ABSTRACT
Introduction@#Valproic acid (VPA) has been used in the treatment of seizures and bipolar disorders. In the present study, we examined how VPA affected PI3K-Akt pathway in response to LPS by using mouse RAW 264.7 macrophage cells.@*Material and methods@#Mouse RAW 264.7 macrophage-like cells cultured and the cell viability checked by MTT and TUNEL assay. In addition, protein expression and protein interaction were detected by immune blotting and immune precipitation, respectively. TLR4 expression on cell surface studied by FACS analysis.@*Results@#The MTT and TUNEL assays demonstrated no significant difference between VPA at 2 mM treated and untreated control cells. VPA attenuated LPS-induced phosphorylation of phosphatidylinositol 3-kinase (PI3K) and Akt, but not nuclear factor (NF)-κB and mitogen activated protein kinases (MAPKs). There was no significant difference in the TLR4 expression on the cell surface between cells treated with or without VPA. VPA inhibited LPS-induced PI3K/Akt signal transduction in a dose dependent manner.@*Conclusion@#VPA at 2mM exhibits nontoxic effect in the RAW 264.7 cells. VPA down regulates LPS-induced phosphorylation of Akt via inhibition of PI3K activation.
ABSTRACT
Background@#The effect of lipopolysaccharide (LPS) on valproic acid (VPA)-induced cell death was examined by using mouse RAW 264.7 macrophage cells. @*Materials and methods, results@#LPS inhibited the activation of caspase 3 and poly (ADP-ribose) polymerase (PARP) and prevented VPA-induced apoptosis. LPS inhibited VPA-induced p53 activation and pifithrin-α as a p53 inhibitor as well as LPS prevented VPA-induced apoptosis. LPS abolished the increase of Bax/Bcl-2 ratio, which is a critical indicator of p53-mediated mitochondrial damage, in response to VPA. The nuclear factor (NF)-κB inhibitors, Bay 11-7082 and parthenolide, abolished the preventive action of LPS on VPA-induced apoptosis. A series of toll-like receptor (TLR) ligands, Pam3CSK4, poly I:C, and CpG DNA as well as LPS prevented VPA-induced apoptosis. @*Conclusion@#Taken together, LPS was suggested to prevent VPA-induced apoptosis via activation of anti-apoptotic NF-κB and inhibition of pro-apoptotic p53 activation.
ABSTRACT
@#BACKGROUND. Prostaglandin E2 (PGE2) is the most abundant prostanoid and a very potent lipid mediator, and is produced predominantly from arachidonic acid by it’s tightly regulated cyclooxygenases (COXs) and prostaglandin E synthases. PGE2 is involved in regulating many different fundamental biological functions, including immune responses. Recently, we have demonstrated that bacterial LPS induces NO production in auditory cells via an inducible NO synthase expression. The LPS-induced production of an excessive NO amount is suggested to cause injury of auditory cells, followed by ototoxicity. Auditory cells injured by such an inflammatory response must be accompanied by tissue repair and remodeling. In order to clarify the production of PGE2 in auditory cells for regulation of inflammatory response or tissue repair AIM: We aimed to examine an effect of LPS on the production of prostaglandin E2 in auditory cells. MATERIALS AND METHODS: The murine auditory cell line HEI-OC1 was established from long-term cultures of immortomouse cochlea and used as conditionally-immortalized auditory cells. HEI-OC1 cells were stimulated with or without LPS. The concentration of PGE2, TNF-α, IL-1β in the culture supernatant was determined with ELISA. COX-2 protein expression and mRNA were measured by immunoblotting and reverse transcription PCR, respectively. The bands were quantified by densitometric analysis using ImageJ software. Statistical analysis was performed using Student’s t-test and P values < 0.05 were considered significant. All experiments were performed independently at least three times. Data represent the mean value of triplicates SD. RESULT: HEI-OC1 auditory cells constitutively produce a small amount of PGE2. LPS augmented the PGE2 production via enhanced cyclooxygenase 2 (COX2) expression. LPS-induced augmentation of COX2 expression was dependent on up-regulation of COX2 mRNA expression. LPS induced the production of TNF-a, but not IL-1b An anti-TNF-α neutralizing Ab significantly inhibited PGE2 production and COX2 mRNA expression in response to LPS. LPS-induced PGE2 production was prevented by a series of pharmacological signaling inhibitors to NF- κB and MAPKs. Pam3CSK4 as a TLR2 ligand, as well as LPS as a TLR4 ligand, augmented the PGE2 production. However, poly I:C as a TLR3 ligand, imiquimod as a TLR7 ligand and CpG DNA as a TLR9 ligand did not augment it. HEI-OC1 cells expressed TLR2, TLR4 and TLR9, but not TLR3 or TLR7. CONCLUSION: The auditory cells produce PGE2 in response to LPS via COX2 expression. The PGE2 production may be involved in tissue repair and remodeling in the organ of Corti. Auditory cells might be important effector cells in host response to infection and inflammation in the organ of Corti and cochleae.