染料木黄酮对脂多糖诱导的RAW264.7细胞炎症因子、腺苷酸激活蛋白激酶磷酸化的影响

EFFECTS OF GENISTEIN ON INFLAMMATORY FACTOR,ADENOSINE MONOPHOSPHATE-ACTIVATED PROTEIN KINASE PHOSPHORYLATION IN LIPOPOLYSACCHARIDES-TREATED RAW264.7 CELLS

目的研究染料木黄酮(genistein,Gen)对脂多糖(LPS)诱导的巨噬细胞炎症因子产生和腺苷酸激活蛋白激酶(AMPK)磷酸化的影响。方法体外培养RAW 264.7小鼠单核巨噬细胞,加入1、5、10、50、100 mol/L的Gen共同培养,MTT法检测其对细胞活性的影响。将RAW264.7细胞随机分为4组:空白对照组不加任何药物,模型组加入终浓度为1 g/ml的LPS进行刺激,Gen高、低剂量组分别加入终浓度为10、5 mol/L的Gen预处理1h后,再加入终浓度为1 g/ml的LPS刺激,24h后收取细胞上清用酶联免疫(ELISA)方法检测TNF-α、IL-6含量,Westernblot检测AMPKα蛋白及其磷酸化的表达水平。结果 100 mol/L的Gen对体外培养RAW264.7细胞的增殖有影响,而其他浓度则无。LPS处理的RAW 264.7细胞与对照组比较,TNF-α、IL-6生成显著增多(P<0.01)、AMPKα磷酸化水平显著降低(P<0.01),而AMPKα总蛋白表达水平无差异(P>0.05)。Gen干预可减少LPS诱导的TNF-α、IL-6生成,上调AMPKα磷酸化水平的表达,与LPS组相比,差异均有统计学意义(P<0.01)。结论 Gen能抑制LPS诱导巨噬细胞的炎症反应,减少TNF-α、IL-6生成,这可能与Gen促进AMPKα磷酸化,从而激活AMPKα有关。

Objective To observe the effect of genistein on（Gen） lipopolysaccharides（LPS） induced inflammation in macrophage cells.Method RAW264.7 cells were cultured with 1,5,10,50,100 μmol/L of Gen and the cell activity was detected by MTT.The cells were randomly dividcd into 4 groups：control group,not treated;model group,treated with 1 μg/ml LPS;high and low dose Gen group,treated with 10 and 5 μmol/L Gen respectively first.then with 1 μg/ml LPS.The production of TNF-α and IL-6 was measured by ELISA.The expression of adenosine monophosphate activated protein kinase α（AMPKα） and its phosphorylation was determined by Western blotting.Results Only 100 μmol/L Gen influenced the activity of RAW 264.7 cells culture in vitro.The concentration of TNF-α and IL-6 was significantly higher in LPS group than those in control group,while the phosphorylation level of AMPKα lower（P0.01）.Gen significantly inhibited LPS-induced TNF-α,IL-6 production,and increased the expression of AMPKα phosphorylation as compared with LPS group（P0.01）.Conclusion Gen could inhibit LPS induced-TNF-α,IL-6 secretion and up-regulate the phosphorylation of AMPKα.