Urolithins Attenuate LPS-Induced Neuroinflammation in BV2Microglia

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Article Cite This: J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Urolithins Attenuate LPS-Induced Neuroinflammation in BV2Microglia via MAPK, Akt, and NF-κB Signaling Pathways Jialin Xu,†,‡ Chunhui Yuan,† Guihua Wang,† Jiaming Luo,† Hang Ma,§ Li Xu,† Yu Mu,† Yuanyuan Li,† Navindra P. Seeram,*,§ Xueshi Huang,*,† and Liya Li*,† †

Institute of Microbial Pharmaceuticals, College of Life and Health Sciences, Northeastern University, Shenyang 110819, People’s Republic of China ‡ Institute of Biochemistry and Molecular Biology, College of Life and Health Sciences, Northeastern University, Shenyang 110819, People’s Republic of China § Bioactive Botanical Research Laboratory, Department of Biomedical and Pharmaceutical Sciences, College of Pharmacy & George and Anne Ryan Institute for Neuroscience, University of Rhode Island, Kingston, Rhode Island 02881, United States S Supporting Information *

ABSTRACT: Emerging data suggest that urolithins, gut microbiota metabolites of ellagitannins, contribute toward multiple health benefits attributed to ellagitannin-rich foods, including walnuts, red raspberry, strawberry, and pomegranate. However, there is limited data on whether the potential neuroprotective effects of these ellagitannin-rich foods are mediated by urolithins. Herein, we evaluated the potential mechanisms of antineuroinflammatory effects of urolithins (urolithins A, B, and C; 8-methyl-O-urolithin A; and 8,9-dimethyl-O-urolithin C) in BV2 murine microglia in vitro. Nitrite analysis and qRT-PCR suggested that urolithins A and B reduced NO levels and suppressed mRNA levels of pro-inflammatory genes of TNF-α, IL-6, IL-1β, iNOS, and COX-2 in LPStreated microglia. Western blot revealed that urolithins A and B decreased phosphorylation levels of Erk1/2, p38 MAPK, and Akt, prevented IκB-α phosphorylation and degradation, and inhibited NF-κB p65 subunit phosphorylation and nuclear translocation in LPS-stimulated microglia. Our results indicated that urolithins A and B attenuated LPS-induced inflammation in BV2 microglia, which may be mediated by inhibiting NF-κB, MAPKs (p38 and Erk1/2), and Akt signaling pathway activation. The antineuroinflammatory activities of urolithins support their role in the potential neuroprotective effects reported for ellagitannin-rich foods warranting further in vivo studies on these ellagitannin gut microbial derived metabolites. KEYWORDS: urolithins, microglia, neuroinflammation, ellagitannins

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models of neuroinflammation.5 LPS is recognized by the toll-like receptor 4 (TLR4) on the surface of microglia. The interaction of LPS with TLR4 results in the initiation of a series of inflammatory cascade, including the activation of the members of mitogenactivated protein kinase (MAPK) family and phosphoinositide 3-kinase (PI3K)/Akt signaling pathway, as well as nuclear factorkappa B (NF-κB) signaling pathway. The activation of these signal transduction pathways promote the mRNA and protein expression of pro-inflammatory enzymes, including inducible nitric oxidesynthase (iNOS) and cyclooxygenase 2 (COX-2), as well as pro-inflammatory cytokines and chemokines.6,7 Therefore, intervention in these pathways are potential methods to treat microgliamediated neuroinflammation and related neurodegenerative diseases.8−10 Ellagitannins (ETs) and ellagic acid (EA) are a group of polyphenols present in a wide variety of plant foods, such as certain nuts and berries, and pomegranate.11,12 Multiple lines of evidence suggest that the consumption of EA or ETs-rich food products has potential beneficial effects in ameliorating brain inflammatory symptoms and improving neurodegenerative diseases. For instance,

INTRODUCTION Microglia, the resident immune macrophages in the central nervous system (CNS), play a crucial role in response to injuries and infections in the brain. Changes in the microenvironment of microglia results in their transformation from a deactivated phenotype (ramified) to activated phenotype (amoeboid) which initiates an inflammatory response to resolve infections and repair tissue damage.1 However, sustained and overactivation of microglia promote the release of excessive pro-inflammatory cytokines, including tumor necrosis factor α (TNF-α) and interleukins (IL-6 and IL-1β), and neurotoxic factors, including reactive oxygen species (ROS), nitric oxide (NO), and prostaglandin-E2 (PGE2), which accelerate neuroinflammation.2,3 A growing body of evidence indicates that microglia-mediated neuroinflammation contributes significantly to the pathogenesis of several neurodegenerative diseases, including Alzheimer’s disease (AD), Parkinson’s disease (PD), amyotrophic lateral sclerosis (ALS), and multiple sclerosis (MS).1,3 Therefore, suppression of the overactivation of microglia is considered as a potential therapeutic strategy for neurodegenerative diseases.4 In the CNS, a wide spectrum of agents has been reported, inducing microglial activation via various molecular mechanisms. Lipopolysaccharide (LPS), an extracellular virulence factor derived from the cells wall of Gram-negative bacteria, is a widely used inflammatory stimulus. Systematic administration of LPS is reported to cause microglial activation and is usually used in experimental © XXXX American Chemical Society

Received: July 17, 2017 Revised: December 20, 2017 Accepted: December 26, 2017

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DOI: 10.1021/acs.jafc.7b03285 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry

caused by abnormal microglial activation. We recently reported that there was a trend in reduction of inflammatory biomarkers in brain tissue of AD mice exposed to an ET-enriched pomegranate extract and urolithins inhibited neuroinflammation in BV-2 microglia cocultured with SH-SY5Y neuronal cells.21 Herein, we sought to investigate the potential mechanisms of antineuroinflammatory action of three major ET colonic-derived metabolites, namely, urolithin A (UA), urolithin B (UB), and urolithin C (UC), along with two urolithin phase II enzyme conjugates, namely, 8-methylO-urolithin A (mUA) and 8,9-dimethyl-O-urolithin C (dmUC) (Figure 1A). LPS-stimulated BV2 microglia were incubated with various concentrations of different urolithins, and the effects of urolithins on mRNA expression of iNOS, COX-2, TNF-α, IL-6, IL-1β, and subsequent production of NO in BV2 microglial cells were evaluated. We also attempted to elucidate the underlying mechanisms of antineuroinflammatory action of the urolithins by investigating the involvement of the NF-κB, MAPKs and PI3K/Akt signaling pathways.

dietary supplementation of pomegranate has been shown to suppress pro-inflammatory cytokines release in the brain of AD mice.13 Our previous research demonstrated that short-term treatment with a pomegranate extract (containing 29.5% punicalagins, the major ET in pomegranate, and 2.3% EA) modulated the processing of amyloid-β precursor protein in an aged transgenic R1.40 AD mice model.14 Walnuts, which contain ETs, have also been found to be capable of improving cognitive and motor performance in aged rats.15 However, while EA and ETs are poorly absorbed in the body, they are extensively metabolized by gut microbiota to urolithins (6H-dibenzo-[b,d]pyran-6-one derivatives) which persist systemically through enterohepatic circulation.12,16 Moreover, urolithins, and not their parent ETs or EA, are detected in the plasma at micromolar concentrations after the intake of pomegranate food-stuffs.11,16 Our recent study suggested that urolithins possess the potential to cross the blood brain barrier (BBB) based on in silico computational studies.12 The anti-inflammatory effects of urolithins have been previously reported in different cell lines (summarized in Table S1), and their beneficial potential against intestinal inflammation, cancer, and cardiovascular disease have been well studied.17−20 However, to date, despite emerging reports on the beneficial effects of ETsrich foods in ameliorating brain inflammatory symptoms and improving neurodegenerative diseases,13−15 there is limited knowledge about whether urolithins could inhibit neuroinflammation

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MATERIALS AND METHODS

Materials and Reagents. UA, UB, UC, mUA, and dmUC were synthesized as previously reported.22,23 The purity of the test compounds was >95% according to UPLC-DAD-MS analyses. Their structures were verified by nuclear magnetic resonance and mass spectrometric data (see Supporting Information, Figures S1−S10). 1D-NMR spectra were collected on an Advance III-600 MHz spectrometer (Bruker Co.,

Figure 1. Effects of urolithins on cell viability of BV2 microglia with or without LPS stimulation. (A) The structures of uroltihins A, B, and C and mUA and dmUC. (B−F) BV2 cells were cotreated with various concentrations of (B) UA, (C) UB, (D) UC, (E) mUA, or (F) dmUC (0.3, 1, 3, 10, 30, 50, 100 μM) with or without LPS (100 ng/mL) for 24 h. Cell viability was determined by sulforhodamine B (SRB) assay. Data for DMSO-treated group without LPS administration was considered as 100% for each compound treatment, respectively. Data presented as mean ± S.E., n = 5. *, P < 0.05 vs DMSO treated with or without LPS administration, respectively. B

DOI: 10.1021/acs.jafc.7b03285 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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(100 ng/mL) for 24 h. The cell culture media was collected and centrifuged for 15 min at 4 °C. The supernatants were reserved and the concentrations of TNF-α, IL-6, and IL-1β were measured with commercial ELISA kits (R&D Systems, Minneapolis, MN, USA; 4A Biotech Co. Ltd., Beijing, China) according to the manufacturer’s protocols. The experiment was performed with three individual samples per treatment. Preparation of Cytosolic and Nuclear Fractions. Nuclear and cytoplasmic extracts were prepared with Nuclear and Cytoplasmic Protein Extract Kit (Beyotime Biotechnology, Shanghai, China). BV2 microglia (5 × 105 cells/mL) were seeded in 10 cm dish and incubated overnight. The next day, cells were treated with UA or UB (3, 10, and 30 μM) for 1 h prior to the administration of LPS (100 ng/mL). After the coincubation for 30 min, cells were washed twice with cold PBS and scraped in 100 μL of cytoplasmic protein extract reagent containing protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO). The cell pellets were vortexed for 5 s and kept on ice for 15 min, followed by an addition of 20 μL of cytoplasmic protein extract reagent B. The nuclear fractions were precipitated by centrifuging at 16 000 g for 15 min at 4 °C. The cytosolic fractions, corresponding to the supernatants, were transferred to an Eppendorf tube and kept at −80 °C until use. The nuclei pellets were resuspended with 50 μL of nuclear protein extract reagent, and then vortexed for 30 s before incubation on ice for another 30 min. During the incubation, the suspension was vortexed every 2 min. Lysates were centrifuged at 12 000 g for 10 min at 4 °C. The supernatants which were nuclear extracts were stored at −80 °C until analyses. Western Blotting. BV2 cells cultured in 6-well plates at a density of 1.5 × 105 cells/mL were pretreated with UA or UB (3, 10, and 30 μM) for 1 h, and then exposure to LPS (100 ng/mL) for 30 min (for detecting phosphorylation levels of MAPKs, Akt, and IκB-α) or 24 h (for detecting iNOS and COX-2), respectively. After that, the cells were lysed with cold RIPA buffer (25 mM Tris-HCl, 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, pH 7.6) and centrifuged at 15 000 g for 15 min at 4 °C. The protein lysates were separated by 12% SDS-PAGE and electrophoretically transferred to PVDF membrane. The membrane was blocked with 5% nonfat milk in TBST, and then incubated with primary antibodies specific to iNOS, COX-2, Erk1/2, p-Erk1/2, p38 MAPK, p-p38 MAPK, Akt, p-Akt, NF-κB p65, p-NF-κB p65, IκB-α, p-IκB-α (Cell Signaling Technology, Danvers, MA, USA) overnight at 4 °C. GAPDH and histone H3 (Cell Signaling Technology, Danvers, MA, USA) were used as a loading control. Chemiluminescence using an ECL detection kit (GE Healthcare, Piscataway, NJ, USA) was detected by using Bio-Rad ChemiDoc XRS+ System (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Immunofluorescence Staining. NF-κB p65 nuclear translocation was examined by immunofluorescence assay. BV2 microglia cells (1 × 104 cells/well) were seeded on glass coverslips in 24-well plates and incubated in serum-free media overnight. The next day, cells were treated with UA or UB for 1 h prior to the administration of LPS (100 ng/mL). After the coincubation for 30 min, BV2 cells were fixed with 4% formalin at 4 °C for 1 h and then permeabilized with 0.2% Triton X-100 for another 1 h. Nonspecific staining was blocked with 1% BSA for 30 min. Cells were then incubated with anti-NF-κB p65 primary antibody at a dilution of 1:500 at 4 °C overnight. Antirabbit fluorescein isothiocyanate (FITC)-conjugated antibody was used as a secondary antibody at a dilution of 1:2000 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA). The cells were sequentially stained for 10 min with 0.5 μg/mL DAPI. Fluorescence staining was visualized on a Leica laser confocal microscope. Statistical Analyses. Quantitative data are presented as average ± SE. Significant differences were determined by a one-way ANOVA followed by a Duncan’s Multiple Range post hoc test. All statistical tests with P < 0.05 were considered significantly different.

Rheinstetten, Germany) with dimethyl sulfoxide (DMSO-d6) as solvent. ESIMS data were obtained on a LC/Triple Quadrupole Mass Spectrometer equipped with an Agilent 1290 analytical HPLC and 6420 triple quadrupole mass spectrometer (Santa Clara, CA, USA). Cell Culture. BV2 cells were a kind gift from Professor Yue Hou from Northeastern University (Shenyang, China), and were routinely cultured in DMEM medium containing 10% FBS, 1% antibiotics, and L-glutamine (Life Technologies, Grand Island, NY, USA) at 37 °C, under a humidified atmosphere containing 5% CO2. The stock solutions of urolithins were prepared in DMSO and the final concentration of DMSO in cell culture media was 0.1%. LPS (Sigma-Aldrich, St. Louis, MO) was used at a concentration of 100 ng/mL for microglia stimulation. Cell Viability. Viability of BV2 microglia after treatment with urolithins was determined by the sulforhodamine B (SRB) assay. A minimum of 5 replicates of 1.5 × 104 BV2 cells per well were seeded in 96-well plates and allowed to adhere to the plate for approximately 24 h, at which time the media was removed and replaced with fresh media containing varying concentrations of urolithins in DMSO (0.3, 1, 3, 10, 30, 50, and 100 μM) with or without LPS. DMSO at a final concentration 0.1% was used as stimulated and nonstimulated controls. Cells were subsequently incubated for an additional 24 h. After the incubation period, cells were fixed with 10% (w/v) trichloroacetic acid for 5 min, and the excess trichloroacetic acid was removed by washing with distilled water for 5 times. The cells were stained with SRB (0.4%, w/v) for 30 min and the excess dye was removed by washing repeatedly with 1% (v/v) acetic acid. The protein-bound dye was dissolved in 10 mM Tris base solution (pH 10.5). Cell viability was determined by measuring the absorbance with a microplate reader at 510 nm. Relative cell viability (%) was displayed using vehicle (0.1% DMSO)-treated samples without LPS incubation as a control. Measurement of Production of Nitric Oxide Species. BV2 cells cultured in 6-well plates at a density of 1.5 × 105 cells/mL were incubated with urolithins (3, 10, and 30 μM) and LPS (100 ng/mL) for 24 h. Nitrite concentration in the supernatant was determined spectrophotometrically by using a nitrate/nitrite assay kit according to the manufacturer’s instructions (Beyotime Biotechnology, Shanghai, China). Briefly, 50 μL of supernatant was mixed with Griess reagent (Reagent I, 50 μL; Reagent II, 50 μL) and then incubated at room temperature for an additional 10 min. Absorbance was measured at 550 nm with 650 nm as a reference filter, and the nitrite concentration was determined using the calibration curve developed from using sodium nitrite as a standard. Each nitrite standard and samples were assayed in triplicate. RNA Extraction and Quantitative Real-Time PCR (qRT-PCR). On reaching the confluence of 80%, BV2 microglial cells cultured in 6-well plates were incubated with urolithins (3, 10, and 30 μM) and LPS (100 ng/mL) for 16 h. Total RNA was extracted by using Trizol (Life Technologies, Grand Island, NY, USA) in accordance with the manufacturer’s instructions. RNA concentration was quantified spectrophotometrically by using Nanodrop 2000 UV−vis spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). Two micrograms of total RNA was reverse transcribed to single-stranded cDNA using the Reverse Transcription System cDNA synthesized kit (Promega, Madison, WI, USA). Relative mRNA levels were quantified by SYBR green chemistry using a Bio-Rad CFX Connect Real-Time system (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Reaction conditions were 10 min at 95 °C, followed by 45 cycles of 10 s at 95 °C, 20 s at 60 °C, 20 s at 72 °C, and then melt curve analysis to identify PCR specificity. The sequences of the primers used for qRT-PCR were listed as following: iNOS, Forward 5′-TGGAGCGAGTTGTGGATTGTC-3′; Reverse 5′-GGTCGTAATGTCCAGGAAG TAG-3′. COX-2, Forward 5′-CCAGCACTTCACCCATCAGT-3′; Reverse 5′-ACACCTCTCCACCAATGACC-3′. IL-1β, Forward 5′-AGCCAAGCTTCCTTG TGCAAGTGT-3′; Reverse 5′-GCTCTCATCAGGACAGCCCAGGT-3′. IL-6, Forward 5′-TGTCTATACCACTTCACAAGTCG-3′; Reverse 5′-GCACAACTC TTTTCTCATTTCCAC-3′. TNF-α, Forward 5′-AGCCCCCAGTCTGTATCCTT-3′; Reverse 5′ACAGTCCAGGTCACTGTCCC-3′. 18S, Forward 5′-AGTCCCTGC CCTTTGTACACA-3′; Reverse 5′-CGATCCGAGGGCCTCACTA-3′. Enzyme-Linked Immunosorbent Assay (ELISA). BV2 cells cultured in 6-well plates at a density of 1.5 × 105 cells/mL were treated with UA or UB (3, 10, and 30 μM) in the presence or absence of LPS

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RESULTS Urolithins A and B Reduce the Release of Nitric Oxide in LPS-Induced BV2Microglia. Nitric oxide (NO) is an important inflammatory mediator involved in the pathogenesis of neuroinflammation and multiple neurodegenerative diseases.24 In order to evaluate the inhibition of urolithins on NO production in LPSstimulated BV2 microglia (100 ng/mL), nitrite (a stable oxidized C

DOI: 10.1021/acs.jafc.7b03285 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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compounds at an equivalent concentration of 30 μM. As shown in Figure 3F, upon LPS administration, UA exhibited the strongest inhibitory effects on the inflammatory response, UB exhibited less inhibitory effects than UA, while UC exhibited almost no effects on the expression of the inflammatory genes. To verify the antineuroinflammatory effects of UA and UB, the production of the pro-inflammatory cytokines, TNF-α, IL-6, and IL-1β in LPS-stimulated BV2 microglia were determined by ELISA. LPS increased TNF-α production by 7-fold (from 14.32 ± 2.2 pg/mL to 302.61 ± 6.81 pg/mL). UA reduced the increase by 91, 93, and 95% at the concentration of 3, 10, and 30 μM, respectively. UB treatment also exhibited similar inhibition on TNF-α production, which by 76, 85, and 91% at the concentration of 3, 10, and 30 μM, respectively (Figure 4A). IL-6 levels were increased by approximately 6-fold (from 56.38 ± 4.85 pg/mL to 396.61 ± 42.75 pg/mL) after LPS induction. And the induction was significantly reduced after incubation with UA or UB (Figure 4B). Similar inhibitory effects was also observed on IL-1β production by the treatment of UA and UB (3−30 μM) when compared with LPS alone (Figure 4C). Overall, the results from measuring NO release, TNF-α, IL-6, and IL-1β production, and pro-inflammatory gene expression in LPS-stimulated BV2 cells suggested that UA and UB were the more active compounds among the urolithins evaluated herein. Therefore, UA and UB were selected for further mechanistic studies (described below). Urolithins A and B Reduce iNOS and COX-2 Protein Levels in LPS-Induced BV2Microglia. iNOS and COX-2 expressed in activated microglial cells play key roles in NO and PGE2 production in CNS response to various stimuli.1 Western blot of cell lysates revealed that iNOS and COX-2 were barely detectable in nonstimulated cells but were strongly expressed in LPS-induced BV2 microglia (Figure 5A and B). UA decreased iNOS and COX-2 protein expression in a dose-dependent manner (3−30 μM). Not surprisingly, UB exhibited weaker inhibition on iNOS and COX-2 protein expression compared with UA (Figure 5A and B), which correlated with the results obtained from iNOS and COX-2 mRNA expression experiments (Figure 5A and B).

product of NO) levels in culture supernatants were determined by using the Griess reagent. None of the urolithins significantly affected the viability of BV2 microglia (at concentrations of 3, 10, and 30 μM) after 24 h incubation compared to control cells, as assessed by the SRB assay (Figure 1B−F), and complementary MTT assay (Figure S11). Thus, the urolithins were further biologically evaluated at these aforementioned nontoxic concentrations. The exposure of BV2 microglia to LPS for 24 h markedly increased nitrite levels by about 3-fold. Treatment of UA inhibited NO release by 28, 55, and 82% at concentrations of 3, 10, and 30 μM, respectively (Figure 2A). UB inhibited NO release by 16 and 59% at concentrations of 10 and 30 μM, respectively (Figure 2B). UC was active only at the high concentration of 30 μM (28% of inhibition) (Figure 2C). No inhibitory effects were observed with the treatment of mUA and dmUC (Figure 2D and E). None of the five urolithins exhibited any effects on NO release by the BV2 microglia without LPS administration (Figure 2F). Urolithins A and B Down-Regulate Pro-Inflammatory Genes Expression and TNF-α, IL-6, and Il-1β Release in LPS-Induced BV2Microglia. To further evaluate the protective roles of the urolithins against neuroinflammation, their inhibitory effects on mRNA levels of inflammatory markers in LPS-stimulated BV2 microglia were determined by qRT-PCR. As shown in Figure 3A−E, the mRNA expression of TNF-α, IL-6, iNOS, COX-2, and IL-1β in BV2 microglia were significantly increased upon LPS stimulation. Both UA and UB inhibited the pro-inflammatory cytokines, TNFα, IL-6, and IL-1β mRNA expression induced by LPS in BV2 microglia in a dosage-dependent manner (3−30 μM) (Figure 3A and B). For the two pro-inflammatory enzymes, the increased COX-2 mRNA expression in BV2 cells by LPS was significantly and dose-dependently attenuated by UA. Furthermore, both UA and UB inhibited iNOS mRNA expression in LPS-stimulated BV2 cells but not in a dosage-dependent manner (Figure 3A and B). UC, mUA, and dmUC did not change the expression of these pro-inflammatory genes at the test concentrations (Figure 3C-E). To further compare the inhibitory activity of UA, UB, and UC on these inflammatory markers gene expression, a further experiment was performed with these

Figure 2. Effects of urolithins on nitric oxide (NO) production in LPS-stimulated BV2 microglia. BV2 cells were cotreated with LPS (100 ng/mL) and various concentrations of (A) UA, (B) UB, (C) UC, (D) mUA, and (E) dmUC (3, 10, 30 μM), or only treated with (F) UA, UB, UC, mUA, and dmUC (30 μM) without LPS for 24 h. The supernatants were collected for the measurement of NO production using the Griess reagent. Data presented as mean ± S.E., n = 3. #, P < 0.05 as compared with the control group. *, P < 0.05 as compared with the LPS-treated group. D

DOI: 10.1021/acs.jafc.7b03285 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Figure 3. Effects of urolithins on the pro-inflammatory genes expression in LPS-stimulated BV2 microglia. BV2 cells were incubated with LPS (100 ng/mL) and various concentrations of (A) UA, (B) UB, (C) UC, (D) mUA, (E) dmUC (3, 10, 30 μM), and (F) UA, UB, and UC (30 μM) for 16 h. The total RNA was extracted. The relative mRNA levels of TNF-α, IL-6, iNOS, Cox2, and IL-1β were measured by using quantitative real-time PCR and normalized with 18S rRNA levels. Data presented as mean ± S.E., n = 3. #, P < 0.05 as compared with the control group. *, P < 0.05 as compared with the LPS-treated group.

disorders.25 Therefore, in the current study, the effects of UA and UB on the phosphorylation levels of Erk1/2 and p38 MAPK, and Akt were determined. Stimulation of BV2 microglia with LPS resulted in increased phosphorylation levels of Erk1/2, p38 MAPK and Akt. Treatment with UA for 1 h significantly attenuated the phosphorylation levels of Erk1/2, p38 MAPKand Akt induced by LPS in a dosage-dependent manner (Figure 7A−C). UB also displayed inhibition on the phosphorylation levels of Erk1/2 and Akt (Figure 7A and C). However, we did not observe significant decrease of p-p38 MAPK in BV2 cells treated with UB (Figure 7B).

Urolithins A and B Suppress the NF-κB Signaling Pathway in LPS-Induced BV2Microglia. To further investigate the anti-inflammatory mechanism of action of urolithins, NF-κB, a key transcription factor modulating a wide range of pro-inflammatory genes expression was examined. As shown in Figure 6A, the p-p65 levels was highly induced upon LPS administration, but the induction was significantly and dosage-dependently abolished by UA and UB treatment (3−30 μM). UB displayed stronger inhibitory activity on p-p65 (Ser536) protein expression than UA. Next, the effects of the uroltihins on classic IκB-NF-κB signaling pathway were measured by Western blot. As shown in Figure 6B, treatment with UA and UB improved the expression of IκB-α and reduced the phosphorylation levels of IκB-α as compared to LPS-stimulated microglia, suggesting that UA and UB suppressed LPS-induced IκB-α phosphorylation and degradation. Furthermore, as shown in Figure 6C, treatment with UA decreased LPS-induced NF-κB p65 nulcear translocation at the concentrations ranging from 3 to 30 μM. UB only exhibited inhibitory effects at the test concentrations of 10 and 30 μM. This finding was supported by immunofluorescence staining of the intracellular NF-κB p65 subunit (Figure 6D). Urolithins A and B Reduce the Phosphorylation Levels of Erk1/2, p38 MAPK and Akt in LPS-Induced BV2Microglia. MAPK and PI3K/Akt signaling cascades have been reported to be highly associated with the response of inflammation and involved in the development of PD and other neurodegenerative

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DISCUSSION Urolithins, gut microbiota metabolites of ETs and EA, have lower molecular weights and improved bioavailability compared to their precursors.16 Emerging data have shown that urolithins are associated with the anti-inflammatory and anticancer effects attributed to dietary EA or ETs.11,16 However, the potential mechanisms of action of urolithins in the context of the neuroprotective effects of EA and ETs-rich food products are not yet well understood. Verzelloni et al. reported that urolithins could protect human neuronal cells in vitro by inhibiting protein glycation.26 GonzalezSarrias et al. reported that urolithins could exert neuroprotective effects by reducing oxidative stress-induced cytotoxicity in human neuroblastoma SH-SY5Y cells.27 To data, limited knowledge is known about the effects of urolithins on abnormal microglial E

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Figure 4. Effects of UA and UB on TNF-α, IL-6, and IL-1β production in LPS-stimulated BV2 microglia. BV2 cells were coincubated with LPS (100 ng/mL) and UA or UB (3, 10, 30 μM) for 24 h. The supernatant was collected and the extracellular levels of (A) TNF-α, (B) IL-6 and (C) IL-1β in culture media were measured using commercial ELISA kit. Data presented as mean ± S.E., n = 3. #, P < 0.05 as compared with the control group. *, P < 0.05 as compared with the LPS-treated group.

Figure 5. Effects of UA and UB on iNOS and COX-2 protein expression in LPS-stimulated BV2 microglia. BV2 cells were pretreated with UA or UB (3, 10, 30 μM) for 1 h and then stimulated with LPS (100 ng/mL) for 24 h. The cells were lysed with RIPA buffer and the protein levels of (A) iNOS and (B) COX-2 were measured by using immunoblot analysis. GAPDH was used as a loading control. And all the experiments have been repeated 3 times independently. Data presented as mean ± S.E., n = 3. #, P < 0.05 as compared with the control group. *, P < 0.05 as compared with the LPS-treated group.

activation. Microglia are considered as the first line of defense in the CNS to initiate immune response to injuries and pathogens. Prolonged activation of microglia is involved in multiple neurodegenerative diseases.3 We recently reported that urolithins could decrease pro-inflammatory cytokins, NO and PGE2 release in LPSstimulated BV2 microglia, but the mechanism of action were unclear.21 In the present study, the effects of UA, UB, UC, along with two urolithin phase II enzyme conjugates, namely, mUA

and dmUC, on inflammatory response in LPS-stimulated BV2 microglia were evaluated. Our results demonstrated that UA and UB inhibited neuroinflammation in LPS-activated BV2 microglial cells by suppressing the production or expression of NO, TNF-α, IL-6, IL-1β, as well as iNOS and COX-2, which were consistent with our previous study.21 Among the five urolithins evaluated herein, UA exhibited the most significant antineuroinflammatory effects, while UC, mUA, F

DOI: 10.1021/acs.jafc.7b03285 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry

Figure 6. Effects of UA and UB on NF-κB signaling pathway in LPS-stimulated BV2 microglia. BV2 cells were pretreated with UA or UB (3, 10, 30 μM) for 1 h and then stimulated with LPS (100 ng/mL) for 30 min. (A) Total p-p65 (Ser536)/total p65 and (B) IκB-α and p-IκB-α were measured by Western blot. GAPDH was used as a loading control. (C) Nuclear and cytosolic extracts were isolated and the levels of p65 in each fraction were determined by Western blot. Histone H3 and GADPH were used as internal controls. (D) The translocation of p65 subunit of NF-κB were visualized by immunofluorescence staining. Scale bar = 20 μm. And all the experiments have been repeated 3 times independently. Data presented as mean ± S.E., n = 3. #, P < 0.05 as compared with the control group. *, P < 0.05 as compared with the LPS-treated group.

suggesting that UA and UB act to reduce NO production by interfering with iNOS enzyme activity in LPS-stimulated BV2 microglia. These observations were consistent with the results obtained by Piwowarski et al. in RAW 264.7 macrophages.17 It should be noted that in a recent research, the mixture of UA and UB (at the concentration of 5 μM) increased nitrite/nitrate levels via up-regulating eNOS expression in human aortic endothelial cells after 24 h treatment.33 The inconsistencies in the effects on NO production by UA and UB may be due to the cell line assayed (BV2 microglial cells vs human aortic endothelial cells). Therefore, it is possible that urolithins may exert different effects in different organs in the body but further research would be required to confirm this. Our data was also consistent with a previous in vivo study on the neuroprotective activities of a pomegranate extract, which reported that pretreatment with the pomegranate extract prior to ischemia/reperfusion could significantly reduce NO, TNF-α, and NF-kB contents in the brain of albino rats.34 Therefore, animal studies using pure urolithins, instead of their precursors, such as EA or ETs-rich foods, to evaluate their antineuroinflammatory activity is warranted, and will be pursued by our group in the future. NF-κB is an important transcription factor to mediate the inflammatory response, and plays crucial roles in regulating the transcription of a wide battery of host genes that controls inflammatory and immune responses during neuroinflammation and neurodegeneration.1,35−38 In resting cells, NF-κB is sequestered in the cytoplasm in an inactive form by inhibitory kappa B (IκB)

and dmUC only showed weak or no effects. Notably, UA has recently been reported to induce auto/mitophagy and prolong lifespan in Caenorhabditis elegans and improve muscle function in rodents.28 Our mechanistic studies revealed, for the first time, that the NF-κB, MAPK, and PI3K/Akt signaling pathways were involved,in part, in the antineuroinflammatory mechanisms of action of UA and UB. Overall, our results support the beneficial effects reported for EA and ETs-rich food products in ameliorating brain inflammatory symptoms and improving neurodegenerative diseases.13−15,29 NO is a free radical produced from L-arginine by the nitric oxide synthases (NOS). NOS could be divided into constitutive and inducible forms. The former is expressed in specific cells, like eNOS in endothelial cells and nNOS in neuron cells. The inducible form (iNOS) is expressed in various cell types, including microglial cells.24 While small amounts of NO exert health beneficial effects, excessive produced NO in CNS will act as a neurotoxic factor through generation of peroxynitrite (ONOO−), which is likely to mediate programmed cell death in brain injury.30 The iNOS expressed in activated microglia cells plays primary roles in NO production in CNS response to various stimuli, including LPS.24 Selective and nonselective inhibitors of NOS have been shown to inhibit inflammation and to reduce neurodegeneration in different animal models.31,32 Our results demonstrated that UA and UB could significantly and dose dependently (3−30 μM) inhibit NO production and down-regulate protein expression levels of iNOS, G

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Figure 7. Effects of UA and UB on the phosphorylation levels of Erk1/2, p38 MAPK and Akt in LPS-stimulated BV2 microglia. BV2 cells were pretreated with UA or UB (3, 10, 30 μM) for 1 h and then stimulated with LPS (100 ng/mL) for 30 min. The cells were lysed with RIPA buffer and the phosphorylation levels of (A) Erk1/2, (B) p38 MAPK, and (C) Akt were measured by using immunoblot analysis. Total Akt, Erk1/2, and p38 MAPK was used as a loading control. And all the experiments have been repeated 3 times independently. Data presented as mean ± S.E., n = 3. #, P < 0.05 as compared with the control group. *, P < 0.05 as compared with the LPS-treated group.

modulate NF-κB signaling via both the classic IκB-dependent pathway and post-transcriptional phosphorylation of p65 with the former mechanism being possibly more relevant. In additional to NF-κB signaling pathway, slight but statistically significant inhibitory effects were also detected in the activation of MAPK signaling pathways, especially p38 MAPK, by UA and UB established on human colon fibroblasts challenged with IL-1β.41 The mixture of EA and urolithins (10 μM EA, 40 μM UA, and 40 μM UB) were reported to interfere in MAPK signaling by significantly reducing phosphorylation level of Erk1/2 in Caco-2 cells.42 Consistent with that previous report, we also found that UA displayed significant inhibition on p38 MAPK phosphorylation in LPS-induced BV2 cells, while no effects was observed with UB treatment (3−30 μM). Both UA and UB lowered the phosphorylation levels of Erk1/2. MAPKs are involved in microglial activation and their phosphorylation directly affect the NF-κB mediated transcription of inflammatory mediators.43 The p38 MAPK is a stress-activated kinase and is considered as one of the most important regulators of inflammatory genes in microglia. The Erk pathway could also be triggered by various cytotoxic stress stimuli in the brain.44 In vivo studies suggest that Erk and p38 MAPK play important roles in harmful microglial activation in chronic

proteins. Upon activation, IκB undergoes phosphorylation via inhibitor of κB kinase (IKK), which leads to its ubiquitination and proteasomal degradation, and resulting in the liberation and nuclear translocation of NF-κB subunits.5,39 Emerging evidence have suggested that post-transcriptional phosphorylation of NF-κB also play important roles in response to inflammatory stimuli.38 An animal study showed that punicalagin, the major ET in pomegranate, is capable of attenuating LPS-induced TNF-α, IL-1β, and IL-6 release in the bronchoalveolar lavage fluid via inhibiting NF-κB signaling pathways in BALB/c mice.40 In the current study, the effects of UA and UB on the classical NF-κB signaling pathway in LPS-stimulated BV2 microglia were investigated. We determined total p-p65 (Ser536) protein expression in LPS-stimulated BV2 cells and while UB was more active than UA (Figure 6A), UA showed stronger inhibition on p65 subunit nuclear translocation (Figures 6C and D). Our result was in agreement with a previous report which demonstrated that UA and UB decreased p65 nuclear translocation and NF-κB DNA binding activity in RAW 264.7 macrophages challenged with LPS, and UA was more active than UB.17 The inhibitory activity of the urolithins against LPSinduced NO production and pro-inflammatory cytokines (TNF-α, IL-6, and IL-1β) release in BV2 microglial cells suggest that they H

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neurodegenerative disease such as AD and PD.25,44 Therefore, the suppression of p38 MAPK and Erk phosphorylation by UA in microglia indicated its potential investigation as a pharmaceutical agent for the treatment of neurodegenerative diseases. We also evaluated the effects of UA and UB on PI3K/Akt pathway in LPS-induced BV2 microglia. It has been reported that PI3K/Akt pathway is involved in catalase, peptidoglycan, or LPS induced COX-2 and iNOS expression in BV2 cells.45−47 As the upstream gene of NF-κB, Akt could modulate IκB degradation via phosphorylation of IKK in activated BV2 microglia.45 In the current study, UA and UB significantly and dose-dependently inhibited the phosphorylation of Akt in LPS-stimulated BV2 cells which may partially contribute to the observed decrease of proinflammatory factors production. Taken together, UA and UB exerted anti-inflammatory activities in LPS-induced BV2 microglia through inhibiting NF-κB, MAPKs, and PI3K/Akt, which are all important inflammatory cascade transduction pathways upon TLR4 activation in LPS-induced microglial cells.7 Thus, the antiinflammatory activities of UA and UB may be mediated by interfering with TLR4 activation. Further mechanistic studies to determine the effect of urolithins on upstream proteins of TLR4 signaling would help to better elucidate the mechanism of antineuroinflammatory actions of these compounds. The metabolism and tissue distribution of urolithins in the body is well established in spite of limited knowledge about the brain tissue disposition.16 Under normal physiological condition, urolithins are mainly transformed to sulfated and glucuronide conjugates by phase II enzymes immediately after absorption. A recent research demonstrated that glucuronide conjugates of uroltihins undergo cleavage by β-glucuronidase in inflammatory conditions and thus would revert back to their intact urolithin aglycon.11 Although it is possible that the penetration of urolithins through the BBB may be limited, urolithins have been detected in the brain of mice after direct intravenous injection of these compounds.48 Our previous research also suggested that UA and UB, instead of their glucuronidated derivatives, fulfill criteria required for BBB penetration based on computational studies.12 Therefore, further animal studies to determine the pharmacokinetics and brain tissue absorption and distribution of urolithins, including in animal models of neurodegenerative diseases would help to better understand the neuroprotective effects of these compounds. In conclusion, our study provides mechanistic insights that urolithins inhibit LPS-induced inflammation of microglia through the inhibition of the NF-κB, MAPK, and PI3K/Akt signaling pathways. Our results warrant further investigation of these compounds as potential dietary and therapeutic agents against neuroinflammatory and neurodegenerative diseases. Therefore, further studies to evaluate the antineuroinflammatory activity of uroltihins using in vivo models are warranted.

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AUTHOR INFORMATION

Corresponding Authors

*Phone/Fax: 001-401-8749367; E-mail: [email protected]. *Phone/Fax: 0086-24-83656106; E-mail: [email protected]. edu.cn. *Phone/Fax: 0086-24-83656122; E-mail: [email protected]. ORCID

Hang Ma: 0000-0001-7565-6889 Navindra P. Seeram: 0000-0001-7064-2904 Xueshi Huang: 0000-0002-1561-8108 Liya Li: 0000-0002-1894-9500 Funding

This work was supported by the National Natural Science Foundation of China [Nos. 31401473, 81570788, and 81341102] and the Fundamental Research Funds for the Central Universities [Nos. N162004004, N130220001, N162004005, and N120820002]. Notes

The authors declare no competing financial interest.

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REFERENCES

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.7b03285. Anti-inflammatory effects of urolithins reported in various models; 1H and 13NMR spectra of urolithins A, B, and C, 8-methyl-O-urolithin A, and 8,9-dimethyl-O-urolithin C; effects of urolithins on cell viability of BV2 microglia with or without LPS stimulation by the MTT assay; expression of pro-inflammatory genes in BV2 cells treated with urolithins alone; and expression of Iba-1 in LPS-stimulated BV2 cells treated with urolithins A and B (PDF) I

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