黄芪皂苷Ⅰ调控PI3K/Akt/NF-κB通路抑制LPS诱导BV-2细胞激活

Astragaloside Ι inhibited the activation of BV-2 cell induced by LPS through modulation of PI3K/Akt/NF-κB pathway

目的:探讨黄芪皂苷Ⅰ(ASTⅠ)对脂多糖(LPS)诱导BV-2小胶质细胞激活的作用及其分子机制。方法:不同浓度的ASTⅠ(25、50、100μmol/L)预处理BV-2细胞2h后,加入LPS(200ng/m L)诱导20h,收集培养基上清及细胞。CCK-8法检测不同浓度的ASTⅠ对BV-2细胞活性的影响;分别采用ELISA和Greiss法检测培养上清中肿瘤坏死因子α(TNF-α)和一氧化氮(NO)的含量;q PCR法检测细胞中CD11b,TNF-α,诱导型一氧化氮合酶(i NOS)和白介素1β(IL-1β)m RNA水平;Western blot法检测细胞PI3K/AKT/NF-κB信号通路蛋白表达;细胞免疫荧光(ICC)法观察p-NFκB的入核情况。结果:ASTⅠ在不影响BV-2细胞存活率的条件下,能显著抑制LPS诱导BV-2细胞TNF-α和NO分泌的增加(P<0.01)。q PCR结果显示,ASTⅠ能够显著抑制TNF-α,i NOS及IL-1β的m RNA生成(P<0.001,P<0.001,P<0.05),但对BV-2细胞CD11b的m RNA水平无显著影响。同时,ASTⅠ能显著抑制BV-2细胞中LPS诱导上调的i NOS,COX-2蛋白表达。ASTⅠ能够降低LPS诱导的p-NF-κB的核转位作用,并且能够减少LPS引起的PI3K/Akt/NF-κB/IκB蛋白的磷酸化作用。结论:ASTⅠ能够抑制LPS诱导BV-2细胞的大量激活,作用机制可能与其抑制PI3K/Akt/NF-κB信号通路有关。

Objective： To investigate the effect and mechanism of astragaloside I（AST I） on the activation of microglial cell line BV-2 induced by LPS. Methods： After pre-treated with AST I（25, 50, 100μmol/L） for 2 h, BV-2 cells were stimulated with LPS（200ng/m L） for 20 h. Thereafter, the cells and the culture medium were collected. Cell viability was measured by CCK-8 method. Concentration of TNF-α was detected by using ELISA kit. Secretion of NO in medium was assayed with Greiss approach. Gene expressions of CD11 b, TNF-α, i NOS, and IL-1β m RNA were detected with quantitative PCR. Protein expression of PI3K/Akt/NF-κB pathway was detected using Western blotting assay. Neuclear translocation of p-NF-κB was monitored with immunocytochemistry. Results： AST I inhibited the secretion of TNF-α and NO on BV-2 cells upon LPS stimulation（P0.001） without affecting cell viability. It could prevent the up-regulation of gene expressions of TNF-α, i NOS and IL-1β（P0.001, P0.001, P0.05） but not that of CD11 b. Meanwhile, AST I suppressed the increase of i NOS and COX-2 protein induced by LPS. Further study disclosed that AST I reduced nuclear translocation of activated NF-κB and deceased phosphorylation of PI3 K, Akt, NF-κB, and IκB. Conclusion： AST I inhibited the activation of BV-2 cells induced by LPS through suppressing the activation of PI3K/Akt/NF-κB pathway, therefore, reduced the nuclear translocation of phosphorylated NF-κB, leading to the down-regulation of the gene expressions of TNF-α, i NOS and IL-1β and thus lessened the production of i NOS, TNF-α and COX-2 protein.