祖师麻醋酸乙酯提取物体外抗炎作用及其机制

In vitro anti-inflammatory effect of ethyl acetate extract from Daphnes Giraldii Cortex and their mechanisms

目的研究祖师麻醋酸乙酯提取物(EAEDGC)的体外抗炎作用及其机制。方法用γ-干扰素(IFN-γ)和脂多糖(LPS)协同制备小鼠RAW264.7细胞炎症模型,Griess反应测定细胞上清液中NO生成量,MTT法测定细胞活力,三价铁还原抗氧化能力测试(FRAP assay)测定细胞总抗氧化能力,RT-PCR检测诱导型一氧化氮合酶(iNOS)、环氧化酶-2(COX-2)、血红素加氧酶-1(HO-1)、白细胞介素-1β(IL-1β)、白细胞介素-6(IL-6)、肿瘤坏死因子-α(TNF-α)mRNA的表达;Western blotting检测iNOS、COX-2、HO-1、p-ERK蛋白表达水平。结果 EAEDGC以剂量依赖方式抑制细胞上清液中NO的生成,提高细胞总抗氧化能力,下调iNOS、IL-1β、IL-6、TNF-αmRNA及iNOS、p-ERK蛋白的表达,上调HO-1 mRNA和蛋白表达,同时不影响细胞生长;但对COX-2 mRNA和蛋白表达影响不大。结论 EAEDGC部分通过丝裂原活化蛋白激酶(MAPK)中的细胞外信号调节激酶(ERK)信号转导通路抑制iNOS基因和蛋白的表达,从而抑制NO的生成,提高细胞总抗氧化能力,同时下调IL-1β、IL-6、TNF-α炎症介质和上调抗炎介质HO-1的表达,进而发挥抗炎作用。

Objective To investigate in vitro anti-inflammatory effect of ethyl acetate extract from Daphnes Giraldii Cortex（EAEDGC） on RAW 264.7 macrophages stimulated with IFN-γ plus LPS and their mechanisms.Methods IFN-γ plus LPS-stimulated RAW 264.7 macrophage has been used as experimental inflammatory model.Griess reaction for nitric oxide（NO） production,FRAP assay for total anti-oxidant capacity,MTT assay for cell proliferation（availability）,RT-PCR for mRNA expression and Western blotting for protein expression examination were performed.Results EAEDGC could concentration-dependently inhibit NO production in stimulated macrophage.The gene and protein expression of inducible nitric oxide synthase（iNOS） were also suppressed by the herb extract.The treatment by EAEDGC significantly attenuated mRNA of inflammatory cytokines,such as IL-1β,IL-6,and TNF-α,while increased HO-1 expression.The herb element elevated anti-oxidant capacity of the cells.Furthermore,phosphorylation of ERK（p-ERK） was markedly inhibited by EAEDGC.Conclusion EAEDGC exerts potential anti-inflammatory effects including the suppression of pro-inflammatory mediators,such as IL-1β,IL-6,TNF-α,and increasing HO-1 expression and total antioxidant capacity.EAEDGC could inhibit the NO production and iNOS expression,which may partially contribute to the regulation of ERK/MAPK pathway.