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

doi:10.1016/j.neuroscience.2019.12.005

Neuroscience

Volume 426, 1 February 2020, Pages 189-200

https://doi.org/10.1016/j.neuroscience.2019.12.005 Get rights and content

Highlights

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HT reduced the production of pro-inflammatory mediators in LPS-stimulated microglia.
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HT reduced the expression of M1 marker CD86, while increased that of M2 marker CD206.
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HT inhibited the LPS-induced NF-κB activation and ERK signaling pathway in BV2 cells.
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In vivo administration of HT inhibited the LPS-induced neuroinflammatory responses.

Abstract

Neuroinflammation has been implicated in the mechanism underlying the progression of neurodegeneration and infectious neuropathology. Growing evidence suggest that hydroxytyrosol (3,4-dihydroxyphenil-ethanol, HT), one of the main polyphenols presented in extra virgin olive oil (EVOO), has shown potential anti-inflammatory 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-κB 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-κB activation and ERK signaling pathway.

Introduction

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 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 olive-oil, 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 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 anti-neuroinflammatory 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-κB 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.

Section snippets

Materials

LPS (Escherichia 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-α and IL-6 were purchased from Multi Sciences (Hangzhou, China). NO assay kit was from Beyotime (Shanghai, China). The antibodies against NF-κB p65, phospho-NF-κB p65, nitric oxide synthase (iNOS), COX-2, p38 mitogen-activated protein kinase (MAPK), phospho-p38 MAPK, ERK1/2,

Effects of HT and LPS on BV2 cell viability

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 μM, and LPS at concentration of 0.5 μg/mL also had no significant adversary effect on cell viability. Therefore, HT at the concentrations of 25, 50 and 100 μM were selected in the subsequent experiments.

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

To determine whether HT exerts anti-inflammatory action, we

Discussion

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

Acknowledgements

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

Highlights

Abstract

Introduction

Section snippets

Materials

Effects of HT and LPS on BV2 cell viability

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

Discussion

Acknowledgements

Exp Neurol

Int Immunopharmacol

Cell

Int Immunopharmacol

Neuroscience

J Mol Biol

Progr Neurobiol

Neurotherapeutics

FEBS Lett

J Nutr Biochem

Food Res Int

J Nutr Biochem

J Nutr Biochem

Mol Immunol

Int Immunopharmacol

The association between Mediterranean diet adherence and Parkinson’s disease

Mov Disord

Inflammation in the nervous system: the human perspective

Glia

Oleuropein aglycone: a possible drug against degenerative conditions. In vivo evidence of its effectiveness against Alzheimer's disease

J Alzheimers Dis

Arginase 1+ microglia reduce Abeta plaque deposition during IL-1beta-dependent neuroinflammation

J Neuroinflamm

Hydroxytyrosol exerts anti-inflammatory and anti-oxidant activities in a mouse model of systemic inflammation

Molecules

The interplay between cyclic AMP, MAPK, and NF-kappaB pathways in response to proinflammatory signals in microglia

BioMed Res Int

Characterization of ameboid microglia isolated from developing mammalian brain

J Neurosci

A basic overview of multiple sclerosis immunopathology

Eur J Neurol

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