Anti-inflammatory mechanism of galangin in lipopolysaccharide-stimulated microglia: Critical role of PPAR-γ signaling pathway

Accepted Manuscript Anti-inflammatory mechanism of galangin in lipopolysaccharide-stimulated microglia: critical role of PPAR-γ signaling pathway Min-Ji Choi, Eun-Jung Lee, Jin-Sun Park, Su-Nam Kim, Eun-Mi Park, Hee-Sun Kim PII: DOI: Reference:

S0006-2952(17)30509-9 http://dx.doi.org/10.1016/j.bcp.2017.07.021 BCP 12884

To appear in:

Biochemical Pharmacology

Received Date: Accepted Date:

29 May 2017 25 July 2017

Please cite this article as: M-J. Choi, E-J. Lee, J-S. Park, S-N. Kim, E-M. Park, H-S. Kim, Anti-inflammatory mechanism of galangin in lipopolysaccharide-stimulated microglia: critical role of PPAR-γ signaling pathway, Biochemical Pharmacology (2017), doi: http://dx.doi.org/10.1016/j.bcp.2017.07.021

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Anti-inflammatory mechanism of galangin in lipopolysaccharide-stimulated microglia: critical role of PPAR-γγ signaling pathway Min-Ji Choi1, Eun-Jung Lee1, Jin-Sun Park1, Su-Nam Kim2, Eun-Mi Park3, Hee-Sun Kim1,* 1

Department of Molecular Medicine, Tissue Injury Defense Research Center, School of

Medicine, Ewha Womans University, Seoul, South Korea, 2Natural Products Research Institute, Korea Institute of Science and Technology, Ganneung, South Korea, 3Department of Pharmacology, Tissue Injury Defense Research Center, School of Medicine, Ewha Womans University, Seoul, South Korea

Running title: Anti-inflammatory mechanism of galangin in microglia

Category: Inflammation and Immunopharmacology *

Correspondence to: Hee-Sun Kim, Department of Molecular Medicine, School of Medicine,

Ewha Womans University, Mok-6-dong 911-1, Yangchun-Ku, Seoul 158-710, South Korea Tel: 82-2-2650-5823 Fax: 82-2-2653-8891 Email: hskimp@ ewha.ac.kr

Abbreviations AD, Alzheimer’s disease; ARE, antioxidant enzyme response element; COX-2, cyclooxygenase-2; CRE, cAMP response element; EMSA, electrophoretic mobility shift assay; H2DCFDA, 2',7'-dichlorohydrofluorescein diacetate; HO-1, hemeoxygenase-1; Iba-1, ionized calcium binding adaptor molecule 1; IL, interleukin; iNOS, inducible nitric oxide synthase; JNK, c-Jun N-terminal kinase; LPS, lipopolysaccharide; MAPK, mitogen-activated protein kinase; NF-κB, nuclear factor-κB; NO, nitric oxide; PD, Parkinson’s disease; PPAR, peroxisome proliferator-activated receptor; PPRE, peroxisome proliferator response element; ROS, reactive oxygen species; TLR, toll-like receptor; TNF, tumor necrosis factor 1

ABSTRACT Since microglia-associated neuroinflammation plays a pivotal role in the progression of neurodegenerative diseases, controlling microglial activation has been suggested as a potential therapeutic strategy. Here, we investigated the anti-inflammatory effects of galangin (3,5,7-trihydroxyflavone) in microglia and analyzed the underlying molecular mechanisms. Galangin inhibited the expression of inducible nitric oxide synthase (iNOS) and proinflammatory cytokines and enhanced the expression of anti-inflammatory interleukin (IL)10 in lipopolysaccharide (LPS)-stimulated BV2 microglia. Galangin also suppressed microglial activation and the expression of pro-inflammatory markers in LPS-injected mouse brains. The results of mechanistic studies have shown that galangin inhibited LPS-induced phosphorylation of p38 mitogen activated protein kinase (MAPK), c-Jun N-terminal kinase (JNK), phosphatidylinositol 3-kinase (PI3K)/Akt, and nuclear factor (NF)-κB activity. On the contrary, galangin increased the activity of transcription factors, such as nuclear factor-E2related factor 2 (Nrf2), cAMP response element-binding protein (CREB), and peroxisome proliferator-activated receptor (PPAR)-γ, known to play an anti-inflammatory role. In addition, galangin showed antioxidant effects by suppressing the expression of NADPH oxidase subunits p47 phox and gp91phox, and by enhancing hemeoxygenase-1. We then investigated whether PPAR-γ was involved in the anti-inflammatory function of galangin. Pretreatment with a PPAR-γ antagonist or siRNA significantly blocked galangin-mediated upregulation of IL-10 and attenuated the inhibition of tumor necrosis factor (TNF)-α, nitric oxide (NO), and IL-6 in LPS-stimulated microglia. Moreover, the PPAR-γ antagonist reversed the effects of galangin on NF-κB, Nrf2, and CREB. Altogether, our data suggest that PPAR-γ plays a key role in mediating the anti-inflammatory effects of galangin by modulating the NF-κB and Nrf2/CREB signaling pathways. 2

Keywords: Galangin, Neuroinflammation, PPAR-γ, NF-κB, Nrf2/CREB signaling

Chemical copmpounds cited in this article: Galangin (PubChem CID: 5281616) Lipopolysaccharide (PubChem CID: 53481793) T0070907 (PubChem CID: 2777391) H2DCFDA (PubChem CID: 77718) DMSO (PubChem CID: 679) Avertin (PubChem CID: 6400) Paraformaldehyde (PubChem CID: 712) 3,3'-Diaminobenzidine tetrahydrochloride (PubChem CID: 23892) Triton X-100 (PubChem CID: 5590) Sodium deoxycholate (PubChem CID: 23668196)

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1. Introduction Microglia are resident immune cells of the brain parenchyma, and play an important role in the phagocytosis of apoptotic cells and neuronal synapses, the regulation of neurogenesis, and the support of neuronal survival during development [1, 2]. Microglia are activated by various forms of stress or brain injury, and produce pro-inflammatory mediators, such as tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), nitric oxide (NO), and reactive oxygen species (ROS), which can exacerbate brain injury [3, 4]. If acute inflammatory state switches to the resolution/anti-inflammatory state, it leads to tissue repair and homeostasis [4, 5]. However, a prolonged and unresolved inflammatory response causes destructive chronic inflammation that results in neuronal cell death and, ultimately, the onset of neurodegenerative diseases [6, 7]. Thus, the development of agents that control microglial activation has been suggested as an important therapeutic strategy for neurodegenerative diseases such as Alzheimer’s disease (AD) and Parkinson’s disease (PD). Peroxisome proliferator-activated receptors (PPARs) belong to the nuclear receptor family of ligand-inducible transcription factors that regulate genes involved in adipogenesis, inflammation, lipid metabolism, mitochondrial biogenesis, and maintenance of metabolic homeostasis [8, 9]. The PPAR receptors can be subdivided into three groups: alpha, beta/delta, and gamma [10]. Among these, PPAR-γ usually forms heterodimers with the retinoid X receptors (RXRs) and binds to peroxisome proliferator response elements (PPREs) at the promoter regions of specific target genes, regulating their transcription [11, 12]. Numerous reports postulate that activation of PPAR signaling, particularly that of PPAR-α and PPAR-γ, can suppress the inflammatory response through inhibition of the NF-κB pathway [13, 14]. In particular, PPAR-γ has been recognized as a pivotal anti-inflammatory regulator that functions primarily by regulating macrophage differentiation and functional polarization [8, 15]. In the brain, PPAR-γ reduces inflammatory responses in microglia and astrocytes and 4

blocks amyloidogenic pathways [14, 16]. Moreover, PPAR-γ exerts neuroprotective effects by inducing neuronal differentiation and neurite outgrowth [17]. Based on these findings, PPAR-γ has been proposed as a promising therapeutic target for the treatment of neurodegenerative diseases. Galangin (3,5,7-trihydroxyflavone) is a polyphenolic compound abundant in honey and medicinal herbs, such as Alpinia officinarum [18, 19]. Several studies have reported the beneficial effects of galangin, including anti-oxidant, anti-mutagenic, anti-tumor, antiinflammatory, anti-microbial, and anti-viral activities in vitro and in vivo [19-24]. Specifically, the anti-inflammatory effects of galangin have been reported in animal models of arthritis, asthma, paw edema, and acute lung injury [19, 25-27]. Additionally, the anti-proliferative or anti-metastatic effects of galangin have been demonstrated in human leukemia, breast cancer, and hepatoma cell lines [18, 22, 28]. Galangin also showed neuroprotective effects in focal cerebral ischemia by improving cortical blood flow and attenuating mitochondrial dysfunction and apoptosis [29]. Moreover, galangin suppressed acetylcholinesterase activity and the expression of β-site amyloid precursor protein-cleaving enzyme-1 (BACE1) in neuronal cells, suggesting a potentially beneficial use of galangin for the treatment of AD [30, 31]. Although many papers have described the pharmacological activities of galangin, the effects of galangin on neuroinflammation have not been investigated. Therefore, in the present study, we examined the effects of galangin under neuroinflammatory conditions in vitro and in vivo and analyzed the underlying molecular mechanisms in detail. Interestingly, we found that PPAR-γ plays a critical role in mediating the anti-inflammatory effects of galangin in activated microglia.

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2. Materials and methods 2.1. Materials Galangin (3,5,7-trihydroxyflavone), LPS (Escherichia coli serotype 055:B5), and PPARγ antagonist (T0070907) were obtained from Sigma-Aldrich (St. Louis, MO). All reagents used for cell culture were purchased from Welgene (Gyeongsan, Korea). Antibodies against iNOS, COX-2, TNF-α, and HO-1 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Antibodies for IL-10 or ionized calcium binding adaptor molecule 1 (Iba1) were purchased from Abcam (Cambridge, UK). Antibodies against phospho-/total forms of p38, JNK/SARK, ERK1/2, and Akt were supplied by Cell Signaling Technology (Beverley, CA). Trizol reagent was obtained from Thermo-Scientific (Waltham, MA). The chemical structure of galangin is shown in Fig. 1.

2.2. Microglial cell culture The immortalized mouse BV2 microglial cell line [32] was grown and maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% heat-inactivated fetal bovine serum, streptomycin (10 µg/mL), and penicillin (10 U/mL) at 37 °C under 5% CO2.

2.3. Measurement of nitric oxide, cytokine, and intracellular ROS levels BV2 cells (1 x 105 cells per well in a 24-well plate) were pre-treated with galangin for 1 h and stimulated with LPS (100 ng/mL). After 16 h, the supernatants were collected and the accumulated nitrite was measured using the Griess reagent (Promega). The concentrations of TNF-α, IL-6, and IL-10 in the supernatants were measured by ELISA, according to the procedure recommended by the supplier (BD Biosciences, San Jose, CA). The intracellular accumulation of ROS was measured with 2',7'-dichlorohydrofluorescein diacetate (H2DCFDA) (Sigma-Aldrich). In brief, microglia were stimulated with LPS for 16 h and stained with 6

20 mM H2DCF-DA in PBS for 1 h at 37 °C. DCF fluorescence intensity was measured at 485-nm excitation and 535-nm emission, using a fluorescence plate reader (Molecular Devices, Sunnyvale, CA).

2.4. Mice Adult male ICR mice (Mus musculus, 28-32 g, 7 weeks old) were purchased from Samtako Inc. (Osan, Korea). All animal experiments were approved by the Institutional Animal Care and Research Ethics Committee at the School of Medicine, Ewha Womans University (IACUC #2014-0274), and were carried out in accordance with the guidelines of the National Institute of Heath’s Guide for the Care and Use of Laboratory Animals. All efforts were made to minimize animal suffering and the number of animals used in the study.

2.5. LPS-induced inflammation and administration of galangin LPS (5 mg/kg) was administered intraperitoneally (i.p.) to induce neuroinflammation in male ICR mice, as previously described [33]. Galangin (50 mg/kg) dissolved in vehicle solution (normal saline containing 1% dimethylsulfoxide; Sigma-Aldrich), was administered daily (i.p) for 4 days before the LPS treatment. The brains were obtained 3 h after LPS administration and analyzed for microglial activation and expression of inflammatory markers.

2.6. Immunohistochemistry Three hours after the LPS treatment, mice were anesthetized with avertin (500 mg/kg) (Sigma-Aldrich) and then perfused transcardially with freshly prepared PBS. The extracted brains were post-fixed in 4% paraformaldehyde (Biosesang, Korea) for 48 h, and 40-µm sections were cut using a freezing microtome (Leica Microsystems, Nussloch GmbH, 7

Nussloch, Germany). H2O2 (3%) (Vector Laboratories, Burlinagam, CA) was used to quench the endogenous peroxidase. Nonspecific binding was blocked by incubating the cells in PBS containing 4% BSA for 60 min at room temperature. After overnight incubation at 4 °C with the primary antibody against Iba-1 (1:1000), the sections were incubated with biotinylated secondary antibody for 1 h at room temperature, washed with 0.1% PBS-Triton X-100, and subsequently incubated with avidin-biotin-HRP complex reagent solution (Vector Laboratories) for 90 min followed by washing with PBS. Then, the peroxidase reaction was performed using 3,3'-diaminobenzidine tetrahydrochloride (Vector Laboratories). Finally, the sections were dehydrated and cover-slipped for light microscopy (Leica, Heidelberg, Germany).

2.7. Traditional and real-time RT-PCR BV2 cells (7.5 × 105 cells per well in a 6-well plate) were treated with LPS in the presence or absence of galangin, and total RNA was extracted using TRI reagent (Thermo Fisher Scientific). Cortical brain tissue was homogenized using a homogenizer (Fisher Scientific, Pittsburgh, PA), and total RNA was extracted using TRI reagent. For RT-PCR, total RNA (1 µg) was reverse-transcribed in a reaction mixture containing 0.1 µg of random primers, 3 mM MgCl2, 0.5 mM dNTP, 1× RT buffer, and 10 U reverse transcriptase (Promega, Madison, WI). The synthesized cDNA was used as a template for the PCR reaction using Go Taq polymerase (Promega) and primers for the target gene. For the amplification of NADPH oxidase subunit genes, real-time PCR was performed. The synthesized cDNAs were amplified with SYBR® Green PCR Master Mix (Applied Bio systems, Foster City, CA). The RT-PCR was carried out using an ABI Prism 7000 Sequence Detection System (Applied Bio systems). The relative amount of the target gene was calculated using the 2(Ct test gene – Ct GAPDH) with GAPDH as an internal control. The primers used in the PCR reaction are shown in 8

Tables 1 and 2.

2.8. Electrophoretic mobility shift assay (EMSA) BV2 cells were pretreated with galangin for 1 h and stimulated with LPS for 3 h. The nuclear extracts from the cells were prepared as previously described [33]. Double-stranded DNA oligonucleotides containing consensus sequences of NF-κB were end-labeled using T4 polynucleotide kinase (New England Biolabs, Beverly, MA) in the presence of [γ-32P] ATP. Nuclear proteins (5 µg) were incubated with a

32

P-labeled probe on ice for 30 min, resolved

on a 5% polyacrylamide gel, and visualized with autoradiography.

2.9. Western blot analysis Whole cell protein lysates were prepared in a lysis buffer (10 mM Tris, pH 7.4, 30 mM NaCl, 1% Triton X-100, 0.1% SDS, 0.1% sodium deoxycholate, and 1 mM EDTA) containing a protease inhibitor cocktail. Protein samples were separated by SDS-PAGE, transferred to nitrocellulose membranes, and incubated with antibodies against the following proteins: the phospho- or the total form of MAP kinase (1:1000), iNOS (1:1000), COX-2 (1:1000), TNF-α (1:200), IL-10 (1:1000), HO-1 (1:1000), and β-actin (1:1000). After thoroughly washing with TBST, HRP-conjugated secondary antibodies (1:2000 dilution in TBST; Bio-Rad, Hercules, CA) were applied, and the blots were developed using an enhanced chemiluminescence detection kit (Thermo Fisher Scientific, Waltham, MA).

2.10. Transient transfection and luciferase assay BV2 cells (2 × 10 5 cells per well on a 12-well plate) were transfected with 1 µg of plasmid DNA ([κB]3-luc, ARE-luc, CRE-luc, or PPRE-luc) using the Convoy Platinum transfection reagent (ACTGene, Inc., Piscataway, NJ) or Metafectene transfection reagent 9

(Biontex, Martinsried/Planegg, Germany). The effect of galangin on reporter gene activity was determined by pre-treating the cells with galangin prior to stimulation with LPS (100 ng/mL) for 6 h. After preparing the cell lysates, the luciferase assay was performed as previously described [34]. siRNA targeting mouse PPAR-γ and non-targeting control siRNA were obtained from Sigma-Aldrich (MISSION siRNA and MISSION siRNA Universal Negative Control, respectively). Transfection of PPAR-γ siRNA into BV2 cells was performed using Metafectene transfection reagent according to the manufacturer’s protocols. The PPAR-γ siRNA sequence was 5'-GACAAAUCACCAUUUGUCA-3'. The cells were harvested 48 h after siRNA transfection, and the expression levels of PPAR-γ were measured with RT-PCR.

2.11. Statistical analysis Data are expressed as mean ± S.E.M., and statistical analyses were performed using a one-way ANOVA followed by Newman-Keuls post-hoc tests or t-tests. The calculations were performed using the GraphPad Prism software (version 4.0; GraphPad Software, Inc., La Jolla, CA). A P-value of less than 0.05 was considered to indicate statistical significance.

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3. Results 3.1. Galangin inhibits LPS-induced expression of iNOS, COX-2, and pro-inflammatory cytokines and increases anti-inflammatory IL-10 in BV2 microglial cells To investigate the anti-inflammatory effects of galangin, BV2 cells were stimulated with LPS (100 ng/mL) in the presence or absence of galangin. It was found that galangin dosedependently suppressed NO, TNF-α, and IL-6 production, induced by LPS. On the contrary, galangin dramatically increased anti-inflammatory IL-10 production (Fig. 2A). Next, RTPCR analysis was performed to determine the effect of galangin on mRNA expression of pro/anti-inflammatory molecules in LPS-stimulated microglia. As shown in Fig. 2B, galangin significantly reduced the mRNA levels of iNOS, TNF-α, IL-1β, IL-6, and COX-2, induced by LPS, while galangin increased IL-10 mRNA expression. Furthermore, western blot analyses showed that galangin suppressed protein expression of iNOS, COX-2, and TNF-α, and increased that of IL-10 (Fig. 2C). These data suggest that galangin regulates the expression of iNOS, COX-2, and several cytokines at the transcriptional level.

3.2. Galangin inhibits microglial activation and expression of pro-inflammatory molecules in LPS-injected mouse brains To verify the effects of galangin under neuroinflammatory conditions in vivo, a dose of 50 mg/kg dosage was determined to be optimal in our preliminary experiment, and was intraperitoneally injected into mice prior to the administration of LPS. Three hours after LPS injection, microglial activation and the expression levels of proinflammatory markers in LPSinjected mouse brains were determined. Microglial activation was investigated by quantifying the immunoreactivity for Iba-1. Systemic LPS administration increased the number of Iba-1positive cells with densely stained amoeboid cell bodies. However, galangin significantly reduced the number of Iba-1-positive cells in the cortex and hippocampus (Fig. 3A). Next, we 11

examined the effects of galangin on various inflammatory parameters, such as cytokines, tolllike receptors (TLRs), and matrix metalloproteinases (MMPs) [35]. RT-PCR analysis showed that galangin inhibited the mRNA expression of iNOS, TNF-α, IL-6, IL-1β, and COX-2 in LPS-injected mouse brains (Fig. 3C and D). Galangin also suppressed the expression of TLR4, a pattern recognition receptor for LPS, but did not significantly inhibit TLR2 expression. Moreover, galangin inhibited the expression of MMP-3 and MMP-9, molecules that play a pro-inflammatory role in activated microglia [34].

3.3. Galangin suppresses LPS-induced NF-κB activity and phosphorylation of p38 MAPK, JNK, and Akt To investigate the mechanisms underlying the anti-inflammatory effects of galangin, we examined the effect of galangin on NF-κB, a key transcription factor modulating gene expression of iNOS and several cytokines [36]. The EMSA data showed that galangin significantly inhibited LPS-induced the DNA binding activity of NF-κB (Fig. 4A). In addition, galangin inhibited NF-κB reporter gene activity (Fig. 4B). Next, we examined the effect of galangin on the phosphorylation of Akt and three types of MAP kinases, the upstream modulators of iNOS, and cytokine expression in activated microglia [37]. We found that galangin significantly inhibited LPS-induced phosphorylation of p38 MAPK, JNK, and Akt, without affecting ERK. Our data collectively suggest that PI3K/Akt, p38 MAPK, JNK, and their downstream NF-κB signaling pathways are involved in the anti-inflammatory mechanism of galangin.

3.4. Galangin shows antioxidant effects by modulating NADPH oxidase subunits and hemeoxygenase-1 expression 12

We examined the effect of galangin on the production of ROS, an early signaling inducer of inflammation [38]. Galangin significantly inhibited LPS-induced ROS production in BV2 cells (Fig. 5A). Next, we examined the effect of galangin on the expression of NADPH oxidase subunits responsible for microglial ROS production. RT-PCR analyses showed that galangin significantly inhibited LPS-induced expression of p47 phox and gp91 phox, but did not alter the expression of p67phox or p22 phox (Fig. 5B). Since hemeoxygenase-1 (HO-1) acts as an anti-inflammatory and antioxidant modulator in microglia [39], we examined the effect of galangin on HO-1 expression. Western blot and RT-PCR analyses showed that galangin upregulated HO-1 expression at the mRNA and protein levels (Fig. 5C and D). In addition, galangin increased the reporter gene activity of ARE-luc (Fig. 5E), that has binding sites for Nrf2, a key transcription factor modulating the expression of HO-1 and other antioxidant enzyme genes. Furthermore, galangin increased CREB-mediated transcriptional activity (CRE-luc), which also acts as an upstream modulator of HO-1 expression (Fig. 5F). Thus, our data suggest that the inhibition of NADPH oxidase subunits and upregulation of HO-1 via the Nrf2/CREB pathway may be at least partly involved in the mechanism underlying the antioxidant/anti-inflammatory effects of galangin in activated microglia.

3.5. Upregulation of PPAR-γ signaling mediates anti-inflammatory effects of galangin in LPS stimulated microglia We then examined the effect of galangin on PPAR-γ, a nuclear receptor that acts as an anti-inflammatory regulator in macrophage and microglial activation [15, 16]. We found that the treatment of BV2 cells with LPS significantly suppressed PPAR-γ expression, as previously reported in macrophages and primary microglia [40, 41]. The treatment with galangin, however, restored PPAR-γ expression to near normal levels (Fig. 6A). Next, to 13

investigate whether galangin increases the transcriptional activity of PPAR-γ, a cell-based reporter gene assay was performed. We found that galangin increased PPRE-luc activity in both the presence and absence of LPS (Fig. 6B). To confirm that the increase in PPRE-luc activity by galangin was mediated by PPAR-γ, the cells were treated with a PPAR-γ antagonist (T0070907) prior to the treatment with galangin. As shown in Fig. 6B, the treatment with T0070907 significantly blocked galangin-mediated upregulation of PPRE-luc activity in the absence or presence of LPS. To determine whether PPAR-γ mediates the anti-inflammatory effects of galangin, BV2 cells were treated with T0070907 before the addition of LPS and/or galangin. As shown in Fig. 6C, treatment with T0070907 significantly blocked galangin-mediated upregulation of IL-10, and the inhibition of IL-6, TNF-α, and NO in LPS-stimulated microglia. In addition, a western blot analysis showed that T0070907 reversed the effects of galangin on IL-10, IL-6, and HO-1 expression at the protein level (Fig. 6D). T0070907 alone did not affect the protein expression of IL-6, IL-10, and HO-1 (data not shown). We further verified the antiinflammatory role of PPAR-γ by performing siRNA knockdown experiments. As shown in Fig. 7, the treatment of BV2 cells with PPAR-γ siRNA reversed the effect of galangin on NO, ROS, TNF-α, IL-6, and IL-10 production, which recapitulates the effects of T0070907. To determine the relationship between PPAR-γ and other transcription factors, we examined the effect of the PPAR-γ antagonist on the reporter gene activities of NF-κB, Nrf2, and CREB. As shown in Fig. 8, T0070907 significantly blocked galangin-mediated inhibition of (κB)3-luc activity, and the upregulation of ARE- and CRE-luc activities. These data collectively suggest that PPAR-γ mediates the anti-inflammatory effects of galangin by inhibiting NF-κB and enhancing Nrf2/CREB signaling.

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4. Discussion In the present study, we have demonstrated the anti-inflammatory effects of galangin under neuroinflammatory conditions in vitro and in vivo. Galangin suppressed the expression of iNOS, COX-2, and proinflammatory cytokines, while it enhanced anti-inflammatory IL-10 in LPS-stimulated microglia. In addition, galangin inhibited microglial activation and the expression of various proinflammatory markers in LPS-injected mouse brains. Detailed mechanistic studies revealed that PPAR-γ plays a critical role in mediating the antiinflammatory effects of galangin by modulating transcription factors, such as NF-κB, Nrf2, and CREB, and its downstream pro-/anti-inflammatory gene expressions. The proposed mechanism underlying the effects of galangin is summarized in Fig. 9. Previous

studies

highlight

the

role

of

PPARs

in

neuroinflammation

and

neurodegenerative processes [14, 42]. PPAR activation has been shown to regulate the inflammatory responses mediated by microglia and astrocytes, protect neurons from damage, reduce oxidative stress, and improve mitochondrial function [14, 43]. In particular, PPAR-γ is mainly expressed in microglia, and it mediates anti-inflammatory activity by inducing microglial polarization into the M2-like phenotype [16]. PPAR-γ activation led to the inhibition of transcription factors, such as NF-κB, AP-1, and STAT-1 and the expression of pro-inflammatory cytokines, chemokines, cell adhesion molecules, such as ICAM-1 and MMP-9 [13, 44]. PPAR-γ activation also had neuroprotective effects after β-amyloid challenge, and improved cognitive performance in AD animal models and patients [45, 46]. Moreover, PPAR-γ agonists exerted neuroprotective effects in PD models by inhibiting apoptosis, oxidative damage, inflammation, and mitochondrial dysfunction [47, 48]. Therefore,

PPAR-γ has been suggested

as a promising therapeutic target for

neurodegenerative diseases. In the present study, we report, for the first time, that galangin 15

induces PPAR-γ activation, and that PPAR-γ plays a role as a master regulator of the antiinflammatory action of galangin in activated microglia. Our data may support the therapeutic potential

of

galangin

for

neurodegenerative

diseases

that

are

associated

with

neuroinflammation. Interestingly, we found that galangin markedly increased the levels of IL-10, a regulator of polarization into the anti-inflammatory M2 phenotype [49, 50]. Treatment with the PPAR-γ antagonist or siRNA almost completely abolished the upregulation of IL-10. These data suggested that the increase of IL-10 by galangin was dependent on PPAR-γ signaling in LPSstimulated microglia. Accordingly, previous studies demonstrated the crosslink between IL10 and PPAR-γ signaling [51]. Pioglitazone, a synthetic PPAR-γ agonist, ameliorated inflammation in a murine model of sepsis by increasing the levels of IL-10. Specifically, these studies showed that IL-10 plays a key role in mediating PPAR-γ inhibition of STAT-1dependent expression of MyD88, the major adaptor protein for TLRs. Therefore, blocking MyD88 through the PPAR-γ/IL-10 axis may inhibit excessive TLR signaling and improve the outcome of inflammation. Further studies are necessary to address whether the TLR/MyD88 pathway is also involved in the anti-inflammatory mechanism of galangin in microglia. We demonstrated that PPAR-γ is also partly involved in galangin-mediated inhibition of TNFα, IL-6, and NO, in LPS-stimulated microglia. NF-κB is a key transcription factor modulating the gene expression of several cytokines and iNOS [36]. In the present study, galangin inhibited DNA binding and transcriptional activities of NF-κB, and the PPAR-γ antagonist partially reversed this inhibition (Fig. 8A). These data suggest that PPAR-γ mediates the inhibition of proinflammatory gene expression by modulating NF-κB signaling. A number of studies have proposed potential mechanisms underlying the regulation of NF-κB by PPAR-γ. PPAR-γ inhibits NF-κB activity by directly binding to NF-κB subunits p50 and 16

p65 [13, 52]. PPAR-γ may indirectly inhibit NF-κB by competing for coactivators, such as p300/CBP, by upregulating the inhibitor kappa B (IκB), or by activating the transcription factor Nrf2, which reduces oxidative molecules required for NF-κB activation [12, 53, 54]. In the present study, we found that galangin increased Nrf2/ARE signaling. Moreover, galangin increased the transcriptional activities of CREB, known to block NF-κB activity by competing for CBP [55]. By performing the treatment with the PPAR-γ antagonist, we demonstrated that PPAR-γ is an upstream modulator of Nrf2 and CREB signaling. Therefore, our data suggest that the concomitant upregulation of Nrf2 and CREB by galangin may contribute to PPAR-γ inhibition of NF-κB in LPS-stimulated microglia. We found that galangin exerts strong antioxidant effects in microglia by modulating the expression of NADPH oxidase subunits and HO-1. NADPH oxidase is a major source of ROS production in microglia, and we observed that galangin inhibited LPS-induced expression of its subunits p47phox and gp91phox. HO-1 plays an antioxidant, anti-inflammatory, and antiapoptotic role via Nrf2/ARE signaling. In addition, our group recently reported that PKA/CREB is an upstream modulator of HO-1 expression [56]. In the present study, we observed that galangin increased HO-1 expression by upregulating Nrf2/ARE and CREB signaling (Fig. 5). Therefore, our data collectively suggest that PPAR-γ mediates antiinflammatory and antioxidant effects of galangin via positive regulation of Nrf2/CREB and negative regulation of NF-κB. In this study, we found that galangin increased the expression of PPAR-γ, but not PPARα or -δ. Moreover, a PPAR-α or -δ antagonist did not significantly affect the antiinflammatory effects of galangin (data not shown). The data suggest that only PPAR-γ, among the three isoforms of PPAR, plays a role in mediating anti-inflammatory effects of galangin in LPS-stimulated microglia. Using a PPAR-γ binding assay, we found that galangin 17

does not directly bind to PPAR-γ (data not shown), suggesting that galangin activates PPREmediated transcriptional activity by increasing PPAR-γ expression, but not by direct binding to PPAR-γ. Recent studies have highlighted the therapeutic potential of galangin in CNS disorders. Galangin showed neuroprotective effects in a model of acute ischemic stroke induced by middle cerebral artery occlusion [29, 57], by restoring regional cortical blood flow, ameliorating mitochondrial dysfunction, and apoptosis [29]. Furthermore, galangin protected the neurovascular unit through Wnt/β-catenin signaling pathways in rat brains subjected to ischemia [57]. Galangin also showed therapeutic potential for Alzheimer’s disease. It inhibited acetylcholine esterase activity [30] and decreased β-amyloid production by selectively inhibiting BACE activity, which may be associated with PPAR-γ activation [58, 59]. A recent study reported that galangin inhibits BACE1 expression via epigenetic mechanisms, such as HDAC1-mediated deacetylation [31]. Currently, galangin is one of the substances under consideration for the treatment of patients with AD [60]. Considering that neuroinflammation is an important pathogenic factor in the progression of neurodegenerative diseases, the anti-neuroinflammatory function of galangin may support its therapeutic potential for the treatment of various neurodegenerative diseases, such as AD, PD, and cerebral ischemia.

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Conflict of interest The authors declare that they have no conflict of interest.

Acknowledgements This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF), funded by the Ministry of Science, ICT, & Future Planning (Grant No. NRF-2010-0027945 & NRF-2015R1A2A2A01005226).

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Figure Legends

Fig. 1 The chemical structure of galangin (3,5,7-trihydroxyflavone)

Fig. 2 Effects of galangin on the expression of pro-/anti-inflammatory molecules in LPSstimulated BV2 microglia (A) Cells were pre-treated with galangin (Gal) at indicated concentrations (10, 30, 50 µM) for 1 h, and then subjected to treatment with LPS (100 ng/mL) for 16 h. The amounts of NO, TNF-α, IL-6, and IL-10 released into the medium were measured using Griess reagent or ELISA. (B) Cells were pre-treated with galangin for 1 h, and then treated with LPS (100 ng/mL) for 6 h. An RT-PCR was performed to measure the mRNA expression of pro-/antiinflammatory molecules. The quantification data are shown in the right panel. (C) Cell lysates were prepared from BV2 cells treated as a panel (A), and western blot analysis was performed to quantify protein expression of pro-/anti-inflammatory molecules. The data are expressed as the mean ± S.E.M. of three independent experiments. *P < 0.05, vs. control samples. #P < 0.05, vs. LPS-treated samples.

Fig. 3 Effect of galangin on microglial activation and mRNA expression of inflammatory markers in LPS-injected mouse brains (A, B) Immunohistochemical staining for Iba-1 and quantification of the number of Iba1positive microglia 3 h after systemic LPS treatment (5 mg/kg, i.p.). Microglial activation in the cortex and hippocampus of LPS-injected mice was reduced by galangin (50 mg/kg) treatment. Representative images (A) and quantification of data (B) are shown (n=5). Scale bars, 50 µm. (C, D) Effects of galangin on mRNA levels of iNOS, COX-2, pro-inflammatory cytokines (IL-6, IL-1β, TNF-α), microglial activation markers (TLR4, TLR2), and MMPs 26

(MMP-3, MMP-9) in the cortex of LPS-injected mice. Representative gels (C), and quantification data (D) are shown (n=5). *P < 0.05, vs. control samples. #P < 0.05, vs. LPStreated samples.

Fig. 4 Effect of galangin on NF-kB activity and the phosphorylation of MAP kinases and Akt in LPS-stimulated BV2 cells (A) Nuclear extracts were prepared from BV2 cells after treatment with LPS (100 ng/mL) for 3 h, in the absence or presence of galangin, and EMSA was performed to determine the DNA binding activity of NF-κB. The arrow indicates the DNA-protein complex of NF-κB. ‘F’ indicates a free probe. (B) BV2 cells transfected with the reporter plasmid ([κB]3-luc) were pretreated with galangin for 1 h prior to LPS treatment. After 6 h, cells were harvested and the luciferase assay was performed. (C) Western blot analysis for MAPKs and PI3K/Akt activities. Cell extracts were prepared from BV2 cells treated with LPS for 15 min, in the absence or presence of galangin, and a western blot analysis was performed using antibodies against phospho- or total forms of three types of MAPKs or Akt. The blots are representative of three independent experiments. (D) Quantification of western blot data. The levels of the phosphorylated forms of MAPKs or Akt were normalized to the levels of each total form and expressed as relative fold changes versus the untreated control group. The results were obtained from three independent experiments and expressed as the mean ± S.E.M. *P < 0.05, vs. control samples. #P < 0.05, vs. LPS-treated samples.

Fig. 5 Galangin inhibits ROS production by suppressing the expression of NADPH oxidase subunits and increasing that of HO-1 (A) BV2 cells were incubated with LPS in the presence or absence of galangin for 16 h. The levels of intracellular ROS were determined by the DCF-DA method. The data are expressed 27

as the mean ± S.E.M. from three independent experiments. *P < 0.05, vs. control samples. #P < 0.05, vs. LPS-treated samples. (B) Real-time PCR for mRNA expression of NADPH oxidase subunits (p47phox, gp91phox, gp22 phox, p67phox) in BV2 cells (n=3). (C, D) BV2 cells were pretreated with galangin for 1 h and incubated with LPS for 6 h. Western blot (C) and RT-PCR (D) analyses were performed to investigate the effects of galangin on protein and mRNA expression of HO-1. Representative gels are shown in the upper panel, and quantitative data are shown in the bottom panel (n=3). (E, F) BV2 cells were transfected with the reporter plasmids ARE-luc (E) or CRE-luc (F), and pretreated with galangin prior to LPS stimulation for 6 h, after which the luciferase assay was performed. *P < 0.05, vs. control samples. #P < 0.05, vs. LPS-treated samples.

Fig. 6 Galangin increases PPAR-γ expression and transcriptional activity, and PPAR-γ antagonist reverses the effect of galangin in LPS-stimulated BV2 cells (A) BV2 microglia were pretreated with galangin for 1 h prior to incubation with LPS (100 ng/mL) for 6 h. The mRNA level of PPAR-γγ was determined by RT-PCR. Quantification data are shown in the bottom panel. (B) The effects of galangin on PPRE-luc reporter gene activity. BV2 cells were transfected with PPRE-luc and treated with galangin with or without LPS. After 6 h, the cells were harvested and the luciferase assay was performed. PPAR-γ antagonist, T0070907 (5 µM) was added to BV2 cells 1 h before the treatment with galangin. *P < 0.05, vs. control samples. **P < 0.05, vs. galangin-treated samples. #P < 0.05, vs. LPStreated samples.

##

P < 0.05, vs. LPS+galangin-treated samples. (C) The effect of T0070907

on the production IL-10, IL-6, TNF-α, and NO in LPS+galangin-treated cells. BV2 cells were pre-treated with the indicated concentrations (1, 3, 5 µM) of T0070907 for 1 h, and then galangin (50 µM) for 1 h, followed by the treatment with LPS (100 ng/mL) for 16 h. The 28

amounts of IL-10, IL-6, TNF-α, and NO released into the medium were determined. (D) The effect of T0070907 on the protein expression of IL-10, IL-6, and HO-1 was determined by western blot analysis. Quantification data are shown in the bottom panel. Data are expressed as mean ± S.E.M. from three independent experiments. *P < 0.05, vs. control samples. #P < 0.05, vs. LPS-treated samples. ##P < 0.05, vs. LPS+galangin-treated samples.

Fig. 7 Knockdown of PPAR-γ reverses the effect of galangin in LPS-stimulated BV2 cells (A) Cells were transfected with PPAR-γ-specific siRNA or control siRNA. Downregulation of PPAR-γ expression by PPAR-γ siRNA was confirmed by RT-PCR analysis. (B - F) BV2 cells were transfected with PPAR-γ siRNA and then treated with LPS in the absence or presence of galangin (50 µM) for 16 h. The amounts of NO (B), TNF-α (C), IL-6 (D), IL-10 (E) released into the media, and the level of intracellular ROS (F) were determined as described in Methods. *P < 0.05, vs. LPS-treated samples. #P < 0.05, vs. control siRNAtransfected cells in the presence of LPS.

Fig. 8 PPAR-γγ antagonist reverses the effect of galangin on reporter gene activities of NF-κ κB, Nrf2, and CREB BV2 cells were transfected with the reporter plasmid [κB]3-luc (A), ARE-luc (B), or (C) CRE-luc and treated with T0070907 (5 µM) for 1 h, followed by galangin (50 µM) and LPS (100 ng/mL). After 6 h, cells were harvested and the reporter gene assay was performed. The data are expressed as the mean ± S.E.M. of three independent experiments. *P < 0.05, vs. control samples. #P < 0.05, vs. LPS-treated samples.

##

P < 0.05, vs. LPS+galangin-treated

samples. 29

Fig. 9 The proposed mechanism underlying the anti-inflammatory action of galangin in LPS-stimulated microglia LPS binds to TLR4 on the surface of microglial cells, and induces intracellular signaling such as the activation of ROS, PI3K/Akt, MAPKs, and NF-κB with the inhibition of PPAR-γ. Galangin blocks these signaling pathways, and results in the inhibition of NF-κB-mediated proinflammatory gene expression. On the contrary, galangin restores PPAR-γ expression and increases PPAR-γ transcriptional activity, subsequently activating Nrf2, CREB, and the expression of their downstream anti-inflammatory targets, such as IL-10 and HO-1. Moreover, PPAR-γ inhibits NF-κB activity and exerts anti-inflammatory effects.

30

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50 (μM)

30

0

D

+ LPS (-)

Gal:

0

50

30

10

+ LPS Gal: (-)

(μM)

10

30

50

(μM) HO-1

β-actin

GAPDH 20

#

HO-1/GAPDH

HO-1/β-actin

0

HO-1

10 8

#

#

6

*

4 2

0 Gal:

(-)

0

10

30

#

#

10

30

50

*

10 5

(-)

0

(μM)

+ LPS

+ LPS

E ▶ Reporter plasmid: ARE-luc

F

▶ Reporter plasmid: CRE-luc

4 3

#

*

2

#

1

(-)

0

10

30

50 (μM)

Fold Induction

8 #

0 Gal:

#

15

0 Gal:

(μM)

50

10

6

+ LPS

# #

4

#

* 2

0 Gal:

(-)

0

10

30

+ LPS

Fig. 5

30

+ LPS

+ LPS

C

#

2

0

1.0

*

4

0

# #

#

5

p22phox mRNA (Fold induction)

*

3

Fold Induction

ROS (Fold induction)

4

*

2

gp91phox mRNA (Fold induction)

B

A

50 (μM)

50 (μM)

A

B

+LPS Gal:

(-)

10

0

50 (μM)

30

▶ Reporter plasmid: PPRE-luc

PPAR- Fold Induction

#

1.0

#

#

*

4 #

*

2

#

*

1

0.5

0

(-)

0

50 (μM)

30

10

LPS Gal (M) T0070907

-

-

+

0 Gal:

*

3

- - 10 30 50 50 - - - +

+ -

+ + + + 10 30 50 50 - - - +

+ +

PPAR-/GAPDH

1.5 #

##

**

5

GAPDH

+ LPS

C

400

* + -

+ + -

+ + 3

+ + 1

D

+ + 5

2500

2000 #

1000

0 LPS Gal (50 M) T0070907(M) -

+ + -

+ -

+ + 1

+ + 3

0 0

50 0

50 1

50 3

50 (μM) 5 (μM)

+ -

+ + -

+ + 1

#

500 + -

+ + -

+ + 1

+ + 3

+ + 5

30 20

0 0

50 0

+ + 3

+ + 5

50 1

#

10

0 LPS Gal (50 M) T0070907(M) -

+ + -

+ -

+ + 1

+ + 3

+ + 5

+ LPS 50 3

50 (μM) 5 (μM)

Gal: (-) T: (-)

0 0

50 0

50 1

50 3

50 (μM) 5 (μM)

IL-10

IL-6

HO-1

(18 kDa)

(21kDa)

(32 kDa)

β-actin

β-actin

β-actin

Fold Induction

*

2

0 LPS Gal (50 M) T0070907(M) -

Gal: (-) T: (-)

4

6

*

2

0 LPS Gal (50 M) T0070907(M) -

##

##

6

#

4

1500

##

*

40

+ LPS

##

6

50

1000

0 LPS Gal (50 M) T0070907(M) -

+ + 5

+ LPS

Gal: (-) T: (-)

##

*

2000

Fold Induction

200

##

*

Nitrite (μM)

#

600

0 LPS Gal (50 M) T0070907(M) -

Fold Induction

3000

TNF-α (pg/ml)

##

IL-6 (pg/ml)

IL-10 (pg/ml)

800

#

+ -

+ + -

+ + 1

Fig. 6

+ + 3

+ + 5

#

4

*

2

0 LPS Gal (50 M) T0070907(M) -

+ -

+ + -

+ + 1

+ + 3

+ + 5

B

C Control siRNA PPAR- siRNA

Con siRNA PPAR- siRNA PPAR- GAPDH

Nitrite (μM)

50 40 30

#

20

*

10

1500 #

1000

0 (-)

(-)

LPS+Gal

1500

#

1000

*

500 0 (-)

LPS

LPS+Gal

LPS

LPS+Gal

F 500

Control siRNA PPAR- siRNA

400

*

300 200 #

3 2 #

100

1

0

0 (-)

LPS

Fig. 7

LPS+Gal

Control siRNA PPAR- siRNA

4 ROS (fold)

Control siRNA PPAR- siRNA IL-10 (pg/ml)

IL-6 (pg/ml)

LPS

E

2000

*

500

0

D

Control siRNA PPAR- siRNA

2000 TNF-α (pg/ml)

A

*

(-)

LPS

LPS+Gal

A

C

B

▶ Reporter plasmid: CRE-luc

▶ Reporter plasmid: ARE-luc

▶ Reporter plasmid: (B)3-luc

##

##

4

10

*

5

0 LPS Gal (50 M) T0070907(5 M)

#

-

+

+ -

+ + -

+ + +

8 #

Fold Induction

##

Fold Induction

Fold Induction

15

3

*

2 1

0 LPS Gal (50 M) T0070907(5 M)

-

+

+ -

Fig. 8

+ + -

+ + +

6

#

4

* 2

0 LPS Gal (50 M) T0070907(5 M) -

+

+ -

+ + -

+ + +

LPS

TLR4 Cytosol

NADPH oxidase PI3K/AKT

ROS

JNK p38

P P P

PPARγ

NF-κB

CREB

: Inhibition by Galangin

GALANGIN

Nrf2

: Activation IL-10, HO-1

: Inhibition

CRE / PPRE / ARE NF-κB

NO, TNF-α, IL-6, MMPs

Anti-inflammatory effects

Fig. 9

Nucleus

LPS

TLR4

NADPH oxidase

ROS

: Inhibition by Galangin : Activation : Inhibition

PI3K/Akt, p38, JNK

NF-κB

PPARγ CREB

GALANGIN

Nrf2

Pro-inflammatory molecules

Anti-inflammatory molecules

(e. g. NO, TNF-α, IL-6, MMPs)

(e.g. IL-10 , HO-1)

Table 1. Primers used in semiquantitative PCR Gene

Forward Primer (5’→3’)

Reverse Primer (5’→3’)

Size

iNOS

GCTTGGGTCTTGTTCACTCC

GGCCTTGTGGTGAAGAGTGT

385 bp

COX-2

TTCAAAAGAAGTGCTGGAAAAGGT

GATCATCTCTACCTGAGTGTCTTT

304 bp

TNF-α

CCTATGTCTCAGCCTCTTCT

CCTGGTATGAGATAGCAAAT

354 bp

IL-1β

GGCAACTGTTCCTGAACTCAACTG

CCATTGAGGTGGAGAGCTTTCAGC

447 bp

IL-6

CCACTTCACAAGTCGGAGGCTT

CCAGCTTATCTGTTAGGAGA

214 bp

IL-10

GCCAGTACAGCCGGGAAGACAATA

GCCTTGTAGACACCTTGGTCTT

185bp

PPAR-γγ

CCGAAGAACCATCCGATT

CGGGAAGGACTTTATGTA

271bp

MMP3

ATTCAGTCCCTCTATGGA

CTCCAGTATTTGTCCTCTAC

245 bp

MMP9

GAAGCCATACAGTTTATCCTGGTC

GTGATCCCCACTTACTATGGAAAC

353 bp

TLR2

TGTAACGCAACAGCTTCAGG

TGCTTTCCTGCTGGAGATTT

197bp

TLR4

AACCAGCTGTATTCCCTCAG

GATGCTTTCTCCTCTGCTGT

399 bp

HO-1

TGTCACCCTGTGCTTGACCT

ATACCCGCTACCTGGGTGAC

209bp

GAPDH

TTCACCACCATGGAGAAGGC

GGCATGGACTGTGGTCATGA

236 bp

31

Table 2. Primers used in quantitative real time PCR Gene

Forward Primer (5’→3’)

Reverse Primer (5’→3’)

Size

p47phox

TTCACCACCATGGAGAAGGC

GGCATGGACTGTGGTCATGA

212 bp

p67phox

CTGGCTGAGGCCATCAGACT

AGGCCACTGCAGAGTGCTTG

214 bp

gp91phox

TTGGGTCAGCACTGGCTCTG

TGGCGGTGTGCAGTGCTATC

185bp

p22phox

GTCCACCATGGAGCGATGTG

CAATGGCCAAGCAGACGGTC

164bp

GAPDH

TTCACCACCATGGAGAAGGC

GGCATGGACTGTGGTCATGA

236 bp

32