A 5-hydroxyoxindole derivative attenuates LPS-induced inflammatory responses by activating the p38-Nrf2 signaling axis

Accepted Manuscript A 5-hydroxyoxindole derivative attenuates LPS-induced inflammatory responses by activating the p38-Nrf2 signaling axis Tomomi Niino, Kenji Tago, Daisuke Yasuda, Kyoko Takahashi, Tadahiko Mashino, Hiroomi Tamura, Megumi Funakoshi-Tago PII: DOI: Reference:

S0006-2952(18)30234-X https://doi.org/10.1016/j.bcp.2018.06.021 BCP 13175

To appear in:

Biochemical Pharmacology

Received Date: Accepted Date:

18 April 2018 14 June 2018

Please cite this article as: T. Niino, K. Tago, D. Yasuda, K. Takahashi, T. Mashino, H. Tamura, M. Funakoshi-Tago, A 5-hydroxyoxindole derivative attenuates LPS-induced inflammatory responses by activating the p38-Nrf2 signaling axis, Biochemical Pharmacology (2018), doi: https://doi.org/10.1016/j.bcp.2018.06.021

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A 5-hydroxyoxindole derivative attenuates LPS-induced inflammatory responses by activating the p38-Nrf2 signaling axis.

Tomomi Niinoa, Kenji Tagob, Daisuke Yasudac, Kyoko Takahashic, Tadahiko Mashino c, Hiroomi Tamuraa, and Megumi Funakoshi-Tagoa

a

Department of Hygienic Chemistry, Faculty of Pharmacy, Keio University, 1-5-30

Shibakoen, Minato-ku, Tokyo 105-8512, Japan b

Division of Structural Biochemistry, Department of Biochemistry, Jichi Medical

University, 3311-1 Yakushiji, Shimotsuke-shi, Tochigi-ken 329-0498, Japan c

Division of Medicinal Chemistry and Bio-organic Chemistry, Faculty of Pharmacy,

Keio University, 1-5-30 Shibakoen, Minato-ku, Tokyo 105-8512, Japan

*Corresponding author: Megumi Funakoshi-Tago Tel.: +81-3-5400-2689, Fax: +81-3-5400-2689, E-mail: [email protected] Department of Hygienic Chemistry, Faculty of Pharmacy, Keio University, 1-5-30 Shibakoen, Minato-ku, Tokyo 105-8512, Japan

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Abbreviations ASK1, apoptosis signal regulating kinase 1; Cul3, cullin 3; DEF, dimethylfumarate; DEM, diethylmaleate; 15d-PGJ2, 15-deoxy-Δ12,14-prostaglandin J2 ; FBS, fetal bovine serum; 5-HI, 5-hydroxyoxindole derivative; HO-1, heme oxygenase-1; iNOS, inducible NO synthase; IL-6, interleukin 6; IRAK-1, Interleukin 1 receptor-associated kinase 1; Keap1, Kelch-like ECH-associated protein 1; LPS, Lipopolysaccharide; MAP kinases, mitogen-activated protein kinases; MyD88, myeloid differentiation factor 88; NAC, N-acetyl cysteine; NQO1, NAD(P)H quinone dehydrogenase 1; NF-κB, nuclear factor kappaB; Nrf2, NF-E2-related factor-2; 9-OA-NO2, 9-nitro-octadec-9-enoic acid; PI3K, phosphatidylinositol 3-kinase; PKC, protein kinase C; SFN, sulforaphane; TAK1, transforming

growth

factor--activated

protein

kinase

1;

tBHQ,

tert-butyl

hydroquinone; TLR4, Toll-like receptor 4; TNF, tumor necrosis factor ; TRAF6, TNF receptor-associated factor 6

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Abstract 5-Hydroxyoxindole is a urinary metabolite of indole that exhibits antioxidant activity. In the present study, we found that a 5-hydroxyoxindole derivative (5-HI) significantly inhibited LPS-induced inflammatory effects in the murine macrophage cell line, RAW264.7. 5-HI induced the expression of the transcription factor, Nrf2, which is typically ubiquitinated by Keap1, an adaptor component of the ubiquitin E3 ligase complex, resulting in its proteasomal degradation. By utilizing Keap1-/- MEFs reconstituted with Keap1 mutants harboring substitutions in their major cysteine residues, we clarified the importance of Cys151 in Keap1 as a sensor for 5-HI in the induction of Nrf2 expression. Furthermore, 5-HI induced the activation of the MKK3/6-p38 pathway, which is required for the transcriptional activation of Nrf2. The knockdown of Nrf2 enhanced the LPS-induced expression of inflammatory mediators, including iNOS, NO, and CCL2, and effectively repressed the inhibitory effects of 5-HI on their expression. Although 5-HI and antioxidant N-acetyl cysteine (NAC) both reduced LPS-induced ROS generation, the treatment with NAC did not affect the LPS-induced expression of inflammatory mediators, suggesting that the anti-inflammatory activity of 5-HI mediated by Nrf2 is independent of redox control. Furthermore, when injected into mice with 5-HI, the expression of Nrf2 was significantly increased, and the LPS-induced mRNA expression of CXCL1, CCL2, TNF and IL-6 were remarkably inhibited in the kidneys, liver, and lungs, and the production of these cytokines in serum was effectively reduced. Collectively, these results suggest that 5-HI has potential in the treatment of inflammatory diseases through the activation of Nrf2.

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Keywords: 5-hydroxyoxindole derivative, LPS, Nrf2, Keap1, anti-inflammatory activity

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1. Introduction Inflammation is a complex immune response that protects the body from infections and tissue injury [1]. Chronic inflammation is closely associated with the pathogenesis of a number of human diseases, such as rheumatoid arthritis, sepsis, cardiovascular diseases, and cancer [2]. Therefore, it is very important to regulate inflammation in order to prevent progression to severe or chronic diseases. Lipopolysaccharide (LPS), which is an endotoxin from Gram-negative bacteria, triggers inflammatory responses through its specific receptor, Toll-like receptor 4 (TLR4) on the surface of macrophages [3]. LPS activates the transcription factor, nuclear factor kappaB (NF-κB) through a series of signaling molecules, such as the adaptor proteins, myeloid differentiation factor 88 (MyD88) and TNF receptor-associated factor 6 (TRAF6), and the serine/threonine kinase, IL-1 receptor-associated kinase 1 (IRAK-1) [4–6]. LPS induces the expression of inducible NO synthase (iNOS) and numerous cytokines and chemokines, including tumor necrosis factor α (TNFα), interleukin 6 (IL-6), CCL2, and CXCL1, through the activation of NF-B [7–11]. The transcription factor NF-E2-related factor-2 (Nrf2), which is essential for protection against oxidative and xenobiotic stresses, has been known to attenuate inflammation. The disruption of the Nrf2 gene exacerbates inflammation in a number of murine models, such as asthma and pleurisy [12-14]. A recent study reported that Nrf2 interfered with the LPS-induced transcriptional up-regulation of the proinflammatory cytokine, IL-6 by binding to the proximity of the IL-6 gene independently of the Nrf2-binding motif and inhibiting the recruitment of RNA Pol II [15]. Under basal unstressed conditions, Nrf2 is constitutively ubiquitinated by Kelch-like ECH-associated protein 1 (Keap1), an adaptor component of the cullin 3 (Cul3)-based 5

ubiquitin E3 ligase complex, resulting in its proteasomal degradation [16-18]. Keap1 also acts as a sensor for oxidative and electrophilic stresses. Nrf2 inducers, such as electrophiles, readily react with the cysteine thiols of Keap1 and the modification of cysteine residues in Keap1 results in the inhibition of the ubiquitin E3 ligase activity of the Keap1-Cul3 complex. As a result, Nrf2 is stabilized and activates its target genes including the detoxification and antioxidant enzymes, heme oxygenase-1 (HO-1) and NAD(P)H quinone dehydrogenase 1 (NQO1) [19-21]. The different roles of Cys151, Cys277, and Cys288 in Keap1 as a sensor for each Nrf2 inducer was clarified utilizing knock-in mice expressing Keap1 mutants harboring Cys151Ser, Cys277Trp, and Cys288Glu. The Cys151 residue is indispensable for the accumulation of Nrf2 in response to tert-butyl hydroquinone (tBHQ), diethylmaleate (DEM), sulforaphane (SFN), and dimethylfumarate (DMF). While the Cys277 residue is critical for the accumulation of Nrf2 in response to 15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2), all three Cys residues are required for 9-nitro-octadec-9-enoic acid (9-OA-NO2) to induce Nrf2 expression [22].

5-Hydroxyoxindole has been identified as a urinary metabolite of the indole, which is produced from tryptophan via the tryptophanase activity of gut bacteria [23, 24]. A previous study reported that 5-hydroxyoxindole is present in circulating blood and in some tissues such as the brain, liver, kidneys, and spleen [25]. It has been shown to selectively inhibit brain monoamine oxidase A activity and exhibit anti-proliferative activity against several cancer cells including human promyelocytic leukemia cell line, HL-60, and neuroblastoma cell line, N1E-115 [26, 27]. We previously reported that 5-hydroxyoxindole exhibited radical scavenging activity and inhibited rat liver microsome/tert-butylhydroperoxide system-induced lipid peroxidation and hydrogen 6

peroxide-induced intracellular oxidative stress [28]. We also showed that the antioxidant activity of 5-hydroxyoxindole was enhanced by the substitution of a 3-hydrogen group to a lipophilic functional group [29].

In the present study, we investigated the anti-inflammatory activities of 5-hydroxyoxindole and several of its derivatives by assessing their effects on the LPS signaling pathway. We found that only the 5-hydroxyoxindole derivative harboring the functional group, benzylidene (=CH-Ph) significantly inhibited LPS-induced inflammatory responses through the induction of Nrf2 expression.

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2. Materials and methods 2.1. Reagents and antibodies 5-Hydroxyoxindole was purchased from Apin Chemical Co. (Oxfordshire OX14 4RU United Kingdom). Six kinds of 5-hydroxyoxindole derivatives were synthesized as previously described [28, 29]. LPS (Escherichia coli 055:B5), aprotinin, pepstatin, and leupeptin were purchased from Sigma-Aldrich (St. Louis, MO, USA). SB203580 was purchased from Tocris Bioscience (Bristol, UK). EcoRI and XhoI were purchased from TOYOBO (Osaka, Japan). Other chemicals were purchased from Nacalai Tesque (Tokyo, Japan). Anti-Nrf2, anti-Keap1, anti-p38, anti-Lamin B, and anti--actin antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-phospho-p38 (Thr180/Tyr182), anti-phospho-MKK3 (Ser189)/MKK (Ser207), anti-MKK3/6, anti-ATF2 (Thr71), and anti-ATF2 antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). Peroxidase-conjugated rabbit anti-mouse, rabbit anti-goat, and goat anti-rabbit secondary antibodies were purchased from Dako-Japan (Tokyo, Japan).

2.2. Cell culture Keap1 -/- MEFs were gifted from Dr. M. Yamamoto (Tohoku University Graduate School of Medicine). The murine macrophage cell line, RAW264.7 and HEK293T cells were purchased from the Riken Cell Bank (Ibaraki, Japan). These cells were cultured at 37 °C under 5% CO2/95% air in DMEM (Nacalai Tesque) supplemented with 10% fetal bovine serum (FBS) (Gibco, Life Technologies, CA, USA) and 1% Penicillin-Streptomycin Mixed Solution (Nacalai Tesque). 8

2.3. Plasmids Full-length Keap1 cDNA with the N-terminal FLAG sequence were generated from the total RNA of RAW264.7 cells by RT-PCR. PCR products were purified and inserted into the EcoRI and XhoI sites of MSCV-Puro. The mutagenesis of amino‐ acid residues in Keap1: C151S, C273W, and C288E was performed using a Site‐ Directed Mutagenesis Kit, according to the manufacturer’s instructions (Clontech, San Francisco, CA). A fusion gene, full-length human Nrf2 and the DNA-binding domain of GAL4 were generated by RT-PCR and inserted into the EcoRI and XhoI sites of MSCV-Puro.

2.4. Retrovirus production and infection HEK293T cells were transfected with a helper retrovirus plasmid together with retroviral plasmids by FUGENE6 Transfection Reagent, according to the manufacturer’s instructions (Roche Diagnostics, Indianapolis, IN). Viruses were harvested 24–60 h post‐ transfection, pooled, and stored on ice. Exponentially growing Keap1‐ deficient MEFs in 100-mm culture dishes were infected three times at 3-h intervals with 2 mLof fresh virus‐ containing supernatant in complete medium containing 10 g/mL polybrene (Nacalai Tesque). In order to select infected cells, 2 g/mL puromycin was used.

2.5. Transfection of siRNA

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Control siRNA and siRNA targeting murine Nrf2 were purchased from Dharmacon GE (GE Healthcare UK Ltd., United Kingdom). siRNA against murine Keap1 was purchased from Santa Cruz Biotechnology. RAW264.7 cells (5×106) were transfected with 10 M siRNA using DharmaFECT (GE Healthcare UK Ltd.) according to the manufacturer’s instructions. Cell lysates and RNA were prepared 48 h post-transfection.

2.6. Transfection and luciferase assay In the Nrf2 transactivation assay, HEK293T cells (5 × 105 cells) and RAW264.7 cells (1 × 106 cells) were transfected with 1 μg of the reporter plasmid pFR-Luc, which contained the luciferase gene controlled by the Gal4 upstream activating sequence (Stratagene, La Jolla, CA), 1 μg of the MSCV-Puro empty vector or MSCV-Puro-GAL4-Nrf2, and 0.2 μg of pRL-TK using polyethylenimine MAX (Polysciences, Inc., Warrington, PA, USA) and the Neon® Transfection System (Life Technologies), respectively. After 36 h, transfected cells were treated with 0.1% DMSO or SB203580 (10 M) for 1 h prior to the stimulation with 5-HI (10 M) at 37 °C for 12 h. Cells were then harvested, and utilized to analyze luciferase activities by the Dual-Luciferase Reporter Assay System (Promega), as previously described [30].

2.7. Measurement of NO RAW264.7 cells (2 × 105 cells) were cultured in a 24-well plate and preincubated with 0.1% DMSO (Nacalai Tesque) or 5-HI at 37 °C for 1 h prior to the stimulation with LPS (1 g/mL) for 16 h. The concentration of nitrate in culture supernatants was measured using Griess reagent (1% sulfanilamide, 0.1% N-naphthylethylenediamine, 2.5% H3PO4) as previously described [30]. 10

2.8. Measurement of cell viability RAW264.7 cells (5 × 105 cells) were cultured in a 24-well plate and preincubated with 0.1% DMSO (Nacalai Tesque) or 5-HI at 37 °C for 1 h prior to the stimulation with LPS (1 g/mL) for 16 h. Cell viability was measured using the trypan blue exclusion method.

2.9. Immunoblotting Cells and murine organs were lysed with Nonidet P-40 lysis buffer (50 mM Tris-HCl pH 7.4, 10% glycerol, 50 mM NaCl, 0.5% sodium deoxycholate, 0.5% NP-40, 20 mM NaF, and 0.2 mM Na3VO4) supplemented with protease inhibitors. In order to prepare nuclear extracts, cells were lysed in buffer A (10 mM HEPES-KOH (pH 7.8), 10 mM KCl, 0.1 mM EDTA, 0.1% Nonidet P-40, 1 mM DTT, 0.5 mM PMSF, 2 μg/mL aprotinin, 2 μg/mL pepstatin, and 2 μg/mL leupeptin). Nuclei were then isolated as a precipitate by centrifugation at 5000 r.p.m. for 2 min. Isolated nuclei were lysed in Nonidet P-40 lysis buffer and homogenized using the ultrasonic homogenizer VP-50 (TAITEC, Japan). Cell lysates and nuclear extracts were then centrifuged at 15,000 r.p.m. at 4 °C for 15 min and the supernatant was mixed with Laemmli’s sample buffer. Denatured samples were resolved by SDS-PAGE and transferred to polyvinylidene difluoride membranes (Millipore, Billerica, MA, USA). Membranes were probed using the designated antibodies and visualized with the ECL detection system (GE Healthcare, Little Chalfont, UK) as described previously [30]. The intensity of each band was quantified by ImageJ software. The phosphorylation levels of MKK3/6 and p38 were normalized with the expression levels of MKK3/6 and p38. In order to show the relative 11

amounts of Nrf2 in the nucleus and Keap1, the band intensities of Nrf2 and Keap1was normalized with the band intensities of Lamin B and -actin, respectively.

2.10. RT-PCR (reverse transcription-polymerase chain reaction) Total RNA was extracted using Trizol (Life Technologies, Waltham, MA, USA). The synthesis of single-strand cDNA was performed using an oligo (dT) 20 primer (TOYOBO) and 1 μg of total RNA for first-strand cDNA synthesis, as previously described [30]. Quantitative real-time PCR was performed using an iCycler iQ™ Real-Time PCR Detection System (Bio-Rad, Berkeley, CA, USA). PCR was performed in a 10-μL volume with the KAPA SYBR® FAST qPCR Kit (KAPA Biosystems, Wilmington, MA, USA). The PCR primer sequences used were shown in Table. 1.

2.11. Enzyme-linked immunosorbent assay (ELISA) RAW264.7 cells (2 × 105 cells) were cultured in a 24-well plate and preincubated with 0.1% DMSO or various concentrations of 5-HI at 37 °C for 1 h prior to the stimulation with LPS (1 μg/mL) for 16 h. Culture supernatants were collected, and the amounts of CCL2 in the supernatants were measured using the Immunoassay Kit (eBioscience, San Diego, CA, USA). The amounts of CCL2, TNF and IL-6 in murine serum were determined using the Immunoassay Kit (eBioscience ) and the amounts of CXCL1 was determined using ELISA kit (R&D Systems, Minneapolis, MN, USA), respectively.

2.12. LPS-induced inflammation in mice

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Six-week-old male C57BL/6 mice obtained from Sankyo Labo Service Corporation, Inc. (Tokyo, Japan) were randomly divided into six groups: control, 5-HI (25 mg/kg), 5-HI (50 mg/kg), LPS, LPS+5-HI (25 mg/kg), and LPS+5-HI (50 mg/kg), and there were 4 mice in each group. Mice were injected intraperitoneally with control vehicle (olive oil) or 5-HI (25 mg/kg, 50 mg/kg). After 1 h, mice were given an intraperitoneal injection of control vehicle (PBS) or LPS (300 g/mouse). Mice were sacrificed 6 h after the LPS injection, and total RNA was extracted from the kidneys, liver, and lungs using Trizol (Life Technologies). To evaluate serum cytokine levels, C57BL/6 mice (6 mice in each group) were injected intraperitoneally with control vehicle (olive oil) or 5-HI (25 mg/kg, 50 mg/kg). After 1 h, mice were given an intraperitoneal injection of control vehicle (PBS) or LPS (300 g/mouse), then 2 h after injection of LPS, blood samples were collected from mice by cardiac puncture. In addition, to investigate the effect of 5-HI in the mice injected with LPS, C57BL/6 mice (6 mice in each group) were injected intraperitoneally with control vehicle (PBS) or LPS (300 g/mouse), then 1 h after injection of LPS, mice were given an intraperitoneal injection of control vehicle (olive oil) or 5-HI (25 mg/kg, 50 mg/kg). After 1h, blood samples were collected from mice by cardiac puncture. Serum samples were separated and stored at − 80 °C until assay. All experimental protocols were approved by the Animal Usage Committee of Keio University (Approval number, 12048-(2)). The methods used were performed in accordance with the approved guidelines.

2.13. Statistical analysis

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All values are expressed as the mean ± SD. A one- or two-way analysis of variance (ANOVA) followed by Tukey’s test was used to evaluate differences between more than three groups. Differences were considered to be significant for values of P < 0.05.

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3. Results 3.1. 5-HI inhibits LPS-induced inflammatory responses. As shown in Fig. 1A, 5-hydroxyindole (compound 1) and 6 types of 5-hydroxyoxindole derivatives with the substitution of the 3-hydrogen group to various functional groups (compounds: 2-7) were synthesized. 5-hydroxyindole and its derivatives at 20 M had no effect on the viability of the macrophage cell line, RAW264.7 in the presence and absence of the LPS stimulation (Fig. 1B). We next investigated the anti-inflammatory activities of 5-hydroxyindole and 6 types of its derivatives at 20 M by evaluating their effects on LPS-induced NO production and CCL2 secretion. As a result, only the 5-hydroxyoxindole derivative harboring the functional group, benzylidene (=CH-Ph) (compound 7) significantly prevented LPS-induced NO production and CCL2 secretion. Then, we named this derivative (compound 7) “5-HI” as shown in Fig. 1A. On the other hand, the 5-hydroxyoxindole derivative harboring the functional group, benzyl (-CH-Ph) (compound 6) failed to inhibit LPS-induced NO production and CCL2 secretion, suggesting that the tertiary structure of the 5-hydroxyoxindole derivative was critical for its anti-inflammatory effects (Fig. 1C, D). In order to further investigate the anti-inflammatory activity of 5-HI, we initially examined the effects of 5-HI at various concentrations on LPS-induced inflammatory responses using RAW264.7 cells. The treatment with 5-HI significantly inhibited LPS-induced NO production in a dose-dependent manner (Fig. 2A). 5-HI only negligibly affected the viability of RAW264.7 cells in the presence and absence of the LPS stimulation (Fig. 2B), indicating that the inhibitory effects of 5-HI on NO production were not due to its cytotoxicity. Since the production of NO is attributed to 15

the expression of inducible NO synthase (iNOS) in immune responses [8], we investigated the effects of 5-HI on the expression of iNOS mRNA by RT-PCR. 5-HI significantly inhibited the LPS-induced expression of iNOS mRNA (Fig. 2C). 5-HI also inhibited the LPS-induced secretion of CCL2 in a dose-dependent manner (Fig. 2D). In addition, 5-HI markedly inhibited the LPS-induced expression of CCL2 mRNA (Fig. 2E), suggesting that 5-HI exhibited anti-inflammatory activities by inhibiting the transcriptional activities of iNOS and CCL2.

3.2. 5-HI inhibits LPS-induced inflammatory responses through Nrf2 activation. In order to elucidate the mechanisms by which 5-HI exhibits anti-inflammatory activity, we examined the effects of 5-HI on the activation of Nrf2, a transcription factor that negatively regulates inflammation [12-14]. The treatment with 5-HI induced the expression of the Nrf2 protein (Fig. 3A). However, Nrf2 mRNA expression levels were slightly reduced by the treatment with 5-HI (Fig. 3B). In RAW264.7 cells, endogenous Nrf2 in RAW264.7 cells was knocked down using an RNA interference technique. 5-HI failed to induce the expression of Nrf2 or HO-1 mRNA and NQO1 mRNA when Nrf2 was knocked down in RAW264.7 cells, confirming that HO-1 and NQO1 are Nrf2 target genes (Fig. 3C, D). 5-HI significantly induced the expression of HO-1 mRNA and NQO-1 mRNA (Fig. 3E). To further investigate the roles of 5-HI-induced Nrf2 activation on LPS-induced inflammatory responses, we analyzed whether 5-HI still exhibits its anti-inflammatory effect in the situation that the expression of Nrf2 was knocked down. The LPS-induced expression of iNOS mRNA was increased and the inhibitory effects of 5-HI on LPS-induced iNOS mRNA expression were reduced by the knockdown of Nrf2 (Fig. 16

4A). LPS-induced NO production consistently increased, and the inhibitory effects of 5-HI on this production were reduced by the knockdown of Nrf2 (Fig. 4B). In addition, the knockdown of Nrf2 increased the LPS-induced expression of CCL2 mRNA and secretion of CCL2 and reduced the inhibitory effects of 5-HI on its expression and secretion (Fig. 4C, D). These results suggest that the 5-HI-induced activation of Nrf2 negatively regulates the LPS-induced expression of iNOS mRNA and CCL2 mRNA.

3.3. The Cys151 residue in Keap1 is indispensable for the accumulation of Nrf2 in response to 5-HI. As described in the Introduction section, Keap1 functions as a component of the ubiquitin E3 ligase complex causing the proteasomal degradation of Nrf2 [16-18]. The ubiquitin ligase activity of Keap1 is regulated by oxidative and electrophilic stresses. Major three cysteine residues, Cys151, Cys273, and Cys288, in Keap1 function as sensors for electrophiles [22]. In order to elucidate the mechanisms by which 5-HI induces the expression of Nrf2, the involvement of Cys residues in Keap1 in 5-HI-induced Nrf2 activation was assessed. We generated seven Keap1 mutants (C151S, C273W, C288E, C151S/C273W, C151S/C288E, C273W/C288E, and C151S/C273W/C288E) harboring mutations in which Cys151, Cys273, and Cys288 were substituted to Ser, Trp, and Glu, respectively (Fig. 5A). Keap1 -/- MEFs were then expressed with wild-type Keap1 and its mutants by retrovirus infection. Not only wild-type Keap1, but also all eight Keap1 mutants maintained the ability to repress the accumulation of Nrf2 (Fig. 5B). Although 5-HI significantly induced the expression of Nrf2 in Keap1 -/- MEFs expressing wild-type Keap1, C273W, C288E, and C273W/C288E, it failed to induce the expression of Nrf2 in Keap1 -/- MEFs expressing 17

C151S, C151S/C273W, C151S/C288E, and C151S/C273W/C288E (Fig. 5C). In Keap1 -/- MEFs infected with an empty virus (-), although the expression of Nrf2 was detected, the treatment with 5-HI failed to induce the expression of HO-1 mRNA and NQO1 mRNA. In addition, 5-HI significantly induced the expression of HO-1 mRNA and NQO1 mRNA in Keap1 -/- MEFs expressing wild-type Keap1, C273W, C288E, and C273W/C288E. On the other hand, 5-HI failed to induce the expression of HO-1 mRNA and NQO1 mRNA in Keap1 -/- MEFs expressing C151S, C151S/C273W, C151S/C288E, and C151S/C273W/C288E (Fig. 5D). These results suggest that Cys151 in Keap1 is critical for the expression of Nrf2 in response to 5-HI. We also investigated the role of Keap1 in RAW264.7 cells using siRNA against Keap1. The knockdown of Keap1 significantly induced the expression of Nrf2 (Fig. 6A, B), however; the expression of HO-1 mRNA and NQO1 mRNA was slightly induced in RAW264.7 cells (Fig. 6C). In addition, although 5-HI drastically induced the expression of HO-1 mRNA and NQO1 mRNA in RAW264 cells transfected with control siRNA (si-control), it failed to induce their expression in RAW264 cells transfected with siRNA against Keap1(si-Keap1) (Fig. 6C). Therefore, it was suggested that Keap1 plays a critical for 5-HI-induced Nrf2 expression also in RAW264.7 cells.

3.4. 5-HI induces the transactivation of Nrf2 through the MKK3/6-p38 pathway. We also examined the effects of 5-HI on the activation of the MAP kinase family members, ERK, JNK, and p38, which are involved in promoting inflammation [31, 32]. Although 5-HI failed to induce the activation of ERK and JNK (data not shown), we found that 5-HI significantly induced the phosphorylation of p38. Furthermore, 5-HI induced the activation of MKK3/6, which are upstream molecules of p38 [33] (Fig. 7A). 18

In order to investigate the role of the activated MKK3/6-p38 pathway by 5-HI, RAW264.7 cells were pretreated with the p38 inhibitor, SB203580 prior to the treatment with 5-HI. SB203580 inhibited the 5-HI-induced phosphorylation of ATF-2, which is a substrate of p38, confirming that the 5-HI-induced activation of p38 was inhibited by the treatment with SB203580 (Fig. 7B). Although the induction of Nrf2 by 5-HI was not affected by the pretreatment with SB203580 (Fig. 7C), the pretreatment with SB203580 markedly inhibited the 5-HI-induced expression of the Nrf2 target genes, HO-1 and NQO1 (Fig. 7D). These results suggest that 5-HI induced not only the expression of Nrf2, but also its activation through the MKK3/6-p38 pathway. In order to further investigate the effects of the 5-HT-induced activation of the MKK3/6-p38 pathway on the transcriptional activation of Nrf2, we assessed the effects of 5-HI and SB203580 on Nrf2-dependent transactivation using the expression plasmid of a fusion protein of Nrf2 and the DNA-binding domain (DBD) of GAL4 transcription factor, named GAL4-Nrf2 (Fig. 8A). HEK293T cells and RAW264.7 cells were co-transfected with pCMV5-GAL4-Nrf2 and a reporter plasmid harboring the GAL4-responsive element (pFR-Luc). The overexpression of GAL4-Nrf2 induced strong transcriptional activity and SB203580 inhibited the transcriptional activation of GAL4-Nrf2 (Fig. 8B, C). Therefore, these results indicate that the 5-HI-induced activation of the MKK3/6-p38 pathway is required for the transactivation of Nrf2.

3.5. 5-HI prevented the generation of ROS in response to LPS. A previous study reported that LPS induced the generation of reactive oxygen species (ROS) [34]. Since we have shown that hydroxyoxindole and its derivatives exhibit potent antioxidant activities [28, 29], we investigated whether the anti-inflammatory 19

activity of 5-HT is mediated by its antioxidant activity. We examined the effects of 5-HI and the antioxidant reagent, N-acetyl cysteine (NAC) on LPS-induced ROS generation by detecting the oxidation of DCFH-DA (2′, 7′-dichlorodihydrofluorescein-DA). The treatment with 5-HI and NAC significantly prevented LPS-induced ROS generation (Fig. 9A). Although the LPS-induced expression of iNOS mRNA, production of NO, expression of CCL2 mRNA, and secretion of CCL2 were effectively inhibited by the treatment with 5-HI, the NAC treatment had no effects on these LPS-induced inflammatory responses (Fig. 9B-E). Therefore, 5-HI appears to exhibit anti-inflammatory activity independently of its own antioxidant activity.

3.6. 5-HI induced the activation of Nrf2 and inhibited the LPS-induced expression of cytokines and chemokines in C57BL/6 mice. We investigated whether 5-HI inhibits the LPS signaling pathway in vivo. C57BL/6 mice were injected with 5-HI (25 mg/kg, 50 mg/kg) prior to the injection of LPS. After 6 h, all mice exhibited no lethality, and we also observed no abnormal alterations in each organ of treated mice, such as necrosis, atrophy and swelling, suggesting that these dosage of 5-HI exhibited no cytotoxicity to the mice. The expression of Nrf2 protein in Kidneys, liver and lungs was determined by immunoblotting. The injection with 5-HI significantly increased the expression level of Nrf2 in all organs regardless the injection with LPS (Fig. 10A). The mRNA expression levels of Nrf2, HO-1, NQO1, CCL2, CXCL1, TNF and IL-6 in the kidneys (Fig. 10B, 11B), liver (Fig. 10C, 11C), and lungs (Fig. 10D, 11D) were evaluated by RT-PCR. Although the expression of Nrf2 mRNA was not altered by the injection of 5-HI and/or LPS, the expression levels of HO-1 mRNA and NQO1 mRNA in all organs were significantly increased by the 20

injection with 5-HI regardless of the LPS injection (Fig. 10B-D). These results suggest that 5-HI induced the activation of Nrf2 in vivo. Furthermore, the LPS-induced expression of CCL2 mRNA, CXCL1 mRNA, TNF mRNA, and IL-6 mRNA in the kidneys (Fig. 11A), liver (Fig. 11B), and lungs (Fig. 11C) was effectively inhibited by the injection with 5-HI in a dose-dependent manner. We further investigated anti-inflammatory effect of 5-HI on the LPS-induced production of serum cytokine and chemokines by giving 5-HI before and after the LPS treatment. The pre-administration of 5-HI significantly decreased serum levels of CCL2, CXCL1, TNF and IL-6 induced by LPS injection (Fig. 12A). Furthermore, when 5-HI (50 mg/mL) was administrated to the mice receiving LPS injection, the production of CCL2, CXCL1, TNF and IL-6 in serum was significantly reduced, although its inhibitory effect was weaker than when pretreated with 5-HI prior to LPS injection (Fig. 12B). Taken together, these results demonstrate that 5-HI significantly inhibits LPS-induced inflammatory responses in vitro and in vivo. In addition, it was suggested that 5-HI exhibits not only curative effect but also more effective prophylactic effect for LPS-induced inflammation.

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4. Discussion Previous studies have shown that a urinary metabolite of the indole, 5-hydroxyoxindole, exhibited antioxidant activity that was enhanced by the substitution of the 3-hydrogen group to a lipophilic functional group [28, 29]. In the present study, we investigated the anti-inflammatory activities of 5-hydroxyindole and 6 types of 5-hydroxyoxindole derivatives by evaluating their effects on LPS-induced NO production and CCL2 secretion and found that only the 5-hydroxyoxindole derivative harboring benzylidene group (5-HI) significantly prevented LPS-induced NO production and CCL2 secretion (Fig. 1C, D). In addition, among these 5-hydroxyoxindole derivatives, only 5-HI induced the expression of Nrf2 target genes, such as HO-1 and NQO-1, suggesting that it exhibited anti-inflammatory effects through the expression of Nrf2 (data not shown). The dissociation of Nrf2 from Keap1 is caused by alternations in the structure of Keap1 as a consequence of Keap1 cysteine thiol modifications [19-22]. In the present study, we demonstrated that Cys151 in Keap1 played a critical role as a sensor for 5-HI in order to induce the expression of Nrf2 utilizing Keap1-/- MEFs reconstituted with Keap1 mutants harboring substitutions in their major cysteine residues (Fig. 5). Since the chemical structure of 5-HI harbors an α, β-unsaturated ketone, 5-HI may interact with the thiol of the cysteine residue at 151 in Keap1 by a nucleophilic reaction (1, 4-addition reaction), indicating that this chemical modification of thiol in Cys151 stabilized Nrf2 (Fig. 13A). The nuclear expression level of Nrf2 in Keap1 -/- MEFs was similar to the 5-HI-induced nuclear expression level of Nrf2 in Keap1 -/- MEFs reconstituting Keap1 (Fig. 5C). In addition, the nuclear expression level of Nrf2 in the RAW264.7 cell that 22

Keap1 was knocked down was similar to the 5-HI-induced nuclear expression level of Nrf2 in RAW264.7 cells treated with 5-HI (Fig. 6B). Since the expression of Nrf2 induced by a loss and knockdown of Keap1 failed to induce the expression of HO-1 and NQO1 (Fig. 5D, 6C), some posttranslational modification to Nrf2 appeared to be necessary for its transcriptional activation. Previous studies reported that the activation of several kinases, such as phosphatidylinositol 3-kinase (PI3K)/Akt, mitogen-activated protein (MAP) kinases, and protein kinase C (PKC), regulates the nuclear translocation and transcriptional activation of Nrf2 [35-37]. Sulforaphane (SFN), a representative isothiocyanate present in broccoli, has been shown to induce the expression of Nrf2-target genes by activating PI3K/Akt- or MEK/ERK-mediated signaling in Caco-2 cells [38]. In addition, the activation of ERK and JNK was found to be involved in the SFN-induced nuclear localization of Nrf2 and ARE-transcription activities in PC3 prostate cancer cells [39]. On the other hand, SFN induced the expression of HO-1 in HepG2 cells by down-regulating p38 MAP kinase, indicating that p38 MAP kinase negatively regulates the activation of Nrf2 [40]. In this study, the overexpression of p38 MAP kinase promoted the interaction between Nrf2 and Keap1 and inhibited the nuclear localization of Nrf2 [41]. However, the glycomacropeptide hydrolysate (GHP)-induced nuclear localization of Nrf2 and expression of HO-1 were clearly inhibited by a specific inhibitor of p38 MAP kinase in RAW264.7 cells [42], and the role of p38 MAP kinase remains controversial. In the present study, we found that the 5-HI-induced activation of the MKK3/6-p38 MAP kinase pathway was required for the transactivation of Nrf2, but not its expression or nuclear localization (Fig. 7, 8). p38 MAPK isoforms have been shown to phosphorylate the purified Nrf2 protein [40], and the PKC-catalyzed phosphorylation of Nrf2 at Ser40 is a critical signaling event leading 23

to antioxidant responsive element (ARE) -mediated cellular antioxidant responses [42]. We observed the phosphorylation of Nrf2 at Ser40 in untreated RAW264.7 cells, and its phosphorylation status was not altered by the treatment with 5-HI and LPS, indicating that p38 MAP kinase phosphorylates different sites of Nrf2 from PKC. p38 and JNK are activated by common upstream MAPKKKs, such as transforming growth factor--activated protein kinase 1 (TAK1), apoptosis signal regulating kinase 1 (ASK1), and MEKK4 [43-45]. Since 5-HI failed to induce the activation of JNK, it currently remains unclear why 5-HI induced the activation of MAPKKK, which activates the p38 and JNK pathways. Therefore, further studies are needed in order to elucidate the mechanisms by which 5-HI induces the activation of MKK3/6. Inflammatory reactions induce the production of ROS, which have been reported to be involved in NF-B-mediated TNF secretion following an LPS stimulation in macrophages [34]. However, a treatment with a representative antioxidant NAC did not exhibit anti-inflammatory activity, suggesting that the 5-HI-induced expression of Nrf2 interfered with the LPS-induced transcriptional up-regulation of proinflammatory cytokines independently of its antioxidant functions (Fig. 9). These results are consistent with recent findings showing that Nrf2 binds to the proximity of the IL-6 gene independent of the Nrf2-binding motif and inhibits RNA Pol II recruitment [15]. In combination with these findings, the present results strongly suggest that Nrf2 functions as the upstream regulator of cytokine production and iNOS expression independently of its antioxidant activity. However, the production of ROS is known to mainly be caused by a dysfunction in mitochondria, and contributes to the initiation and progression of multiple neurological diseases, such as Parkinson’s disease. Since 5-HI harbors the ability to stimulate the p38-Nrf2 antioxidant response pathway, 5-HI has potential as a 24

neuroprotectant. We also investigated whether 5-HI inhibits the LPS signaling pathway in vivo by giving 5-HI to C57/BL6 mice. Previous reports showed that an intraperitoneal injection of 2.5 mg / kg in rats induces a decrease in brain monoamine oxidase (MAO) activity after 2 hr [26]. However, all mice exhibited no lethality, and no abnormal behaviors after the injection with 5-HI, suggesting that 5-HI exhibited no toxicity to the mice. Administration of 5-HI significantly decreased serum levels of cytokines and chemokines induced by LPS injection and mRNA expression of cytokines and chemokines induced by LPS in various organs, suggesting that 5-HI exhibits anti-inflammatory activity in vivo (Fig. 11, 12). Although 5-HI significantly inhibited the LPS-induced expression of various cytokines and chemokines in RAW264.7 cells and mice, their inhibitory effects were modest in both in vitro and in vivo. To effectively treat the LPS-induced inflammation, it is thought that 5-HI should be given more frequently or that the derivatives harboring more effective anti-inflammatory activity should be synthesized by utilizing 5-HI as a lead compound.

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Conflict of Interest Statement The authors have declared no conflict of interest.

Acknowledgment We thank Dr. M. Yamamoto (Tohoku University Graduate School of Medicine) for the gifted Keap1 -/- MEFs. This work was supported in part by grants from the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan (17K08286).

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Fig. 1 The effects of 5-hydroxyindole and 6 kinds of its derivatives on LPS-induced inflammatory responses. (A) The structures of 5-hydroxyoxindole (1) and 6 kinds of its derivatives (2-7). Compound 7 is named as 5-HI. (B-D) RAW264.7 cells were pretreated with DMSO (0.1%)s, 5-hydroxyoxindole or its derivatives (compounds 1-7) (20 M) for 1 h prior to the stimulation with LPS (1 g/mL) for 24 h. (B) Cell viability was assessed by the trypan blue exclusion method. (C) Nitrate concentrations in culture supernatants were measured using Griess reagent. Values are given as the mean ± S.D. of three independent experiments. ***P < 0.001 significantly different from control cells treated with LPS. (D) The amounts of CCL2 in supernatants were evaluated by ELISA. Values are given as the mean ± S.D. of three independent experiments. ***P < 0.001 significantly different from control cells treated with LPS.

Fig. 2 The 5-hydroxyoxindole derivative inhibits LPS-induced inflammatory responses. (A, B, D) RAW264.7 cells were pretreated with DMSO (0.1%) or 5-hydroxyoxindole derivative (5-HI) (2.5, 5, 10, 20 M) for 1 h prior to the stimulation with LPS (1 g/mL) for 24 h. (A) Nitrate concentrations in culture supernatants were measured using Griess reagent. Values are given as the mean ± S.D. of three independent experiments. (B) Cell viability was assessed by the trypan blue exclusion method. (C) RAW264.7 cells were pretreated with DMSO (0.1%) or 5-HI (2.5, 5, 10, and 20 M) for 1 h prior to the stimulation with LPS (1 g/mL) for 12 h. Total RNA was prepared and the expression of iNOS mRNA was assessed by RT-PCR. GAPDH mRNA was analyzed as an internal control. Values are the mean ± S.D. of three independent experiments. (D) 34

The amounts of CCL2 in supernatants were evaluated by ELISA. Values are given as the mean ± S.D. of three independent experiments. (E) RAW264.7 cells were pretreated with DMSO (0.1%) or 5-HI (2.5, 5, 10, and 20 M) for 1 h prior to the stimulation with LPS (1 g/mL) for 1.5 h. Total RNA was prepared and the expression of CCL2 mRNA was assessed by RT-PCR. GAPDH mRNA was analyzed as an internal control. Values are the mean ± S.D. of three independent experiments. *P <0.05, **P < 0.01, ***P <0.001 significantly different from control cells treated with LPS.

Fig. 3 The 5-hydroxyoxindole derivative induces the expression of Nrf2. (A, B, E) RAW264.7 cells were treated with the 5-hydroxyoxindole derivative (5-HI, 10 M) for the indicated periods. (A) Nuclear extracts were immunoblotted with an anti-Nrf2 or anti-Lamin B antibody. The relative expression levels of Nrf2 in the nucleus are shown in the graphs. Values are given as the mean ± SD of three independent experiments. **P < 0.01 significantly different from untreated control cells. (B) Total RNA was extracted and the expression of Nrf2 mRNA was assessed by RT-PCR. GAPDH mRNA was analyzed as an internal control. Values are the mean ± S.D. of three independent experiments. *P < 0.05 significantly different from untreated control cells. (C, D) RAW264.7 cells were transfected with control siRNA (si-control) or siRNA against Nrf2 (si-Nrf2). (C) Forty-eight hours after transfection, RAW264.7 cells were treated with 5-HI (10 M) for 1 h and cell lysates were immunoblotted with an anti-Nrf2 or anti-LaminB antibody. (D) Forty-eight hours after transfection, total RNAs were prepared from treated RAW264.7 cells with 5-HI (10 M) for 4 h. (D, E) Total RNA was extracted and the expression of HO-1 mRNA and NQO1 mRNA was assessed by RT-PCR. GAPDH mRNA was analyzed as an internal control. Values are 35

the mean ± S.D. of three independent experiments. *, **, and *** indicate P <0.05, P <0.01, and P <0.001, respectively.

Fig. 4 Knockdown of Nrf2 reduces inhibitory effects of the 5-hydroxyoxindole derivative on LPS-induced inflammatory responses. RAW264.7 cells were transfected with control siRNA (si-control) or siRNA against Nrf2 (si-Nrf2). (A) Forty-eight hours after transfection, RAW264.7 cells were pretreated with DMSO (0.1%) or the 5-hydroxyoxindole derivative (5-HI,10 M) for 1 h prior to the stimulation with LPS (1 g/mL) for 12 h. Total RNAs were prepared from treated RAW264.7 cells and the expression of iNOS mRNA was assessed by RT-PCR. GAPDH mRNA was analyzed as an internal control. Values are the mean ± S.D. of three independent experiments. (B, D) Forty-eight hours after transfection, RAW264.7 cells were pretreated with DMSO (0.1%) or 5-HI (10 M) for 1 h prior to the stimulation with LPS (1 g/mL) for 24 h. (B) Nitrate concentrations in culture supernatants were measured using Griess reagent. Values are given as the mean ± S.D. of three independent experiments. (C) Forty-eight hours after transfection, RAW264.7 cells were pretreated with DMSO (0.1%) or 5-HI (10 M) for 1 h prior to the stimulation with LPS (1 g/mL) for 1.5 h. Total RNA was prepared from treated RAW264.7 cells and the expression of CCL2 mRNA was assessed by RT-PCR. GAPDH mRNA was analyzed as an internal control. Values are the mean ± S.D. of three independent experiments. (D) The amounts of CCL2 in supernatants were evaluated by ELISA. Values are given as the mean ± S.D. of three independent experiments. *P < 0.05, *** P <0.001 significantly different from untreated control cells.

36

Fig. 5 The 5-hydroxyoxindole derivative induces the expression of Nrf2 through Cys151 in Keap1. (A) Scheme of Keap1. The cysteine residues, C151, C273, and C288 in Keap1 function as sensors for each Nrf2 inducer. (B-D) Keap1 -/- MEFS were infected with an empty virus (-) and retroviruses encoding Keap1 and its mutants (C151S, C273W, C288E, C151S/C273W, C151S/C288E, C273W/C288E, and C151S/C273W/C288E). (B) Whole cell lysates were immunoblotted with an anti-Flag, anti-Keap1, or anti--actin antibody. Nuclear extracts were immunoblotted with an anti-Nrf2 or anti-Lamin B antibody. The relative expression levels of Nrf2 in the nucleus are shown in the graph. Values are given as the mean ± SD of three independent experiments. *** indicates P < 0.001. (C) Transduced Keap1-/- MEFs were treated with the 5-hydroxyoxindole derivative (5-HI, 10 M) for 1 h. Nuclear extracts were prepared and immunoblotted with an anti-Nrf2 or anti-Lamin B antibody. The relative expression levels of Nrf2 in the nucleus are shown in the graph. Values are given as the mean ± SD of three independent experiments. *** indicates P < 0.001. (D) Transduced Keap1-/- MEFs were treated with 5-HI (10 M) for 4 h. Total RNA was extracted and the expression of HO-1 mRNA and NQO1 mRNA was assessed by RT-PCR. GAPDH mRNA was analyzed as an internal control. Values are the mean ± S.D. of three independent experiments. *** indicates P<0.001.

Fig. 6 Knockdown of Keap1 inhibits the 5-hydroxyoxindole-induced Nrf2 activation in RAW264.7 cells. RAW264.7 cells were transfected with control siRNA (si-control) or siRNA against Keap1 (si-Keap1). (A, B) Forty-eight hours after transfection, transfected RAW264.7 37

cells were treated with DMSO (0.1%) or the 5-hydroxyoxindole derivative (5-HI,10 M) for 1 h. (A) Whole cell lysates were immunoblotted with anti-Keap1 or anti--actin antibody. The relative expression levels of Keap1are shown in the graph. Values are given as the mean ± S.D. of three independent experiments. *** indicates P<0.001. (B) Nuclear extracts were immunoblotted with an anti-Nrf2 or anti-Lamin B antibody. The relative expression of Nrf2 in the nucleus is shown in the graph. Values are given as the mean ± SD of three independent experiments. *** indicates P<0.001. (C) Forty-eight hours after transfection, transfected RAW264.7 cells were treated with DMSO (0.1%) or the 5-hydroxyoxindole derivative (5-HI,10 M) for 4 h. Total RNAs were prepared and the expression of HO-1mRNA and NQO1 mRNA was assessed by RT-PCR. GAPDH mRNA was analyzed as an internal control. Values are the mean ± S.D. of three independent experiments. *P < 0.05, ***P<0.001 significantly different from control cells.

Fig. 7 The 5-Hydroxyoxindole derivative induces the activation of Nrf2 through the MKK3/6-p38 pathway. (A) RAW264.7 cells were treated with the 5-hydroxyoxindole derivative (5-HI, 10 M) for the indicated periods. Whole cell lysates were immunoblotted with an anti-phospho-MKK3/6, anti-MKK3/6, anti-phospho-p38, anti-p38, or anti--actin antibody. The relative phosphorylation levels of MKK3/6 and p38 are shown in the graphs. Values are given as the mean ± SD of three independent experiments. **P < 0.01, ***P<0.001 significantly different from untreated control cells. (B, C) RAW264.7 cells were treated with DMSO (0.1%) or SB203580 (10 M) for 1 h prior to the treatment with 5-HI (10 M) for 1 h. (B) Whole cell lysates were immunoblotted with 38

an anti-phospho-ATF2 or anti-ATF2 antibody. The relative phosphorylation levels of ATF2 are shown in the graph. Values are given as the mean ± SD of three independent experiments. ** indicates P < 0.01. (C) Nuclear extracts were immunoblotted with an anti-Nrf2 or anti-Lamin B antibody. The relative expression of Nrf2 in the nucleus is shown in the graph. Values are given as the mean ± SD of three independent experiments. *P < 0.05 significantly different from the control cells treated with DMSO. (D) RAW264.7 cells were treated with DMSO (0.1%) or SB203580 (10 M) for 1 h prior to the treatment with 5-HI (10 M) for 4 h. Total RNA was extracted and the expression of HO-1 mRNA and NQO1 mRNA was assessed by RT-PCR. GAPDH mRNA was analyzed as an internal control. Values are the mean ± S.D. of three independent experiments. ***indicates P<0.001.

Fig. 8 The 5-Hydroxyoxindole derivative induces the transactivation of Nrf2 through the MKK3/6-p38 pathway. (A) Scheme of GAL4-Nrf2. (B) HEK293T cells (5 × 105 cells) and (C) RAW264.7 cells (1 × 106 cells) were transfected with 1 μg of pFR-Luc, 1 μg of the MSCV-Puro empty vector or MSCV-Puro-GAL4-Nrf2, and 0.2 μg of pRL-TK. After 36 h, transfected cells were treated with 0.1% DMSO or SB203580 (10 M) for 1 h prior to the stimulation with the 5-hydroxyoxindole derivative (5-HI, 10 M) at 37 °C for 12 h. Luciferase activity was normalized to the activity of the constitutively expressed Renilla luciferase. Values are the mean ± S.D. of three independent experiments. * and ** indicate P < 0.05 and P< 0.01, respectively. Whole cell lysates were immunoblotted with an anti-Nrf2 or anti--actin antibody.

39

Fig. 9 The 5-hydroxyoxindole derivative reduces LPS-induced ROS generation. (A) RAW264.7 cells were pretreated with DMSO (0.1%), the 5-hydroxyoxindole derivative (5-HI, 10 M), or N-acetyl cysteine (NAC) (200 M) for 1 h prior to the stimulation with LPS (1 g/mL) for 4 h. Intracellular ROS levels were measured in a flow cytometric analysis using DCFH-DA. Values are given as the mean ± SD of three independent experiments. (B-E) RAW264.7 cells were pretreated with DMSO (0.1%), 5-HI (10 M), or NAC (200 M) for 1 h prior to the stimulation with LPS (1 g/mL) for 12 h (B), 24 h (C, E), and 1.5 h (D). (B, D) Total RNA was prepared and the expression of iNOS mRNA and CCL2 mRNA was assessed by RT-PCR. GAPDH mRNA was analyzed as an internal control. Values are the mean ± S.D. of three independent experiments. (C) Nitrate concentrations in culture supernatants were measured using Griess reagent. Values are given as the mean ± S.D. of three independent experiments. (E) The amounts of CCL2 in supernatants were evaluated by ELISA. Values are given as the mean ± S.D. of three independent experiments. ** and *** indicate P < 0.01 and ***p<0.001, respectively.

Fig. 10 Administration of the 5-hydroxyoxindole derivative increases the activation of Nrf2 in mice. (A) C57BL/6 mice (3 mice per group) were injected intraperitoneally with control vehicle (olive oil) or the 5-hydroxyoxindole derivative (5-HI, 25 and 50 mg/kg). After 1h, mice were injected intraperitoneally with LPS (300 g/mouse). Four hours after the LPS stimulation, homogenates of kidneys, liver and lungs were prepared and immunoblotted with anti-Nrf2 or anti--actin antibody. The relative expression levels of Nrf2 are shown in the graphs. Values are given as the mean ± SD of three independent 40

experiments. *P<0.05, **P < 0.01 significantly different from control group. (B-D) C57BL/6 mice (4 mice per group) were injected intraperitoneally with control vehicle (olive oil) or the 5-hydroxyoxindole derivative (5-HI, 25 and 50 mg/kg). After 1h, mice were intraperitoneally injected with LPS (300 g/mouse).Six hours after the LPS injection, total RNA was prepared from the kidneys (B), liver (C), and lungs (D). The mRNA expression of Nrf2, HO-1, and NQO1 was assessed by RT-PCR. GAPDH mRNA was analyzed as an internal control. Values are the mean ± S.D. of four independent experiments. *, **, and *** indicate P<0.05, P<0.01, and P<0.001, respectively.

Fig. 11 Administration of the 5-hydroxyoxindole derivative significantly prevents the LPS-induced expression of CCL2, CXCL1, TNF, and IL-6 in mice. C57BL/6 mice (4 mice per group) were injected intraperitoneally with control vehicle (olive oil) or the 5-hydroxyoxindole derivative (5-HI, 25 and 50 mg/kg) or prior to the intraperitoneal injection of LPS (300 g/mouse). Six hours after the LPS injection, total RNA was prepared from the kidneys (A), liver (B), and lungs (C). The mRNA expression of CCL2, CXCL1, TNF, and IL-6 was assessed by RT-PCR. GAPDH mRNA was analyzed as an internal control. Values are the mean ± S.D. of four independent experiments. *, **, and *** indicate P<0.05, P<0.01, and P<0.001, respectively.

Fig. 12 Administration of the 5-hydroxyoxindole derivative significantly reduced LPS-induced serum level of CCL2, CXCL1, TNF, and IL-6 in mice. (A) C57BL/6 mice (6 mice per group) were injected intraperitoneally with control 41

vehicle (olive oil) or the 5-hydroxyoxindole derivative (5-HI, 25 and 50 mg/kg) or prior to the intraperitoneal injection of LPS (300 g/mouse). Two hours after the LPS injection, blood samples from mice were collected by cardiac puncture. (B) C57BL/6 mice (6 mice per group) were intraperitoneally injected with LPS (300 g/mouse). One hour after injection of LPS, the mice were intraperitoneally injected with control vehicle (olive oil) or the 5-hydroxyoxindole derivative (5-HI, 25 and 50 mg/kg). One hour later, blood samples from mice were collected by cardiac puncture. (A, B) The amounts of CCL2, CXCL1, TNF, and IL-6 in murine serum were measured by ELISA. *, **, and *** indicate P<0.05, P<0.01, and P<0.001, respectively.

Fig. 13 Inhibitory effects of the 5-hydroxyoxindole derivative on LPS-induced inflammatory responses via the activation of Nrf2. (A) The chemical model of reaction between Keap1 and 5-hydroxyoxindole derivative (5-HI). (B) The 5-hydroxyoxindole derivative (5-HI)-induced expression of Nrf2 through the cysteine residue at 151 in Keap1. 5-HI also induced the transactivation of Nrf2 through the MKK3/6-p38 pathway. As a result, 5-HI inhibited the LPS-induced expression of iNOS and CCL2, leading to the inhibition of LPS-induced inflammatory responses.

42

Figure 1

Fig. 1 A 1

2

3

4

6

7

5

5-HI

C

***P<0.001

120

160

100

140

NO (µM)

Viability (%)

B

80 60

40

120 100 80 60

***

40 20

LPS

0

Compound

CCL2 (ng/mL)

D

20

-+

-+

-+

-+

-+

-+

-+

-+

(-)

1

2

3

4

5

6

7

***P<0.001 140 120 100 80 60

***

40 20

LPS

0

Compound

-+

-+

-+

-+

-+

-+

-+

-+

(-)

1

2

3

4

5

6

7

LPS

0

Compound

-+

-+

-+

-+

-+

-+

-+

-+

(-)

1

2

3

4

5

6

7

Figure 2

Fig. 2

*P<0.05 **P<0.01 ***P<0.001

B

160

NO (µM)

*

120

**

100 80

***

60

***

40

Viability (%)

120

140

100 80 60 40 20

20 0

LPS 5-HI (mM)

C Relative expression of iNOS mRNA

A

-+

-+

-+

-+

-+

0

2.5

5

10

20

D

0

LPS 5-HI (mM)

-+

-+

-+

-+

0

2.5

5

10

20

E

*

80

**

60

***

40 20 0

LPS 5-HI (mM)

-+

-+

-+

-+

-+

0

2.5

5

10

20

**P<0.01 ***P<0.001

Relative expression of CCL2 mRNA

CCL2 (ng/mL)

100

500 400 300

**

200

*** 100

0

LPS 5-HI (mM)

12 10

*

8

**

6

***

4 2 0

-+

*P<0.05 **P<0.01 ***P<0.001 120

*P<0.05 **P<0.01 ***P<0.001 14

***

-+

-+

-+

-+

-+

0

2.5

5

10

20

LPS 5-HI (mM)

-+

-+

-+

-+

-+

0

2.5

5

10

20

Figure 3

5-HI (h)

0

0.5

1

Fig. 3

B

2

*P<0.05

Relative expression of Nrf2 mRNA

A

IB: Nrf2

**P<0.01

6

**

**

**

4

1

*

0.5

0 5-HI (hr)

0

0.5

1

2

2

0 5-HI (hr)

0

0.5

1

2

C

D si-control si-Nrf2 -

5-HI

-

+

***P<0.001

Relative expression of HO-1 mRNA

Relative expression of the Nrf2 protein (Nrf2/Lamin B)

IB: Lamin B

1.5

+

IB: Nrf2 IB: Lamin B

8

***

6 4 2 0

-

5-HI

+

-

si-control

+ si-Nrf2

Relative expression of NQO1 mRNA

Relative expression of Nrf2 protein (Nrf2/Lamin B)

**P<0.01, ***P<0.001

**

4 3 2 1 0

-

5-HI

*** -

+

4

***

3 2 1 0

-

5-HI

+

+

-

si-control si-control

+ si-Nrf2

si-Nrf2

E 4

***

3 ***

2

***

1

0 5-HI (h)

0

4

8

12

Relative expression of NQO1 mRNA

Relative expression of HO-1 mRNA

*P<0.05, **P<0.01, ***P<0.001

8 6

*

***

**

4

8

4 2

0 5-HI (h)

0

12

Figure 4

Fig. 4

A 20

B

***P<0.001

***

***

250

***

15

***

300

NO (µM)

Relative expression of iNOS mRNA

***P<0.001

10

200 150 100

5 50 0

5-HI LPS

-

+

-

+

-

+ si-control

+ -

-

+

+ si-Nrf2

C

0

5-HI LPS

-

+

-

+

-

+ si-control

+ -

-

+

+ si-Nrf2

D ***P<0.001 250

*

600

CCL2 (ng/mL)

Relative expression of CCL2 mRNA

*P<0.05, ***P<0.001 700

500

**

400 300 200 100 0

5-HI LPS

***

200

***

150 100 50 0

-

+

-

+

+ si-control

-

+ -

-

+

+ si-Nrf2

5-HI LPS

-

+

-

+

+ si-control

-

+ -

-

+

+ si-Nrf2

A

S151

W273

E288

C151

C273

C288

Flag-

BTB

Fig. 5 DGR/Kelch

IVR

Cul3 binding Dimerization

Nrf2 binding

B

C151S/C273W/C288E

C273W/C288E

C151S/C288E

C151S/C273W

C288E

C273W

C151S

(-)

Whole cell lysate

Keap1

Keap1-/- MEF

IB: Flag IB: Keap1 IB: b-actin Nuclear extract IB: Nrf2 IB: Lamin B

1.2 ***P<0.001

1 0.8 0.6

***

***

***

*** ***

C288E

C151S/C273W

C151S/C288E

***

C273W

***

C151S

0.2

Keap1

0.4

***

Keap1-/- MEF

C151S/C273W/ C288E

C273W/C288E

0

(-)

Relative expression of Nrf2 (Nrf2/Lamin B)

Figure 5

C

Keap1-/- MEF Keap1 C151S C273W C288E

(-)

5-HI

-

+

-

+

-

+

-

+

-

C151S C151S C151S C273W C273W C273W C288E C288E C288E

+

-

+

-

+

-

+

IB: Nrf2

1.5 ***P<0.001

1 0.5

C288E

- +

- +

***

***

- +

***

***

4 2

- +

- +

- +

- +

- +

- +

C288E

C151S C273W

C151S C288E

*** - +

*** - + C151S C273W C288E

- +

C273W

***

C273W C288E

***

***

C151S

***

Keap1

***

(-)

***

***P<0.001

5 ***

4

***

***

***

3 2 ***

- +

- +

- +

- +

- +

- +

- +

- + C273W C288E

*** C151S C288E

*** C151S C273W

*** C288E

*** C273W

*** C151S

*** Keap1

0 5-HI

*** - + C151S C273W C288E

1

(-)

Relative expression of HO-1 mRNA

- +

*** *** C151S C273W C288E

- +

C273W C288E

- +

C151S C288E

- +

C151S C273W

- +

*** *** *** *** ***

***P<0.001

6

0 5-HI

Relative expression of NQO1 mRNA

D

***

C273W

- +

*** *** *** C151S

*** Keap1

0 5-HI

(-)

Relative expression of Nrf2 (Nrf2/Lamin B)

IB: Lamin B

-

+

Figure 6

Fig. 6

A

B

si-control si-Keap1 -

5-HI

-

+

si-control si-Keap1

+

-

5-HI

IB: Keap1

IB: Nrf2

IB: b-actin

IB: Lamin B

1

***

***

-

+

0

-

5-HI

+

+

***P<0.001

Relative expression of Nrf2 protein (Nrf2/Lamin B)

Relative expression of Keap1 protein (Keap1/b-action)

***P<0.001 1.5

0.5

-

+

6

***

***

-

+

***

4 2 0

-

5-HI

si-control si-Keap1

+

si-control si-Keap1

*P<0.05, ***P<0.001 8

***

6

n.s.

4

*

2

*

0

5-HI

-

+

-

+

si-control si-Keap1

*P<0.05, ***P<0.001

Relative expression of NQO1 mRNA

Relative expression of HO-1 mRNA

C 5

***

4

n.s.

3 2

*

*

-

+

1 0

5-HI

-

+

si-control si-Keap1

A 5-HI (min)

0

15 30 60 120 180

IB: p-MKK3/6 IB: MKK3/6

Relative phosphorylation of MKK3/6 (p-MKK3/6/pMKK3/6)

Figure 7

Relative phosphorylation of p38 (p-p38/p38)

IB: p38 IB: b-actin

-

-

+

0

0 15 30 60 120 180 **P<0.01, ***P<0.001

80

***

60 40

0

0 15 30* 60** 120 180

DMSO SB203580 5-HI

+ IB: Nrf2

IB: ATF2

IB: Lamin B Relative expression of Nrf2 (Nrf2/Lamin B)

Relative phosphorylation of ATF2 (p-ATF2/ATF2)

**P<0.01

** **

4 3 2 1 0

5-HI

-

-

+

+

Relative expression of NQO1 mRNA

Relative expression of HO-1 mRNA

***

1.5 1 0.5

5-HI

-

+

DMSO

-

-

+

+

*P<0.05

6

*

*

4 2 0

-

+

-

+

DMSO SB203580

D

0

-

5-HI

DMSO SB203580

2

**

***

20

IB: p-ATF2

5

**

**

10

C

DMSO SB203580 5-HI

*** ***

20

5-HI (min)

B

**

30

5-HI (min)

IB: p-p38

Fig. 7

**P<0.01, ***P<0.001

40

+

SB203580

4

***

***P<0.001

3 2 1 0

5-HI

-

+

DMSO

-

+

SB203580

Figure 8

Fig. 8

A

GAL4-Nrf2 1

147 154 (1)

GAL4 DBD

758 (605) aa

Nrf2 Basic motif Leucin-Zipper (499-518) (522-529)

Luciferase activity (Fold induction)

B

**P<0.01 ** **

10 8 6

4 2 0

-

SB203580

+

-

-

5-HI

+

-

+

+

-

-

MSCV

+ +

MSCV-GAL4-Nrf2

IB: Nrf2

GAL4-Nrf2

IB: b-actin

Luciferase activity (Fold induction)

C

*P<0.05

10

SB203580 5-HI

*

8

* 6 4 2 0

-

+

-

-

+ +

MSCV

-

+ -

-

+ +

MSCV-GAL4-Nrf2

Figure 9

Fig. 9 A

***P<0.001

NAC+LPS

Mean fluorescence

100

5-HI+LPS

DMSO

DMSO+LPS

80 60 40 20 0

- + -

LPS

***

***

- + 5-HI

- + NAC

ROS generation

C **P<0.01

**

10

***P<0.001

***

150

8 NO (µM)

Relative expression of iNOS mRNA

B

6 4 2

50 0

0

LPS

100

- +

- +

- +

-

5-HI

NAC

- +

- +

- +

-

5-HI

NAC

E ***P<0.001

40 *** 30 20

10 0 LPS

- + -

- + 5-HI

- + NAC

***P<0.001

***

80 CCL2 (ng/mL)

Relative expression of CCL2 mRNA

D

LPS

60 40 20 0 LPS

- + -

- + 5-HI

- + NAC

Figure 10

A

Fig. 10

Kidneys LPS 5-HI (mg/kg)

+

25

0

50

25

0

50

IB: Nrf2 IB: b-actin

Relative expression of Nrf2 (Nrf2/b-actin)

Kidneys 3

* 2 1 0

5-HI (mg/kg)

0

5-HI (mg/kg)

+

0

25

50

0

25

50

IB: Nrf2

Relative expression of Nrf2 (Nrf2/b-actin)

LPS

25

50

+

Liver *

4

**

3

**

**

2 1

5-HI (mg/kg)

LPS

+

0

25

50

0

25

50

Relative expression of Nrf2 (Nrf2/b-actin)

Lungs

IB: b-actin

0

5

0

25

50

0

-

LPS

IB: Nrf2

50

0

IB: b-actin

5-HI (mg/kg)

25 -

LPS

Liver

*

25

50

+

Lungs 4

*

*

3 2 1 0

5-HI (mg/kg) LPS

0

25 -

50

0

25 +

50

*P<0.05 **P<0.01

Kidneys HO-1 Relative expression

Relative expression

5

1 0.5 0

2

**

1

-

+

0 25 50

-

LPS

6

**

**

*P<0.05 **P<0.01 ***P<0.001

4 2

**

*

0

5-HI (mg/kg) 0 25 50

+

5-HI (mg/kg) 0 25 50

0 25 50

-

LPS

+

Liver Nrf2

HO-1

1 0.5 0

-

2

***

***

3

*

*

1

+

0 25 50

-

LPS

Nrf2

2

5

1.5

4

1 0.5 0

3

2

+

*P<0.05 **P<0.01 ***P<0.001

***

1

+

5-HI (mg/kg) 0 25 50

+

NQO1 **

*** **

0 25 50

-

LPS

*

1

5

0

5-HI (mg/kg) 0 25 50 0 25 50

***

*

HO-1

2

-

** 3

0

5-HI (mg/kg) 0 25 50

Relative expression

Relative expression

Lungs

LPS

4

0

5-HI (mg/kg) 0 25 50 0 25 50 LPS

NQO1

4

Relative expression

Relative expression

1.5

D

***

Relative expression

C

3

0

5-HI (mg/kg) 0 25 50 0 25 50 LPS

***

***

4

NQO1

8

Relative expression

Nrf2 1.5

Relative expression

B

***

4 3

*

** *

2 1 0

5-HI (mg/kg)

0 25 50

LPS

-

0 25 50 +

5-HI (mg/kg) 0 25 50 LPS

-

0 25 50 +

*P<0.05 **P<0.01 ***P<0.001

Figure 11

*

300

***

200 100

5-HI (mg/kg) 0 25 50 0 25 50 + LPS

40

* 20

**

0

IL-6

300 250 200 150 100 50 0

*

*P<0.05 **P<0.01 ***P<0.001

Liver CXCL1

***

0

***

200

***

0

5-HI (mg/kg) 0 25 50 0 25 50 + LPS

30

60

*

40

**

20

Relative expression

500

400

80

Relative expression

**

IL-6

TNFa

600

Relative expression

0

20

* 10

*P<0.05 **P<0.01 ***P<0.001

0

5-HI (mg/kg) 0 25 50 0 25 50 5-HI (mg/kg) 0 25 50 0 25 50 5-HI (mg/kg) 0 25 50 0 25 50 + + + LPS LPS LPS

Lungs CXCL1

400

** 200 0

5-HI (mg/kg) 0 25 50 0 25 50 + LPS

Relative expression

800

30

600 400 200 0

** ***

20 10 0

IL-6

TNFa

200

* ***

Relative expression

CCL2 600

Relative expression

Relative expression

**

200

CCL2

Relative expression

*

TNFa

5-HI (mg/kg) 0 25 50 0 25 50 5-HI (mg/kg) 0 25 50 0 25 50 5-HI (mg/kg) 0 25 50 0 25 50 + + + LPS LPS LPS

1000

C

60

0

0

B

400

CXCL1

Relative expression

600

Relative expression

CCL2

400

Relative expression

Fig. 11

Kidneys

Relative expression

A

150 100

**

50 0

5-HI (mg/kg) 0 25 50 0 25 50 5-HI (mg/kg) 0 25 50 0 25 50 5-HI (mg/kg) 0 25 50 0 25 50 + + + LPS LPS LPS

*P<0.05 **P<0.01 ***P<0.001

Figure 12

Fig. 12

***

20 0

200

1500

** ***

100 0

1000 500 0

5-HI (mg/kg) 0 25 50 0 25 50 + LPS

5-HI (mg/kg) 0 25 50 0 25 50 + LPS

20 0

5-HI (mg/kg) 0 25 50 0 25 50 + LPS

*P<0.05 **P<0.01

1000

300

** 500

0

5-HI (mg/kg) 0 25 50 0 25 50 + LPS

200

1500

*

100 0

5-HI (mg/kg) 0 25 50 0 25 50 + LPS

IL-6 (pg/mL)

*

** ***

(Post-treatment with 5-HI)

60

CCL2 (ng/mL)

500

***

5-HI (mg/kg) 0 25 50 0 25 50 + LPS

CXCL1 (pg/mL)

Serum

40

**

0

5-HI (mg/kg) 0 25 50 0 25 50 + LPS

B

300

IL-6 (pg/mL)

*

1000

TNFa (pg/mL)

60 40

*P<0.05 **P<0.01 ***P<0.001

(Pretreatment with 5-HI)

TNFa (pg/mL)

CCL2 (ng/mL)

Serum

CXCL1 (pg/mL)

A

1000

**

500 0

5-HI (mg/kg) 0 25 50 0 25 50 + LPS

Figure 13

Fig. 13 A

B

LPS

5-HI Macrophage

TLR4 Keap1

P

MKK3/6

Cys151

Cys151

Keap1 p38

Nrf2 Ub Ub Ub

Degradation

Nrf2 Expression

Activation

NO CCL2

Transactivation NF-kB

P

iNOS mRNA CCL2 mRNA

Inflammation

Table 1. Nrf2

PCR primer sequences 5’-CCAGAAGCCACACTGACAGA-3’ (upstream) 5’-GGAGAGGATGCTGCTGAAAG-3’ (downstream)

HO-1

5’-CACGCATATACCCGCTACCT-3’ (upstream) 5’-CCAGAGTGTTCATTCGAGCA-3’ (downstream)

NQO1

5’-TTCTCTGGCCGATTCAGAGT-3’ (upstream) 5’-GGCTGCTTGGAGCAAAATAG-3’ (downstream)

iNOS

5′-TCTGCGCCTTTGCTCATGAC-3′ (upstream) 5′-TAAAGGCTCCGGGCTCTG-3′ (downstream)

TNFα

5′-TACTGAACTTCGG GGTGATCGGTCC-3′ (upstream) 5′-CAGCCTTGTCCCTTGAAGAGAACC-3′ (downstream)

IL-6

5′-CCAGAGATACAAAGAAATGATGG-3′ (upstream) 5′-ACTCCAGAAGACCAGAGGAAAT-3′ 43

(downstream) CCL2

5′-TGAGGTGGTTGTGGAAAAGG-3′ (upstream) 5′-CCTGCTGTTCACAGTTGCC-3′ (downstream)

CXCL1

5′-TGGGGACACCTTTTAGCATC-3′ (upstream) 5′-GCCCATCGCCAATGAGCTG-3′ (downstream)

GAPDH

5′-ACTCCACTCACGGCAAATTC-3′ (upstream) 5′-CCTTCCACAATGCCAAAGTT-3′ (downstream)

44

LPS

5-HI Macrophage

TLR4 Keap1

P

MKK3/6

Cys151

Cys151

Keap1 p38

Nrf2 Ub Ub Ub

Degradation

Nrf2 Expression

Inflammation

Activation

NO CCL2

Transactivation NF-kB

P

iNOS mRNA CCL2 mRNA