The anti-inflammatory effects of eugenol, methyl eugenol, estragole, and pulegone through the regulation of NF-κB and Nrf2 signaling pathways in LPS-stimulated RAW 264.7 cells.

Abstract

Eugenol, methyl eugenol, estragole, and pulegone are naturally occurring organic compounds obtained from essential oils of 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 RAW 264.7 cells. The production of nitric oxide (NO) was detected by Griess reaction and ELISA. The expression of iNOS and COX-2 was detected by Western blot. The expression of NF-κB and MAPK was also detected by Western blotting analysis. Furthermore, the intracellular ROS level was measured using fluorescent probe DCFH-DA. Then the expression of HO-1 and Nrf-2 was detected by western blotting analysis. Additionally, molecular docking studies were performed to evaluate binding affinities and binding sites of eugenol, methyl eugenol, estragole, and pulegone towards iNOS. Results revealed that those four compounds significantly inhibited NO production as well as iNOS and COX-2 expression. Meanwhile, western blot analysis showed that pulegone, estragole, methyl eugenol, and eugenol inhibited LPS-induced NF-κB and MAPK activation in RAW 264.7 cells. Furthermore, all those compounds suppressed LPS-induced intracellular ROS production in RAW 264.7 cells, while the expression of stress response gene HO-1 was up-regulated via the activation of transcription factor Nrf-2. In addition, molecular docking results showed that pulegone exhibits higher binding affinity in iNOS inhibitory complex compared to others tested compounds. Collectively, these findings provide that pulegone, estragole, methyl eugenol, and eugenol inhibit the LPS-induced expression of inflammatory mediators via the down-regulation of iNOS, COX-2, NF-κB, and MAPKs pathways as well as up-regulation of Nrf-2/HO-1 pathway indicating that all four compounds have a potential therapeutic and preventive application in various inflammatory diseases