Elsevier

Epilepsy Research

Volume 126, October 2016, Pages 126-133
Epilepsy Research

Glycyrrhizin ameliorates oxidative stress and inflammation in hippocampus and olfactory bulb in lithium/pilocarpine-induced status epilepticus in rats

https://doi.org/10.1016/j.eplepsyres.2016.07.007Get rights and content

Highlights

Abstract

Glycyrrhizin (GL) is a triterpene present in the roots and rhizomes of Glycyrrhiza glabra that has anti-inflammatory, hepatoprotective and neuroprotective effects. Recently, it was demonstrated that GL produced neuroprotective effects on the postischemic brain as well as on the kainic acid injury model in rats. In addition to this, GL also prevented excitotoxic effects on primary cultures. The aims of the present study were to evaluate GL scavenging properties and to investigate GL’s effect on oxidative stress and inflammation in the lithium/pilocarpine-induced seizure model in two cerebral regions, hippocampus and olfactory bulb, at acute time intervals (3 or 24 h) after status epilepticus (SE).

Fluorometric methods showed that GL scavenged three reactive oxygen species: hydrogen peroxide, peroxyl radicals and superoxide anions. In contrast, GL was unable to scavenge peroxynitrite, hydroxyl radicals, singlet oxygen and 2,2-diphenil-1-picrylhydrazyl (DPPH) radicals suggesting that GL is a weak scavenger.

Additionally, administration of GL (50 mg/kg, i.p.) 30 min before pilocarpine administration significantly suppressed oxidative stress. Moreover, malondialdehyde levels were diminished and glutathione levels were maintained at control values in both cerebral regions at 3 and 24 after SE. At 24 h after SE, glutathione S-transferase and superoxide dismutase activity increased in the hippocampus, while both glutathione reductase and glutathione peroxidase activity were unchanged in the olfactory bulb at that time.

In addition, GL suppressed the induction of the proinflammatory cytokines interleukin-1 beta (IL-1β) and tumor necrosis factor alpha (TNF-α) in both cerebral regions evaluated. These results suggest that GL confers protection against pilocarpine damage via antioxidant and anti-inflammatory effects.

Introduction

Temporal lobe epilepsy (TLE) is initiated by an injury such as head trauma, hypoxia, complex febrile seizures or status epilepticus (SE) (Delgado-Escueta et al., 1999, Kandratavicius et al., 2014). In SE, overproduction of reactive oxygen species (ROS) and reactive nitrogen species generate oxidative stress (Waldbaum et al., 2010, Freitas et al., 2005) and inflammation (Vezzani and Rüegg, 2011) that are involved in tissue damage. The exaggerated release of cytokines or their protracted presence in tissue is associated with neuronal dysfunctions (Vezzani and Viviani, 2015). Interleukin-1 beta (IL-1β) affects the excitability of the central nervous system and suppresses the induction of long-term potentiation in the CA region and dentate gyrus of the rat hippocampus (Yu et al., 2012). IL-1β induces changes in GABAergic neurotransmission in the forebrain. It also inhibits the astrocytic reuptake of glutamate, which may increase glutamate release from glia via production of tumor necrosis factor-α (TNF-α), thus resulting in elevated extracellular glutamate levels; both defects are able to promote hyperexcitability, which has been reported in pathologic conditions associated with neuroinflammation such as in epilepsy (Vezzani and Viviani, 2015).

In regards to oxidative stress, diverse research suggests that this process is both a cause and a consequence of epileptic seizures in humans and in animal models (Patel, 2004, Menon et al., 2012, Keskin Guler et al., 2016). In a TLE model, an increased steady-state levels of ROS and impaired glutathione (GSH) redox status (Patel et al., 2001, Patel et al., 2008) were found in hippocampus. Strategies to fight oxidative stress in epilepsy include synthetic (Pearson et al., 2015) and natural (MacGregor et al., 1996, Khamse et al., 2015) compounds with antioxidant properties.

Glycyrrhizin (GL), a triterpene saponin, is a major active constituent of licorice Glycyrrhiza glabra root. GL is composed of a molecule of glycyrrhizic acid and two molecules of glucuronic acid (chemical structure shown in Fig. 1) (Kao et al., 2014). Licorice has demulcent, antacid, antiulcer, anti-inflammatory, expectorant, tonic, diuretic, laxative and sedative properties (Asl and Hosseinzadeh, 2008). GL also possesses antipyretic, antimicrobial, antiherpes and anxiolytic activities. GL has hepatoprotective activities and is commonly used in Asia to treat patients with chronic hepatitis (Xiong et al., 2015, Hsiang et al., 2015, van Rossum et al., 2001). GL confers neuroprotection through anti-inflammatory and antiexcitotoxic effects in a kainic acid (KA)-induced neuronal death model (Luo et al., 2013). GL attenuated rat ischemic spinal cord injury by suppressing inflammatory cytokine induction and high-mobility group box 1 (HMGB1) secretion (Gong et al., 2012). In the KA-injected mouse model, GL has neuroprotective effects on the brain that might be attributed to the inhibition of HMGB1 induction and release, which in turn mitigates the inflammatory process (Luo et al., 2014).

In this study, we investigated whether GL protects rat hippocampus and olfactory bulb from pilocarpine-induced oxidative and inflammatory damage. We also compared the effect of GL on both cerebral regions.

Section snippets

Reagents

3-O-(2-O-β-d-glucopyranuronosyl-α-d-glucopyranuronosyl)-18β-glycyrrhetinic acid ammonium salt (Glycyrrhizin >70% and >90%), (3S,4R)-4,5-dihydro-3-ethyl-4-(1-methyl-1H-imidazol-5-ylmethyl)-2(3H)-furanone hydrochloride (Pilocarpine hydrochloride), lithium chloride (LiCl), (−)Scopolamine methyl nitrate, 5,5′-dithio-bis(2-nitrobenzoic acid) (DNBT), 2-vinylpyridine (2-VP), glutathione reduced form (GSH), l-glutathione oxidized form (GSSG), nitroblue tetrazolium (NBT), 1-chloro-2,4-dinitrobenzene

GL shows antioxidant activity

In our work, we evaluated the ability of GL to scavenge ROS, including superoxide anions, hydrogen peroxide, peroxyl radicals, peroxynitrite, hydroxyl radicals, singlet oxygen and DPPH radicals. We observed that the IC50 value (the concentration of the compound that caused 50% inhibition of a specific radical species) of GL was in mM range: 1.24 ± 0.1 for hydrogen peroxide, 0.78 ± 0.22 for superoxide anions and 0.134 ± 0.05 for peroxyl radicals. GL was unable to scavenge peroxynitrite, hydroxyl

Discussion

In this work we evaluated GL effect in SE. This compound has diverse uses in treatments of illness in traditional oriental medicine. However, it is important to determine its action mechanisms. We first investigated if GL scavenges radicals and with this its role such as good antioxidant. Our results agree with some reports that show GL scavenging species. Nevertheless, conflicting results have been reported regarding whether GL itself scavenges radicals at all. In one report, GL only slightly

Conflicts of interest

The authors declare no conflicts of interest.

Acknowledgments

We would like to thank doctor Laura Elena Cordóva Dávalos for her help to PCR assays, M. Sci. Omar Noel Medina Campos on interpretation of scavenging ROS experiments and Mrs. Josefina Bolado, for editing the English-language version of this manuscript.

This study was supported by CONACyT 152613, PAPIIT IN211913-3. S G-R fellowship for postdoctoral by DGAPA.

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