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

Purpose: To determine whether the methanol extract of Myelophycus caespitosus

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(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-κB (NF-κB) 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-κB and nuclear translocation of NF-κB subunits p65 and p50 via the inhibition of IκBa phosphorylation and degradation. MEMC promoted dephosphorylation of Akt that subsequently suppressed the DNA-binding activity of NF-κB 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-κB activity.

Keywords
Myelophycus caespitosus, Nitric oxide, Prostaglandin E2, Nuclear factor-κB