Ethyl ferulate contributes to the inhibition of the inflammatory responses in murine RAW 264.7 macrophage cells and acute lung injury in mice

Background: Ethyl ferulate (EF) is a derivative of ferulic acid (FA), which is a monomeric component purified from the traditional medicinal herb Ferula, but its effects have not been clear yet. The purpose of this study was to evaluate whether EF can reduce inflammation levels in macrophages by regulating the Nrf2-HO-1 and NF-кB pathway.

Methods: The LPS-induced raw 264.7 macrophage cells model was used to determine the anti-inflammatory and anti-oxidative stress effects of EF. The levels of IL-1β, IL-6, TNF-α and PGE2 were analyzed by ELISA. The mRNA and protein of COX-2, iNOS, TNF-α, IL-6, HO-1 and Nrf2 were identified by RT-PCR analysis and western blotting. Intracellular ROS levels were assessed with DCFH oxidation staining. The expressions of NF-кB p-p65 and Nrf2 were analyzed by immunofluorescence assay. The inhibitory effect of Nrf2 inhibitor ML385 (2μM) on mediatation of antioxidant activity by raw 264.7 macrophage cells was evaluated. The effect of EF was confirmed in acute lung injury mice model.

Results: In our research, EF reduced the expression of iNOS, COX2 and the production of PGE2. EF could inhibit the production of pro-inflammatory cytokines (IL-1β, IL-6 and TNF-α) in lipopolysaccharide (LPS) stimulated macrophages and decreased expression of IL-6 and TNF-α in LPS stimulated macrophages. Furthermore, EF inhibited NF-кB p65 from transporting to the nucleus, decreased the expression of p-IкBα, significantly decreased the level of intracellular Reactive Oxygen Species (ROS) and activated Nrf2/HO-1 pathways. EF could attenuate the degree of leukocyte infiltration, reduced MPO activity, mRNA levels and secretion of TNF-α and IL-6 in vivo. EF exhibited potent protective effects against LPS-induced acute lung injury in mice.

Conclusions: Collectively, our data showed that EF relieved LPS-induced inflammatory responses by inhibiting NF-κB pathway and activating Nrf2/HO-1 pathway, known to be involved in the regulation of inflammatory responses by Nrf2.