铁皮石斛多糖对RAW264.7细胞分泌TNF-α的影响

Effects of polysaccharides from Dendrobium Officinale on the production of TNF-α by RAW264.7 cells

目的研究铁皮石斛多糖(polysaccharides from Den-drobium officinale,DOP)对小鼠巨噬细胞系RAW264.7细胞分泌TNF-α的影响并探讨其作用机制。方法 MTT法检测细胞活性;ELISA法检测TNF-α的分泌水平;反转录聚合酶链反应(RT-PCR)检测TNF-αmRNA表达量;Western blot技术检测胞质I-κBα蛋白的表达量。结果 DOP在20～160mg·L-1范围内明显促进RAW264.7细胞增殖,无细胞毒性;与空白对照组比较,DOP剂量依赖性地促进TNF-α的分泌;上调TNF-αmRNA表达;Western blot结果显示DOP能明显降低胞质内I-κBα蛋白水平。结论 DOP能增强RAW264.7细胞分泌TNF-α,其作用机制可能与降低胞质内I-κBα蛋白水平,活化核转录因子NF-κB,诱导TNF-αmRNA的表达有关。

Aim To study the effects of polysaccharides from dendrobium officinale（DOP） on the production of TNF-α in RAW264.7 cells and its possible mechanism.Methods Cell proliferation was measured by MTT assay;Concentration of TNF-α in supernatants was determined by ELISA kit;Expression of TNF-α mRNA was measured by reverse transcription polymerase chain reaction（RT-PCR）;Production of I-κBα protein was detected by Western blot.Results DOP（20～160 mg·L-1） possessed the stimulation effect on macrophages proliferation without cytotoxic.The production of TNF-α was increased in a dose-dependent manner when pretreated with DOP.Compared with control group,expression of TNF-α mRNA was significantly up-regulated.DOP markedly decreased intracellular I-κBα level as demonstrated by Western blot assay.Conclusion DOP induces TNF-α gene expression followed by TNF-α production in macrophages via decreasing intracellular I-κBα level,activating transcription factor,NF-κB,suggesting that the immunoregulation mechanism of DOP may be related to the induction of TNF-α production in macrophages.