PI3K/PTEN/Akt/mTOR通路在浒苔多糖粗提物调节小鼠巨噬细胞RAW264.7免疫功能中的作用

摘要

Objective: To verify the up-regulation of the immunity of mouse macrophage RAW264.7 is achieved with the crude extract of enteromorpha polysaccharide and preliminarily explore whether it is achieved through PI3K/PTEN/Akt/mTORpathway. Methods: To interfere RAW264.7cells with crude extract of different concentration of enteromorpha polysaccharide ,after intervention of differernt time, to determine the proliferation rate of macrophage RAW264.7 by methyl thiazolyl tetrazolium (MIT) assay;to test the level of interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) with ELISA assay;to test express level of mTOR mRNA by real-time fluorescent quantitative PCR;to interfere RAW264.7cells with rapamycin of 100nmol/L,crude extract of enteromorpha polysaccharide of 500μg/ml,and crude extract of enteromorpha polysaccharide of 500μg/ml+rapamycin of 100nmol/L,to test secretion level of TNF-α.Results: Optimized proliferation rate would reach 53.47%with the intervention of crude extract of enteromorpha polysacchride for 12 hours. The higher the concentration of crude extract of enteromorpha polysacchride is, the higher the IL-6 secretion level of RAW264.7 cell would become, and it would reach the peak when it has been interfered for 24 hours, with the extension of time,the secretion level of TNF-αwould become higher and higher (F=311.63,P<0.001) and at the same time it would also go up with the rising of the concentration of intervention (F=51.80,P<0.001);after the intervention of enteromorpha polysaccharide of 12 or 24 hours, the express level of mTOR goes up (F=4.256,P=0.029;F=20.606,P<0.001); compared with the group of crude extract of enteromorpha polysaccharide of 500μg /ml, IL-6 level of the group of rude extract of enteromorpha polysaccharide of 500μg /ml+ rapamycin of 100nmol/L declines by 44.58%, the level TNF-α declines by 65.96%(average P<0.001).Conclusion: Crude extract of enteromorpha polysaccharide promotes the proliferation of macrophage RAW264.7 and regulate the release of cytokine, which may be relative to PI3K/PTEN/Akt/mTOR pathway.%目的：验证浒苔多糖粗提物上调小鼠巨噬细胞RAW264.7免疫功能，并初步探讨其是否通过PI3K/PTEN/Akt/mTOR通路实现。方法：以不同浓度浒苔多糖粗提物干预RAW264.7细胞，干预不同时间后四甲基偶氮噻唑蓝（MTT）比色法测定巨噬细胞RAW264.7增殖率；ELISA法测定白介素-6（IL-6）和肿瘤坏死因子-α（TNF-α）水平；实时荧光定量PCR检测mTORmRNA的表达水平；以100nmol/L雷帕霉素、500μg/ml浒苔多糖粗提物、500μg/mL浒苔多糖粗提物+100nmol/L雷帕霉素干预RAW264.7细胞，测定细胞因子IL-6、TNF-α分泌水平。结果：500μg/mL浒苔多糖粗提物干预12h，可获得最佳增殖率53.47%；RAW264.7细胞分泌IL-6的水平随着浒苔多糖粗提物浓度的增大而增加，且在24h达到分泌高峰，TNF-α分泌水平随着时间延长而增加（F=311.63，P＜0.001），同时随着干预浓度的提高而增加（F=51.80，P＜0.001）；浒苔多糖粗提物干预12、24h后，mTOR表达水平增高（F=4.256，P=0.029；F=20.606，P＜0.001）；较之500μg/mL浒苔多糖粗提物组，500μg/mL浒苔粗提物+100nmol/L雷帕霉素组IL-6水平下降44.58%，TNF-α水平下降65.96%（P均＜0.001）。结论：浒苔多糖粗提物促进巨噬细胞RAW264.7增殖，调节其细胞因子释放，可能与PI3K/PTEN/Akt/mTOR通路有关。