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KMID : 0861020170320020077
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Korea Journal of Herbology
2017 Volume.32 No. 2 p.77 ~ p.85
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The study of anti-inflammatory effect of Hyeonto-dan extract in RAW 264.7 macrophage
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Kim Ma-Ryong
Kang Ok-Hwa
Kong Ryong
Seo Yun-Soo
Tian Zhou
Kim Sang-A
Kim Eun-Su
Sin Min-A
Lee Young-Seob
Kwon Dong-Yeul
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Abstract
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Objectives : This study aimed to investigate the unknown mechanisms behind the anti- inflammatory activity of Hyeonto-dan (HT) 70% ethanol extract on LPS-stimulated RAW 264.7 cells.
Methods : Cells were treated with Hyeonto-dan 1 h prior to addition of 200 ng/mL of LPS. Cell viability was measured by the MTS assay. Nitric oxide levels were determined by the Griess assay. PGE2 were measured using EIA kit. Proinflammatory cytokine production was measured by the enzyme-linked immunosorbent assay (ELISA). The expression of COX-2, iNOS, and MAPKs was investigated by Western blot, qRT-PCR. NF-κB/p65 localization and interaction of the TLR-4 receptor with LPS was examined by immunofluorescence assays.
Results : Hyeonto-dan had no cytotoxicity at the measured concentration. Hyeonto-dan inhibited NO production and pro-inflammatory cytokines such as IL-6, TNF-α, and PGE2 as well as the protein and mRNA expression of iNOS and COX-2. Moreover, Hyeonto-dan inhibited the interaction between LPS and TLR-4 in murine macrophages. It suppressed phosphorylation of extracellular signal-regulated kinase (ERK 1/2), c-jun N-terminal kinase (JNK 1/2) and p38. Finally, it inhibited translocation of NF-κB in response to competitive LPS.
Conclusions : Based on the results of this study, Hyeonto-dan inhibited the binding of TLR-4 receptor to LPS and inhibited the phosphorylation of extracellular signaling pathway MAPKs. These inhibitory effects are thought that the amount of NF-κB delivered to the nucleus was decreased and the inflammatory reaction was prevented by decreasing the production of LPS-induced PGE2,NO, IL-6 and TNF-α.
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KEYWORD
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Hyeonto-dan, Anti-inflammatory effect, RAW 264.7 cells, TLR-4.
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