The anti-inflammatory and anti-oxidative effect of a classical hypnotic bromovalerylurea mediated by the activation of NRF2

Abstract

The Kelch-like ECH-associated protein 1-nuclear factor erythroid 2-related factor 2 (KEAP1-NRF2) system plays a central role in redox homeostasis and inflammation control. Oxidative stress or electrophilic compounds promote NRF2 stabilization and transcriptional activity by negatively regulating its inhibitor, KEAP1. We have previously reported that bromovalerylurea (BU), originally developed as a hypnotic, exerts anti-inflammatory effects in various inflammatory disease models. However, the molecular mechanism underlying its effect remains uncertain. Herein, we found that by real-time multicolor luciferase assay using stable luciferase red3 (SLR3) and green-emitting emerald luciferase (ELuc), BU potentiates NRF2-dependent transcription in the human hepatoblastoma cell line HepG2 cells, which lasted for more than 60 h. Further analysis revealed that BU promotes NRF2 accumulation and the transcription of its downstream cytoprotective genes in the HepG2 and the murine microglial cell line BV2. Keap1 knockdown did not further enhance NRF2 activity, suggesting that BU upregulates NRF2 by targeting KEAP1. Knockdown of Nfe2l2 in BV2 cells diminished the suppressive effects of BU on the production of pro-inflammatory mediators, like nitric oxide (NO) and its synthase NOS2, indicating the involvement of NRF2 in the anti-inflammatory effects of BU. These data collectively suggest that BU could be repurposed as a novel NRF2 activator to control inflammation and oxidative stress.

Keywords: Abbreviations: ARE, antioxidant responsive element; BU, bromovalerylurea; CCL2, C-C motif chemokine 2; DMF, dimethyl fumarate; GCLC, glutamate–cysteine ligase catalytic subunit; GCLM, glutamate–cysteine ligase modifier subunit; GSS, glutathione synthetase; GSH, glutathione; Hmox-1, heme oxygenase-1; IL-1β, interluekin-1β; IL-6, interluekin-6; JAK, Janus kinase; KEAP1, Kelch-like ECH-associated protein; NO, nitric oxide; NOS2, NO synthase 2; NRE, NF-κB responsive element; NQO-1, NAD(P)H quinone dehydrogenase; NRF2, nuclear factor erythroid 2-related factor 2/nuclear factor erythroid-derived 2-like 2; TXNRD, thioredoxin–disulfide reductase; ROS, reactive oxygen species; KEAP1–NRF2; anti-inflammation; anti-oxidant oxygen; bromovalerylurea; drug action toxins/drugs/xenobiotics.

Graphical Abstract

Fig. 1

The molecular structure of BU. From left to right, the structural formula, the ball-and-stick model, and the pseudo-color presentation of the electron density. Warm color represents strong electrophilicity.

Fig. 2

BU enhances ARE-dependent transcriptional activity in a dose-dependent manner. A. Effects of BU on ARE-dependent transcription (upper panels), which was calculated as log₂ (ARE-TK-SLR3 (% of control)/TK-ELuc (% of control)). TK-ELuc (% of control) was shown in lower panels. B–D. Effects of BU on NRE-dependent (B), ERSE-dependent (C), HSE-dependent (D) transcription. Dose dependencies are summarized in the areas under the curves (AUC) at the right-most panel. Data are shown as means ± standard deviations (n = 3).

Fig. 3

BU enhances NRF2 accumulation and transcriptional activity by inhibiting its negative regulator, KEAP1. A–F. HepG2 cells were treated with siRNA against Keap1 and/or BU. Immunoblot analyses of NRF2 protein levels in nuclear fractions (A) and qPCR measurement of the factors related to NRF2 (B, Nfe2l2; C, Keap1; D, Gclc; E, Gclm; and F, Txnrd1) were executed. The mRNA levels were represented as ratios of Gapdh (B–F). ^*P < 0.05, ^**P < 0.01, ^***P < 0.001, and ^****P < 0.0001 between indicated conditions.

Fig. 4

BU enhances NRF2 stabilization and transcriptional activity in the murine microglial cell line, BV2. Immunoblotting analysis and quantification of NRF2 protein (arrows) levels under BU and LPS stimulation in entire cell lysates (A) and nuclear extracts (B) of BV2 cells. Quantifications were executed using β-actin and histone 2A (H2A) blots as loading controls for whole cell lysates and nuclear extracts, respectively. Effects of BU on anti-oxidative NRF2 activity during LPS-mediated proinflammatory reactions in BV2 cells were examined and ARE activity measured by the luciferase reporter enhancer assay (C); mRNA levels of Nqo1 (D); Hmox1 (E); Gclc (F); Gclm (G); Gss (H); cellular levels of GSH (I); and binding of NRF2 to the ARE motif in the Nqo1 promoter(J); Rpl30 Intron 2 (negative locus) (K) measured by the ChIP-qPCR assay, as described in Methods. The mRNA levels were represented as ratios of Gapdh, as shown in Fig. 2. The black circle plot in the quantification represents BU (−) and the blue triangle plot, BU (+). ^*P < 0.05, ^**P < 0.01 and ^***P < 0.001 vs. BU (−).

Fig. 5

BU acts as an anti-inflammatory agent by inhibiting the transcription of pro-inflammatory genes. A–O. Effects of BU on LPS-mediated pro-inflammatory reaction in BV2 cells. mRNA expression of Nos2 (A); Il1-β (B); Il-6 (C); Ccl2 (D); protein levels of NOS2 (E); cellular levels of nitrite (F); promoter activities of Nos2 measured by the luciferase reporter enhancer assay (G), as described in Methods. β-actin blot was utilized as loading control for the quantification of NOS2 protein levels in E. H–K. Effects of BU on the mRNA levels of the pro-inflammatory genes under the LTA-mediated pro-inflammatory reaction (H, Nos2; I, Il1b; J, Il6; K Ccl2). L–O. Effects of BU on the mRNA levels of the pro-inflammatory genes under the Poly (I:C)-mediated pro-inflammatory reaction (L, Nos2; M, Il1b; N, Il6; O Ccl2). ^**P < 0.01, ^***P < 0.001, and ^****P < 0.0001 vs. BU (−).

Fig. 6

Anti-oxidative effects of BU require NRF2. A. Irr: BV2 cell stably expressing irrelevant sequence shRNA, and KD: BV2 cell expressing shRNA against NRF2. The reactivity to LPS and BU was assessed by measuring the expression of NRF2 and NOS2 proteins using immunoblot analyses with β-actin reblotting as the loading control. Quantification of the NRF2 signal normalized by β-actin is shown to the right of the immunoblot images. B–F. The mRNA levels of the downstream factors of NRF2 (B, Nqo1; C, Hmox1; D, Gclc; E Gclm and F, Gss), measured by qPCR, during LPS stimulation in the absence of NRF2, are represented as ratios of Gapdh as shown in Fig. 3. NRF2 binding to the ARE motif in the Nqo1 promoter during LPS stimulation in the absence of NRF2 was examined by ChIP-qPCR (G). ^*P < 0.05, ^**P < 0.01, ^***P < 0.001 and ^****P < 0.0001 between the indicated conditions.

Fig. 7

Anti-inflammatory effects of BU require NRF2. The expression of pro-inflammatory genes was measured using qPCR (A, Nos2; B, Il1- β; C, Il-6 and D, Ccl2, each left panel), and the rates of reduction accompanied by the knockdown of NRF2 upon LPS stimulation (irr vs. KD of [% of Gapdh {(LPS –LPS + BU)/LPS}]) are plotted in each right panel. The promoter activities of the Nos2 gene (E), NOS2 protein expression, as shown by the quantification of the NOS2 immunoblotting signal normalized to β-actin in Fig. 6A (F), and nitrite production (G) during LPS stimulation in the absence of NRF2 were also assessed. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001 and ^****P < 0.0001 between the indicated conditions.