Departamento de Bioquímica, Instituto de Ciências Básicas da Saúde, UFRGS, Porto Alegre, RS, Brazil
J Neuroimmunol. 2012 Aug 15;249(1-2):8-15. doi: 10.1016/j.jneuroim.2012.04.004. Epub 2012 May 3.
In this study we investigated the anti-inflammatory effects of chronic ethanol (EtOH) treatment on lipopolysaccharide (LPS)-stimulated C6 glioma cells. The cells were chronically treated with 200mM EtOH; coincubation with LPS and EtOH was obtained upon addition of 2μg/ml LPS to the incubation medium in the last 24h of EtOH exposure. We found that EtOH prevented the LPS-induced production of tumor necrosis factor α (TNFα) without decreasing cell viability. Either LPS treated or EtOH plus LPS treated cells presented upregulated glial fibrillary acidic protein (GFAP) and downregulated vimentin levels characterizing a program of reactive astrogliosis. Also, EtOH plus LPS stimulation greatly increased the oxidative stress generation evaluated by DCF-DA measurement, while either EtOH alone or LPS alone was unable to induce oxidative stress. Western blot analysis indicated that either EtOH, LPS or EtOH plus LPS treatments are unable to affect Akt/GSK3β signaling pathway. However, LPS alone and EtOH plus LPS co-treatment inhibited Erk phosphorylation. A dramatic loss of stress fibers was found in EtOH exposed cells, evaluated by cytochemistry using phalloidin-fluorescein. However, LPS alone was not able to disrupt actin organization. Furthermore, cells co-incubated with LPS and EtOH presented reversion of the disrupted stress fibers provoked by EtOH. Supporting this action, RhoA and vinculin immunocontent were upregulated in response to EtOH plus LPS. Interestingly, EtOH suppresses the inflammatory cascade (TNFα production) in response to LPS. Concomitantly it sustains Erk inhibition, increases oxidative stress generation and induces reactive astrogliosis in the presence of LPS, conditions associated with neurotoxicity. The effects observed were not supported by actin reorganization. Altogether, these findings suggest that Erk signaling inhibition could play a role in both suppressing TNFα production and inducing oxidative stress generation and astrogliosis, therefore modulating a dual action of EtOH plus LPS in glial cells.
在这项研究中，我们研究了慢性乙醇（EtOH）处理对脂多糖（LPS）刺激的 C6 神经胶质瘤细胞的抗炎作用。细胞用 200mM EtOH 进行慢性处理；在 EtOH 暴露的最后 24 小时，将 2μg/ml LPS 添加到孵育培养基中，以获得 LPS 和 EtOH 的共孵育。我们发现 EtOH 可预防 LPS 诱导的肿瘤坏死因子 α（TNFα）的产生，而不会降低细胞活力。LPS 处理或 EtOH 加 LPS 处理的细胞均呈现出胶质纤维酸性蛋白（GFAP）上调和波形蛋白下调的特征，表现出反应性星形胶质细胞增生的程序。此外，EtOH 加 LPS 刺激大大增加了通过 DCF-DA 测量评估的氧化应激生成，而 EtOH 单独或 LPS 单独均不能诱导氧化应激。Western blot 分析表明，EtOH、LPS 或 EtOH 加 LPS 处理均不能影响 Akt/GSK3β信号通路。然而，LPS 单独和 EtOH 加 LPS 共处理抑制了 Erk 磷酸化。用鬼笔环肽-荧光素通过细胞化学染色发现 EtOH 暴露的细胞中应力纤维明显丢失。然而，LPS 单独不能破坏肌动蛋白组织。此外，与 LPS 和 EtOH 共孵育的细胞呈现出由 EtOH 引起的破坏的应力纤维的逆转。支持这一作用，RhoA 和 vinculin 免疫含量上调以响应 EtOH 加 LPS。有趣的是，EtOH 抑制 LPS 引起的炎症级联（TNFα 产生）。同时，它在 LPS 存在下维持 Erk 抑制、增加氧化应激生成并诱导反应性星形胶质细胞增生，这些条件与神经毒性相关。观察到的作用不受肌动蛋白重组的支持。总的来说，这些发现表明 Erk 信号抑制可能在抑制 TNFα 产生和诱导氧化应激生成和星形胶质细胞增生中起作用，从而调节 EtOH 加 LPS 在神经胶质细胞中的双重作用。
