Inhibition of Nitric Oxide and Prostaglandin E 2 Expression by Methanol Extract of Polyopes affinis in Lipopolysaccharide-stimulated BV2 Microglial Cells through Suppression of Akt-dependent NF-kB Activity and MAPK Pathway

Purpose: To determine whether the methanol extract of Polyopes affinis (MEPA) down-regulates the expression of pro-inflammatory mediators in lipopolysaccharide (LPS)-stimulated BV2 microglial cells. Methods: The production of nitric oxide (NO) and prostaglandin E-2 (PGE(2)) was measured by the Griess reagents and enzyme-linked immunosorbent assay (ELISA), respectively. Expression levels of mRNA and protein in LPS-stimulated BV2 microglial cells were assessed by reverse transcription-polymerase (RT-PCR) and Western blot analysis. Activation of nuclear factor-kappa B (NF-kappa B) was detected by electrophoretic mobility shift assay (EMSA). Results: MEPA inhibited the expression of LPS-induced pro-inflammatory mediators, NO and PGE(2), as well as their respective genes, iNOS and COX-2, at both protein and mRNA levels, without any accompanying cytotoxicity. Moreover, treatment with MEPA significantly suppressed the LPS-induced DNA-binding activity of NF-kappa B, which is known as a main transcription factor for the regulation of pro-inflammatory genes, as well as the nuclear translocation of its subunit p65 and p50, by degrading I kappa B alpha. MEPA increased Akt dephosphorylation which leads to suppression of the DNA-binding activity of NF-kappa B in LPS-stimulated BV2 microglial cells and suppressed phosphorylation of ERK and JNK, which are involved in the mitogen-activated protein kinase (MAPK) signaling pathway for regulating pro-inflammatory genes. Conclusion: Our results indicate that MEPA down-regulates pro-inflammatory mediators such as NO and PGE(2) by suppressing Akt-dependent NF-kappa B activity as well as phosphorylation of ERK and JNK in LPS-stimulated BV2 microglial cells.