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, 26 (6), 935-40

Chamomile: An Anti-Inflammatory Agent Inhibits Inducible Nitric Oxide Synthase Expression by Blocking RelA/p65 Activity

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Chamomile: An Anti-Inflammatory Agent Inhibits Inducible Nitric Oxide Synthase Expression by Blocking RelA/p65 Activity

Natarajan Bhaskaran et al. Int J Mol Med.

Abstract

Chamomile has long been used in traditional medicine for the treatment of inflammation-related disorders. In this study we investigated the inhibitory effects of chamomile on nitric oxide (NO) production and inducible nitric oxide synthase (iNOS) expression, and explored its potential anti-inflammatory mechanisms using RAW 264.7 macrophages. Chamomile treatment inhibited LPS-induced NO production and significantly blocked IL-1β, IL-6 and TNFα-induced NO levels in RAW 264.7 macrophages. Chamomile caused reduction in LPS-induced iNOS mRNA and protein expression. In RAW 264.7 macrophages, LPS-induced DNA binding activity of RelA/p65 was significantly inhibited by chamomile, an effect that was mediated through the inhibition of IKKβ, the upstream kinase regulating NF-κB/Rel activity, and degradation of inhibitory factor-κB. These results demonstrate that chamomile inhibits NO production and iNOS gene expression by inhibiting RelA/p65 activation and supports the utilization of chamomile as an effective anti-inflammatory agent.

Figures

Figure 1
Figure 1
(A) HPLC chromatogram of aqueous chamomile extract demonstrates apigenin 7-O-glucoside as major constituent (B) standardization of chamomile extract with apigenin 7-O-glucoside concentration (C) effect of chamomile on endogenous nitrite levels in culture medium of RAW 264.7 macrophages. Bars represent mean ± SEM of at least 3 independent experiments each performed in triplicate **P<0.05 (ANOVA) and (D) effect of chamomile on cell viability as determined by MTT assay and lower panel shows photograph of macrophages (a–d) after treatment with 0, 10, 20 and 40 μg/mL chamomile extract. Details are described in ‘Materials and methods’ section.
Figure 2
Figure 2
Effect of chamomile on NO production in culture medium of RAW 264.7 cells (A) RAW 264.7 cells activated with 1μg/mL LPS (B) 1μg/mL IL-6, (C) 1μg/mL IL-1β , and (D) 1μg/mL TNFα in the absence and presence of chamomile (10 and 20 μg/mL) for 16 h. Bars represent mean ± SEM of at least 3 independent experiment each performed in triplicate, **P<0.001 (ANOVA), compared to LPS-challenge group. Details are described in ‘Materials and methods’ section.
Figure 3
Figure 3
Effect of chamomile on NO production in the culture medium of RAW 264.7 macrophages (A) time-dependent effect using 1 μg/mL LPS in the absence and presence of 10 μg/mL chamomile for indicated times. (B) dose-dependent effect using 1μg/mL LPS for 16 h in the presence and absence of chamomile at indicated doses. L-NMMA (1 mM), a non-selective inhibitor of all NOS isoforms was used as positive control. Bars represent mean ± SEM of at least 3 independent experiments each performed in triplicate, **P<0.001 (ANOVA) compared to LPS challenge group. Details are described in ‘Materials and methods’ section.
Figure 4
Figure 4
Effect of chamomile on iNOS expression in RAW 264.7 macrophages. (A) Western blot for iNOS protein expression, and (B) mRNA expression of iNOS in RAW 264.7 macrophages stimulated with 1 μg/mL LPS and LPS and chamomile as indicated. Graph represents protein and mRNA levels of iNOS corrected to the corresponding controls. L-NMMA, a non-selective inhibitor of all NOS isoforms was used as positive control. Details are described in ‘Materials and methods’ section.
Figure 5
Figure 5
Effect of chamomile on NF-κ B activity. (A) Western blot analysis for protein expression of p- IKKα /β , Iκ Bα and its phosphorylation, and RelA/p65 in the cytosolic fraction of RAW 264.7 macrophages stimulated with 1μg/mL LPS and LPS and chamomile for indicated doses, (B) RelA/p65 expression in the nuclear fraction, (C) EMSA assay. EMSA was performed to determine the effect of chamomile on the nuclear translocation of NF-κ B dimers and their binding to DNA. Controls: #1 Biotin-EBNA control DNA, #2 Biotin-EBNA control DNA+EBNA extract, #3 Biotin-EBNA control DNA + EBNA extract + 20-fold molar excess of unlabeled EBNA DNA. L-NMMA, a non-selective inhibitor of all NOS isoforms was used as positive control. The details are described in ‘Materials and Methods’ section.

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