蔓越莓提取物对脂多糖诱导RAW246.7细胞炎症反应的抑制作用

Inhibitory Effect of Cranberry Extract on LPS Induced Inflammatory Response in RAW246.7 Cells

研究蔓越莓提取物对脂多糖(lipopolysaccharide,LPS)诱导RAW246.7细胞炎症反应的抑制作用及其作用机制。筛选LPS浓度建立细胞炎症模型;采用噻唑蓝[3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide,MTT]法测定蔓越莓提取物对RAW246.7细胞活力的影响;利用4,6-联脒-2-苯基吲哚(4′,6-diamidino-2-phenylindole,DAPI)染色法观察蔓越莓提取物对细胞核形态的影响;采用荧光分析法测定一氧化氮合酶(nitric oxide synthase,NOS)的活力;酶联免疫吸附剂测定(enzyme linked immunosorbent assay,ELISA)试剂盒测定IL-1、IL-6、TNF-α细胞因子的水平;蛋白质印迹法(Western-Blot)法检测Nrf2/NF-κB通路相关蛋白的水平。实验结果显示5μg/m L的LPS建立炎症模型,LPS与细胞共培养24 h时炎症水平达到最高,蔓越莓提取物在5μg/m L^400μg/m L范围内对RAW246.7细胞无显著性毒性作用,细胞核形态完整,无明显损伤;与模型组比较,蔓越莓提取物在无毒性浓度范围内显著降低LPS诱导RAW246.7细胞炎症反应的NOS活力和IL-1、IL-6、TNF-α水平,且呈现出剂量依赖关系。Western-Blot实验结果显示蔓越莓提取物量效依赖性地降低Keap1、IKKα/β、NF-κBp65蛋白表达的水平,而上调了Nrf2、HO-1的蛋白表达水平。结果证明蔓越莓提取物能够有效地抑制LPS诱导的炎症反应,其作用机制可能与经典的抗氧化通路Keap1/Nrf2/HO-1和炎症通路NF-κBp65蛋白表达有关。

To study the anti-inflammatory effect of cranberry extract on inflammation suppression induced by lipopolysaccharide,and explore its mechanism. Cell inflammatory model was established with RAW264.7 cells treated with lipopolysaccharide. Cell viability of RAW264.7 cells treated with cranberry extract were analyzed by 3-（4,5-Dimethylthiazol-2-yl）-2,5-diphenyl-2 H-tetrazolium bromide（MTT） assay. The effect of cranberry extract on nucleus was observed by 4′,6-diamidino-2-phenylindole（DAPI）staining. The activity of nitric oxide synthase（NOS）was determined by fluorescence analysis. Enzyme-linked immunosorbent assay（ELISA）for determination of IL-1β,IL-6 and TNF-α. RAW264.7 cells were treated with cranberry extract for24 h,and the expression of Keap1,Nrf2,HO-1,IKKα/β and NF-κBp65 were detected by Western blotting. The result showed that the inflammatory model was established by 5 μg/m L lipopolysaccharide,the highest level of inflammation was reached at 24 hours. There was no significant toxic effect on RAW246.7 cells in the range of5 μg/m L-400 μg/m L,and the cell nucleus was intact and without obvious damage. Compared with the model group,cranberry extract could significantly inhibit the activity of NOS and decreased the content of IL-1β,IL-6,TNF-α with the increase of dose. The Western blot result showed that cranberry extract inhibited the expression of Keap1,IKKα/β,NF-κBp65 and increase the expression of Nrf2 and HO-1 protein levels. These results suggest that cranberry extract can inhibit the inflammatory response induced by lipopolysaccharide,and its mechanism may be related to activation of Keap1/Nrf2/HO-1 signaling pathway and NF-κBp65.