An in vitro inflammation model to study the Nrf2 and NF-κB crosstalk in presence of ferulic acid as modulator.

The aim of this study was to evaluate the crosstalk between Nrf2 and NF-κB signaling pathways and to explore the modulating activity actuated by ferulic acid. In the inflammation process, a key player is the nuclear factor-κB (NF-κB) transcription factor pathway. On the contrary, the activation of Nrf2 inhibits inflammation and impairs degenerative disease providing an interface between redox and anti-inflammatory responses. Recent studies have demonstrated that protein phosphorylation of IKK complex is a potential mechanism for the activation of both Nrf2 and NF-κB pathways. The IKK complex is as an integration point for signals emanating from these different pathways. In this study, we demonstrated that ferulic acid is able to regulate NF-κB and Nrf2 activities. Interestingly, we showed that ferulic acid mimics the potent IKK inhibitor such as BMS, down-regulating the NF-κB response, TAK 1 activation and turning off Nrf2 activities in LPS-stimulated RAW 264.7 cells. Immunoblot data showed that the release of Nrf2 from Keap1 is maintained at low levels also in the presence of LPS stimulus. Nrf2 controls the expression of many antioxidant and detoxification genes, by binding to antioxidant response elements (AREs) that are commonly found in the promoter region of antioxidant (and other) genes. We demonstrated that in the pARE-Luc transfected cells the pre-treatment with FA significantly reduced LPS-induced (p<0.01) and BMS-induced (p<0.01) transcriptional activities. Analysis of well-known Nrf2 transcriptional targets showed that mRNAs expression of Nrf2-dependent antioxidant and phase II enzymes such as dehydrogenase quinone1 (NQO1) and glutathione S-transferase A2 (GSTA2) were up-regulated by BMS and significantly increase more by association with LPS, but are down-regulated in the presence of FA. Interestingly, cells depleted of Keap1 showed increased response of the Nrf2 transcriptional activity also in the presence of FA, strongly suggesting its modulating role in Keap1-Nrf2 signaling pathway.