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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 study the anti-inflammatory effect and mechanism of EAEDGC in vitro. Methods The mouse RAW264.7 cells were induced by γ-interferon (IFN-γ) and lipopolysaccharide (LPS), the NO production was measured by Griess reaction, the viability was measured by MTT assay, The total antioxidant capacity of cells was determined by FRAP assay. The expressions of iNOS, COX-2 and heme oxygenase-1 The expression of HO-1, IL-1β, IL-6 and TNF-α mRNA were detected by ELISA. The expressions of iNOS, COX-2, HO-1, p-ERK protein expression levels. Results EAEDGC inhibited NO production, increased total cellular antioxidant capacity, decreased iNOS, IL-1β, IL-6, TNF-αmRNA and iNOS, p-ERK protein expression in a dose- 1 mRNA and protein expression, while not affecting cell growth; but had little effect on COX-2 mRNA and protein expression. Conclusion EAEDGC partially inhibits the production of nitric oxide synthase (iNOS) and enhances the total anti-oxidative capacity of cells by down-regulating the expression of iNOS gene and protein through the extracellular signal-regulated kinase (ERK) signaling pathway in mitogen activated protein kinase (MAPK) IL-1β, IL-6, TNF-αinflammatory mediators and up-regulate the expression of anti-inflammatory mediators HO-1, and then exert anti-inflammatory effects.