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. 2016 Aug 18;21(8):1083.
doi: 10.3390/molecules21081083.

Rosmarinic Acid Methyl Ester Inhibits LPS-Induced NO Production via Suppression of MyD88- Dependent and -Independent Pathways and Induction of HO-1 in RAW 264.7 Cells

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Rosmarinic Acid Methyl Ester Inhibits LPS-Induced NO Production via Suppression of MyD88- Dependent and -Independent Pathways and Induction of HO-1 in RAW 264.7 Cells

Yangkang So et al. Molecules. .

Abstract

In this study, we investigated the anti-inflammatory effect of rosmarinic acid methyl ester (RAME) isolated from a mutant cultivar of Perilla frutescens (L.) Britton. We found that RAME inhibits lipopolysaccharide (LPS)-induced nitric oxide (NO) production, with an IC50 of 14.25 µM, in RAW 264.7 cells. RAME inhibited the LPS-induced expression of pro-inflammatory cytokines including interleukin (IL)-1β, IL-6, IL-10, monocyte chemoattractant protein-1, interferon-β, and inducible nitric oxide synthase (iNOS). Moreover, RAME suppressed the activation of nuclear factor kappa B. These results suggest that the downregulation of iNOS expression by RAME was due to myeloid differentiation primary response gene 88 (MyD88)-dependent and -independent pathways. Furthermore, RAME induced the expression of heme oxygenase-1 (HO-1) through activation of nuclear factor-erythroid 2-related factor 2. Treatment with tin protoporphyrin, an inhibitor of HO-1, reversed the RAME-induced suppression of NO production. Taken together, RAME isolated from P. frutescens inhibited NO production in LPS-treated RAW 264.7 cells through simultaneous induction of HO-1 and inhibition of MyD88-dependent and -independent pathways.

Keywords: MyD88-dependent and -independent pathways; Perilla frutescens; anti-inflammation; heme oxygenase-1; rosmarinic acid methyl ester.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Chemical structure of RAME and its effects on cell viability. (a) Chemical structure of RAME; (b) Purity of RAME from a mutant cultivar of P. frutescens measured by HPLC analysis; (c) Cell viability. # p < 0.05 vs. control.
Figure 1
Figure 1
Chemical structure of RAME and its effects on cell viability. (a) Chemical structure of RAME; (b) Purity of RAME from a mutant cultivar of P. frutescens measured by HPLC analysis; (c) Cell viability. # p < 0.05 vs. control.
Figure 2
Figure 2
Effects of RAME on NO production and iNOS expression levels in RAW 264.7 cells. RAW 264.7 cells were treated with various concentrations of RAME for 2 h prior to incubation with LPS (1 μg/mL) for 18 h. (a) Inhibition of NO production; (b) mRNA expression levels of iNOS measured by real-time PCR; (c) Protein expression levels of iNOS measured by Western blot. # p < 0.05 vs. control group, * p < 0.05 vs. LPS-treated.
Figure 2
Figure 2
Effects of RAME on NO production and iNOS expression levels in RAW 264.7 cells. RAW 264.7 cells were treated with various concentrations of RAME for 2 h prior to incubation with LPS (1 μg/mL) for 18 h. (a) Inhibition of NO production; (b) mRNA expression levels of iNOS measured by real-time PCR; (c) Protein expression levels of iNOS measured by Western blot. # p < 0.05 vs. control group, * p < 0.05 vs. LPS-treated.
Figure 3
Figure 3
Effects of RAME on the NF-κB pathway. # p < 0.05 vs. control group , * p < 0.05 vs. LPS-treated group.
Figure 4
Figure 4
Effects of RAME on IL-1β, IL-10, and IL-6 mRNA expression levels in LPS-treated RAW 264.7 cells. The expression level of pro-inflammatory cytokine genes were measured by real-time PCR. RAW 264.7 cells were treated RAME (12.5, 25, and 50 µM) with LPS (1 µg/mL) and incubated for an additional 18 h. mRNA expression level of genes was normalized with β-actin. (a) IL-1β mRNA expression level; (b) IL-6 mRNA expression level; (c) IL-10 RNA expression level. The results are presented as the means ± SDs of three replicates of one representative experiment. # p < 0.05 vs. control group , * p < 0.05 vs. LPS-treated group.
Figure 5
Figure 5
Effects of RAME on the IFN-β pathway. (a) RAW 264.7 cells were treated with RAME for 2 h prior to incubation with LPS (1 μg/mL) for 4 h. IFN-β levels were detected in the culture supernatant using an ELISA kit. # p < 0.05 vs. control group, * p < 0.05 vs. LPS-treated group; (b) RAW 264.7 cells were treated with various concentrations of RAME for 2 h prior to incubation with IFN-β (100 units/mL) for 8 h. MCP-1 levels were detected in the culture supernatant using an ELISA kit. # p < 0.05 vs. control group , * p < 0.05 vs. IFN-β-treated group.
Figure 6
Figure 6
Effects of RAME on HO-1 expression. (a) RAW 264.7 cells were incubated with various concentrations of RAME for 6 h. The HO-1 mRNA expression level was determined by quantitative real-time PCR. HO-1 protein expression levels were measured by western blot analysis. # p < 0.05 vs. control group; (b) RAW 264.7 cells were treated with 25 μM RAME for 0, 2, 4, 6 and 8 h. Total RNA was isolated and used to measure HO-1 mRNA expression levels by quantitative real-time PCR. # p < 0.05 vs. control group; (c) Cytosolic and nuclear protein fractions were extracted after treatment with RAME (50 μM) for 0, 1, 3, 6 and 12 h. Protein levels were normalized to those of β-tubulin and lamin B; (d) RAW 264.7 cells were treated with either RAME or RAME plus SnPP, an HO-1 inhibitor, for 2 h prior to incubation with LPS (1 μg/mL) for 18 h. # p < 0.05 vs. control group, * p < 0.05 vs. LPS-treated group and ** p < 0.05 vs. LPS + RAME group.
Figure 7
Figure 7
Possible model for the inhibition of iNOS or NO production by RAME in LPS-treated RAW 264.7 cells.

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References

    1. Kim K.N., Heo S.J., Yoon W.J., Kang S.M., Ahn G., Yi T.H., Jeon Y.J. Fucoxanthin inhibits the inflammatory response by suppressing the activation of NF-κB and MAPKs in lipopolysaccharide-induced RAW 264.7 macrophages. Eur. J. Pharmacol. 2010;649:369–375. - PubMed
    1. Guzik T.J., Korbut R., Adamek-Guzik T. Nitric oxide and superoxide in inflammation and immune regulation. J. Physiol. Pharmacol. 2003;54:469–487. - PubMed
    1. Nathan C. Points of control in inflammation. Nature. 2002;420:846–852. - PubMed
    1. Walsh N.C., Crotti T.N., Goldring S.R., Gravallese E.M. Rheumatic diseases: The effects of inflammation on bone. Immunol. Rev. 2005;208:228–251. - PubMed
    1. Lee H.J., Hyun E.A., Yoon W.J., Kim B.H., Rhee M.H., Kang H.K., Cho J.Y., Yoo E.S. In vitro anti-inflammatory and anti-oxidative effects of Cinnamomum camphora extracts. J. Ethnopharmacol. 2006;103:208–216. - PubMed

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