用膜联蛋白 V(Annexin V)-异硫氰酸荧光素( FITC)然和碘化丙啶( PI)凋亡和坏死检测法结合流式分析技术检测细胞凋亡，用实时荧光定量聚合酶链式反应( qRT-PCR)和酶联免疫吸附测定( ELISA)分别检测细胞炎症因子转录水平表达和释放，用蛋白质印迹法检测凋亡因子、 TLR4/NF-κB信号通路蛋白表达和 NF-κB核转移。结果 MTT结果证明 200 μmol/L七叶苷给药处理能够显著增加脂多糖诱导后 RAW264.7细胞的增殖活性( 1.524±0.223)。流式分析结果证明 200 μmol/L七叶苷显著抑制脂多糖诱导的细胞凋亡和坏死［(10.68±3.69)%］，且免疫印迹结果证实 200 μmol/L七叶苷显著促进 B细胞白血病 /淋巴瘤 2(Bcl-2)［( 1.981±0.026)倍］和抑制 Bcl-2相关 X蛋白( Bax)［( 1.750±0.016)倍］的蛋白表达。 200 μmol/L七叶苷显著降低 RAW264.7细胞中炎症因子肿瘤坏死因子 α(TNF-α)［( 1.59±0.14)倍，(267.0±25.5)ng/L］、白细胞介素( IL)-1β［( 1.28±0.22)倍，(126.0±19.4)ng/L］和 IL-6［(1.26±0.13)倍，(113.0±18.4)ng/L］的转录水平表达量和培养上清液中旁分泌量。 200 μmol/L七叶苷能抑制脂多糖诱导的 RAW264.7细胞中 TLR4蛋白表达和核因子 -κB p65(NF-κB p65)、 NF-κB抑制因子( IκB)的磷酸化以及 NF-κB p65核转录。结论七叶苷通过抑制 TLR4/NF-κB信号通路传导和 NF-κB p65核转移来抑制脂多糖诱导的 RAW264.7细胞凋亡、坏死和炎症反应。 |
| 英文摘要: |
| Objective To investigate the inhibitory effect of esculin on lipopolysaccharide (LPS) induced RAW264.7 macrophages in-flammation by regulating Toll-like receptor 4/ nuclear factor κB (TLR4/NF-κB) pathway.Methods RAW264.7 macrophages were cul-tured and treated with LPS to induce inflammatory injury from October 2022 to June 2023. The cells were divided into normal group,LPS-stimulated model group, and esculin low, medium, and high concentration groups (drug concentrations of esculin were 25, 100 and200 μmol/L, respectively). RAW264.7 macrophages was treated with esculin for 12 hours and then induced by LPS for 12 hours. Thecell viability was detected by thiazole blue tetrazolium bromide (MTT) method. Apoptosis was detected by Annexin V-fluorescein iso-thiocyanate (FITC) and propyl iodide (PI) apoptosis and necrosis assay combined with cell flow analysis. The expression and release ofinflammatory factors were detected by real-time fluorescence quantitative polymerase chain reaction (qRT-PCR) and enzyme-linked im-munosorbent assay (ELISA), respectively. Protein expression of apoptosis factors, TLR4/NF-κB signaling pathway and nuclear translo-cation of NF-κB were detected by Western blot.Results MTT assay showed that 200 μmol/L esculin treatment significantly increasedthe proliferation activity of RAW264.7 cells induced by LPS (1.524±0.223). Flow analysis showed that esculin significantly inhibitedLPS-induced apoptosis and necrosis [(10.68±3.69)%], and western blot showed that 200 μmol/L esculin significantly promoted B-cell leukemia/lymphoma 2 (Bcl-2) [(1.981±0.026) fold] and promoted Bcl-2-associated X (Bax) protein [(1.750±0.016) fold] expression. 200 μmol/L esculin significantly decreased the transcription levels of inflammatory factors tumor necrosis factor α (TNF-α) [(1.59±0.14) fold, (267.0±25.5) ng/L] , interleukin-1β (IL-1β) [(1.28±0.22) fold, (126.0±19.4) ng/L] , and interleukin-6 (IL-6) [(1.26±0.13) fold,(113.0±18.4) ng/L] in RAW264.7 cells and the paracrine levels in the culture supernatant. 200 μmol/L esculin inhibited LPS-induced TLR4 protein expression , phosphorylation of nuclear factor-κB p65 (NF-κB p65) , pNF-κB inhibitor (IκB), and NF-κB p65 nuclear transcription in RAW264.7 cells.Conclusion Esculin inhibits LPS-induced apoptosis, necrosis and inflammation in RAW264.7 cells by inhibiting TLR4/NF-κB signaling pathway and NF-κB p65 nuclear translocation. |
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