Nat Prod Sci.  2018 Mar;24(1):28-35. 10.20307/nps.2018.24.1.28.

Pulegone Exhibits Anti-inflammatory Activities through the Regulation of NF-κB and Nrf-2 Signaling Pathways in LPS-stimulated RAW 264.7 cells

Affiliations
  • 1Department of Food and Life Science, Pukyong National University, Busan 608-737, Republic of Korea. choijs@pknu.ac.kr
  • 2Department of Pharmaceutical Engineering, Sangji University, Wonju 220-702, Republic of Korea.
  • 3Department of Food Science and Human Nutrition, Chonbuk National University, Jeonju 561-756, Republic of Korea. jungha@jbnu.ac.kr

Abstract

Pulegone is a naturally occurring organic compound obtained from essential oils from a variety of plants. The aim of this study was to investigate the anti-inflammatory effects through the inhibitory mechanism of inducible nitric oxide synthase (iNOS), cyclooxygenase (COX-2), nuclear factor kappa B (NF-κB), mitogen-activated protein kinases (MAPK) pathways and the activation of nuclear factor erythroid 2-related factor 2 (Nrf2)/ heme oxygenase (HO)-1 pathways in lipopolysaccharide (LPS)-stimulated RAW264.7 cells. Results revealed that pulegone significantly inhibited NO production as well as iNOS and COX-2 expressions. Meanwhile, western blot analysis showed that pulegone down-regulated LPS-induced NF-κB and MAPKs activation in RAW 264.7 cells. Furthermore, the selected compound suppressed LPS-induced intracellular ROS production in RAW 264.7 cells, while the expression of stress response gene, HO-1, and its transcriptional activator, Nrf-2 was upregulated upon pulegone treatment. Taking together, these findings provided that pulegone inhibited the LPS-induced expression of inflammatory mediators via the down-regulation iNOS, COX-2, NF-κB, and MAPKs signaling pathways as well as up-regulation of Nrf-2/HO-1 indicating that pulegone has a potential therapeutic and preventive application in various inflammatory diseases.

Keyword

Pulegone; Anti-inflammation; HO-1; Nrf-2; NF-κB

MeSH Terms

Blotting, Western
Down-Regulation
Heme Oxygenase (Decyclizing)
Mitogen-Activated Protein Kinases
NF-kappa B
Nitric Oxide Synthase Type II
Oils, Volatile
Prostaglandin-Endoperoxide Synthases
RAW 264.7 Cells*
Up-Regulation
Heme Oxygenase (Decyclizing)
Mitogen-Activated Protein Kinases
NF-kappa B
Nitric Oxide Synthase Type II
Oils, Volatile
Prostaglandin-Endoperoxide Synthases

Figure

  • Fig. 1 The structure of pulegone.

  • Fig. 2 Cell viability of pulegone measured by the MTT assay. Values represent the mean ± SD of three independent experiments.

  • Fig. 3 Inhibitory effects of pulegone on the production of NO in LPS-stimulated RAW264.7 cells. Cells were pretreated with different concentrations of pulegone for 2 h and then stimulated with LPS (1.0 µg/ml) for 24 h. The culture media were used to measure the amount of nitrite to determine NO production. Data are presented as mean ± SD of three independent experiments. ###p < 0.001 indicates significant difference from the control group. *p < 0.05, **p < 0.01, and ***p < 0.001 indicate significant difference from the LPS-treated group.

  • Fig. 4 Inhibitory effects of pulegone on the expression of iNOS and COX-2 in LPS-stimulated RAW264.7 cells. Cells were pretreated with the indicated concentration of pulegone for 2 h and stimulated with LPS (1.0 µg/ml) for 18 h. The expression of iNOS, COX-2, and β-actin was detected by Western blot using corresponding antibodies. The results presented are representative of three independent experiments. #p < 0.05 indicates a significant difference from the control group. *p < 0.05 indicates a significant difference from the LPS-treated group.

  • Fig. 5 Inhibitory effects of pulegone on the expression of NF-κB in LPS-stimulated RAW264.7 cells. Cells were pretreated with the indicated concentration of pulegone for 2 h and stimulated with LPS (1.0 µg/ml) for 18 h. The expression of NF-κB and β-actin was detected by Western blot using corresponding antibodies. The results presented are representative of three independent experiments. #p < 0.05 indicates a significant difference from the control group. *p < 0.05 indicates a significant difference from the LPS-treated group.

  • Fig. 6 Inhibitory effects of pulegone on the expression of MAPKs in LPS-stimulated RAW264.7 cells. Cells were pretreated with the indicated concentration of pulegone for 2 h and stimulated with LPS (1.0 µg/ml) for 18 h. The expression of MAPKs was detected by Western blot using corresponding antibodies. The results presented are representative of three independent experiments. #p < 0.05 indicates a significant difference from the control group. *p < 0.05 indicate a significant difference from the LPS-treated group.

  • Fig. 7 Inhibitory effects of pulegone on the production of ROS in LPS-stimulated RAW264.7 cells. Cells pretreated with different concentration of pulegone for 2 h and stimulated with LPS (1.0 µg/ml) for 24 h. ROS levels were measured by fluorescence analysis of DCFH-DA. Data are presented as mean ± SD of three independent experiments. ###p < 0.01 indicates a significant difference from the control group. *p < 0.05, **p < 0.01, and ***p < 0.001 indicate significant differences from the LPS-treated group.

  • Fig. 8 Inhibitory effects of pulegone on the expression of HO-1 (A) and Nrf2 (B) in LPS-stimulated RAW264.7 cells. Cells were pretreated with indicated concentration of pulegone for 2 h and stimulated with LPS (1.0 µg/ml) for 18 h. The expression of HO-1 and Nrf2 was detected by Western blot using corresponding antibodies. The results presented are representative of three independent experiments. #p < 0.05 indicates a significant difference from the control group. *p < 0.05 indicates a significant difference from the LPS-treated group.


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