Aqueous Extract of Oldenlandia diffusa Suppresses LPS-Induced iNOS, COX-2 and TNF-α Expression in RAW 264.7 Cells via the NF-kB Activity

Purpose: To elucidate the anti-inflammatory mechanisms of aqueous extract of Oldenlandia diffusa (AEOD) in LPS-stimulated RAW 264.7 cells. Methods: We evaluated the mRNA and protein expression of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2) and tumor necrosis factor (TNF)-α using RT-PCR and Western blot analyses. Expressions of IkBα, phospho-IkBα and p65 were analyzed by Western blot analysis. The level of nitric oxide (NO) production was analyzed using Griess reaction. The release of prostaglandin E2 (PGE2) and tumor necrosis factor (TNF)-α was determined using sandwich ELISA. Results: AEOD significantly suppressed nitric oxide (NO) production in LPS-stimulated RAW 264.7 cells without direct cytotoxicity. AEOD decreased the production of prostaglandin E2 (PGE2) and TNF-α in LPS-stimulated RAW 264.7 cells. LPS-induced mRNA and protein expression of iNOS, COX-2 and TNF-α were attenuated by treatment with AEOD. These data imply that AEOD tightly regulates the expression of these inflammatory mediators at the transcriptional level. Therefore, we determined the effects of AEOD on nuclear factor-kB (NF-kB) activity, which has been considered to be a potential transcriptional factor for regulating the expression of iNOS, COX-2 and TNF-α. As expected, AEOD suppressed the LPS-induced degradation and phosphorylation of IkBα and sustained the expression of p65 in the cytosol. Furthermore, AEOD substantially inhibited the LPS-induced DNA binding activity of NF-kB. These data show that AEOD may control NO, PGE2 and TNF-α production via the suppression of NF-kB activity. Conclusion: Our results suggest that AEOD has a high potential activity for regulating LPS-induced inflammation.