芫花总黄酮的抗炎机制研究

目的研究芫花总黄酮（TFDG）对脂多糖（LPS）协同干扰素γ（IFN-γ）诱导鼠RAW264.7巨噬细胞炎症模型中亚硝酸盐含量、诱导型合酶和细胞因子表达的影响,并从ERK/MAPK通路探讨其抗炎机制。方法细胞随机分为芫花总黄酮处理组,阳性药物组（I-NIL50μM）、炎症模型组和正常对照组;Griess反应测定TFDG25、50、100mg/L3个剂量和预刺激、同时加入、预处理3个处理时间及I-NIL对激活细胞上清液中亚硝酸盐的影响,并测定TFDG3个剂量及I-NIL对静息细胞上清液中亚硝酸盐产量的影响;MTT法测定TFDG3个剂量和I-NIL分别对激活细胞和静息细胞的细胞活力的影响;RT-PCR检测TFDG50、100mg/L预处理1h对激活细胞中诱导型合酶iNOS、COX-2、细胞因子IL-1β、IL-6、TNF-α基因表达的影响;Western blot检测TFDG50、100mg/L预处理1h对激活细胞中诱导型合酶iNOS、COX-2和p-ERK蛋白表达的影响。结果模型组中一氧化氮（NO）产物亚硝酸盐含量、诱导型合酶iNOS和COX-2基因和蛋白表达,细胞因子IL-1β、IL-6、TNF-α基因表达,ERK蛋白磷酸化水平均明显高于正常对照组（P〈0.01,P〈0.05）,细胞活力显著下降（P〈0.01）;TFDG呈剂量和时间依赖性抑制激活细胞上清液中亚硝酸盐含量（P〈0.01）,提高炎症损伤的细胞活力（P〈0.01）;TFDG对静息细胞上清液中亚硝酸盐含量无影响,无细胞毒性且于100mg/L剂量促进细胞增殖（P〈0.05）。阳性药物I-NIL于50μM剂量的抑制率为35.20%（P〈0.01）,提高炎症损伤后的细胞活力（P〈0.01）,但对静息细胞呈细胞毒性（P〈0.05）。与模型组比较,芫花总黄酮下调iNOS基因和蛋白表达（P〈0.01,P〈0.05）,抑制COX-2基因表达（P〈0.01）,对COX-2蛋白表达仅在高剂量100mg/L具抑制效果（P〈0.01）;下调细胞因子IL-1β、IL-6、TNF-α的基因表达（P〈0.01）,同时能抑制ERK蛋白的磷酸化水平（P〈0.05）。结论芫花总黄酮抑制激活细胞中iNOS基因和蛋白的表达从而降低NO稳定产物亚硝酸盐的产量,抑制COX-2基因和蛋白的表达,同时抑制细胞因子IL-1β、IL-6、TNF-α的基因表达,部分机制为抑制ERK/MAPK信号通路中ERK蛋白磷酸化而达到多靶点抗炎效果。

Anti-inflammatory effect of total flavones from Daphne genkwa Sieb.et Zucc on stimulated macrophages

Objective Investigating the anti-inflammatory effect of total flavones from Daphne genkwa Sieb.et Zucc（TFDG） on accumulation of nitrite,expression of inducible synthases and cytokines in RAW 264.7 macrophages stimulated with interferon-γ（IFN-γ） plus lipopolysaccharide（LPS） and revealing its mechanisms.Methods Cell randomly divided into TFDG treated group,model group which stimulated with IFN-γ plus LPS,I-NIL（50 μM） group and normal control group.Griess reaction for nitrite production with TFDG 25,50,100 mg/L dosage and different treatment time（-6 h,0 h,＋6 h）;MTT assay for cell availability in stimulated and resting cell with TFDG 25,50,100mg/L;I-NIL（50 μM） as positive drug control;RT-PCR for iNOS,COX-2,IL-1β,IL-6 and TNF-α mRNA expression and Western blot for iNOS,COX-2,p-ERK protein expression and phosphorylation examination with TFDG pre-treatment for 1h at 50,100mg/L dosage prior to stimulation.Results Compared with control group,the inflammatory mediators expression stimulated by LPS and IFN-γ including nitrite,iNOS,COX-2,IL-1β,IL-6 and TNF-α,p-ERK significantly increased（P〈0.01,P〈0.05）while the cell viability decreased.Compared with model group,TFDG exerted concentration and time-dependent inhibitory effect on nitrite production,showed some protection to stimulated cell and no cytotoxicity in resting cell,on the other hand,I-NIL at 50μm inhubited nitinte production by 35.20%（P〈0.01）,and protected the altivated cells from injurg（P〈0.01）,but in resting cells I-NIL displayed some cytotoxicity（P〈0.05）.TFDG reduced gene and protein expression of iNOS and COX-2（P〈0.01,P〈0.05）,while suppressed the inflammatory cytokines expression of IL-1β,IL-6 and TNF-α mRNA（P〈0.01）.Furthermore,the phosphorylation of ERK were markedly inhibited by TFDG（P〈0.05）.Conclusion These results suggest that TFDG may provide potential anti-inflammatory effects against attenuating excessive NO,suppressing gene expression of pro-inflammatory cytokines IL-1β,IL-6 and TNF-α.TFDG inhibited iNOS and COX-2 expression may partially contribute to regulation of extra cellular regulated protein kinases（ERK/MAPK） pathway.