芫花根中菖蒲烷型倍半萜4-epi-15-hydroxyacorenone抗炎机制研究

Study on the Anti-Inflammatory Mechanism of Acorane Sesquiterpene 4-epi-15-hydroxyacorenone from the Roots of Daphne Genkwa Sieb.et Zucc.

目的:对从芫花根中分离的菖蒲烷型倍半萜4-epi-15-hydroxyacorenone进行抗炎活性研究,探讨其抗炎分子机制,为芫花药用资源的合理开发利用提供理论依据。方法:采用RAW 264.7巨噬细胞建立炎症模型,分为空白组、模型组、给药组。通过Griess法检测4-epi-15-hydroxyacorenone对脂多糖(LPS)诱导的RAW 264.7巨噬细胞释放炎症介质一氧化氮(NO)的抑制活性,运用噻唑蓝(MTT)比色法检测细胞毒性,酶联免疫吸附测定法(ELISA)检测化合物对炎症介质前列腺素E2(PGE2)、炎症因子肿瘤坏死因子-α(TNF-α)以及白细胞介素-6(IL-6)的抑制作用,蛋白免疫印迹法(Western Blot)检测化合物对炎症蛋白iNOS和COX-2高表达的影响,对核转录因子κB(NF-κB)信号通路中IκB-α蛋白的降解以及丝裂原活化蛋白激酶(MAPKs)信号通路中ERK、JNK、p38蛋白磷酸化的抑制作用。结果:与空白组相比,模型组经LPS刺激后炎症因子、炎症介质的释放,炎症蛋白的表达均显著增加(P<0.01),与模型组相比,给药组菖蒲烷型倍半萜4-epi-15-hydroxyacorenone可阻断ERK、JNK蛋白磷酸化(P<0.01),抑制iNOS和COX-2的高表达并且呈良好的剂量依赖关系(P<0.01),显著降低炎症因子TNF-α、IL-6以及炎症介质NO、PGE2的释放(P<0.01),但对NF-κB信号通路的激活无任何影响。结论:4-epi-15-hydroxyacorenone主要通过调控MAPKs信号通路的激活,下调LPS诱导的炎症蛋白的高表达,抑制RAW 264.7巨噬细胞中炎症介质以及炎症因子的释放发挥抗炎作用。

Objective:To study the anti-inflammatory activity of the acorane sesquiterpene 4-epi-15-hydroxyacorenone isolated from the roots of Daphne genkwa,and to explore the molecular mechanism of its anti-inflammatory activity,so as to provide a theoretical basis for the rational development and utilization of the medicinal resources of Daphne genkwa.Methods:RAW 264.7 macrophages were used to establish the inflammation model,which was divided into blank group,model group and drug administration group.The inhibitory activity of 4-epi-15-hydroxyacorenone on the release of inflammatory mediator nitric oxide(NO)by lipopolysaccharide(LPS)-induced RAW 264.7 macrophages was detected by Griess assay,and the cytotoxicity was detected by MTT assay.The inhibitory effects of active compounds on inflammatory mediators prostaglandin E_(2)(PGE_(2)),inflammatory cytokines tumor necrosis factor-α(TNF-α)and interleukin-6(IL-6)were detected by enzyme-linked immunosorbent assay(ELISA).Western blotting was used to detect the effects of the compound on the expression of inflammatory proteins iNOS and COX-2,the degradation of IκB-αin nuclear factor-κB(NF-κB)signaling pathway,and the inhibition of ERK,JNK and p38 protein phosphorylation in mitogen-activated protein kinases(MAPKs)signaling pathway.Results:Compared with the blank group,the release of inflammatory cytokines,inflammatory mediators and the expression of inflammatory proteins in the model group were significantly increased after LPS stimulationP<0.01.Compared with the model group,the acorane sesquiterpene 4-epi-15-hydroxyacorenonein the administration group could block the phosphorylation of ERK and JNK proteins(P<0.01),inhibited the high expression of iNOS and COX-2 in a good dose-dependent manner(P<0.01),significantly reduced the release of inflammatory cytokines TNF-α,IL-6,and inflammatory mediators NO and PGE_(2)(P<0.01),but had no effect on the activation of NF-κB signaling pathway.Conclusion:4-epi-15-hydroxyacorenone plays an anti-inflammatory role by regulating the activation of MAPKs signaling pathway,down-regulating the expression of LPS-induced inflammatory proteins,and inhibiting the release of inflammatory mediators and inflammatory factors in RAW 264.7 macrophages.