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目的:观察穿心莲内酯对脂多糖激活的小鼠腹腔巨噬细胞细胞外信号调节激酶1/2(extracellularsignal-regulated kinase1/2,ERK1/2)信号转导通路及肿瘤坏死因子α(tumor necrosisfactor-α,TNF-α)的影响。方法:以穿心莲内酯作用于脂多糖活化的小鼠腹腔巨噬细胞后,细胞计数试剂盒8测定穿心莲内酯对巨噬细胞的细胞毒作用,蛋白质印迹法检测巨噬细胞ERK1/2、p38和JNK蛋白磷酸化水平以及IκBα蛋白表达水平,逆转录聚合酶链反应法检测巨噬细胞TNF-αmRNA的表达水平,酶联免疫吸附测定法检测巨噬细胞培养上清液中TNF-α蛋白的表达水平。结果:1~100μg/mL穿心莲内酯对脂多糖活化的小鼠腹腔巨噬细胞无细胞毒作用;穿心莲内酯能抑制脂多糖活化小鼠腹腔巨噬细胞的ERK1/2磷酸化,随着穿心莲内酯浓度增加,抑制作用增强(P<0.01);1~25μg/mL穿心莲内酯不能抑制脂多糖活化小鼠腹腔巨噬细胞的p38和JNK磷酸化,对脂多糖引起的IκBα降解也无影响。12μg/mL穿心莲内酯和20μmol/LPD98059(ERK1/2信号转导通路抑制剂)能降低巨噬细胞TNF-αmRNA的表达和其上清液中TNF-α的分泌水平(P<0.01)。结论:穿心莲内酯能阻断ERK1/2信号转导通路,并在抑制TNF-α的表达中可能起作用。
Objective: To observe the effects of andrographolide on lipopolysaccharide-induced extracellular signal-regulated kinase1 / 2 (ERK1 / 2) signal transduction pathway and tumor necrosis factor- α, TNF-α). Methods: After andrographolide was administered to lipopolysaccharide-activated mouse peritoneal macrophages, cytotoxicity of andrographolide to macrophages was determined by kit 8, Western blotting was used to detect macrophages ERK1 / 2, p38 And JNK protein phosphorylation levels and IκBα protein expression levels, macrophages TNF-αmRNA expression levels detected by reverse transcription polymerase chain reaction, macrophage cell culture supernatants TNF-α protein by enzyme-linked immunosorbent assay The expression level. RESULTS: 1 ~ 100μg / mL andrographolide had no cytotoxic effect on lipopolysaccharide-activated mouse peritoneal macrophages. Andrographolide could inhibit phosphorylation of ERK1 / 2 induced by lipopolysaccharide in mouse peritoneal macrophages, (P <0.01); 1 ~ 25μg / mL andrographolide could not inhibit phosphorylation of p38 and JNK in peritoneal macrophages activated by LPS and had no effect on the degradation of IκBα induced by lipopolysaccharide . 12μg / mL andrographolide and 20μmol / LPD98059 (ERK1 / 2 signal transduction pathway inhibitor) decreased TNF-αmRNA expression in macrophages and secretion of TNF-α in supernatant (P <0.01). Andrographolide can block ERK1 / 2 signal transduction pathway and may play an important role in inhibiting the expression of TNF-α.