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雷公藤为卫矛科雷公藤属植物雷公藤Tripterygium wilfordii Hook.f.的干燥根,是一种公认的同时具有较强药效和较强毒性的中药材,广泛用于治疗类风湿性关节炎、肾病综合征、系统性红斑狼疮等疾病[1]。但其毒性较大,不良反应高发且严重,常使患者不能耐受[2]。雷公藤的主要有效成分为二萜类、三萜类和生物碱类,研究表明各种成分均具有不同程度的抗炎、抗肿瘤和免疫抑制等活性[3-4],其中生物碱的毒性要小于二萜和三萜类成分,临床应用具有疗效好,不良反应较小的特点[5]。雷公藤次碱是雷公藤生物碱中含量较高的一种倍半萜类单体化合物。目前研究显示其具有良好的杀虫活性,其他药理活性和机理研究较少,特别是抗炎和免疫抑制活性方面。
本研究主要通过建立脂多糖(LPS)诱导的RAW264.7细胞炎症模型,探讨雷公藤次碱对LPS诱导的炎性因子NO,IL-1β,TNF-α和IL-6释放的影响,用免疫印迹法考察雷公藤次碱对TRAF6、IRAK、NF-κB、IκBα、JNK、ERK、p38等关键蛋白表达或磷酸化的影响,探讨其可能的抗炎作用机制,为促进雷公藤次碱的应用研究提供基础。
Wilforine inhibits LPS-induced inflammatory response in RAW264.7 cells by regulating the TLR4/MyD88/TRAF6 signaling pathway
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摘要:
目的 探讨雷公藤次碱抗炎活性及其作用机制。 方法 用细胞计数盒-8 (CCK-8)法考察雷公藤次碱对小鼠单核巨噬细胞白血病细胞RAW 264.7 增殖活性的影响,用酶联免疫吸附法(ELISA)检测雷公藤次碱对脂多糖(LPS)诱导的RAW264.7细胞分泌细胞因子一氧化氮(NO)、白细胞介素-1β(IL-1β)、 肿瘤坏死因子α(TNF-α)和白细胞介素6(IL-6)的影响,用免疫印迹法考察雷公藤次碱对白介素受体相关激酶(IRAK)、肿瘤坏死因子受体相关蛋白6(TRAF6)、核因子κB抑制因子α(IκBα)、核因子κB(NF-κB p65)、分裂原激活的蛋白激酶p38(p38)、c-Jun氨基末端激酶(JNK)、细胞外调节蛋白激酶(ERK)在LPS刺激的RAW264.7细胞中的表达及其磷酸化的影响。 结果 雷公藤次碱在25、50、100 μmol/L浓度下对RAW264.7细胞无显著毒性,并可显著抑制细胞因子NO、IL-1β、TNF-α和IL-6含量。免疫印迹法检测结果显示雷公藤次碱可显著抑制IRAK及TRAF6的表达;显著抑制ERK、P38和JNK的磷酸化;抑制IκBα的降解,降低NF-κB p65的核转运水平。 结论 雷公藤次碱具有体外抗炎活性,其作用机制可能介导TLR4/MyD88/TRAF6信号通路。 Abstract:Objective To investigate the anti-inflammatory effect and mechanism of wilforine. Methods Anti-inflammatory activity of wilforine was investigated in LPS-induced RAW264.7 cells. The cytokines production of RAW264.7 cells was analyzed by ELISA assay and the cell viability was assessed by CCK-8 method. The expression of TRAF6, the phosphorylation of IRAK, p38, ERK and JNK, the degradation of inhibitory κBα (IκBα) and the nuclear translocation of NF-κB p65 were further investigated by western blot. Results Triptolide had no significant toxicity to RAW264.7 cells at concentrations of 25, 50 and 100 μmol/L. and could significantly inhibit the contents of cytokines NO, IL-1β, TNF-α and IL-6. Wilforine significantly decreased the expression of TRAF6 and phosphorylation of IRAK, and inhibited the phosphorylation of ERK, p38, and JNK and degradation of IκBα, and reduced the level of nuclear translocation of NF-κB p65. Conclusion The anti-inflammatory activity of wilforine of LPS-induced RAW264.7 cells is probably via TLR4/MyD88/TRAF6 signaling pathway. -
Key words:
- Tripterygium wilfordii /
- wilforine /
- anti-inflammation /
- TLR4 /
- MyD88 /
- NF-κB
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