Beneficial Effects of Hesperetin in a Mouse Model of Temporal Lobe Epilepsy

Temporal lobe epilepsy (TLE), which belongs to a chronic neurological disease, is common in adults with localization-related epilepsy. ^1,2 Many patients with TLE have pathologically abnormal features, including granule cell dispersion (GCD) in the dentate gyrus (DG) and neuroinflammation. ^{3 –5} These features are thought to be related to status epilepticus and the progression of epilepsy. ^6,7 Mammalian target of rapamycin complex 1 (mTORC1) can mediate cellular changes involved in epileptogenesis, and in some studies mTORC1 activation was found in the hippocampus of murine models of epilepsy and in TLE patients. ^{8 –10} Thus, these observations suggest that controlling GCD by inhibiting mTORC1 activation in the hippocampus may be useful for preventing epileptogenesis. In addition, neuroinflammation, including glial activation and increases in neurotoxic inflammatory cytokines, also contributes to the recurrence and precipitation of seizure in epileptic patients and in animal models. ^{5,7,11 –13}

Hesperetin (3′,5,7-trihydroxy-4′-methoxyflavanone), extracted from citrus fruit, is a flavonoid. It easily passes through the blood–brain barrier into the brain and exerts neuroprotective effects, possibly due to its radical scavenging property and by attenuating caspase-3 activity in vitro. ^14,15 In addition, hesperetin could reduce neuronal cell death through antioxidant properties. ¹⁶ However, a preventive effect of hesperetin on pathological changes induced by kainic acid (KA) and epileptic seizures in vivo has not been established yet. ¹⁷ In this study, we investigated the effects of hesperetin on seizure onset, GCD formation, and neuroinflammation, in a KA-induced mouse model of epilepsy.

Mice were administered orally with hesperetin (Abcam, Cambridge, United Kingdom) (5, 10, or 20 mg/kg/day) for 8 days, starting 1 day before KA injection. ^17,18 The degree of GCD was measured as the average width of the granule cell layer (GCL) in the middle and upper quadrant at top of the DG. Then, the experimental group's ipsilateral GCL was normalized to the corresponding side in the control group. ^17,19 In addition, the phosphorylated form of eukaryotic translation initiation factor 4E (elF-4E)-binding protein 1 (4E-BP1) and p70 ribosomal S6 kinase (p70S6K), the markers for mTORC1 activation, and inflammatory molecules such as tumor necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1β), and inducible nitric oxide synthase (iNOS) were analyzed by western blotting and immunohistochemical staining 2 days after KA treatment. ^17,18 For KA administration, C57BL/6 male mice (∼22 g, 8 weeks old), purchased from Daehan Biolink (Eumseong, Korea), were anesthetized with ketamine/xylazine solution and unilaterally injected with KA into the hippocampal CA1 region (anterior-posterior: −0.2 cm; medial-lateral: −0.12 cm; dorsal-ventral: −0.15 cm), using a stereotaxic instrument (David Kopf Instruments, Tujunga, CA, USA). ^17,18 All surgical experiments were conducted according to the Animal Care Committee of the Kyungpook National University guidelines (No. KNU 2016-42). Data were presented as mean and standard error of the mean. Significance testing was performed using a Student's t-test and one-way analysis of variance followed by Tukey's post hoc between groups (SigmaPlot 12.0; Systat Software, San Leandro, CA, USA).

As shown in Figure 1, mice that had received 20 mg/kg hesperetin exhibited a significant delay in seizure onset compared to mice that received KA injections only (Fig. 1A; ^# P < .05 vs. KA only group). Furthermore, treatment with 20 mg/kg hesperetin significantly narrowed the width of the KA-induced GCD (Fig. 1B, C; *P < .05 and ^# P < .05 vs. CON and KA only group, respectively), suggesting that hesperetin might have anticonvulsant properties and that it contributes to the maintenance of a normal DG structure following epileptic insults.

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FIG. 1.

Effect of hesperetin on the seizure latency and GCD in the DG of KA-injected mice. (A) To study the onset of seizure behaviors, we monitored each group, following KA injection in the hippocampal CA1 layer. Hesperetin (5, 10, or 20 mg/kg) was orally administered 1 h before KA injection. ^#P < .05 versus KA alone (one-way analysis of variance and Tukey's post hoc test). (B) Representative sections of Nissl-stained DG 7 days after KA injection in the hippocampus. Scale bar, 200 μm. (C) Quantification of ipsilateral GCD in comparison with the contralateral side for each sample. The GCD in the DG of KA-treated mice was significantly lower in the 20 mg/kg hesperetin-treated group than that treated with KA alone. *P < .001 versus CON; ^#P < .05 versus KA alone (one-way analysis of variance and Tukey's post hoc test). All values are expressed as the mean ± standard error of the mean. Hsp(5), (10), and (20) are 5, 10, and 20 mg/kg hesperetin, respectively. CON, intact controls; PBS, phosphate buffered saline; KA, kainic acid; GCD, granule cell dispersion; DG, dentate gyrus.

Next, to clarify whether 20 mg/kg hesperetin alleviates KA-induced mTORC1 activation, we sacrificed the mice 2 days after KA injection and inspected their DG. Figure 2A shows that while p-4E-BP1 expression was increased in the DG treated with KA, it was attenuated in the DG of mice that had received hesperetin before KA administration. In line with the immunostaining data, western blot results suggest that hesperetin significantly decreases both p-4E-BP1 expression (Fig. 2B; **P < .001 and ^## P < .01 vs. CON and KA only group) and p-p70S6K levels (Fig. 2B; *P < .05 and ^# P < .05 vs. CON and KA only group).

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FIG. 2.

Inhibitory effect of hesperetin on KA-induced mammalian target of rapamycin complex 1 activation in the DG. (A) Representative sections of DG immunostained with anti-p-4E-BP1. Treatment with 20 mg/kg hesperetin attenuated the expression of p-4E-BP1 induced by KA injection in the DG in comparison with KA alone. Scale bar, 100 μm. (B) Western blotting of p-4E-BP1 and p-p70S6K at 2 days after KA injection in the DG, and a quantitative analysis based on the density of the p-4E-BP1 (**P < .001 and ^## P < .01 vs. CON and KA alone, respectively) and p-p70S6K bands (*P < .05 and ^# P < .05 vs. CON and KA alone, respectively), normalized to the β-actin, for each group. 4E-BP1, eukaryotic translation initiation factor 4E (elF-4E)-binding protein 1; p70S6K, p70 ribosomal S6 kinase.

Neuroinflammation is a natural physiological response to brain disease, and inflammatory processes strongly contribute to the pathology of chronic epilepsy, as well as to the generation of epileptic seizures. ¹¹ Thus, in addition to investigating the beneficial effects of hesperetin on GCD and mTORC1 activity, we used immunofluorescence staining two days post-KA treatment to examine whether it could attenuate the increase of TNF-α, IL-1β, and iNOS produced by activated microglia (Fig. 3A–C). Reduced levels of pro-inflammatory cytokines and iNOS in the hesperetin receiving groups were quantitatively confirmed by western blot analysis (Fig. 3D).

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FIG. 3.

Inhibitory effect of hesperetin on KA-increased levels of pro-inflammatory cytokines and iNOS in the activated microglia.. (A–C) Double-immunofluorescence staining for Iba1 (red) and IL-1β (green), Iba1 (red) and TNF-α (green), and Iba1 (red) and iNOS (green) in the hippocampus 2 days following KA treatment. Scale bar, 100 μm. (D) Western blotting of IL-1β, TNF-α, and iNOS in the hippocampus 2 days after KA treatment. The density of each band for IL-1β, TNF-α, and iNOS was normalized to β-actin for each sample. *P < .05, **P < .05, and ***P < .001 versus CON; ^# P < .05 versus KA alone (one-way analysis of variance and Tukey's post hoc analysis). ^& P = .011 versus KA alone (t-test). TNF-α, tumor necrosis factor-alpha; IL-1β, interleukin-1 beta; iNOS, inducible nitric oxide synthase.

A previous in vitro study using electrophysiology showed that hesperetin has anticonvulsant effects in convulsant-superfused hippocampal slices. ²⁰ However, the antiepileptic effects of hesperetin had not been investigated in vivo before. In this study, we demonstrated that hesperetin attenuated epileptic progression in a KA-treated animal model of seizure and that it could suppress GCD by inhibiting mTORC1 activation. Thus, our results show that hesperetin has the potential to effectively prevent hippocampal-onset epilepsy in vivo.