α-Ecdysone suppresses inflammatory responses via the Nrf2 pathway in lipopolysaccharide-stimulated RAW 264.7 cells

The search for new biologically active compounds is a topic of current research because of their ubiquitous availability and low toxicity. Plants of the Silene genus contain secondary metabolites known as phytoecdysteroids that reportedly have various biological activities. α-Ecdysone is a phytoecdysteroid with biological activity that has not been thoroughly investigated to date. Therefore, we investigated the immunomodulatory and anti-inflammatory effects of α-ecdysone on LPS-treated RAW264.7macrophage cells and in a zebrafish model. To explore these activities, RAW264.7 cells were pretreated with α-ecdysone (0.1-10 μM) for 24 h and then with LPS to induce inflammation. We assayed membrane fluidity, lysosomal enzyme activity, and superoxide generation to determine the immunomodulatory activity. Using ELISA, we examined the levels of the pro-inflammatory cytokines prostaglandin (PGE₂) and interleukin-1β (IL-1β), as well as the protein expression of cyclooxygenase-2 (COX-2), tumor necrosis factor-alpha (TNF-α) and heme‑oxygenase-1 (HO-1) by immunoblotting. We also investigated the subcellular localization of the nuclear transcription factor (NF-κB) subunits and expression of the mitogen-activated protein kinase (MAPK) pathway. We found that α-ecdysone is a potent immunostimulator that enhances membrane fluidity and lysosomal enzyme activity and generates superoxide anions. Simultaneously, α-ecdysone inhibited nitric oxide levels and suppressed the levels of pro-inflammatory mediators and cytokines. Furthermore, α-ecdysone increased HO-1 and nuclear factor erythroid 2-related factor (Nrf2) production, mitigated NF-κB subunit proteins in the nucleus and decreased MAPKs and Akt activation. These results suggest that α-ecdysone is a good immunostimulator with anti-inflammatory effects that occur via inhibition of pro-inflammatory mediators and cytokines through stimulation of HO-1 and Nrf-2 production.