Immunomodulatory effects of palm oil-derived delta-tocotrienol on microglia responses

Microglia are the main immunocompetent cells of the central nervous system (CNS). The chronic inflammatory responses of microglia can lead to significant neuronal damage. Thus, strategies to control microglia activation provide an alternative therapeutic approach for neuroinflammatory diseases. Vitamin E, namely the tocopherols and tocotrienols, are potent antioxidant and anti-inflammatory compounds. Importantly, tocotrienols confer higher protection against glutamate- induced neurotoxicity compared to tocopherols. BV2 microglia pre- treated with delta-tocotrienol (-tocotrienol) showed highest reduction in NO production compared to treatment with alpha- (-) and gamma- (-) isomers. This current study explores the modulatory function of - tocotrienol on microglial inflammatory responses, mainly by using primary mouse microglia cultures. Primary mouse microglia cells were treated with -tocotrienol at various concentrations (10, 12.5, 15, 17.5 and 20 g/mL) 24 hrs prior to co- stimulation with lipopolysaccharide (LPS)/interferon-gamma (IFN-) (0.5 g/mL; 50 ng/mL). The two highest concentrations of -tocotrienol (17.5 and 20 g/mL) were found to reduce NO levels most by 50% and 58% (p<.05), respectively ence, these doses were chosen for downstream experiments. The effects of -tocotrienol on the inflammatory phenotype of primary microglia including the production of inflammatory cytokines, eicosanoids as well as expression of the CD40 co-stimulatory molecule, were determined. Primary mouse microglia cells were treated with -tocotrienol at various concentrations (10, 12.5, 15, 17.5 and 20 g/mL) 24 hrs prior to co- stimulation with lipopolysaccharide (LPS)/interferon-gamma (IFN-) (0.5 g/mL; 50 ng/mL). The two highest concentrations of -tocotrienol (17.5 and 20 g/mL) were found to reduce NO levels most by 50% and 58% (p<.05), respectively ence, these doses were chosen for downstream experiments. The effects of -tocotrienol on the inflammatory phenotype of primary microglia including the production of inflammatory cytokines, eicosanoids as well as expression of the CD40 co-stimulatory molecule, were determined. It was found that the NO reduction by -tocotrienol was not attributed to NO scavenging, but to the down-regulation of inducible nitric oxide synthase (iNOS) mRNA (-3.7 fold; p<.05) in primary microglia. In BV2 microglia, -tocotrienol reduced iNOS protein expression by 3-fold (p<.05). -tocotrienol also exhibited prominent inhibitory effects on primary microglial production of interleukin-1 (IL-1) by 77.6%, which was coupled with down-regulation of IL-1 mRNA expression by 6.6-fold (p<.05). However, primary microglial expression of both tumour necrosis factor-alpha (TNF-) and IL-6 was not affected by -tocotrienol, be it at the mRNA or protein level. Production of the anti-inflammatory cytokine (IL-10) was not detectable. Production of an important eicosanoid, prostaglandin E2 (PGE2), was significantly reduced by -tocotrienol in activated microglia by 78.2%. This was accompanied by a down- regulation of cyclooxygenase (COX)-2 mRNA expression by 3.3-fold (p<.05), but not the expression of COX-1. Despite the undetectable levels of leukotriene B4 (LTB4) in primary microglia, -tocotrienol significantly down-regulated the mRNA expression of 5-lipoxygenase (5-LOX) in activated primary microglia cells by 3.6-fold (p<.05). Unexpectedly, - tocotrienol increased the CD40 microglial activation marker in primary microglia, indicating the heterogeneous effects of -tocotrienol on microglial responses. Finally, HPLC analysis revealed that -tocotrienol uptake by BV2 microglia was detected as early as five mins after administration, and relatively low amounts of intracellular -tocotrienol (14% of the administered -tocotrienol) were required to exert the observed beneficial effects on microglia observed. These findings indicate that fast incorporation and retention of -tocotrienol in microglia are most likely to be main factors for all modulatory effects of -tocotrienol observed in this study. Taken together, the findings from this project revealed the ability of -tocotrienol in modulating microglial inflammatory responses via reduction of the pro-inflammatory mediators NO, IL-1 and PGE2. The modulatory effects of -tocotrienol did not include reduction of the pro-inflammatory cytokines, TNF- and IL-6 nor increased the anti- inflammatory cytokine IL-10. This indicates -tocotrienol is a potentially therapeutic substance for neuroinflammatory diseases.

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