Validating the antiseizure effects of vitexin and related flavone glycosides in zebrafish

Abstract

Current epilepsy treatments often fail to provide sufficient control over seizures, highlighting the need for new therapeutic agents. Vitexin, a flavone with antioxidant, anti-inflammatory, and neuroprotective properties, was previously shown to suppress seizure activity in rodent models. Utilizing zebrafish, this study further evaluates the antiseizure properties of vitexin and for the first time, examines the related flavone glycosides: isovitexin, vitexin 2-O-rhamnoside, vitexin-4-O-glucoside and saponarin. We initially tested the ability of the compounds to reduce behavioral seizures stimulated by the GABA_A receptor antagonists (pentylenetetrazole: PTZ and picrotoxin: PTX) and spontaneous seizures in a genetic epilepsy model (Dravet syndrome, scn1lab ^-/- zebrafish larvae). Seizure behavior was quantified in 5-day old larvae via automated tracking with a DanioVision monitoring chamber linked to EthoVision XT 15 software. Microelectrode array electrophysiology (MEA) was then used to examine the effects on PTZ-induced seizure-like brain activity. While having no effect on basal locomotion, vitexin and isovitexin significantly reduced seizure activity in PTZ-treated zebrafish. None of the flavones exhibited antiseizure effects in the PTX-induced epilepsy model. Additional studies with vitexin demonstrated that though it did not suppress spontaneous seizure behaviors in our genetic model of epilepsy, it did significantly inhibit PTZ-induced electrographic activity. These findings support the continued exploration of the translational potential of the vitexin scaffold. This work advances our search for safer, more effective antiseizure drugs and could pave the way for vitexin-based treatments for epilepsy and related disorders.

Keywords: behavior; epilepsy; flavonoids; multielectrode array; vitexin; zebrafish.

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FIGURE 1

Structure and basal effects of flavones under investigation. (A) Chemical structures of compounds: vitexin (1), isovitexin (2), vitexin-2-O-rhamnoside (3), vitexin-4-O-glucoside (4), saponarin (5). (B) Heat map depicts mean movement (distance, mm) after 5 dpf larvae were treated with 0, 0.1, 1, 10, 100, 500 µM of compounds 1–5, prior to exposure to PTZ or PTX. N = 15–29 larvae per concentration. Analyses For 500 µM saponarin (5) vs. 0 μM control: Pre-PTZ, F_{(6, 118)} = 5.99, ****p < 0.0001 (one-way ANOVA with Dunnett’s test); Pre-PTX, ***p < 0.001 (Kruskal–Wallis with Dunn’s test).

FIGURE 2

Dose-response effects of compounds 1–5 on PTZ-induced seizure activity. Five dpf zebrafish larvae were treated with 0-500 µM of each compound and 50 µM stiripentol (stir) for 1 h followed by a 15-min exposure to 10 mM PTZ. Behavior was then tracked for 15 min and total distance traveled was extracted to assess seizure activity. Bar graphs represent the mean ± SEM (n = 14–29 per group). (A,C,E) One-way ANOVA: F_{(8, 124)} = 11.64; F_{(8, 125)} = 18.70; F_{(8, 143)} = 20.14 (all p < 0.0001), with Dunnett’s post hoc test: *p < 0.05, **p < 0.01, ****p < 0.0001 vs. 0 μM vitexin + PTZ control. (B,D) Kruskal–Wallis with Dunn’s post hoc test: *p < 0.05, ****p < 0.0001 vs. 0 μM vitexin + PTZ control.

FIGURE 3

Dose-response effects of compounds 1-5 on PTX-induced seizure activity. Five dpf zebrafish larvae were treated with 0-500 µM of each compound for 1 h followed by a 15-min exposure to 1 mM PTX. Behavior was then tracked for 15 min and total distance traveled was extracted to assess seizure activity. Bar graphs represent the mean ± SEM (n = 13–18 per group). (A) Kruskal–Wallis with Dunn’s post hoc test: ****p < 0.0001 vs. 0 μM vitexin + PTX control. (B–E) One-way ANOVA: F_{(8, 153)} = 27.28; F_{(8, 134)} = 48.35; F_{(8, 144)} = 24.71; F_(8,153) = 142.8 (all p < 0.0001). Post hoc Dunnett multiple comparison test, ****p < 0.0001 vs. 0 µM vitexin + PTX control.

FIGURE 4

Effect of vitexin (1) on spontaneous seizures in zebrafish DS model. Scn1lab ^+/− breeders were in-crossed and scn1lab ^−/− larvae were identified at 5 dpf via their black pigmentation. In 96 well-plates, the larvae were exposed to 0–1,000 µM of vitexin or 100 µM stiripentol (stir) and 15-min recordings were taken at (A) t = 30 min and (B) t = 60 min. Seizure behavior was quantified from maximum velocity measurements. Bars are shown as mean ± SEM (n = 12–18 per concentration). Kruskal-Wallis with Dunn’s multiple comparisons test: **p < 0.01 and ****p < 0.0001 (compared to 0 µM treatment).

FIGURE 5

Effect of vitexin (1) on PTZ-induced electrographic events. Larvae were treated with 0 µM vitexin (1% DMSO), 500 µM vitexin or 25 µM stiripentol (stir) for 1 h and then embedded in separate wells of the MEA plate. Egg water or 10 mM PTZ was then added to achieve the following four conditions: 0 µM vitexin (N = 6), 0 µM vitexin + PTZ (N = 8), 500 µM vitexin + PTZ (N = 6), 25 µM stir + PTZ (N = 6). (A) Number of spikes and (B) mean firing rate were quantified using the Axion MEA system. Data are represented in bar graphs as mean ± SEM. One-way ANOVA with Post hoc Dunnett multiple comparison test: spike; F_{(3, 22)} = 70.48, ****p < 0.0001 and firing rate; F_{(3, 22)} = 11.14, ***p < 0.001 (compared to 0 µM + PTZ treatment).