Aqueous Extract of &lt;i&gt;Oldenlandia diffusa&lt;/i&gt; Suppresses LPS-Induced iNOS, COX-2 and TNF-α Expression in RAW 264.7 Cells via the NF-κB Activity

Rgtp Jayasooriya,Yh Cho,Ws Ko,G-Y Kim,C-H Kang,I-W Choi

doi:10.4314/tjpr.v10i4.5

Abstract

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 IκBα, phospho-IκBα 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-κB (NF-κB) 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 IκBα and sustained the expression of p65 in the cytosol. Furthermore, AEOD substantially inhibited the LPS-induced DNA binding activity of NF-κB. These data show that AEOD may control NO, PGE2 and TNF-α production via the suppression of NF-κB activity. Conclusion: Our results suggest that AEOD has a high potential activity for regulating LPS-induced inflammation. Keywords: Oldenlandia diffusa , NO, iNOS, COX-2, PGE2, TNF-α, NF-κB

Highlights

Activated macrophages provide an immediate defense against extracellular foreign agents and are important in maintaining a balance in the immune system.They play a crucial role in inflammatory processes such as lipopolysaccharide (LPS)-induced shock, asthma, inflammatory bowel disease, and atherosclerosis [1]
We found that aqueous extract of Oldenlandia diffusa (AEOD) downregulates the expression of inducible nitric oxide synthase (iNOS), COX2 and tumor necrosis factor (TNF)-α mRNA and attenuates the corresponding mediators in vitro in RAW 264.7 macrophage cells
The expression levels of prostaglandin E2 (PGE2) and TNF-α were measured by an enzyme immunosorbent assay (ELISA) kit (R&D Systems, Minneapolis, USA) according to the manufacturer’s instructions

Summary

Introduction

Activated macrophages provide an immediate defense against extracellular foreign agents and are important in maintaining a balance in the immune system.They play a crucial role in inflammatory processes such as lipopolysaccharide (LPS)-induced shock, asthma, inflammatory bowel disease, and atherosclerosis [1]. We investigated the effects of AEOD on the expression of NO, PGE2, and TNF-α in LPS-stimulated RAW 264.7 macrophage cells. We found that AEOD downregulates the expression of iNOS, COX2 and TNF-α mRNA and attenuates the corresponding mediators in vitro in RAW 264.7 macrophage cells. The expression levels of PGE2 and TNF-α were measured by an enzyme immunosorbent assay (ELISA) kit (R&D Systems, Minneapolis, USA) according to the manufacturer’s instructions.

Results

Conclusion