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.
The production of nitric oxide (NO) and prostaglandin E2
) 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-κB (NF-κB) was detected by
electrophoretic mobility shift assay (EMSA).
MEPA inhibited the expression of LPS-induced pro-inflammatory mediators, NO and PGE2
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-κB, which is known as a main transcription factor for the regulation of proinflammatory
genes, as well as the nuclear translocation of its subunit p65 and p50, by degrading IκBα.
MEPA increased Akt dephosphorylation which leads to suppression of the DNA-binding activity of NF-
κB 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 proinflammatory
Our results indicate that MEPA down-regulates pro-inflammatory mediators such as NO
by suppressing Akt-dependent NF-κB activity as well as phosphorylation of ERK and JNK in
LPS-stimulated BV2 microglial cells.