Nutr Res Pract.  2014 Aug;8(4):352-359.

Anti-inflammatory effect of methanol extract from Erigeron Canadensis L. may be involved with upregulation of heme oxygenase-1 expression and suppression of NFkappaB and MAPKs activation in macrophages

• Sung J
• Sung M
• Kim Y
• Ham H
• Jeong HS
• Lee J

Abstract

BACKGROUND/OBJECTIVES
In this study, we determined the anti-inflammatory activities and the underlying molecular mechanisms of the methanol extract from Erigeron Canadensis L. (ECM) in LPS-stimulated RAW264.7 macrophage cells.
MATERIALS/METHODS
The potential anti-inflammatory properties of ECM were investigated by using RAW264.7 macrophages. We used western blot assays and real time quantitative polymerase chain reaction to detect protein and mRNA expression, respectively. Luciferase assays were performed to determine the transactivity of transcription factors.
RESULTS
ECM significantly inhibited inducible nitric oxide synthase (iNOS)-derived NO and cyclooxygenase-2 (COX-2) derived PGE2 production in LPS-stimulated RAW264.7 macrophages. These inhibitory effects of ECM were accompanied by decreases in LPS-induced nuclear translocations and transactivities of NFkappaB. Moreover, phosphorylation of mitogen-activated protein kinase (MAPKs) including extracellular signal-related kinase (ERK1/2), p38, and c-jun N-terminal kinase (JNK) was significantly suppressed by ECM in LPS-stimulated RAW264.7 macrophages. Further studies demonstrated that ECM by itself induced heme oxygenase-1 (HO-1) protein expression at the protein levels in dose-dependent manner. However, zinc protoporphyrin (ZnPP), a selective HO-1 inhibitor, abolished the ECM-induced suppression of NO production.
CONCLUSIONS
These results suggested that ECM-induced HO-1 expression was partly responsible for the resulting anti-inflammatory effects. These findings suggest that ECM exerts anti-inflammatory actions and help to elucidate the mechanisms underlying the potential therapeutic values of Erigeron Canadensis L.

Keyword

Erigeron Canadensis L.; anti-inflammation; nitric oxide; heme oxygenase-1; NFkappaB

MeSH Terms

Blotting, Western
Cyclooxygenase 2
Dinoprostone
Erigeron*
Heme Oxygenase-1*
JNK Mitogen-Activated Protein Kinases
Luciferases
Macrophages*
Methanol*
Nitric Oxide
Nitric Oxide Synthase Type II
Phosphorylation
Phosphotransferases
Polymerase Chain Reaction
Protein Kinases
RNA, Messenger
Transcription Factors
Up-Regulation*
Zinc
Cyclooxygenase 2
Dinoprostone
Heme Oxygenase-1
JNK Mitogen-Activated Protein Kinases
Luciferases
Methanol
Nitric Oxide
Nitric Oxide Synthase Type II
Phosphotransferases
Protein Kinases
RNA, Messenger
Transcription Factors
Zinc

Figure

• 

  Fig. 1 Inhibitory effects of ECM on nitrite and PGE2 production in LPS-stimulated RAW264.7 macrophages. Cells were co-incubated with indicated concentrations of ECM and LPS for 24 h. The treated culture media were used to measure the amounts of nitrite production (A) and PGE2 production (B). Cytotoxic effect of ECM was measured by MTT assay (A). Each value is expressed as a mean ± standard error (n = 3). #P < 0.05 compared to control, *P < 0.05 compared to LPS.

• 

  Fig. 2 Inhibitory effects of ECM on iNOS (A) and COX-2 (B) expression in LPS-stimulated RAW264.7 macrophages. Cells were co-incubated with indicated concentrations of ECM and LPS for 24 h. Each value is expressed as a mean ± standard error (n = 3). #P < 0.05 compared to control, *P < 0.05 compared to LPS.

• 

  Fig. 3 Inhibitory effects of ECM on NFκB translation (A) and promoter activity (B) in LPS-stimulated RAW264.7 macrophages. Cells were co-incubated with indicated concentrations of ECM and LPS for 1 h. Each value is expressed as a mean ± standard error (n = 3). #P < 0.05 compared to control, *P < 0.05 compared to LPS.

• 

  Fig. 4 Inhibitory effects of ECM on phosphorylation of MAPKs in LPS-stimulated RAW264.7 macrophages. Cells were co-incubated with indicated concentrations of ECM and LPS for 30 minutes and whole-cell lysates were analyzed by Westernblot analysis using various antibodies against activated MAPKs such as (A) ERK, (B) JNK, and (C) p38. Each value is expressed as a mean ± standard error (n = 3). #P < 0.05 compared to control, *P < 0.05 compared to LPS.

• 

  Fig. 5 Inductions by ECM of HO-1 protein expression in RAW264.7 cells. Cells were treated (A) without or (B) with LPS and indicated concentrations of ECM for 24 h. The Westernblot is a representative of three independent experiments. Each value is expressed as a mean ± standard error (n = 3). #P < 0.05 compared to control, *P < 0.05 compared to LPS.

• 

  Fig. 6 HO-1 mediates the suppression of LPS-induced NO production in RAW 264.7 cells. Cells were pre-incubated with 200 µg/ml ECM for 30 min before exposure 30 µM ZnPP and LPS (1 µg/ml) for 24 h. The treated culture media were used to measure the amount of nitrite production. Each value is expressed as a mean ± standard error (n = 3). #P < 0.05 compared to LPS, *P < 0.05 compared to LPS + ECM.

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