Vitexin Alleviates Kainic Acid-Induced Seizure Through Inhibiting P2X7R/NLRP3 Signaling Pathway

Abstract

Epilepsy, a prevalent chronic brain disorder, remains inadequately controlled by current antiepileptic drugs. Neuroinflammation is considered a crucial component in the process of epileptogenesis. Vitexin (VT), primarily derived from medicinal plants, is a flavonoid monomer with significant biological activity that has demonstrated benefits in various neurological disorders by suppressing neuroinflammation. However, the role and underlying mechanism of VT in epileptic seizures are incompletely understood. We herein investigate VT’s antiseizure potential and the related mechanism utilizing a mouse model of status epilepticus induced by kainic acid (KA). Our findings indicate that VT pre-treatment exerts an antiseizure effect in a dose-dependent manner and attenuates KA-induced hippocampal neuronal damage in mice. Moreover, VT post-treatment (administration after the appearance of generalized seizures) can also alleviate KA-induced seizure and neuronal damage. Furthermore, VT suppresses the activity of hippocampal P2X7R and NLRP3 inflammasome in KA-treated mice. A438079, a specific P2X7R antagonist, inhibits NLRP3 inflammasome activation and reduces seizure severity and hippocampal neuronal damage. Conversely, the P2X7R activator BzATP negates the antiseizure and neuroprotective effects of VT. These findings demonstrate that VT can alleviate KA-induced seizure and neuronal damage by inhibiting P2X7R-NLRP3 inflammasome signaling in mice, potentially providing a novel strategy for prevention and treatment of acute seizures.

Introduction

Epilepsy is a neurological disease with high prevalence that affects approximately 1% of the global population [1–3]. Temporal lobe epilepsy (TLE), which often originates in the amygdala or hippocampus, is among the most common forms of epilepsy, affecting approximately 60% of patients [4]. Without proper intervention, TLE may worsen the neurological outcomes caused by epileptic seizures, aggravating impairments in cognitive functions including spatial learning and memory [5]. Currently, pharmacological therapies for epilepsy are effective in roughly two-thirds of all individuals with the condition. However, the majority of TLE patients are non-responsive to antiepileptic medications [6]. Consequently, developing new therapeutic targets and drugs is of pivotal significance in the treatment of epilepsy and its associated comorbidities.

Numerous studies have demonstrated that brain inflammation can enhance neuronal hyperexcitability and promote seizures [7]. Neuroglial immunoinflammatory dysfunction is recognized as a common factor in epilepsy patients and experimental models [8]. Therefore, understanding the neuroinflammatory mechanisms of epilepsy is crucial as it may facilitate the elucidation of epilepsy etiopathogenesis, thereby promoting treatment target identification. Recently, the Nod-like receptor family pyrin domain-containing protein 3 (NLRP3) inflammasome, comprising NLRP3, Caspase-1 and apoptosis-associated speck-like protein containing a CARD (ASC), has been implicated in epilepsy [9–11]. Activated NLRP3 inflammasome can trigger inflammatory reactions by releasing IL-1β and IL-18 [12]. Conversely, NLRP3 suppression prevents status epilepticus and exhibits neuroprotective effects in the amygdala-kindling model [9], suggesting that NLRP3 inflammasome-mediated inflammatory reaction is involved in epilepsy. Additionally, extracellular ATP exposure can activate the purinergic P2X receptor-7 (P2X7R), subsequently causing microglial activation and intracellular K⁺ efflux, which triggers the assembly of NLRP3 inflammasome [13]. However, the effect of P2X7R on NLRP3 inflammasome in the pathological process of epilepsy requires further investigation.

Vitexin (apigenin-8-C-β-D-glucopyranoside, VT) is a flavone glycoside prevalent in numerous types of natural materials, including dried hawthorn leaves, bamboo, and Ficus deltoidea [14]. The compound was implicated with anti-inflammation, neuroprotective, cardioprotective, antioxidant, and anti-cancer activities [15–20]. Furthermore, Vitexin (VT) demonstrates benefits in fat reduction, glucose metabolism, and hepatoprotection [21–23]. Additionally, VT exerts neuroprotective effects through suppressing N-methyl-D-aspartic acid (NMDA) receptors on mouse cerebral cortex neurons [24]. While NMDAR plays pivotal roles in the pathophysiology of epilepsy. A recent study indicates that VT can mitigate hypoxic ischemia-induced seizures in the neonatal brain [25]. Nonetheless, whether VT can alleviate seizures triggered by activators for glutamate receptor, including NMDA and kainic acid (KA), as well as the associated mechanisms, await detailed investigation.

Herein, we initially established a mouse model of status epilepticus employing KA, and subsequently investigated the antiseizure effect of VT and its underlying mechanism. Our findings demonstrate that VT mitigated KA-triggered seizures and neuronal damage in mice, possibly via suppressing the P2X7R-NLRP3 axis.

Materials and methods

Animals

Eight-week-old C57BL/6J male mice, weighing 21–23 g, were acquired from the Experimental Animal Center of Anhui Medical University and utilized as experimental animals. The mice were reared in an institutional SPF-level animal room, where they received standard animal feed and tap water (autoclaving) in accordance with established animal care protocols. All procedures related to mouse breeding, identification, and experimentation in this study adhered to directives issued by the Animal Experiment Ethics Committee of Anhui Medical University (permit number: LLSC20241176, Approval Date: 2024–03–07).

Chemicals

Vitexin (V113960) was procured from Aladdin Industrial Corporation. Antibodies for Caspase-1 (ab1872), P2X7R (ab259942), and CD68 (ab283654) were obtained from Abcam (USA). Rabbit anti-ASC (67824 S) and NLRP3 (15101 S) primary antibodies were provided by Cell Signaling Technology (CST, USA). Affinity (Australia) supplied the rabbit polyclonal antibody to Cleaved-caspase3 (Asp175). IL-1β (bs-6319R) was acquired from Bioss (China). WANLEIBIO (China) provided Bax (WL01637) and Bcl-2 (WL01556). Valproate (VPA) (HY-10585 A), A438079 (HY-15488), and BzATP (HY-136254) were obtained from Med Chem Express (MCE, USA). Additional reagents were commercially sourced.

Acute Seizure Induction and Drug Treatment

The acute seizure induction protocol followed established methods [26]. In brief, mice were anesthetized with isoflurane (5% in O₂) and secured in a stereotaxic apparatus. A guide cannula (26-gauge stainless steel) was intracerebroventricularly inserted into the position with dorsoventral (DV), anteroposterior (AP), and mediolateral (ML) coordinates of −2.0, −0.2, and + 1.0 mm, respectively. After a minimum recovery period of 3 days post-surgery, KA (5 µl of artificial CSF containing 0.15 µg of KA, Tocris) was injected into lateral ventricle (LV) via the cannula using a microsyringe pump. A modified Racine scale was employed for evaluation of epileptic seizures: (I) freezing; (II) rigid posture with raised tail; (III) repetitive head bobbing and forepaw clonus; (IV) occasional rearing, falling, and jumping; (V) continuous rearing, falling, and jumping; (VI) loss of posture and tonic-clonic seizures. Also, latency to seizure (time from KA injection to the onset of stage III seizures), seizure duration (time spent in stages III and above) and mortality rate were recorded to evaluate the severity of seizure. For appraising VT’s antiseizure and neuroprotective efficacy, the mice were randomized into a control group (saline injection), a KA group (KA injection), a KA + VT group (KA injection 30 min after VT administration or VT administration 15 min after KA injection), and a KA + VPA group (KA injection 30 min after VPA administration or VPA administration 15 min after KA injection).

Western Blot Analysis

Following behavioral tests, animals were euthanized by an overdose of isoflurane. The hippocampal tissues were isolated and prepared into lysates with RIPA buffer. After a 15-min centrifugation at 12, 000 g, 4 °C, the soluble fractions were quantified for protein content employing a Pierce (USA) BCA kit. Following SDS–PAGE separation, the proteins were electrotransferred onto PVDF membranes. These membranes were then subjected to a 90-min incubation at ambient temperature in 5% BSA dissolved with Tris-buffered saline supplemented with 0.1% Tween-20 (TBST), immersed first in primary antibodies (1:1 000 Cleaved-Caspase 3, Bax, P2X7R, Bcl2, NLRP3, ASC, IL-1β and 1:500 Caspase-1) at 4 °C for 16 h, and then in matched secondary antibodies for 90 min at 37 °C. Western blot images were visualized by enhanced chemiluminescence agents using a Bio-Rad (USA) imaging system.

Quantitative Reverse Transcription PCR

Following behavioral tests, mice were decapitated for hippocampi isolation. TRIzol reagent (Invitrogen) was employed for extraction of total RNA. After obtaining cDNA utilizing the RevertAid First Strand cDNA Synthesis kit (Takara), gene-specific primers P2X7R-F (5-CCACAGAGCAAAGGAATCCAGAC-3), P2X7R-R (5-CAGTAGGAC ACCAGGCAGAGAC-3), NLRP3-F (5-GCTGCGATCAACAGGCGAGAC-3), NLRP3-R (5-CCATCCACTCTTCTTCAAGGCTGTC-3), ASC-F (5-GGACGGAGT GCTGGATGCTTTG-3), ASC-R (5-CATCTTGTCTTGGCTGGTGGTCTC-3), Caspase-1-F (5-ATACAACCACTCGTACACGTCTTGC-3), Caspase-1-R (5-TCCTCC AGCAGCAACTTCATTTCTC-3), IL-1β-F (5-CACTACAGGCTCCGAGATGAACA AC-3), IL-1β-R (5-TGTCGTTGCTTGGTTCTCCTTGTAC-3), β-actin-F (5-TTCCTT CCTGGGTATGGAAT-3), and β-actin-R (5-GAGGAGCAATGATCTTGAT C-3) were adopted for real-time PCR. The 2^−ΔΔCT algorithm was applied for evaluation of target gene mRNA abundance relative to that of β-actin. All samples involved no less than three biological replicates.

Fluoro-Jade C Staining

Neuronal degeneration was detected using Fluoro-Jade C (FJC). In brief, brain slices were equilibrated to ambient temperature and air-dried. After PBS washes, the slides were incubated for 5 min within a 1% NaOH + 80% EtOH solution. They were subsequently rinsed with 70% EtOH and ddiH₂O in sequence. Following a 15-min incubation period with a 0.006% potassium permanganate solution, brain slices were rinsed with ddiH₂O. The slides were then immersed for 30 min in a 0.001% FJC (Millipore, USA) dissolved with 0.1% hydrogen acetate. Slides were subsequently air dried, covered with a coverslip, and dehydrated and cleaned using xylenes. FJC-positive cells per section were quantified using ImageJ (National Institutes of Health) by blinded investigators.

Nissl Staining

Coronal hippocampal Sect. (5 μm) were deparaffinized and washed with ddiH₂O. The sections were subsequently immersed for 25 min within Nissl staining solution (Beyotime) at 37 °C. Following water rinsing, the samples were rinsed for 5 min with 95% ethanol and allowed to air dry. The sections then underwent two 5-min xylene washes. Upon completion of mounting with neutral balsam, the dyed tissue specimens were analyzed and imaged employing a computerized scanning system (Pannoramic MIDI, 3D HISTECH). Nissl-positive cells were quantified using ImageJ (National Institutes of Health) by blinded investigators.

Hematoxylin-Eosin (HE) Staining

Coronal hippocampal Sect. (5 μm) from three mice per group underwent deparaffinization, washing with ddiH₂O, and staining with hematoxylin for 1 min, followed by three ddiH₂O washes. Subsequently, the samples were subjected to acidic liquid alcohol differentiation for 30 s, then immersed in eosin for 50 s, followed by treatment with 95% and dehydrated ethyl alcohol. Lastly, the sections were cleared with xylene and mounted using non-acidic resins. The scanning system utilized in HE staining assays was employed to examine and view the stained sections. The survival neurons were quantified using ImageJ (National Institutes of Health) by blinded investigators.

Immunofluorescence Staining

Following comprehensive anesthesia with isoflurane, mice underwent perfusion with 30 ml of isotonic saline (0.9% NaCl) followed by an equivalent volume of phosphate-buffered 4% paraformaldehyde (PFA) solution. Cerebral tissues were carefully dissected and immersed over a 16-h period in 4% PFA for post-fixation at 4 °C. Following fixation, the specimens were immersed for 48 h in a 30% sucrose-PBS solution kept under 4 °C. The samples were further processed into 14-µm-thick slices, which underwent triple 5-min rinsing in PBS and a 90-min incubation inside 0.3% Triton X-100-supplemented 5% BSA at 25 °C. Following overnight incubation within diluted antibodies for CD68 (1:200, abcam) and Iba1 (1:500, abcam) under 4 °C, the specimens were rinsed with PBS and subjected to 90-min incubation within diluted secondary antibodies (1:350 Alexa Fluor 488-labelled and 1:200 Alexa Fluor 594, Jackson) at ambient temperature. The immunofluorescently labeled specimens were subsequently captured using the computerized scanning system mentioned above. Iba-1 and CD68-positive cells were quantified using ImageJ (National Institutes of Health) by blinded investigators. The statistical analysis was normalized and the results are expressed as relative numbers of cells.

Statistical Analysis

We employed GraphPad Prism 8.2 to analyze statistical results. The distribution normality of data was determined by Shapiro-Wilk test. Data from two groups were compared with the Mann-Whitney U or t-test, while those from ≥ 3 groups were compared by one- or two-way ANOVA, following which Tukey’s or Bonferroni’s post hoc test was implemented. Data are presented as mean ± standard error of the mean (SEM). We designated 0.05 as the threshold P value for determining statistical significance.

Results

VT Alleviates the Severity of KA-Induced Seizure in Mice

To appraise VT’s antiseizure efficacy, a status epilepticus model was constructed by intracerebroventricular injection of KA (5 µl) in mice. Intraperitoneal VT (5, 10, 20 mg/kg) administration was carried out 30 min preceding KA injection. Sodium valproate (VPA, 250 mg/kg), a commonly used antiseizure medication for generalized onset tonic-clonic seizures, served as a positive control and was also administered intraperitoneally 30 min before KA injection. Seizure behavior was observed for 2 h following KA infusion (Fig. 1A). As illustrated in Fig. 1B-C, both VT and VPA pre- treatments significantly reduced seizure scores (n = 9–10, F_5,138= 255.8, P < 0.01) and average seizure scores (n = 9–10, F_4,43= 162.7, P < 0.01) compared to the KA group. Additionally, VT and VPA pre-treatments resulted in significant retardation of generalized seizure onset (n = 9–10, F_4,42= 16.82, P < 0.01 or P < 0.05) and considerably decreased seizure duration (n = 9–10, F_4,43= 119.2, P < 0.01) (Fig. 1D-E). Moreover, mortality rates were lower in VT and VPA groups relative to the KA group (n = 9–15) (Fig. 1F). Particularly, 20 mg/kg VT demonstrated a potent antiseizure effect comparable to that of VPA. Consequently, the 20 mg/kg concentration of VT was selected for subsequent studies. Finally, to assess the effect of VT post-treatment on KA-induced seizure, 20 mg/kg VT is administrated 15 min after KA injection and generalized seizure is usually elicited at that time (Fig. 1G). Our results showed that VT post-treatment also decreased seizure scores (n = 6, F_{2, 69}= 14.55, P < 0.001), duration (n = 6, F_{2, 15}= 9.623, P < 0.05 or P < 0.01) and mortality rates in mice (Fig. 1H-K). It is consistent with the results from VT pre-treatment. Collectively, these findings showcase that VT could mitigate KA-induced seizures in mice.

Fig. 1.

VT alleviates KA-induced seizure in mice. A Schematic of the experimental design and representative cannula implanting image. B Seizure scores of VT and VPA pre-treatment in KA mice. n = 9–10, F_5,138 = 255.8. C Average seizure scores of VT and VPA pre-treatment in KA mice. n = 9–10, F_4,43 = 162.7. D Effects of VT and VPA pre-treatment on the latency to seizure onset in KA-induced seizure model. n = 9–10, F_4,42 = 16.82. E The generalized seizure duration after VT and VPA pre-treatment in KA-induced mice. n = 9–10, F_4,43 = 119.2. F Effects of VT and VPA pre-treatment on the mortality in KA mice. n = 9–15. G Schematic of the experimental design and representative cannula implanting image. H Seizure scores of VT and VPA post-treatment in KA mice. n = 6, F_{2, 69} = 14.55. I Average seizure scores of VT and VPA post-treatment in KA mice. n = 6, F_{2, 15} = 32.06. J The generalized seizure duration after VT and VPA post-treatment in KA-induced mice. n = 6, F_{2, 15} = 9.623. K Effects of VT and VPA post-treatment on the mortality in KA mice. Data are expressed as means ± SEM. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001. One-way ANOVA with Tukey’s post-hoc test

VT Attenuates KA-Induced Neuronal Damage in Mice

Subsequently, the effects of VT on KA-induced neuronal damage were investigated. Three days after KA infusion, the mice were euthanized, and FJC staining was employed to identify dead and dying neurons. The results revealed that KA treatment increased the number of FJC-positive neurons in the hippocampal CA3 region, the effect was mitigated by VT administration (Fig. 2A) (n = 3, F_{2, 6}= 23.52, P < 0.05 or P < 0.001), suggesting that VT pre-treatment attenuates KA-induced neuronal injury. To further validate this observation, Nissl staining was conducted three days after KA injection. Consistently, KA treatment induced neuronal loss and cellular structural damage in the hippocampal CA3 region, while VT pre-treatment significantly reduced neuronal degeneration and increased the number of surviving neurons (n = 3, F_{2, 6}= 12.57, P < 0.05 or P < 0.01) (Fig. 2B). Moreover, VT post-treatment also decreased cell apoptosis and increased the number of surviving neurons (n = 3, F_{3, 8}= 20.83, P < 0.05 or P < 0.001) (Fig. 2C). Furthermore, KA pre-treatment elevated hippocampal Cleaved Caspase-3 (n = 6, F_{2, 15} = 81.70, P < 0.05 or P < 0.01) and Bax levels while decreasing Bcl-2 levels and the Bcl-2/Bax ratio (n = 6, F_{2, 15}= 3.421, P < 0.01). These effects were reversed by VT administration (Fig. 2D-F), indicating that VT mitigates hippocampal neuronal damage following KA-induced status epilepticus.

Fig. 2.

VT attenuates hippocampal neuronal damage in KA mice. A Representative images and quantification of FJC⁺ neurons showed that VT pre-treatment attenuates neuronal damage in the hippocampus. Scale bars = 100–200 μm. n = 3, F_{2, 6} = 23.52. B Nissl staining showed that VT pre-treatment alleviated KA-induced neuronal damage in the hippocampal CA3 region. Scale bars = 100–500 μm. n = 3. F_{2, 6} = 12.57. C Nissl staining showed that VT post-treatment alleviated KA-induced neuronal damage in the hippocampal CA3 region. n = 3, F (3, 8) = 20.83. Scale bars = 100–500 μm. D Representative immunoblots of hippocampal Cleaved Caspase-3, Bax, Bcl2 and β-actin 3 days after KA injection. E, F Relative protein expression of hippocampal Bcl-2/Bax (E, n = 6, F_{2, 15}= 3.421) and Cleaved Caspase-3 (F, n = 6, F_{2, 15} = 81.70). Data are shown as Means ± SEM. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001. One-way ANOVA with Tukey’s post-hoc test

VT Suppresses the Activation of Microglia and NLRP3 Inflammasome in KA Mice

Neuroinflammation is considered a crucial component in the development of epilepsy [27]. Microglia, the brain’s resident immune cells, can drive inflammatory responses and are significantly implicated in the etiology of epilepsy [28]. To determine whether VT influences microglial activation, hippocampal CD68, a marker of activated and phagocytic microglia, was assessed. The study found that KA treatment markedly increased CD68 levels in the hippocampal CA3 region (n = 4, F_{2, 9} = 24.68, P < 0.05 or P < 0.01), while VT pre-treatment inhibited this effect (Fig. 3A-C), suggesting that VT prevents KA-induced microglial activation in the hippocampus. Previous research has shown that NLRP3 is primarily present in microglia, and activated NLRP3 inflammasome is involved in epilepsy [29]. Consequently, the study examined whether VT influences NLRP3 inflammasome activation in the KA model. As anticipated, KA treatment caused a significant increase in the protein levels of hippocampal NLRP3 (n = 6, F_{2, 15} = 11.30, P < 0.05 or P < 0.01), Caspase-1 (n = 6, F_{2, 15} = 5.720, P < 0.05), ASC (n = 6, F_{2, 15} = 20.85, P < 0.05 or P < 0.01), and IL-1β (n = 6, F_{2, 15}= 12.47, P < 0.01), while VT substantially inhibited this effect (Fig. 3D-H). Similarly, VT also prevented KA-induced increases in the mRNA levels of hippocampal NLRP3 (n = 6, F_{2, 15}= 13.24, P < 0.01), Caspase-1 (n = 6, F_{2, 15}= 8.236, P < 0.01), ASC (n = 6, F_{2, 15}= 11.63, P < 0.05 or P < 0.01), and IL-1β (n = 6, F_{2, 15}= 28.61, P < 0.01) (Fig. 3I-L). Collectively, these findings suggest that VT may alleviate KA-induced seizures and hippocampal neuronal damage by preventing the activation of microglia and NLRP3 inflammasome.

Fig. 3.

VT exerts inhibitory effect on microglia and NLRP3 inflammasome in KA mice. A Typical immunostaining images of hippocampal Iba1 (red) and CD68 (green). Scale bars = 50–200 μm. B Relative numbers of CD68⁺ microglia. n = 4, F_{2, 9} = 24.68. C Ratio of CD68⁺ microglia in the total microglial cells. n = 4, F_{2, 9} = 8.412. D Representative immunoreactive bands of hippocampal NLRP3, IL-1β, ASC, Caspase-1 and β-actin. E-H Relative protein level of hippocampal NLRP3 (E, n = 6, F_{2, 15} = 11.30), ASC (F, n = 6, F_{2, 15} = 20.85), Caspase-1 (G, n = 6, F_{2, 15} = 5.720) and IL-1β (H, n = 6, F_{2, 15}= 12.47), respectively. I-L The relative mRNA levels of hippocampal NLRP3 (I, n = 6, F_{2, 15}= 13.24), ASC (J, n = 6, F_{2, 15}= 11.63), Caspase-1 (K, n = 6, F_{2, 15}= 8.236) and IL-1β (L, n = 6, F_{2, 15}= 28.61), respectively. Data are expressed as means ± SEM. ^*P < 0.05, ^**P < 0.01. One-way ANOVA with Tukey’s post-hoc test

P2X7R Contributes To VT’s Antiseizure and Neuroprotective Effects

P2X7R is considered a potential molecular biomarker for epilepsy and is associated with various inflammatory and immunological responses [30–32]. Consequently, we investigated whether P2X7R is involved in VT ‘s antiseizure effect. Our findings revealed that VT significantly reduced the KA-induced increase in hippocampal P2X7R expression at both mRNA (n = 6, F_{2, 15}= 16.68, P < 0.01) and protein levels (n = 6, F_{2, 15}= 12.19, P < 0.01) (Fig. 4A-B), suggesting P2X7R’s potential involvement in VT ‘s antiseizure effect. To further validate this hypothesis, a specific P2X7R inhibitor, A438079 (20 µM, 5 µl), was administered into the lateral ventricle 30 minutes before KA injection in mice (Fig. 4C). The results demonstrated that A438079 significantly lowered seizure scores (n = 10, F_{2, 69}= 575.5, P < 0.001) and extended the latency to seizure onset (n = 10, t = 6.185, P < 0.001) compared to the KA group (Fig. 4D-F). Moreover, A438079 mitigated KA-induced increases in the duration of generalized seizures (n = 10, t = 21.63, P < 0.001) and mortality rates (n = 10) (Fig. 4G-H). Additionally, while KA microinjection substantially reduced the number of surviving neurons in the hippocampal CA3 region, A438079 counteracted this effect (Fig. 4I-L) (n = 3, F_{2, 6}= 29.17 (K), F_{2, 6} = 19.95 (L), P < 0.01). These findings suggest that P2X7R inhibition may contribute to the antiseizure and neuroprotective effects of VT.

Fig. 4.

P2X7R contributes to VT’s antiseizure and neuroprotective effects. A Relative protein expression of P2X7R. n = 6, F_{2, 15} = 12.19. B Relative mRNA level of P2X7R. n = 6, F_{2, 15}= 16.68. C Schematic representation of the experimental timeline. D Seizure scores of A438079 pre-treatment in KA mice. n = 10, F_{2, 69}= 575.5. E Average seizure scores of A438079 pre-treatment in KA mice. n = 10, t = 23.15. F Effects of A438079 pre-treatment on the latency to seizure onset in KA-induced seizure model. n = 10, t = 6.185. G The generalized seizure duration after A438079 pre-treatment in KA mice. n = 10, t = 21.63. H Effects of A438079 pre-treatment on the mortality in KA mice. n = 10. I Typical immunohistochemical images of Nissl staining. Scale bars = 100–500 μm. J Representative images of H.E. staining. Scale bars = 100–500 μm. K Effects of A438079 pre-treatment on the Nissl positive neurons in CA3 area in KA mice. n = 3, F_{2, 6}= 29.17. L Effects of A438079 pre-treatment on neuronal damage in CA3 area in KA mice. n = 3, F_{2, 6} = 19.95. Data are shown as Means ± SEM. ^**P < 0.01, ^***P < 0.001. One-way ANOVA with Tukey’s post-hoc test was used in A, B, D, K and L. Unpaired two-tailed Student’s t-test was used in E-G

A438079 Suppresses NLRP3 Inflammasome Activation in KA Mice

Subsequently, we investigated the influence of A438079 on NLRP3 inflammasome activation in KA-induced mouse model of status epilepticus. Our findings revealed that KA significantly increased hippocampal NLRP3 (n = 6, F_{2, 15} = 73.08, P < 0.01) and P2X7R (n = 6, F_{2, 15} = 7.961, P < 0.05 or P < 0.01) protein expression, while A438079 significantly inhibited this effect (Fig. 5A-C). Similarly, A438079 also attenuated the KA-induced elevation in hippocampal Caspase-1 (n = 6, F_{2, 14} = 7.611, P < 0.05), ASC (n = 6, F_{2, 15}= 108.0, P < 0.01) and IL-1β (n = 6, F_{2, 15} = 6.961, P < 0.05) protein levels (Fig. 5D-F). Furthermore, KA induced a substantial increase in the mRNA levels of hippocampal P2X7R (n = 3, F_{2, 6}= 9.168, P < 0.05), NLRP3 (n = 3, F_{2, 6} = 28.99, P < 0.01), Caspase-1 (n = 3, F_{2, 6} = 7.693, P < 0.01), ASC (n = 3, F_{2, 6} = 18.26, P < 0.01) and IL-1β (n = 3, F_{2, 6} = 7.716, P < 0.05 or P < 0.01), and the effects that were significantly reversed by A438079 (Fig. 5G-K). These results demonstrate that A438079 inhibited NLRP3 inflammasome activation in KA-treated mice, suggesting that P2X7R/NLRP3 inflammasome signaling may contribute to the antiseizure and neuroprotective effects of VT.

Fig. 5.

A438079 suppresses NLRP3 inflammasome activation in KA mice. A Representative immunoreactive bands of hippocampal NLRP3, P2X7R, IL-1β, ASC, Caspase-1. B-F Relative protein level of hippocampal P2X7R (B, n = 6, F_{2, 15} = 7.961), NLRP3 (C, n = 6, F_{2, 15} = 73.08), ASC (D, n = 6, F_{2, 15}= 108.0), Caspase-1 (E, n = 6, F_{2, 14} = 7.611) and IL-1β (F, n = 6, F_{2, 15} = 6.961), respectively. G-K Relative mRNA levels of hippocampal P2X7R (G, n = 3, F_{2, 6}= 9.168), NLRP3 (H, n = 3, F_{2, 6} = 28.99), ASC (I, n = 3, F_{2, 6} = 18.26), Caspase-1 (J, n = 3, F_{2, 6} = 7.693) and IL-1β (K, n = 3, F_{2, 6} = 7.716), respectively. ^*P < 0.05, ^**P < 0.01. Data are expressed as means ± SEM. One-way ANOVA with Tukey’s post-hoc test

P2X7R Activation Abolishes VT’s Antiseizure and Neuroprotective Effects in KA Mice

To further elucidate the role of P2X7R in VT ‘s antiseizure and neuroprotective effects, BzATP (10 µM, 5 µl), an agonist of P2X7R, was administered into the lateral ventricle 30 min prior to KA injection in mice (Fig. 6A). The results indicated that BzATP significantly elevated seizure scores and prolonged the duration of generalized seizures in KA-treated mice, suggesting P2X7R activation exacerbates KA-induced seizures. Moreover, BzATP counteracted the antiseizure effect of VT in KA-treated mice (Fig. 6B-E) (n = 7, F_{3, 92} = 40.51 (B), F_{3, 24} = 127.4 (C), F_{3, 24} = 84.2 (D), F_{3, 24} = 54.81 (E), P < 0.05 or P < 0.01). However, it did not affect the mortality rate of KA-treated mice (Fig. 6F). Additionally, VT substantially reduced the number of FJC-positive neurons in the hippocampal CA3 region of KA-treated mice, but this effect was significantly attenuated by BzATP (Fig. 6G-H) (n = 3, F_{3, 8} = 92.83, P < 0.01 or P < 0.001). These findings demonstrate that P2X7R activation aggravates KA-induced seizures and neuronal damage, thereby negating the antiseizure and neuroprotective effects of VT.

Fig. 6.

P2X7R activation abolishes VT’s antiseizure and neuroprotective effects in KA mice. A Schematic of the experimental design. B Seizure scores of BzATP and VT pre-treatment in KA mice. n = 7, F_{3, 92} = 40.51. C Average seizure scores of BzATP and VT pre-treatment in KA mice. n = 7, F_{3, 24} = 127.4. D The generalized seizure duration after BzATP and VT pre-treatment in KA-induced mice. n = 7, F_{3, 24}= 84.20. E Effects of BzATP and VT pre-treatment on the latency to seizure onset in KA-induced seizure model. n = 7, F_{3, 24} = 54.81. F Effects of BzATP and VT pre-treatment on the mortality in KA mice. n = 7 mice/group in B-F. G Representative images of FJC staining. Scale bars = 100–200 μm. (H) Effects of BzATP and VT pre-treatment on the FJC positive neurons in CA3 area in KA mice. n = 3, F_{3, 8} = 92.83. Data are shown as means ± SEM. ^*P < 0.05, ^**P < 0.01. Two-way ANOVA followed by Bonferroni’s post hoc test

BzATP Abolishes VT’s Inhibitory Effect on NLRP3 Inflammasome in KA Mice

Subsequently, we investigated whether hippocampal P2X7R/NLRP3 inflammasome signaling mediates VT’s effects in KA mice. As illustrated in Fig. 7A-F, KA significantly increased the protein expression of hippocampal NLRP3, P2X7R, Caspase-1, ASC and IL-1β. VT markedly attenuated this effect, but BzATP administration counteracted its inhibitory actions on these proteins (n = 6, F_{3, 20} = 22.09 (B), F_{3, 20} = 31.03 (C), F_{3, 20} = 46.36 (D), F_{3, 20} = 17.53 (E), F_{3, 20} = 11.87 (F), P < 0.05 or P < 0.01). Correspondingly, we observed that VT suppressed KA-induced elevation in the mRNA levels of hippocampal NLRP3, P2X7R, Caspase-1, ASC and IL-1β. These effects were also significantly mitigated by BzATP (Fig. 7G-K) (n = 4, F_{3, 12} = 15.24 (G), F_{3, 12} = 11.25 (H), F_{3, 12} = 168.8 (I), F_{3, 12} = 74.90 (J), F_{3, 12} = 29.67 (K), P < 0.05 or P < 0.01), suggesting that P2X7R activation negates VT’s inhibitory effects on the NLRP3 inflammasome. Collectively, our results indicate that hippocampal P2X7R/NLRP3 inhibition contributes to VT’s antiseizure and neuroprotective properties.

Fig. 7.

BzATP abolishes VT’s inhibitory effect on NLRP3 inflammasome. A Representative immunoreactive bands of hippocampal NLRP3, P2X7R, IL-1β, ASC, Caspase-1 and β-actin. B-F Relative protein level of hippocampal P2X7R (B, n = 6, F_{3, 20} = 31.03), NLRP3 (C, n = 6, F_{3, 20} = 22.09), ASC (D, n = 6, F_{3, 20} = 17.53), Caspase-1 (E, n = 6, F_{3, 20} = 46.36) and IL-1β (F, n = 6, F_{3, 20} = 11.87), respectively. G-K Relative mRNA level of P2X7R (G, n = 4, F_{3, 12} = 11.25), NLRP3 (H, n = 4, F_{3, 12} = 15.24), ASC (I, n = 4, F_{3, 12} = 74.90), Caspase-1 (J, n = 4, F_{3, 12} = 168.8) and IL-1β (K, n = 4, F_{3, 12} = 29.67), respectively. ^*P < 0.05, ^**P < 0.01. Data are shown as Means ± SEM. Two-way ANOVA followed by Bonferroni’s post hoc test

Discussion

Epilepsy is a complex neurological disorder that remains inadequately controlled by current treatment approaches due to its multifaceted pathogenesis [33]. This study demonstrates that VT mitigates KA-induced seizures and reduces hippocampal neuronal damage in mice. Furthermore, VT inhibits the activation of hippocampal P2X7R and NLRP3 inflammasome. The P2X7R antagonist A438079 was found to suppress NLRP3 inflammasome activation and alleviate KA-induced seizures and neuronal damage. Conversely, the P2X7R activator BzATP counteracted VT’s effects on acute seizures and NLRP3 inflammasome activation. These findings suggest that VT attenuates KA-induced seizures by inhibiting the P2X7R/NLRP3 signaling pathway.

Accumulating evidence from experimental animal studies and patients with epilepsy suggests that neuroinflammation is a common feature of epilepsy. Pro-inflammatory cytokine levels are elevated in the brains of epileptic animals and patients [34, 35]. Increased inflammation can activate Toll-like receptor 4 and NMDA receptors, resulting in neuronal hyperexcitability and subsequent epileptic seizures [36, 37]. Conversely, anti-inflammatory drugs or NMDA inhibition demonstrate antiseizure and neuroprotective effects [38, 39]. As a flavonoid compound, VT exhibits potent anti-inflammatory activity [40]. It inhibits brain inflammation induced by high-fat diets [41] and alleviates chronic cerebral hypoperfusion injury by suppressing inflammatory responses [42]. Additionally, it can mitigate neuronal damage by inhibiting NMDAR activation [24]. A recent study reported that VT exerts an anticonvulsant effect in a PTZ kindled rat model but does not block seizures induced by NMDA and KA [43]. However, contrary to this report, our findings indicate that both VT pre-treatment and post-treatment can alleviate the severity of KA-induced seizures. Moreover, VT’s efficacy was comparable to that of the traditional antiseizure medication VPA (Fig. 1). It is important to note that the maximum dose of VT in the aforementioned study was 5 mg/kg. Consistent with the previous report, we also observed that 5 mg/kg VT had no influence on KA-induced seizures. However, our findings revealed that 10 and 20 mg/kg VT had a significant antiseizure effect, suggesting that the antiseizure effect of VT varies in different animal models and may be more sensitive to GABAergic than Glutamatergic neurotransmission.

The hippocampus plays a crucial role in learning, memory, and emotional behavior [44]. Recurrent seizures can lead to hippocampal damage, resulting in cognitive impairments [38]. Previous research has demonstrated that KA-induced neuropathological changes closely resemble those observed in patients with TLE, particularly in the hippocampal region [45]. Additionally, VT has shown neuroprotective effects in both vitro and vivo studies [14]. However, the potential protective role of VT against KA-induced hippocampal neuronal damage remains unclear. In this study, we observed that VT pre-treatment substantially mitigated KA-induced hippocampal neuronal loss and damage. However, it should be noted that the vitexin pre-treated mice might have no sufficient seizure activity to elicit neurological damage. Interestingly, VT post-treatment (administration after the appearance of generalized seizures) also increased the number of surviving neurons and attenuated neuronal damage in the hippocampus. Moreover, VT decreased the protein levels of cleaved-caspase-3 and Bax while increasing Bcl-2 levels in the hippocampus (Fig. 2), suggesting that VT exhibits potent anti-apoptotic and neuroprotective effects in KA-treated mice.

Microglia play a pivotal role in regulating immune reactions and maintaining brain homeostasis [46]. They can be rapidly activated following an epileptic seizure, subsequently producing numerous pro-inflammatory cytokines that result in neuronal hyperexcitability and a low seizure threshold [47]. Inhibition of inflammatory microglia exhibits significant antiepileptic and neuroprotective effects in experimental epilepsy models [48]. However, microglia are also reported to have beneficial functions in epilepsy [26]. Our previous study demonstrated that microglia ablation aggravated KA-induced seizures, indicating that microglia deletion can worsen seizures, while inflammatory microglia inhibition may have an anti-seizure effect [26]. NLRP3, a widely studied and well-characterized inflammasome, is highly expressed in microglia and is a crucial regulator contributing to proinflammatory cytokine release [49, 50]. NLRP3 suppression can decrease microglia activation and inflammatory response [51]. Increasing evidence shows that NLRP3 is activated in both epileptic animals and patients. Blockage of NLRP3 inflammasome activation can alleviate the severity of seizures and produce neuroprotective effects [29, 48]. Our findings suggest that VT prevented the activation of microglia and NLRP3 inflammasome in KA mice (Fig. 3), indicating that interfering with the activity of microglia and NLRP3 inflammasome could contribute to VT’s antiseizure and neuroprotective effects.

P2X7R, a member of the ATP-gated ionotropic P2XR family, is predominantly expressed in immune cells, including microglia, macrophages, and monocytes. It is considered a major regulator of inflammatory reactions, contributing to microglial activation and pro-inflammatory cytokine release [52]. Mounting evidence indicates elevated P2X7R levels in the brains of epileptic animals and patients, with P2X7R blockade or deletion demonstrating antiseizure and neuroprotective effects [31]. P2X7R has also been identified as a potent activator of the NLRP3 inflammasome. Activated P2X7R triggers K⁺ efflux and Ca²⁺ influx, serving as a promoter of NLRP3 inflammasome assembly [53]. The P2X7R/NLRP3 signaling pathway has been implicated in various brain disorders, including depression, ischemic brain injury, Alzheimer’s disease, and migraine [54]. In this study, we observed that VT blocked the KA-induced increase in P2X7R expression. A438079, a specific P2X7R antagonist, inhibited NLRP3 inflammasome activation and exhibited antiseizure and neuroprotective effects in the KA-induced mouse model of status epilepticus (Figs. 4 and 5). Conversely, the P2X7R activator BzATP exacerbated acute seizures and negated the antiseizure and neuroprotective effects of VT (Fig. 6). Furthermore, BzATP reversed VT’s inhibitory effects on the NLRP3 inflammasome in KA-treated mice (Fig. 7). These results suggest that VT alleviates KA-induced seizures and neuronal damage by inhibiting the P2X7R/NLRP3 inflammasome signaling pathway. It should be noted that NMDAR inhibition and antioxidation have been reported to involve in VT’s neuroprotective effects, while NMDAR and oxidative stress are closed to epilepsy [24, 55, 56]. Thus, VT’s effects on seizure could be related to regulating NMDAR and oxidative stress. Further research should be performed to clarity this point in the future.

Although our findings reveal VT’s antiseizure and neuroprotective effects, there are still some limitations in this study. Firstly, despite establishing causality in the KA-induced mouse model of status epilepticus, translational validation in human biospecimens and alternative epilepsy models (e.g., electrical kindling, genetic epilepsies) is required to assess pathway conservation across etiologies. Secondly, we only observed the effect of VT on male epileptic seizure mice, whether sex difference needs to be examined in future research. Lastly, the precise molecular mechanisms of VT’s effects were not fully elucidated. Vitro studies (e.g., primary microglia cultures) are needed to further validate molecular mechanisms and explore human biomarker validation to support clinical relevance. Therefore, future efforts will focus on addressing these limitations and making VT develop into a viable option for seizure treatment.

Conclusion

In conclusion, this study demonstrates that VT mitigates KA-induced seizures and neuronal damage in mice. Furthermore, VT suppresses the activation of the P2X7R and the NLRP3 inflammasome. Notably, the effects of VT can be counteracted by a P2X7R agonist (Fig. 8). Collectively, our findings indicate that VT exhibits antiseizure and neuroprotective properties via inhibiting the P2X7R/NLRP3 signaling pathway in the KA-triggered mouse model of status epilepticus. This study proposes a potential seizure treatment strategy via VT, but further studies are needed to confirm VT’s clinical efficacy in the future.

Fig. 8.

The graphical abstract of the mechanism by which VT exhibits antiseizure and neuroprotective effects though inhibiting P2X7R/NLRP3 signaling pathway in KA-induced mouse model of status epilepticus

Author Contributions

W-NW designed the study. RC, S-MC H-CW performed the experiments. KD, H-LS and M-MS analyzed the data. RC, BG and W-NW wrote the manuscript. All authors have reviewed and approved the final version of the manuscript.

Funding

This research was funded by grants from the National Natural Science Foundation of China (No. 82171449) and the Scientific Research Promotion Plan of Anhui Medical University (No. 2020xkjT004).

Data Availability

No datasets were generated or analysed during the current study.

Declarations

Ethics Approval

All animal care and experimental procedures adhered to guidelines approved by the Committee for Experimental Animal Use and Care of Anhui Medical University (permit number: LLSC20241176, Approval Date: 2024–03–07).

Consent for Publication

All authors agree to publish the finalized version of this paper.

Competing interests

The authors declare no competing interests.