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. 2015;2015:474509.
doi: 10.1155/2015/474509. Epub 2015 Sep 6.

Anti-Inflammatory Effects of Cumin Essential Oil by Blocking JNK, ERK, and NF-κB Signaling Pathways in LPS-Stimulated RAW 264.7 Cells

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Free PMC article

Anti-Inflammatory Effects of Cumin Essential Oil by Blocking JNK, ERK, and NF-κB Signaling Pathways in LPS-Stimulated RAW 264.7 Cells

Juan Wei et al. Evid Based Complement Alternat Med. .
Free PMC article

Abstract

Cumin seeds (Cuminum cyminum L.) have been commonly used in food flavoring and perfumery. In this study, cumin essential oil (CuEO) extracted from seeds was employed to investigate the anti-inflammatory effects in lipopolysaccharide- (LPS-) stimulated RAW 264.7 cells and the underlying mechanisms. A total of 26 volatile constituents were identified in CuEO by GC-MS, and the most abundant constituent was cuminaldehyde (48.773%). Mitochondrial-respiration-dependent 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) reduction assay demonstrated that CuEO did not exhibit any cytotoxic effect at the employed concentrations (0.0005-0.01%). Real-time PCR tests showed that CuEO significantly inhibited the mRNA expressions of inducible nitric oxide synthase (iNOS), cyclooxygenase (COX-2), interleukin- (IL-) 1, and IL-6. Moreover, western blotting analysis revealed that CuEO blocked LPS-induced transcriptional activation of nuclear factor-kappa B (NF-κB) and inhibited the phosphorylation of extracellular signal regulated kinase (ERK) and c-Jun N-terminal kinase (JNK). These results suggested that CuEO exerted anti-inflammatory effects in LPS-stimulated RAW 264.7 cells via inhibition of NF-κB and mitogen-activated protein kinases ERK and JNK signaling; the chemical could be used as a source of anti-inflammatory agents as well as dietary complement for health promotion.

Figures

Figure 1
Figure 1
GC-MS total ion chromatogram of CuEO. (a) Retention time was from 0 to 35 min; (b) retention time was from 9 to 14 min. The compound labels in the chromatogram correspond to the Arabic numeral given in Table 1.
Figure 2
Figure 2
Cytotoxicity of CuEO at different concentrations on the viability of RAW 264.7 cells. Cells (1 × 104 cells/well) plated on 96-well plates were treated with various concentrations of CuEO at 37°C for 24 h. Cytotoxicity of CuEO was assessed by MTT assay. Values are expressed as mean ± SD, n = 6.
Figure 3
Figure 3
Effect of CuEO at different concentrations on the mRNA expression of LPS-induced iNOS (a) and COX-2 (b) of RAW 264.7 cells. The cells (1 × 105 cells/well in a six-well plate) were pretreated with CuEO (0.001% to 0.01%) for 1 h and then stimulated with LPS (1 ug/mL). After 24 h incubation, total RNA was isolated and the mRNA expression was determined by real-time RT-PCR. Data represent the mean ± SD of three different experiments. p < 0.05 and ∗∗ p < 0.01 versus LPS-treated alone.
Figure 4
Figure 4
Effect of CuEO at different concentrations on the mRNA expression of LPS-induced IL-1β (a) and IL-6 (b) of RAW 264.7 cells. The cells (1 × 105 cells/well in a six-well plate) were pretreated with CuEO (0.001% to 0.01%) for 1 h and then stimulated with LPS (1 ug/mL). After 24 h incubation, total RNA was isolated and the mRNA expression was determined by real-time RT-PCR. Data represent the mean ± SD of three different experiments. p < 0.05 and ∗∗ p < 0.01 versus LPS-treated alone.
Figure 5
Figure 5
Effect of CuEO on NF-κB p65 nuclear translocation in LPS-stimulated RAW 264.7 cells. (a) The bands of NF-κB p65 proteins tested by western blotting. (b) Data quantification of NF-κB p65. The bands of western blotting were quantified and expressed as the ratio of p65/β-actin intensity. The cells (3 × 106 cells per dish) were pretreated with CuEO (0.001% and 0.01%) for 1 h and then stimulated with LPS (1 ug/mL). After 30 min incubation, cells were collected and cytoplasmic and nuclear proteins were extracted. Actin was used as an internal control. Data represent the mean ± SD of three different experiments. p < 0.05 and ∗∗ p < 0.01 versus LPS-treated alone.
Figure 6
Figure 6
Effect of CuEO on the protein phosphorylation levels of MAPK pathways in LPS-stimulated RAW 264.7 cells. (a) The bands of MAPK proteins tested by western blotting. (b) Data quantification of p-ERK. (c) Data quantification of p-JNK. (d) Data quantification of p-p38. The bands of western blotting were quantified and expressed as the ratio of p-ERK/ERK, p-JNK/JNK, and p-p38/p38 intensity. The cells (3 × 106 cells per dish) were pretreated with CuEO (0.001% and 0.01%) for 1 h and then stimulated with LPS (1 ug/mL). After 30 min incubation, cells were collected and total cellular proteins were extracted. Data represent the mean ± SD of three different experiments. p < 0.05 and ∗∗ p < 0.01 versus LPS-treated alone.

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