The Anti-Inflammatory Activity of Ferulic Acid on NF-κBDepends on Keap1

an broad tram track, a central region and a series of six C-terminal Kelch repeats. Abstract Nrf2 and NF-κB are the two master transcriptional factors activated by different cellular signals turned to counteract the deleterious effects of pathological cellular processes linked to inflammation and oxidative stress.Several recent studies have highlighted a molecular connection between NF-κB and Keap1/Nrf2 pathways. The Keap1 protein seems to be the central player in this interaction, as it is involved in both IKKβ-NF-κB and Nrf2 modulation. Ferulic acid (FA) is a well-known antioxidant and anti-inflammatory agent, able to relieveinflammatory response viaNF-κB/IKK kinase, but until now the complete molecular network under its action is not all clear. Immunoblot data conducted on LPS-treated macrophage-like RAW264.7 cells transfected with si-Keap1 show that the FA anti-inflammatory and modulatory effects on NF-κB are abolished. Luciferase assay conducted in human A549 cell line, in which Keap1 protein is partially inactive, highlights that NF-κB activation induced by LPS is refractory to FA inhibition. This study proved that Keap1 and IKK together are important modulators of NF-κB and their activity is essential for FA anti-inflammatory effectiveness.


Introduction
The process of inflammation leads to the onset of a state of oxidative stress and a series of cascade reactions that are associated with chronic diseases such as cancer, autoimmune disorders, and metabolic diseases [1]. NF-κB is the master transcriptional factor mainly involved in the activation of inflammation and immunity, whose excessive upregulation is associated with many human diseases, including inflammatory disease and cancer [2,3].
External pro-inflammatory stimuli activate NF-κB that induces the expression and the release of a set of inflammatory mediators such as IL-6, a pleiotropic cytokine contributing to switch the acute to chronic phase of inflammation [4,5]. NF-κB is mainly regulated by IKK kinase, which in presence of pro-inflammatory stimuli phosphorylates the inhibitory protein IkB, that dissociates from NF-κB with subsequent nuclear translocation [6]. Recently, many studies have highlighted the interaction between NF-κB/ IKK kinase and Keap1protein [7,8]. Our previous studies showed that Keap1 is a modulator both of Nrf2 and NF-κB pathways [9]. In physiological conditions, Keap1 maintains Nrf2 levels low. In conditions of oxidative stress, Keap1 is oxidized on the reactive cysteine residues and inactivated, so that Nrf2 moves into the nucleus. Nrf2 (nuclear factor erythroid2-related factor2), belongs to the basic leucine zipper (bZIP) transcription factor and heterodimerizes with small Maf proteins [10], and it is the primary player in the inducible cell defense system. It binds to the promoter region of genes involved in redox regulation, proteostasis, DNA repair, prevention of apoptosis, iron and heme metabolism, and phase I, II, and III drug/xenobiotic metabolism [11]. The activation of this factor is controlled at the transcriptional and post-transcriptional level through the regulation of its stability, the post-transcriptional changes, and the availability of its binding partners [12,13]. In response to different stimuli, Nrf2 moves to the nucleus where it activates the transcription of its target genes, such as HO-1. Nrf2 is a modular protein presenting seven domains of homology to Nrf2-ECH (Neh), each of which performs a different function. In particular, the Neh2 domain binds Keap1 in the Kelch repeats [14]. In addition, Keap1 has five major domains: an N-terminal broad complex, tram track, and bric-a-brac (BTB) domain; a central intervening region (IVR) and a series of six C-terminal Kelch repeats.
The IVR and BTB domains are required for the redox-sensitive regulation of Nrf2 through a series of reactive cysteines. The 624 amino acids of murine Keap1 include 25 cysteines [15,16]. All cysteine residues are conserved and cysteines C257, C273, C288, and C297, located in the intervening region (IVR) domain, give Keap1 the molecular sensor able to respond to such a diverse array of chemical stimuli [17]. Early studies have identified IKKβ and Keap1 as the key drivers of inflammatory response [18,19]. IKK kinase is the main target of several anti-inflammatory molecules and kinase inhibitors are very effective in the control of many diseases [20][21][22][23]. In fact, LPS-signalling that is activated in macrophages via TLR4 involves a number of kinases. After binding to its receptor, The MyD88-dependent pathway converges into IKK (IκB kinase) and MAPK (Mitogen-activated protein kinases) activation and subsequent activation of NF-κB and activator protein AP-1. In LPS-activated THP-1 cells, a human monocytic cell line, has been reported thanks to bioactive compounds such as flavonoids, hydroxycinnamic acids, tannins and in particular ferulic acid (FA), a hydroxycinnamic acid derivative, an effective inhibition of NF-κB activation as well as a decrease in the expression of proinflammatory cytokines TNF-α and IL-1β [24,25]. Furthermore, FA has a wide range of therapeutic effects like anti-inflammatory, antidiabetic, neuroprotective and hepatoprotective properties in others cellular models and in animals [26]. The present study carried out in RAW264.7 cells shows that the ferulic acid exerts its anti-inflammatory activity when Keap1 and IKK kinase are functionally active.

RT-Qpcr
RAW 264.7 cells were cultured (1 ×10 6 cells/well) in a 6-well plate overnight. Cells were treated with 100 ng/ml LPS or without Transfection and luciferase reporter assays A549 cells (3 × 10 4 cells/well) were seeded in 96-well plates and allowed to adhere for 24 hours. The cells were then cotransfected with pKEAP1Vector, pIL-6FL and Renilla-Luc plasmids (Promega) using the Lipofectamine 3000 (Invitrogen) according to the manufacturer's instructions. After that, the cell culture medium was removed and replaced with fresh medium containing 100μM of FA for 1-hour, followed by co-incubation with 100ng/ml of LPS for 4 hours. Then, the cells were harvested, the luciferase activity was quantified by using the Dual-Glo Luciferase Assay System

Sirna transfection
The RAW264.7 cells (5×10 5 cells/well) were seeded in 6-well plates for 24 hours. Briefly, the siRNA pool for Keap1 (Qiagen) and After vortexing for 10s, the lysates were centrifuged at 1250g for 15min; the supernatants (cytoplasmic extract) were immediately transferred to a clean pre-chilled tube and put on ice. The pelleted nuclei were re-suspended in a hypertonic buffer (5% glycerol,

Statistics
Statistical analysis and graphical presentation were performed using the statistical package GraphPad Prism Version 8 (GraphPad Software, Inc,USA). Data were expressed as mean ± SD and were evaluated using unpaired t-test calculator or one-way analysis of variance (ANOVA) for multiple comparisons. The differences were considered statistically significant when p values were <0.05.

Keap1 silenced abolishes ferulic acid modulation on NF-κB-IKK pathway in LPS-treated RAW 264.7 cells
Our previous study [9] has proved that in Keap1silenced RAW264.7 cells, the LPS treatment induced a higher proinflammatory cytokines mRNA expression compared with nc-siRNA transfected cells and moreover FA treatment resulted not able to reduce cytokines mRNA expression induced by LPS. Other authors [27] showed that depletion of Nrf2 induced the activation of NF-κB and a higher expression of TNF-α, IL-1β, IL-6 cytokines.
In addition, it was reported that Keap1 is identified as an IKKβ interacting protein, involved in IKKβ phosphorylation [28]. To verify a potential Keap1 role in the FA modulation on NF-κB pathway, we analyzed by Western blot analysis NF-κB-IKK pathway in depleted Keap1 and FA-LPS treated RAW 264.7 cells. Cells were transfected with siRNAs for Keap1 and, after 24h, exposed to 100ng/ml LPS for 4h. One hour before LPS, cells were treated or not with 100μM FA, a known inhibitor of the NF-κB-signalling pathway in LPS-activated RAW 264.7 [29]. Cytoplasmic and nuclear extracts were prepared and analyzed in immunoblotting for the NF-κB p65. Figure 1A (panel a) shows that in Keap1-silenced and LPS-treated cells there is a significant increase of p65 in the nuclear fraction, compared to non LPS treated cells, and in accordance with our previous evidences [9], pre-treatment with FA does not decrease NF-κB translocation.

264.7cells Keap1 overexpressed
The overexpression of a specific protein is a common method for investigating the specific biological function and the mechanism of action. In order to confirm the data observed previously, we decided to over-express Keap1and to analyze the FA modulation on NF-κB/IKKβ activation. In our previous paper we described the FA effectiveness on inhibition of NF-κB activity [29]. We transfected  Figure   2A. We conducted an immunoblot analysis on proteins extracted from nuclear and cytoplasmic fractions. Figure 2B shows that LPS induces p65 translocation and FA-modulation occurs (0,5fold FA+LPS vs 6,8-fold LPS p<0.0001), unlike in Keap1 silencing cells.

A549 cells a useful model to analyze FA effect on NF-kB activities
To confirm the results obtained by molecular approach by silencing the messenger for Keap1, we used A549 cell line. A549 is a lung carcinoma cell line that has an inactivating mutation on Keap1 allele, which translates into a protein with a minor binding capacity towards Nrf2 [30]. This cell line is a useful model to study Keap1 loss of functionality. We transfected A549 cells with pIL6-Luc, containing the IL6 promoter region and examined the effects of FA pre-treatment on luciferase activity and ferulic acid capability to alleviate inflammatory response via NF-κB pathway. Figure 3A shows that the up-regulation of luciferase activity LPS induced is not modulated by pre-treatment with FA (171fold FA+LPS vs 111fold LPS p<0.0001). Immunoblot for IKKα/β in Figure 3B shows that the effectiveness of FA is lost, in fact there is not a significant decrease of IKK protein level when LPS and FA treatment occurs. Moreover, to verify the level of Keap1 protein in A549 cells we conducted an Immunoblot on total proteins extracted from A549 treated or no with FA and LPS. Figure 3C shows that there are not significantly differences in Keap1 level in cells treated or not. These results follow the Keap1 silencing data indicate that Keap1and IKK are involved in the ferulic acid anti-inflammatory effectiveness. Figure 3A: Analysis of ferulic acid modulation of IL6-promoter activity in A549 cells treated with LPS and FA. A549 cells (3×105 cells/well) were transfected with IL6-Luc reporter or vector for 24h and co-transfected with pRK-Renilla for internal normalization. Twenty-four hours after transfection, cells were pretreated with or without FA (100 μM) for 1h, then LPS was added (100ngr/ml). After 4h of activation, total cells were lysed and Luciferase activity was measured with a dual-luciferase system. The graph shows the relative light emission level of IL-6 promoter activation, expressed as difference between means±SEMof three experiments each one performed in triplicate.Difference between means (LPS+FA-LPS)±SEM 60,00±3,367, difference between means (LPS-C)±SEM 11,00±1,732 are significantly different (p<0.05).

Figure 3B
: IKK kinase protein level in A549 cell line. A549 cells were pre-treated with or without FA 100µM for 1h and then with LPS 100ngr/ml for 4h. Total proteins were extracted and immunoblot analysis of IKKα/β protein level was effectuated. Densitometric analysis is shown in the graph. Difference between means (FA+LPS-LPS) ± SEM 0,4167±0,1271 is significantly different (P < 0.05).The data shown represents three independent experiments. β-actin immunolabelling was used as a loading control Figure 3C: Keap1 protein level in A549 cell line. A549 cells were treated as described above and total proteins were extracted and immunoblot analysis of Keap1 protein was conducted. The data shown represents three independent experiments.Analysis by one-way Anovashows thatSDs are not significantly different(P<0.05).

Discussion
FA, a phenolic phytochemical found in many traditional Chinese medicines such as Angelica Radix and Chuanxiong Rhizoma, appears to be a potential therapeutic agent for treating various inflammatory disorders, although the molecular mechanism underlying the effectiveness is not entirely known. The pharmacological activity of FA in vascular endothelial cells is demonstrated, in fact, capable of inhibiting the expression of adhesion molecules in HUVECs [31]. Therefore, it was demonstrated that free and SLNs-loaded FA recover cell viability in neuroblastoma cells LAN5 [32]. but also tumorigenesis and apoptosis. There seems to be many the ways in which the two pathways interact, and Keap1 is one of the main [34]. Our results showed that Keap1 protein is the key point of the regulation carried out by FA on NF-κB pathway through IKK kinase. Recently, an ETGE motif (NQE 36 TGE 39 ) similar to that of Nrf2 (DEE 79 TGE 82 ) has been identified in IKKβ and so it is reasonable to suppose that Keap1 is an IKK modulator, regulating the ubiquitination and subsequent degradation [12,13,14].  when LPS treatment occurs. Remarkably, Keap1 silencing does not inactivate NF-κB pathway, but the FA modulation is lost. In our previous study, we demonstrated that IKK is an upstream target of ferulic acid in LPS-induced NF-κB activation [29]. It can be hypothesized that Keap1 plays an important role in the NF-κB/ IKK modulation FA mediated. Yet, to confirm the data observed previously, we overexpress Keap1 in RAW264.7 cells and we analyze FA modulation on NF-κB/IKK pathway after treatment with LPS. Immunoblot data confirm that LPS stimulus activates NF-κB/ IKK pathway and FA modulation occurs.
Therefore, Keap1 and IKK kinase proteins are very important for the FA effectiveness. To confirm the results obtained by molecular approach by silencing the messenger for Keap1, we used A549 cell line to examine the biological effect of Keap1 protein mutation on the NF-κB/IKK modulation by FA. A549, in fact, is a lung carcinoma cell line that has an inactivating mutation on Keap1 allele, and the level of the Keap1 protein is the same in non-treated and LPS treated cells. In this cell line the Nrf2 pathway is dysfunctional, because the NRF2-Keap1interaction is altered [30]. The Luciferase assay data show that LPS induces up-regulation of IL-6 promoter in LPS and in FA+LPS treated cells. It is probable so the Keap1 evolvement in NF-κB/IKK FA-modulation. Recent results have revealed that the cysteine residues Cys-151, Cys-273 and Cys-288 have been identified as direct sensors of electrophiles and oxidants [17].
Keap1 acts as a molecular sensor of ROS, NO and oxidant species and the interaction with oxidizing species results in an activation signal towards Nrf2. Nrf2 is an important mammalian member of the CncC family, which has been studied in numerous cellular systems and represents an important target for drug discovery in different diseases. Nrf2 is distributed in the cytoplasm together with Keap1 normally [12,13,14]. Under oxidative stress, Nrf2 was released and migrated to the nucleus, inducing HO-1 expression.
HO-1, an antioxidant enzyme in the stress response, catalyzing the degradation of heme, biliverdin, and carbon monoxide (CO), also plays a key role in inflammation and oxidation responses.
Our previous study showed that the pre-treatment with FA carried out on LPS-treated RAW264.7 cells determined a decrease of HO-1 protein level [9]. Until now there are not any data demonstrating IKK involvement in Nrf2 modulation, so to verify the FA effectiveness and IKK/Keap1 involvement we analyze the HO-1 expression in Keap1silenced RAW264.7 cells.
The results showed that pretreatment with FA decreased HO-1 mRNA expression in Keap1silenced RAW264.7 cells. Therefore, the results obtained agree with Kim et al [28] who identified Keap1 as IKK interacting protein and highlight that the Keap1 protein underlie the FA effectiveness on NF-κB modulation. The network Keap1-Nrf2-NF-κB/IKKβ is very interesting: it has many implications, being involved in inflammation, chemoresistance and cellular homeostasis [16,17,18]. Further studies are necessary to clarify the role of Keap1 in the NF-κB/ IKKβ dialogue. Our results also show that the Keap1 is an attractive target for therapeutic purposes, especially to improve the efficacy of anticancer and antiinflammatory molecules.

Conflict of Interest
The authors declare that there are no conflicts of interest.