Methanol Extract of Myelophycus caespitosus Inhibits the Inflammatory Response in Lipopolysaccharidestimulated BV2 Microglial Cells by Downregulating NF-kB via Inhibition of the Akt Signaling Pathway

Abstract

Purpose: To determine whether the methanol extract of Myelophycus caespitosus (MEMC) downregulates the expression of pro-inflammatory mediators in lipopolysaccharide (LPS)-stimulated BV2 microglial cells.
Methods: Reverse transcription-polymerase chain reaction (RT-PCR) together with Western blot analysis was used to evaluate the expression of pro-inflammatory mediators such as nitric oxide (NO) and prostaglandin E2
(PGE2) as well as their regulatory genes such as inducible NO synthase (iNOS) and cyclooxygenase-2 (COX-2), in LPS-stimulated BV2 microglial cells. The level of NO production was analyzed using Griess reaction.
The release of PGE2 was determined using sandwich enzyme-linked immunosorbent assay. The DNA-binding activity of nuclear factor-êB (NF-êB) was measured by electrophoretic mobility shift assay.
Results: MEMC inhibited LPS-induced pro-inflammatory mediators, NO and PGE2, as well as their respective genes, iNOS and COX-2, at both protein and mRNA levels, without any significant cytotoxicity. Treatment with
MEMC also substantially reduced the LPS-induced DNA-binding activity of NF-êB and nuclear translocation of NF-êB subunits p65 and p50 via the inhibition of IêBá phosphorylation and degradation. MEMC promoted
dephosphorylation of Akt that subsequently suppressed the DNA-binding activity of NF-êB in LPS-stimulated BV2 microglial cells.
Conclusion: Collectively, these data suggest that MEMC attenuates expression of pro-inflammatory mediators such as NO and PGE2 by suppression of their regulatory genes through the inhibition of Aktmediated NF-êB activity.

Keywords: Myelophycus caespitosus, Nitric oxide, Prostaglandin E2, Nuclear factor-êB.