Anti-inflammatory activity of Leonotis nepetifolia leaf essential oil in LPS-stimulated RAW 264.7 cells and its molecular mechanism of action

The anti-inflammatory activity and the underlying molecular mechanisms of action of the leaf essential oil of Leonotis nepetifolia (LNLEO), traditionally used in folk and ethnomedicine against inflammatory diseases, are evaluated in this study through an in vitro LPS-induced RAW 264.7 macrophage model. GC-MS analysis revealed the presence of 44 constituents in LNLEO, germacrene D, β-caryophyllene, α-humulene, and phytol being the predominant terpenoids. Based on the MTT assay, the non-toxic doses of 12.5 and 50 µg/mL of LNLEO were used for in vitro studies. Incubation of LNLEO significantly reduced the expression of proinflammatory cytokines (TNF-α, IL-1β, IL-6), inflammatory enzymes (iNOS, COX-2), and intracellular ROS, while enhancing the levels of endogenous antioxidant enzymes (SOD, GSH, GPx, and CAT). LNLEO alleviated oxidative stress by attenuating Keap1 and upregulating Nrf2, HO-1 expression in a concentration-dependent manner. The RT-qPCR analysis revealed that LNLEO treatment inhibited TLR4 and MyD88 expression. Confocal microscopy and RT-qPCR analysis showed a considerable reduction in both nuclear localization and transcriptional activity of NF-κB p65 in LPS-activated macrophages. ELISA studies also indicated that LNLEO suppressed inflammation by limiting phosphorylation of MAPKs that are implicated in the activation of NF-κB and other transcription factors. Based on these results, it can be concluded that LNLEO has the potential to modulate several key signaling pathways, which are intricately involved in the inflammatory response and oxidative stress, and they can be effectively targeted for therapeutic benefits. However, in vivo studies, as well as clinical trials, will be helpful in validating the results for future applications.