title: Dual action of chronic ethanol treatment on LPS-induced response in C6 glioma cells.
abstract: 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.
authors.affiliation: Departamento de Bioquímica, Instituto de Ciências Básicas da Saúde, UFRGS, Porto Alegre, RS, Brazil
authors.forename: Samanta Oliveira
authors.initials: SO
authors.lastname: Loureiro
authors.forename: Luana
authors.initials: L
authors.lastname: Heimfarth
authors.forename: Bárbara Ortiz
authors.initials: BO
authors.lastname: de Lima
authors.forename: Marina C
authors.initials: MC
authors.lastname: Leite
authors.forename: Maria Cristina
authors.initials: MC
authors.lastname: Guerra
authors.forename: Carlos Alberto
authors.initials: CA
authors.lastname: Gonçalves
authors.forename: Regina
authors.initials: R
authors.lastname: Pessoa-Pureur
mesh_terms: D000818:Animals; D000893:Anti-Inflammatory Agents / Q000494:pharmacology*; D015153:Blotting, Western; D045744:Cell Line, Tumor; D002470:Cell Survival; D004591:Electrophoresis, Polyacrylamide Gel; D000431:Ethanol / Q000494:pharmacology*; D005911:Gliosis / Q000139:chemically induced / Q000378:metabolism / Q000473:pathology; D007150:Immunohistochemistry; D007249:Inflammation / Q000139:chemically induced / Q000378:metabolism; D008070:Lipopolysaccharides / Q000633:toxicity; D020935:MAP Kinase Signaling System / Q000187:drug effects*; D009457:Neuroglia / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D018384:Oxidative Stress / Q000187:drug effects*; D051381:Rats; D022502:Stress Fibers / Q000187:drug effects / Q000378:metabolism; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism
chemical_list: D000893:Anti-Inflammatory Agents; D008070:Lipopolysaccharides; D014409:Tumor Necrosis Factor-alpha; D000431:Ethanol