Hydroxytyrosol Inhibits LPS-Induced Neuroinflammatory Responses via Suppression of TLR-4-Mediated NF-κB P65 Activation and ERK Signaling Pathway

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RESEARCH ARTICLE L. Zhang et al. / Neuroscience xxx (2018) xxx–xxx

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Hydroxytyrosol Inhibits LPS-Induced Neuroinflammatory Responses via Suppression of TLR-4-Mediated NF-jB P65 Activation and ERK Signaling Pathway

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Lanqiu Zhang, a* Jinlu Zhang, a Xiaolin Jiang, a Lei Yang, a Qi Zhang, a Botao Wang, b Lihua Cui a and Ximo Wang a*

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a Tianjin Key Laboratory of Acute Abdomen Disease Associated Organ Injury and ITCWM Repair, Institute of Acute Abdominal Diseases, Tianjin Nankai Hospital, Tianjin 300100, China

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b

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Graduate School, Tianjin Medical University, Tianjin 300070, China

Abstract—Neuroinflammation has been implicated in the mechanism underlying the progression of neurodegeneration and infectious neuropathology. Growing evidence suggest that hydroxytyrosol (3,4-dihydroxyphenilethanol, HT), one of the main polyphenols presented in extra virgin olive oil (EVOO), has shown potential antiinflammatory and neuroprotective effects. However, the potential anti-neuroinflammation activity and underlying mechanism of HT remain poorly understood. The present study aimed to investigate the effects of HT on lipopolysaccharide (LPS)-induced inflammation in both in vitro and in vivo models and the associated molecular mechanism. Our results revealed that HT significantly reduced the production of pro-inflammatory mediators in BV2 microglia and primary microglia cells. Phenotypic analysis showed that HT significantly reduced M1 marker CD86 expression and increased M2 marker CD206 expression. In addition, HT significantly decreased the levels of phospho-NF-jB p65 and phospho-extracellular signal-regulated kinase (ERK) in a dose-dependent manner. Moreover, HT suppressed the LPS-induced Toll like receptor 4 (TLR4) in BV2 microglia. In vivo administration of HT following LPS injection significantly reduced some proinflammatory mediator levels and microglia/astrocyte activation in the brain. Together, these results suggest that HT suppressed the LPS-induced neuroinflammatory responses via modulation of microglia M1/M2 polarization and downregulation of TLR-4 mediated NF-jB activation and ERK signaling pathway. Ó 2019 IBRO. Published by Elsevier Ltd. All rights reserved. Key words: hydroxytyrosol, microglia, LPS, anti-neuroinflammation, Iba-1, GFAP.

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INTRODUCTION

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Neuroinflammation is a characteristic feature of various neurological disorders including Alzheimer’s disease (AD), Parkinson’s disease (PD), multiple sclerosis and acute traumatic brain injury as well as infectious neuropathology (Grigoriadis et al., 2015, Latta et al., 2015, Rocha et al., 2015, Bergold, 2016). Microglia are the major resident immune cells in the brain that contribute to immune surveillance and regulate the homeostasis of the central nervous system (CNS) (Nimmerjahn et al., 2005). However, over-activated

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microglia secrete proinflammatory cytokines and oxygen free radicals, often resulting in cerebral injury under various pathologic conditions. Therefore, inhibition of microglia activation has been regarded as one of prime targets for treatment of diverse neuropathological conditions (Glass et al., 2010). Hydroxytyrosol (3,4-dihydroxyphenil-ethanol, HT), which is a small phenolic molecule derived from oliveoil, has shown strong anti-oxidant, anti-inflammatory and antithrombotic activities. For example, Fuccelli et al. recently found that HT demonstrated anti-inflammatory and anti-oxidant abilities in a mouse model of systemic inflammation induced by LPS (Fuccelli et al., 2018). HT also reduced liver inflammation and oxidative stress by reducing the production of oxygen species and lipid peroxidation (Pirozzi et al., 2016). In vitro, HT inhibited the production of inflammatory mediators such as COX-2 and PGE2 in human isolated peripheral blood monocytes (Rosignoli et al., 2013). In addition, some evidence showed that HT prevented the inflammatory progress of atherosclerosis by decreasing the concentration of proinflammatory cytokines, inhibiting the endothelial activation

*Corresponding authors. Address: Tianjin Key Laboratory of Acute Abdomen Disease Associated Organ Injury and ITCWM Repair, Institute of Acute Abdominal Diseases, Tianjin Nankai Hospital, 6 Changjiang Road, Tianjin 300100, China. E-mail addresses: [email protected] (L. Zhang), wangximonkyy @126.com (X. Wang). Abbreviations: AD, Alzheimer’s disease; EDTA, ethylenediaminetetraacetic acid; ELISA, Enzyme-linked immunosorbent assay; ERK, extracellular signal-regulated kinase; EVOO, extra virgin olive oil; iNOS, nitric oxide synthase; LPS, lipopolysaccharide; MAPK, mitogen-activated protein kinase; PD, Parkinson’s disease; TLR4, Toll like receptor 4. https://doi.org/10.1016/j.neuroscience.2019.12.005 0306-4522/Ó 2019 IBRO. Published by Elsevier Ltd. All rights reserved. 1

Please cite this article in press as: Zhang L et al. Hydroxytyrosol Inhibits LPS-Induced Neuroinflammatory Responses via Suppression of TLR-4-Mediated NF-jB P65 Activation and ERK Signaling Pathway. Neuroscience (2019), https://doi.org/10.1016/j.neuroscience.2019.12.005

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and expression of chemotactic and adhesion molecules (Souza et al., 2017). Recently, several clinical trials and population studies indicated that the main polyphenols such as HT, oleuropein, and tyrosol, are mainly responsible for the neuroprotection effect in neurodegenerative disease such as AD and PD, as well as improvement of cognitive performance (Alcalay et al., 2012, Casamenti et al., 2015, Peyrol et al., 2017, Robles-Almazan et al., 2018). However, the effects of HT on neuroinflammation in microglial cells have not been reported yet. In the present study, we investigated the antineuroinflammatory effects of HT on LPS-stimulated microglial cells, along with underlying signaling mechanisms. Our results showed that HT potently inhibited proinflammatory cytokine production associated with the downregulation of TLR-4 mediated NF-jB activation and ERK signaling pathway. In addition, we found that HT modulated the polarization of microglia by down-regulating the expression of M1 marker CD86 and up-regulating that of M2 marker CD206. In vivo administration of HT significantly suppressed microglia and astrocyte activation induced by LPS and decreased levels of proinflammatory mediators. Those findings strongly indicate that HT is a potential therapeutic candidate for inflammation related neurodegenerative diseases or acute brain injury.

EXPERIMENTAL PROCEDURES

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Materials

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LPS (E scherichia coli 0111: B4), DAPI, IL-4, and MTT were obtained from Sigma-Aldrich (St Louis, MO, USA). HT were purchased from Aladdin (Shanghai, China). Enzyme-linked immunosorbent assay (ELISA) kit for TNF-a and IL-6 were purchased from Multi Sciences (Hangzhou, China). NO assay kit was from Beyotime (Shanghai, China). The antibodies against NF-jB p65, phospho-NF-jB p65, nitric oxide synthase (iNOS), COX2, p38 mitogen-activated protein kinase (MAPK), phospho-p38 MAPK, ERK1/2, phosphor-ERK1/2, JNK, and phospho-JNK were purchased from Cell Signaling (Danvers, MA, USA). The PerCP anti-CD86 and PE anti-CD206 antibodies were purchased from Sungene Biotech (Tianjin, China). The antibodies against TLR-4 and GAPDH were obtained from Abcam (Cambridge, UK). Alexa FluorTM 546 Goat Anti-Rabbit IgG (H + L) were obtained from Invitrogen (Carlsbad, CA, USA). Trizol reagent was obtained from Takara (Takara; Shiga, Japan), and RT-PCR primers were obtained from Sangon Biotech (Shanghai, China).

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BV2 microglia cell culture

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The murine BV2 microglia cells were obtained from the China Infrastructure of Cell Line Resources (Beijing, China). The cells were cultured in DMEM supplemented with 10% FBS and 1% penicillin/streptomycin at 37 °C in a humidified incubator under 5% CO2.

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Cell viability

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Cell viability was determined using the MTT following the manufacturer’s instruction. Briefly, cells (8
104 cells/ mL) were seeded into 96-well plates and treated with different concentrations of HT for 1 h before treatment with or without LPS (0.5 lg/mL) for 24 h. The cells were then incubated with 10 ll MTT (5 mg/mL) for 4 h. 100 ll DMSO was added to each well to dissolve the formazan crystals, and the absorbance was determined at 490 nm using a microplate reader.

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Primary microglia isolation and culture

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Isolated mouse primary microglia cultures were prepared as previously described (Giulian and Baker, 1986). In brief, primary mixed glial cell cultures were prepared from cerebral cortices of neonatal mice (postnatal day 1–3). After mechanical and chemical dissociation, glial cells were seeded in 75 cm2 tissue culture flask and maintained in DMEM/F12 containing 10% FCS, 1% penicillin/ streptomycin, and 1% glutamine at 37 °C under 5% CO2. The medium was replaced every 4–5 days and confluency was achieved after 10 –14 days. Microglia were isolated and purified with a trypsin solution (0.25% trypsin, 1 mM EDTA) diluted 1:3 in DMEM/F12 (Saura et al., 2003). The mild trypsinization resulted in the separation of a detached upper layer of astrocytes from the highly enriched microglia population attached to the bottom of the well. The attached microglia cells were then removed by trypsinization and cultured in new plates for subsequent experiments. The purity of primary microglia cells was determined by flow cytometry with anti-CD11b antibody and was always >95%.

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Animals

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Male C57BL/6 mice were purchased from the Experimental Animal Center of Military Medical Sciences (Beijing, China). All experiments were performed in accordance with the animal protocols and guidelines approved by the Committee of Animal Care of Tianjin Nankai Hospital. In this study, 25 C57BL/6 mice (8–12 weeks) were used and divided into three groups (control, LPS, and HT + LPS group). Briefly, mice were pretreated with HT (100 mg/kg) by gavage daily for 2 days. 1 h after the last gavage, all animals received intraperitoneal (i.p.) injections of PBS or 10 mg/ kg LPS. After LPS or PBS injection, mice were anesthetized with choral hydrate and perfused with icecold PBS for RNA isolation or ice-cold 4% paraformaldehyde for immunostaining.

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ELISa

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BV2 microglia cells (8
104 cells/mL) were seeded in 96well plate and incubated for 24 h. The cells were incubated with different concentrations of HT for 1 h, and co-cultured with LPS (0.5 lg/mL) for additional 24 h. Then, cell-free supernatants were collected and stored at 80 °C. The concentrations of TNF-a and IL-6 were determined with ELISA kits according to the manufacturer’s instructions. The concentration of NO

Please cite this article in press as: Zhang L et al. Hydroxytyrosol Inhibits LPS-Induced Neuroinflammatory Responses via Suppression of TLR-4-Mediated NF-jB P65 Activation and ERK Signaling Pathway. Neuroscience (2019), https://doi.org/10.1016/j.neuroscience.2019.12.005

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Fig. 1. Effects of HT on the viability of BV2 microglial cells. Cells were treated with different concentration of HT (0, 25, 50, 100 and 200 lM) in the absence or presence of LPS (0.5 lg/mL) for 24 h. Cell viability was determined by MTT assay. Data were presented as mean ± SEM from three independent experiments.

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was determined using a commercially available NO assay kit according to the manufacturer’s instruction.

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RNA isolation and quantitative RT-PCR

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Total RNA was extracted from BV2 cells or primary microglia cells with Trizol reagent according to manufacturer’s protocol. 1 lg of RNA was reverse transcribed using RevertAid First strand cDNA synthesis kit (Thermo, Waltham, MA). Real-time quantitative PCR was performed using a SYBR Green qPCR Mater Mix (Thermo) on a Bio-Rad CFX96 Detection System. The three-step amplification conditions consist of (95 °C, 10 s; 60 °C, 40 s; 72 °C, 30 s). The relative expression

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of mRNA was normalized against the GAPDH mRNA and calculated using the 2(44C(T)) method.

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Western blot analysis

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BV2 cells were harvested and lysed in RIPA buffer (50 mM Tris-HCl pH 7.4, 1% NP-40, 150 mM NaCl, 1 mM Na3VO4, 1 mM NaF, 1 mM EDTA) containing protease inhibitors, 10 lM PMSF, and phosphatase inhibitors. The protein concentration was measured using a BCA kit. The proteins (30 lg) were separated by sodium dodecyl sulfate polyacrylamide gels and transferred to polyvinylidene difluoride (PVDF) membrane. After blocking with 5% non-fat milk for 1 h in TBS-T (0.20 M Tris, 1.37 M NaCl, pH 7.6, 0.1% Tween20), the membrane was incubated with the primary antibodies at 4 °C overnight. Then, the membrane was washed with TBS-T and incubated with anti-rabbit or anti-mouse horseradish peroxidase-conjugated secondary antibodies (1:5000) for 1 h at room temperature. After washing with TBS-T, the blots were developed by enhanced chemiluminescence kit (Thermo) and detected with a chemiluminescence detection system (ChemiDoc Bio-Rad, Berkeley, CA, USA). The Quantity one (Bio-Rad) was used to analyze the images to obtain the grey-scale value of signals.

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Immunofluorescence assay

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The BV2 cells were seeded in 24-well plates containing microscope cover glasses. Then, cells were pretreated with HT for 1 h and treated with LPS (0.5 lg/mL) for 1 h, fixed with 4% paraformaldehyde for 10 min. After washing with PBS, cells were permeabilized using 0.5% Triton X 100 in PBS for 10 min and blocked with 10% goat serum for 1 h at room temperature. Cells were washed and incubated with NF-jB p65 primary antibody (1: 800) at 4 °C overnight. After washed with PBS, the cells were incubated with Alexa FluorTM 546-conjugated secondary antibodies for 1 h at room temperature. The nuclei were stained with 2 lg/ml DAPI for 10 min at room temperature. Finally, the images were captured using a fluorescent microscope (DM4000B, Leica., Germany), and the image data were analyzed by Image J (National Institute of Health, USA) for quantitative information. For staining brain samples, mice were perfused and fixed with 4% paraformaldehyde solution, and Fig. 2. Effects of HT on proinflammatory cytokines and COX-2 mRNA levels in LPS-stimulated BV2 the brains were carefully microglia cells. Cells were pretreated with HT (0, 25, 50, and 100 lM) for 1 h, followed by treatment removed. Afterwards, the brains with PBS or LPS (0.5 lg/mL) for 6 h, and RT-PCR was performed. Data were presented as mean were fixed in 4% ± SEM from three independent experiments. ###p < 0.001 vs. control group; *p < 0.05, **p < 0.01, *** p < 0.001 vs. LPS group. paraformaldehyde solution for Please cite this article in press as: Zhang L et al. Hydroxytyrosol Inhibits LPS-Induced Neuroinflammatory Responses via Suppression of TLR-4-Mediated NF-jB P65 Activation and ERK Signaling Pathway. Neuroscience (2019), https://doi.org/10.1016/j.neuroscience.2019.12.005

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24 h at 4 °C and transferred to 30% sucrose solution for 48 h. Brains were then embedded in OCT, frozen on dry ice, and stored at 80 °C until further use. Coronal sections (5 lm) were prepared using Leica Cryostat. The brain sections were permeabilized in 0.3% Triton X-100 PBS solution for 5 min and then blocked with 1% BSA for 30 min at room temperature. The specimens were subsequently incubated with antiIba-1 (1:400, Affinity Biosciences, USA) or anti-GFAP antibody (1: 1000, Abcam) at 4 °C overnight. The next day, the sections were incubated with Alexa fluor-488conjugated F (ab0 ) two fragment goat anti-rabbit IgG (Invitrogen) or Alexa fluor-546-conjuated donkey anti-goat IgG (Invitrogen) for 1 h at room temperature. Fig. 3. Effects of HT on LPS-induced proinflammatory cytokines and COX-2 production. BV2 microglia cells were pretreated with HT at the indicated concentration for 1 h, followed by treatment with PBS or LPS (0.5 lg/mL) for 24 h. The accumulated levels of IL-6 (A) and TNF-a (B) in the media were measured by ELISA. (C, D) COX-2 protein expression was measured by western blotting. Data were presented as mean ± SEM from at least three independent experiments. ###p < 0.001 vs. control group; *p < 0.05, **p < 0.01, ***p < 0.001 vs. LPS group.

Fig. 4. Effects of HT on LPS-induced NO production and iNOS expression. (A) BV2 microglia cells were pretreated with HT at the indicated concentration for 1 h, followed by treatment with PBS or LPS (0.5 lg/mL) for 24 h. Nitrite content was measured by the Griess reaction. (B, C) iNOS protein expression was determined by western blotting. (D) BV2 microglia cells were pretreated with the indicated concentration of HT 1 h before LPS (0.5 lg/mL) treatment for 6 h. RT-PCR was performed to examine the level of iNOS mRNA. Data were presented as mean ± SEM from at least three independent experiments. ##p < 0.01, ###p < 0.001 vs. control group; *p < 0.05, ***p < 0.001 vs. LPS group.

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Flow cytometry

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BV2 cells were seeded in 12-well plate and incubated for 24 h. The cells were then pretreated with HT for 24 h followed by LPS (0.5 lg/mL) for an additional 24 h. To determine the expression of membrane protein CD86 and CD206, the cells were incubated with PerCP anti-CD86 and PE anti-CD206 antibodies (1:200) for 1 h in the dark. The cells were then washed with PBS. Finally, each sample was detected and analyzed using NovoCyte flow cytometry and NovoExpress software (ACEA Biosciences, USA) respectively.

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Statistical analysis

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The values were expressed as the mean ± SEM. All of experiments were repeated at least three times. Statistical analyses were performed by GraphPad Prism 6 software to identify differences between groups using one-way ANOVA followed by Tukey’s multiple comparison test. Values of p < 0.05 were considered statistically significant.

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Fig. 5. Effects of HT on proinflammatory cytokines in LPS-stimulated primary microglia cells. Cells were pretreated with HT (0, 25, 50, and 100 lM) for 1 h, followed by treatment with PBS or LPS (0.5 lg/mL) for 6 h, and RT-PCR was performed. Data were presented as mean ± SEM from three independent experiments. ###p < 0.001 vs. control group; *p < 0.05, **p < 0.01, ***p < 0.001 vs. LPS group.

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Effects of HT on LPS-induced NO production and iNOS expression

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The effect of HT on the proinflammatory mediator NO was conducted by the Griess reagent assay. In LPSstimulated cells, a significant increase in the production of NO was observed. However, HT suppressed the LPS-induced NO production dose-dependently (Fig. 4A). To determine whether the inhibitory effect of HT on NO production was due to decreased iNOS expression, we further examined the effect of HT on LPS-induced iNOS expression. As shown in Fig. 4B–D, 100 lM HT significantly inhibited the iNOS expression stimulated by LPS.

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Effects of HT on the LPS-induced proinflammatory cytokines and COX-2 production

Effects of HT on the LPS-induced proinflammatory cytokines in primary microglial cell

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To determine whether HT exerts anti-inflammatory action, we examined the effects of HT on the production of proinflammatory mediators in BV2 cells. Firstly, RT-PCR was used to test the effects of HT treatment on the mRNA levels of proinflammatory cytokines. As shown in Fig. 2, HT significantly reduced the mRNA levels of proinflammatory mediator TNF-a, IL-6, IL-1b, and COX2 induced by LPS in a dose-dependent manner. To further confirm the anti-inflammatory effects of HT, the protein levels of TNF-a and IL-6 were measured by ELISA. The results showed that LPS significantly increased TNF-a and IL-6 production when compared with control group. However, HT significantly suppressed the increases in TNF-a and IL-6 secretion induced by LPS (Fig. 3A, B). In addition, we investigated the COX-2 protein levels by western blotting, showing that HT also significantly inhibited the LPS-induced COX-2 expression (Fig. 3C, D).

Next, we determined the effects of HT on LPS-induced proinflammatory responses in primary microglial cells. RT-PCR analysis revealed that HT treatment significantly downregulated the LPS-induced increase in the mRNA levels of proinflammatory cytokines TNF-a, IL-6, IL-1b, and iNOS (Fig. 5).

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Effects of HT on the expression of Cd86 and Cd206 in LPS-stimulated Bv2 cells

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To determine whether the anti-inflammatory effect of HT is associated with regulation of the expression of M1/M1 phenotype specific surface marker, we analyzed the CD86 and CD206 expression by flow cytometry in LPSstimulated BV2 cells. As shown in Fig. 6, HT pretreatment significantly reduced the CD86 positive cell populations (Fig. 6A, B), but increased the CD206 expression in LPS-stimulated BV2 cells (Fig. 6C, D).

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RESULTS

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Effects of HT and LPS on BV2 cell viability

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To determine the cytotoxic effects of HT and LPS on BV2 cells, the cells were treated with HT in the presence or absence of LPS for 24 h. As shown in Fig. 1, HT did not show significant cytotoxic effects at the concentrations up to 200 lM, and LPS at concentration of 0.5 lg/mL also had no significant adversary effect on cell viability. Therefore, HT at the concentrations of 25, 50 and 100 lM were selected in the subsequent experiments.

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Please cite this article in press as: Zhang L et al. Hydroxytyrosol Inhibits LPS-Induced Neuroinflammatory Responses via Suppression of TLR-4-Mediated NF-jB P65 Activation and ERK Signaling Pathway. Neuroscience (2019), https://doi.org/10.1016/j.neuroscience.2019.12.005

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Fig. 6. Effects of HT on CD86 and CD206 expression in LPS-stimulated BV2 cells. Cells were pretreated with HT (0, 25, 50, and 100 lM) or IL-4 (10 ng/mL) for 24 h, followed by treatment with LPS (0.5 lg/mL) for 24 h. The CD86 (A, B) and CD206 (C, D) expression were measured by flow cytometry. The results were expressed as mean ± SEM from at three independent experiments. ##p < 0.01 vs. control group; *p < 0.05, ** p < 0.01 vs. LPS group.

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Effects of HT on LPS-induced inflammatory signaling

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A large amount of evidence demonstrated that MAPK (e.g., ERK, JNK, p-P38), AKT, and NF-jB play important roles in modulating proinflammatory genes expression in glia cells (Siebenlist et al., 1994, Liu et al., 2005, Yu et al., 2015). Thus, the effects of HT on the LPS-induced phosphorylation of ERK1/2, p38, JNK, AKT, and NF-jB p65 were investigated by western blot analysis. As shown in Fig. 7, LPS treatment resulted in significant increases in the phosphorylation of NF-jB p65, ERK1/2, JNK, and AKT, while pretreatment with HT significantly attenuated the LPS-induced the phosphorylation of NF-jB p65 and ERK1/2. However, the LPSinduced phosphorylation of JNK and AKT were unaffected

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by HT. To further confirm the effect of HT on NF-jB activation, we explored whether HT prevented p65 nuclear translocation induced by LPS. Immunofluorescence images showed that cytosolic p65 translocated to the nucleus after LPS stimulation, and this translocation was markedly prevented by the pretreatment of HT (Fig. 8).

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Effects of HT on TLR4 expression in LPS-stimulated BV2 cells

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It has been shown that LPS interacts with TLR4 to initiate inflammatory response, and TLR4 is predominantly expressed on microglia. In addition, TLR4 expression is involved in the proinflammatory molecules production

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Fig. 7. Effects of HT on LPS-induced activation of NF-jB, ERK1/2, p38MAPK, JNK, and AKT in BV2 cells. Cells were pretreated with increasing dose of HT for 1 h, followed by treatment with LPS (0.5 lg/ mL) for 30 min. Cell extracts were analyzed using western blotting with antibodies that specifically recognized phosphorylated and non-phosphorylated NF-jB p65 (A, B), ERK1/2 (C, D), p38 MAPK (E, F) JNK (G, H), and AKT (I, J) antibodies. Data were presented as mean ± SEM from three independent experiments. #p < 0.05, ##p < 0.01 vs. control group; *p < 0.05, **p < 0.01, *** p < 0.001 vs. LPS group.

through NF-jB activation. Thus, we examined the effects of HT on LPS-induced TLR4 expression. As shown in Fig. 9, HT treatment dose-dependently decreased the expression of TLR4 in LPSstimulated BV2 cells. These results suggested that TLR4 was involved in the antineuroinflammatory action of HT in LPS-stimulated BV2 microglia cells.

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Effects of HT on proinflammatory responses in LPS-challenged mice

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To test the effects of HT on neuroinflammation in vivo, mice were pretreated with HT daily for 2 days and then administrated with LPS. As shown in Fig. 10, LPS injection significantly increased the expression of proinflammatory genes in the mouse brain. However, HT treatment significantly damped the LPS-induced increase in the mRNA levels of TNF-a, IL-6, IL1b, iNOS, and COX-2.

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Effects of HT on LPS-induced microglia and astrocyte activation in the mouse brain

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Given activated microglia and astrocytes are shown to release proinflammatory mediators, we examined whether HT affects LPS-induced activation of microglia and astrocytes in vivo. After LPS injection for 24 h, brain sections were prepared and immunostained with anti-Iba-1 (microglia marker) or anti-GFAP (astrocyte marker) antibodies. As shown in Fig. 11A, B, HT treatment significantly inhibited microglia activation in the hippocampus and cerebral cortex. HT also significantly inhibited the activation of astrocytes in the hippocampus and corpus callosum induced by LPS (Fig. 11C, D).

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DISCUSSION

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In the present study, we investigated potential inhibitory effects of HT on LPS-induced neuroinflammation using both

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Please cite this article in press as: Zhang L et al. Hydroxytyrosol Inhibits LPS-Induced Neuroinflammatory Responses via Suppression of TLR-4-Mediated NF-jB P65 Activation and ERK Signaling Pathway. Neuroscience (2019), https://doi.org/10.1016/j.neuroscience.2019.12.005

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Fig. 8. Effects of HT on LPS-induced NF-jB p65 translocation in BV2 cells. (A) Cells were pretreated with increasing dose of HT for 1 h, followed by treatment with LPS (0.5 lg/mL) for 1 h. NF-jB p65 translocation was determined by immunocytochemistry (red, Alexa Fluor 546; blue, DAPI). (B) Quantification of the data shown in (A). Data were presented as mean ± SEM from three independent experiments. ###p < 0.001 vs. control group; **p < 0.01, ***p < 0.001 vs. LPS group. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

in vitro and in vivo models. Our results demonstrated that HT potently inhibits LPS-induced proinflammatory responses in microglia by suppressing TLR-4 mediated NF-jB activation and ERK signaling pathway. To our knowledge, this is the first report of such effects of HT on LPS-induced neuroinflammation in microglia cells and the molecular mechanism underlying these effects. HT is one of the main polyphenols presented in extra virgin olive oil (EVOO) and its concentrations range from 50 to 200 mg/kg oil for EVOO (Visioli and Bernardini, 2011). The absorption of HT occurs via passive transport in the intestine (Manna et al., 2000), and the degree of absorption for HT is remarkable being higher (>40%) (Tuck and Hayball, 2002). Numerous studies have indicated that olive phenolic compounds are strongly associated with the neuroprotective activity by acting against oxidation and inflammation and improving cognitive deficits (Hornedo-Ortega et al., 2018). Several studies have also showed that HT has anti-inflammatory effects in macrophages cell lines. Maiuri et al. firstly reported that HT inhibited the expression of iNOS and COX-2 in LPS-stimulated J774 cells by suppressing the NF-jB, STAT1a, and IRF-1 activation (Maiuri et al., 2005). Moreover, HT has been shown to inhibit the NO and PGE2 production in LPS-treated RAW 264.7 cells (Richard et al., 2011). Most recently, Bigagli et al. observed similar results, showed that HT inhibited the production of NO and PGE2 at nutritional relevant concentration of HT (10 and 50 lM) (Bigagli et al., 2017). However, the study of the anti-neuroinflammatory effects of HT in microglia cell lines remains unexplored. In this study, we investigated the antineuroinflammatory activity of HT and the underlying mechanism in LPS-stimulated BV2 microglia cells and primary microglia cells. Normally, microglia cells function as phagocytes to swamp dead cells and tissue debris to maintain homeostasis of CNS. However, over-stimulated microglia cells

Please cite this article in press as: Zhang L et al. Hydroxytyrosol Inhibits LPS-Induced Neuroinflammatory Responses via Suppression of TLR-4-Mediated NF-jB P65 Activation and ERK Signaling Pathway. Neuroscience (2019), https://doi.org/10.1016/j.neuroscience.2019.12.005

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neuroinflammatory effects by regulating the microglia activation. Similarly to macrophages, activated microglia polarized to an M1 phenotype in response to LPS and expressed high levels of proinflammatory cytokines, such as TNF-a, IL-6, and IL-1b (Saijo and Glass, 2011). Conversely, the anti-inflammatory cytokines IL-4 promotes the formation of a protective M2 phenotype microglia Fig. 9. Effects of HT on TLR4 expression in LPS-stimulated BV2 cells. BV2 microglia cells were (Cherry et al., 2015). M1 phenopretreated with HT at the indicated concentration for 1 h, followed by treatment with PBS or LPS type microglia tend to induce neu(0.5 lg/mL) for 24 h. (A) TLR-4 protein expression was measured by western blotting. (B) ronal death; however, M2 Quantification of the data shown in (A). Data were presented as mean ± SEM from at least three microglia release antiindependent experiments. #p < 0.05 vs. control group; *p < 0.05, **p < 0.01 vs. LPS group. inflammatory factors to suppress inflammation, strongly promoting significantly increase neuroinflammation, resulting in neuronal survival and tissue repair pathogenesis by secreting various proinflammatory (Xiang et al., 2018). Based on these findings, it is conceivmediators. LPS is a well-known strong stimulator of able that mediators regulating microglia polarization could microglia activation that can cause harmful affect the promotion or resolution of neuroinflammation. neuroinflammatory responses by producing TNF-a, IL-6, Thus, we examined the effect of HT on controlling the IL-1b, iNOS, and COX-2 (Long-Smith et al., 2009, Lull polarization of microglia using IL-4 as a positive control. and Block, 2010). Therefore, in this study, we examined We found that HT treatment resulted in the downregulathe production of TNF-a, IL-6, IL-1b, iNOS, and COX-2 tion of M1 marker CD86 and upregulation of M2 marker after treatment of microglia cells with HT in order to invesCD206 on the BV2 microglia cell membranes. Therefore, tigate its inhibitory effects on LPS-induced increase in the our findings indicate that HT may act as a neuroprotectant levels of pro-inflammatory mediators. We found that HT that suppresses inflammation via modulation of microglia significantly inhibited LPS-induced increase in the levels M1/M2 polarization. of TNF-a, IL-6, IL-1b, iNOS and COX-2 in a doseThe transcription factor NF-jB is an important dependent manner, suggesting that HT exerted antiregulator of the immune responses induced by LPS

Fig. 10. Effects of HT proinflammatory responses in LPS-challenged mice. C57BL/6 mice were pretreated with HT (100 mg/kg) by gavage daily for 2 days, followed by injection with LPS (10 mg/kg) or PBS. 3 h after the injection of LPS or PBS, brains were collected to examine the gene expression by RT-PCR. Data were presented as mean ± SEM from four mice. ##p < 0.01, ###p < 0.001 vs. control group; **p < 0.01, ***p < 0.001 vs. LPS group.

Please cite this article in press as: Zhang L et al. Hydroxytyrosol Inhibits LPS-Induced Neuroinflammatory Responses via Suppression of TLR-4-Mediated NF-jB P65 Activation and ERK Signaling Pathway. Neuroscience (2019), https://doi.org/10.1016/j.neuroscience.2019.12.005

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(Ghosh et al., 2015). Previous research has identified the NF-jB binding sites on proinflammatory genes, such as TNF-a, COX-2, and iNOS (Rahman and Fazal, 2011). Our results demonstrated that HT significantly attenuated LPS-induced phosphorylation of NF-jB and the nuclear translocation of NF-jB p65. The results suggest that HT-mediated inhibition of the activation of NF-jB is involved in the regulation of LPS-induced inflammatory responses. In addition, we further evaluated the effects of HT on the MAPK and AKT signaling pathway. As shown by Johnson et al., MAPK pathway is directly involved in the downregulation of TNF-a, iNOS, and COX2 expression induced by LPS (Johnson and Lapadat, 2002). Moreover, phosphorylated AKT promotes the expression of inflammatory mediators iNOS and COX2 in BV2 microglia cells (Zhao et al., 2017). In this study, we found that LPS significantly increased the phosphorylation levels of ERK1/2, JNK, and AKT. The pretreatment with HT suppressed the LPSinduced phosphorylation of ERK1/2, but not that of JNK and AKT, suggesting that the ERK signaling pathway is a main effector in the HT-induced antineuroinflammatory responses. Our results are consistent with a previous study that indicates the ERK pathway to be an important regulator in LPS-stimulated RAW 264.7 cells (Jung et al., 2010). More recently, Xiang et al. demonstrated that JNK might be a critical mediator in LPS-induced inflammation (Xiang et al., 2018). The discrepancies among these studies may result from the different binding site of each chemical. TLR family members are mainly expressed on immune cells and have also been identified on all major glia cells, including microglia, astrocytes and oligodendrocytes (Hanke and Kielian, 2011). TLR-4 is predominantly expressed on microglia and responses to LPS through its coreceptor, myeloid differentiation protein-2 (MD-2) (Shimazu et al., 1999). The interaction between

Please cite this article in press as: Zhang L et al. Hydroxytyrosol Inhibits LPS-Induced Neuroinflammatory Responses via Suppression of TLR-4-Mediated NF-jB P65 Activation and ERK Signaling Pathway. Neuroscience (2019), https://doi.org/10.1016/j.neuroscience.2019.12.005

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LPS and TLR4 leads to the activation of NF-jB, which promotes the release of pro-inflammatory cytokines (Lester and Li, 2014, Jiang et al., 2016, Li et al., 2016). Therefore, abnormal expression of TLR4 may cause abnormal neuroinflammatory responses, ultimately resulting in the damage of neighboring neurons. Interestingly, we found that pretreatment with HT decreased the expression of TLR4 induced by LPS. Thus, it appears that HT prevented the NF-jB signaling pathway by downregulating the expression of TLR4 on microglia cells, resulting in suppressing the production of pro-inflammatory mediators. We further evaluated the anti-neuroinflammation effects of HT in vivo with systemic injection of LPS. Previous studies have demonstrated that LPS administration promotes the levels of proinflammatory cytokines, nitric oxide, and other proinflammatory mediators in the brain (Sugita et al., 2002, Park et al., 2016). Consistent with these results, our data showed significant increases in the mRNA levels of TNF-a, IL-6, IL1b, iNOS, and COX-2 after systemic injection of LPS and HT treatment strongly inhibited the increased expressions of these proinflammatory mediators. Activated microglia and astrocytes have been demonstrated to be associated with the release of proinflammatory mediators in the brain parenchyma (Bauer et al., 2001, YanguasCasas et al., 2014). Moreover, systemic LPS inducedneuroinflammation has been evidenced by increased microglia and astrocyte activation (Hoogland et al., 2015). Our findings showed similar results, suggesting that HT has potential to treat neuroinflammation-related disease. There are several limitations in this study. First, we did not examine the effects of HT alone on the expression of pro-inflammatory mediators in the microglia cells; however, we did evaluate the effect of HT alone on the expression of M1 marker CD86 and M2 marker CD206 and found no influence on these markers (data not shown). Future studies will explore whether HT itself has effects on inflammatory responses and the molecular mechanism responsible for its effects in the absence of LPS. In addition, we did not establish the relationship between NF-jB activation and ERK in the effects of HT on the LPS-induced neuroinflammation. In the future, we will use specific NF-jB or ERK inhibitor to investigate their relationship in HT-mediated antineuroinflammation effects. In the present study, we showed that HT suppressed the LPS-induced neuroinflammatory responses by inhibiting proinflammatory mediators in microglia cells. In addition, our results demonstrated that HT exhibited potential anti-inflammatory effects via modulation of microglia M1/M2 polarization and downregulation of

TLR-4 mediated NF-jB activation and ERK signaling pathway. These data suggested that HT could be a promising therapeutic candidate for the treatment of neuroinflammation in the neurodegenerative conditions or brain injury.

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This work was supported by the National Natural Science Foundation of China (81470263), Tianjin Science and Technology Project, China (13RCGFSY19300), and by the Clinical Medicine Research Centre for ITCWM Acute Abdomen Diseases of Tianjin Municipal Science and Technology Commission (15ZXLCSY00030).

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3 Fig. 11. Effects of HT on microglia and astrocyte activation in LPS-challenged mouse brain. C57BL/6 mice were pretreated with HT (100 mg/kg) by gavage daily for 2 days, followed by injection with LPS (10 mg/kg) or PBS. 24 h after LPS or PBS injection, brain section (5 lm thick) were prepared and immunostaining for Iba-1 and GFAP were performed. Representative images of labeling for Iba-1 ((A), hippocampus and cerebral cortex) and GFAP ((C), hippocampus and corpus callosum) are presented, along with quantification results of Iba-1 (B) and GFAP (D). Data were presented as mean ± SEM from four mice. #p < 0.05, ##p < 0.01, ###p < 0.001 vs. control group; *p < 0.05, ***p < 0.001 vs. LPS group.

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(Received 17 August 2019, Accepted 3 December 2019) (Available online xxxx)

Please cite this article in press as: Zhang L et al. Hydroxytyrosol Inhibits LPS-Induced Neuroinflammatory Responses via Suppression of TLR-4-Mediated NF-jB P65 Activation and ERK Signaling Pathway. Neuroscience (2019), https://doi.org/10.1016/j.neuroscience.2019.12.005

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