JC124 confers multimodal neuroprotection in epilepsy by suppressing NLRP3 inflammasome activation: evidence from animal and human neuronal models

Abstract

Background

Despite continuous development of antiseizure medications (ASMs), global seizure control rates remain unsatisfactory, highlighting the urgent need for novel ASMs targeting distinct pathophysiological mechanisms. Inhibition of NLRP3 inflammasome activation represents an emerging strategy to simultaneously attenuate seizures and associated neuropsychiatric comorbidities. Therefore, this study investigates whether JC124, a novel NLRP3 inflammasome inhibitor, exerts neuroprotective effects in kainic acid (KA)-induced epileptic mice and in human induced pluripotent stem cell (hiPSC)-derived neurons stimulated by lipopolysaccharide (LPS) and adenosine triphosphate (ATP).

Methods

Summary-based Mendelian Randomization (SMR) was used to analyze the association between NLRP3 alleles and epilepsy susceptibility. NLRP3 knockout mice were generated, and then epileptic mice induced by intrahippocampal KA injection were administered JC124 (50 mg/kg, intraperitoneal) once daily for 28 days. The spontaneous recurrent seizures, hippocampal local field potential, depressive-like behavior, cognitive dysfunction, and locomotor ability of mice were evaluated. The brain tissues of the mice were collected for Western blotting, immunohistochemistry, immunofluorescence labeling, enzyme-linked immunosorbent assay, transmission electron microscopy, and morphological staining. The binding capacity of JC124 to the human NLRP3 protein was assessed using molecular docking and molecular dynamics simulations. hiPSC-derived neurons were used to explore the neuroprotective effects of JC124 against inflammatory injury in human neurons.

Results

In this study, a positive correlation was identified between the expression of the NLRP3 gene and the susceptibility to epilepsy through SMR analysis. JC124 intervention markedly inhibited seizures and improved depressive-like behavior and cognitive dysfunction. It also reduced hippocampal neuronal loss, neuronal pyroptosis, microgliosis, and astrogliosis. Importantly, the neuroprotective effects of JC124 in KA-induced epileptic mice were mediated through the inhibition of the NLRP3 inflammasome. JC124 inhibited neuroinflammation and oxidative stress in KA-treated NLRP3 wild-type mice, but not in KA-treated NLRP3 knockout mice. Furthermore, JC124 bound directly to the human NLRP3 protein and alleviated neuroinflammation and oxidative stress in hiPSC-derived neurons stimulated by LPS and ATP.

Conclusions

These findings indicate that inhibiting the activation of the NLRP3 inflammasome with JC124 represents a potentially safe and innovative therapeutic strategy to mitigate neuroinflammation and oxidative stress in epilepsy and to alleviate seizures and associated neuropsychiatric comorbidities.

Graphical Abstract

Schematic illustration of the mechanisms by which JC124 exerts neuroprotective effects in epilepsy

Supplementary Information

The online version contains supplementary material available at 10.1186/s12964-025-02239-3.

Background

Epilepsy is a chronic disorder resulting from abnormal neuronal discharges in the brain. People with epilepsy often have depression and cognitive deficits that seriously affect their quality of life. It is estimated that more than 70 million patients suffer from epilepsy worldwide [1, 2]. Antiseizure medications (ASMs) are the primary treatment for managing seizures. However, the majority of ASMs have a limited therapeutic index and a variety of adverse effects, such as skin rashes, liver and kidney damage, teratogenic consequences, exacerbation of cognitive impairment, and promotion of depression [2, 3]. Furthermore, although several second-generation ASMs have been on the market since 1989, it is disappointing that the rate of seizure control has not significantly increased [4]. Consequently, developing new epilepsy treatments is critically important.

Ample evidence has indicated that there is a vicious cycle among neuroinflammation, oxidative stress, and seizures, in which any one factor can exacerbate the others [5, 6]. It has been proven that inhibition of neuroinflammation and oxidative stress is an essential treatment method for epilepsy and neuropsychiatric comorbidities [5–10].

The activation of the NOD-, LRR- and pyrin domain-containing protein 3 (NLRP3) inflammasome is widely perceived as an essential event that initiates neuroinflammation in epilepsy [11–13]. In addition, a recent study has indicated that NLRP3 inflammasome activation also promotes the degradation of the nuclear factor erythroid 2-related factor 2 (Nrf2), a core antioxidant and anti-inflammatory molecule [14–16]. Interestingly, this alteration is blocked by NLRP3 deletion [15]. From a clinical perspective, inhibition of NLRP3 inflammasome activation reduces seizures, neurobehavioral comorbidities, and neuronal damage in animal models of epilepsy [17–20]. These findings indicate that targeting the NLRP3 inflammasome is a viable strategy to discover effective treatments for epilepsy. However, the development of potent, selective, and safe NLRP3 inflammasome inhibitors remains an unmet therapeutic challenge.

Through structural optimization of glyburide, JC124, a specific NLRP3 inflammasome inhibitor, was developed [21, 22]. Animal studies have shown that JC124 has therapeutic potential in myocardial infraction, Alzheimer's disease (AD) and traumatic brain injury (TBI) through its anti-inflammatory effects [21–24]. However, the potential neuroprotective effects of JC124 on kainic acid (KA)-induced epileptic mice and neurons derived from human induced pluripotent stem cells (hiPSC) are not yet clear and warrant further investigation.

The present study identified a significant association between the expression of the NLRP3 gene and an increased susceptibility to epilepsy, as determined through Summary-based Mendelian Randomization (SMR) analysis. JC124 reduced seizures; alleviated depressive-like behavior and cognitive deficits; mitigated hippocampal neuronal loss, neuronal pyroptosis, and gliosis; and inhibited neuroinflammation and oxidative stress in KA-induced epileptic mice by suppressing NLRP3 inflammasome activation. Importantly, JC124 directly interacted with the human NLRP3 protein and alleviated neuroinflammation and oxidative stress in neurons derived from hiPSCs that were stimulated by lipopolysaccharide (LPS) and adenosine triphosphate (ATP). These findings highlight JC124's potential as a novel therapeutic agent for epilepsy.

Materials and methods

SMR analysis

This SMR study utilized publicly available summary data from genome-wide association study (GWAS) and expression quantitative trait loci (eQTL) study [25]. The summary-level eQTL data were obtained from the eQTLGen Consortium (https://www.eqtlgen.org/). We selected common (minor allele frequency > 1%) single-nucleotide polymorphisms that were significantly (P < 5.0 × 10^–8) associated with the expression of the NLRP3 gene in blood [26, 27]. Only cis-eQTLs (eQTLs located within 1 Mb on either side of the encoded gene) were included to generate genetic instruments for this study. A multi-ancestor GWAS dataset on epilepsy was obtained from the International League Against Epilepsy [28]. The association between NLRP3 alleles and epilepsy susceptibility was analyzed using SMR software 1.3.1 (https://yanglab.westlake.edu.cn/software/smr) [27]. See the Supplementary Table 1 (Additional File 1) for further details.

Animals

Seven- to eight-week-old male C57BL/6 J mice, weighing 22–25 g, were provided by Chongqing Medical University. Male NLRP3^−/− mice, aged 7–8 weeks and weighing 22–25 g, were sourced from the Shanghai Model Organisms Center. There was a 12-h cycle between light and darkness, and all the mice had access to food and water ad libitum in a pathogen-free environment. The genotypes of NLRP3^−/− and NLRP3^WT mice were determined following the kit instructions (YK-MG-100, UBIGENE, Guangzhou, China). See the Supplementary materials and methods (Additional File 1) for further details.

KA-induced epilepsy mouse model and JC124 treatment

According to our previous study [29], mice were anesthetized with pentobarbital (50 mg/kg, intraperitoneal injection) and then fasted on a stereotaxic apparatus. KA (Sigma‒Aldrich, MO, USA) was administered into the Cornus Ammonis (CA)1 region of the right hippocampus at the following coordinates: −2.0 mm anteroposterior, −1.5 mm mediolateral, and −1.5 mm dorsoventral from the bregma. A 0.5-μL Hamilton syringe delivered 50 nL (200 ng, 10 nL/min) of KA. Then, the syringe was held for 5 min after injection to prevent reflux. Sham mice received an equivalent volume of saline solution via identical injection methods.

To assess the neuroprotective effects of JC124 on seizures and associated neurobehavioral comorbidities in a KA-induced epilepsy mouse model, wild-type mice were randomly assigned to one of five groups: sham + vehicle, sham + JC124, post-SE + vehicle, post-SE + JC124, and post-SE + valproic acid (VPA, selected as a positive control), with 25 mice in each group. To elucidate whether the neuroprotective effects of JC124 in a KA-induced epilepsy mouse model are mediated through NLRP3 inhibition, NLRP3^−/− mice were generated, while wild-type littermate mice were used as controls. The mice were randomly allocated into six distinct groups: NLRP3^WT + sham + vehicle, NLRP3^WT + post-SE + vehicle, NLRP3^WT + post-SE + JC124, NLRP3^−/− + sham + vehicle, NLRP3^−/− + post-SE + vehicle, and NLRP3^−/− + post-SE + JC124, with 25 mice in each group.

In accordance with previous studies [22, 30] and our preliminary experiments, JC124 (CSNpharm, IL, USA; 50 mg/kg, intraperitoneally) or VPA (TargetMol, MA, USA; 200 mg/kg, intraperitoneally) was administered once daily for 28 days. The vehicle groups received an identical volume of solvent, consisting of 10% DMSO in saline and PEG (TargetMol).

Seizure analysis and hippocampal local field potential (LFP) measurement

During epilepsy modeling in mice, seizure grades were estimated using Racine′s scale. The latency to the first occurrence of grade 0–2, 3, 4, and 5 seizures, the latency to the onset of status epilepticus (SE), and the duration of SE were recorded [31]. Mice were video-monitored for seizures during the 4-week administration of JC124. The latency to the first nonconvulsive spontaneous recurrent seizure (SRS), the latency to the first convulsive SRS, the number of nonconvulsive SRSs, and the number of convulsive SRSs, were analyzed [31–34]. For hippocampal LFP measurement, a nickel‒chromium alloy electrode (Plexon, HK) was implanted into the CA1 region of the right hippocampus (coordinates as previously described) after KA injection and then fixed with bone cement. After the completion of JC124 treatment, the hippocampal LFP was recorded as previously reported [35]. Seizure-like events (SLEs), including their total number, mean duration, and cumulative duration, were evaluated via NeuroExplorer software (Nex Technologies, MA, USA) [29]. See the Supplementary materials and methods (Additional File 1) for further details.

Behavioral tests

Ethology tests were conducted in a quiet, clean, and well-lit room (110 lx). The animals were allowed to acclimatize for at least three hours before testing began. The obtained data were analyzed with ANY Maze software (Stoelting, IL, USA). The first animal behavioral test was performed after completion of the LFP recording and at intervals of 24 h between different ethology tests.

Sucrose preference test (SPT)

This classical paradigm was selected to assess anhedonia (a core symptom of depressive behavior in rodents). Mice were acclimated to a 1% sucrose solution for 48 h prior to the formal experiment (free access to two identical bottles, one containing sucrose and one with water) [36]. Then, the mice were given a 1% sucrose solution and water. After 12 h, the positions of the sucrose solution and water bottles were interchanged, and the consumption of both sucrose solution and water by each mouse was recorded 24 h later. Finally, sucrose preference values were evaluated [36, 37].

$$\mathrm{sucrose}\mathrm{preference}\left(\%\right)=\frac{\mathrm{sucrose}\mathrm{consumption}}{\left(\mathrm{sucrose},,+,,\mathrm{water},,\mathrm{consumption}\right)}\times 100\%$$

Open field test (OFT)

This test was used to evaluate ​​anxiety-like behavior​​ and general locomotor activity of mice [38, 39]. The mice were positioned in the central area of the apparatus (50 cm × 50 cm × 40 cm). After an adaptation period of 30 s, the mice moved freely for 5 min, during which the movement time and distance of each mouse in both the central and surrounding areas were recorded [37]. The apparatus was wiped clean with 75% alcohol before each testing trial.

Morris water maze (MWM)

This classical test was performed to assess ​​spatial learning and memory​​ of mice [40]. The MWM apparatus was a 122-cm diameter, 51-cm high white polyvinyl chloride pool with an 8-cm diameter circular platform (in the middle of the southwest quadrant of the MWM), 1 cm underwater. The water temperature of the MWM was maintained at 23 ± 1 °C, and the large black and white symbols affixed to the walls of the four quadrants of the MWM were used as directional markers for the mice. During the spatial acquisition test, 4 trials were conducted each day for 5 consecutive days. Each training session lasted 60 s, and the entry position was randomly selected. During the experiment, the mouse was carefully lifted by the tail and allowed to enter the water. When the mouse climbed onto the platform and stayed there for 5 s, the experiment ended. The spatial memory of the mice was measured by escape latency (the time to find a hidden platform), and the locomotor ability of the mice was measured by swimming speed. In the probe trial, the platform was removed, and the mice entered the water from the northeast quadrant. The number of platform crossings, as well as the movement distance and movement time in the southwest quadrant within 60 s were recorded [40].

Differentiation of hiPSC-derived neurons

hiPSCs were differentiated into neurons according to our previously reported methods [41]. hiPSCs, neural stem cells (NSCs), and hiPSC-derived neurons identification were confirmed by immunofluorescence staining with specific antibodies: anti-OcT4/anti-SSEA4, anti-PAX6/anti-Nestin, and anti-MAP2, respectively. The Supplementary materials and methods (Additional File 1) include a comprehensive protocol.

Inflammatory stimulation of hiPSC-derived neurons and JC124 treatment

hiPSC-derived neurons were cultured in 96-well plates. These neurons were treated with varying concentrations of JC124 (0.1, 0.3, 1, 3, 10, or 30 μM) for a duration of 24 h. Cell viability of hiPSC-derived neurons was assessed in accordance with the protocol provided by the CCK-8 kit (C0041, Beyotime, Shanghai, China). hiPSC-derived neurons were pretreated with JC124 (3 or 10 μM) for 1 h, after which the neurons were subjected to LPS (100 ng/mL, Sigma‒Aldrich) priming for a duration of 3 h. Subsequently, a 30-min stimulation with ATP (5 mM, MedChemExpress, NJ, USA) was performed [42–45]. The supernatant and whole cell lysate from the culture of hiPSC-derived neurons were harvested for subsequent biological experiments.

Tissue collection

The process for tissue collection has been described previously [24]. Mice were anesthetized with pentobarbital (50 mg/kg, intraperitoneal injection). After the brains were carefully removed, the total protein or deoxyribonucleic acid (DNA) in the hippocampi of the mice was rapidly extracted for biological experiments. For Nissl staining, immunohistochemistry, and immunofluorescence labeling, brain tissues were fixed, dehydrated, embedded, and sliced.

Western blotting (WB)

The protocol for western blotting was described in our previous studies [33, 46]. Hippocampal total protein was extracted following the kit instructions (BC3710, Solarbio, Beijing, China). The antibodies utilized included anti-NLRP3 (1:1500, #15101, Cell Signaling Technology (CST), MA, USA), anti-ASC (1:1200, #67824, CST), anti-Caspase-1 (1:1200, #3866, CST), anti-Caspase-1 p10 (1:1200, AF4022, Affinity, OH, USA), anti-GSDMD (1:800, 20770-1-AP, Proteintech, Wuhan, China) and anti-GAPDH (1:10,000, 60004-1-Ig, Proteintech). See the Supplementary materials and methods (Additional File 1) for the detailed procedures.

Immunohistochemistry

The procedures for immunohistochemistry were previously reported [47]. After deparaffinization, antigen retrieval and serum blocking, the paraffin slices were treated with the following antibodies: anti-NLRP3 (1:150, bs-10021R, BIOSS, Beijing, China), anti-ASC (1:200, #67824, CST), anti-Caspase-1 p10 (1:150, AF4022, Affinity), anti-GSDMD (1:150, 20770-1-AP, Proteintech), anti-IL-1β (1:150, 26048-1-AP, Proteintech), anti-IL-18 (1:150, 10663-1-AP, Proteintech), anti-Nrf2 (1:150, bs-1074R, BIOSS), and anti-HO-1 (1:150, bs-2075R, BIOSS). Images of the hippocampal CA1 region were acquired (OLYMPUS, Tokyo, Japan), and the mean optical densities (OD) of the immunohistochemical images were quantified using Image-Pro Plus 6.0 software. See the Supplementary materials and methods (Additional File 1) for the detailed procedures.

Immunofluorescence staining

Similar to our previous studies [33, 48], after antigen retrieval, permeabilization and serum blocking, coronal hippocampal slices or hiPSC-derived neurons were treated with the following antibodies: anti-NLRP3 (1:150, bs-10021R, BIOSS), anti-ASC (1:400, #67824, CST), anti-Caspase-1 p10 (1:150, AF4022, Affinity), anti-GSDMD (1:150, 20770-1-AP, Proteintech), anti-GFAP (1:800, sc-33673, Santa Cruz, CA, USA), anti-Iba1 (1:350, GT10312, GeneTex, CA, USA), anti-NeuN (1:450, MAB377, Millipore, MA, USA), anti-OcT4 (1:100, 381,335, ZENBIO, Chengdu, China), anti-SSEA4 (1:100, ab16287, Abcam, Cambridge, UK), anti-PAX6 (1:200, 67529-1-Ig, Proteintech), anti-Nestin (1:100, R381211, ZENBIO), and anti-MAP2 (1:200, 250,035, ZENBIO). Brain sections and cell slides were mounted with mounting medium containing DAPI (Southern Biotech, AL, USA), and fluorescence images were obtained (Nikon, A1R HD25, NY, USA). The numbers of microglia and astrocytes in the hippocampal CA1 region were analyzed using ImageJ 1.8.0 software. See the Supplementary materials and methods (Additional File 1) for the detailed procedures.

Enzyme-linked immunosorbent assay (ELISA)

ELISA kits (EMC001b, EMC011, EHC002b, EHC127, NeoBioscience, Shenzhen, China) were used to measure the IL-1β and IL-18 concentrations in mouse hippocampal lysates and culture supernatants of hiPSC-derived neurons. The level of 8-hydroxy-2′-deoxyguanosine (8-OHdG) in the mouse hippocampus and hiPSC-derived neurons was measured using an assay kit (EM1636, EU2548, FineTest, Wuhan, China).

Measurement of superoxide dismutase (SOD), catalase (CAT), malondialdehyde (MDA), and protein carbonylation

The levels of SOD, CAT and MDA in mouse hippocampal lysates and hiPSC-derived neurons were detected with assay kits (S0103, S0051, S0131, Beyotime). The protein carbonylation content in mouse hippocampal homogenates and hiPSC-derived neurons was quantified using a commercially available kit (E-BC-K117, Elabscience, Wuhan, China).

Nissl staining

The paraffin sections were deparaffinized and then stained with 0.5% toluidine blue (KEYGEN Biotech, Jiangsu, China) for 5 min, followed by a 0.1% glacial acetic acid differentiation step, dehydration, and mounting with neutral resin (BOSTER, Wuhan, China). Images of the hippocampal CA1 region were obtained (OLYMPUS). The quantification of Nissl-stained neurons was performed using ImageJ 1.8.0 software.

Golgi staining

The procedures for Golgi staining were previously reported [49]. The classical hippocampal region was isolated from anesthetized mice, brain tissues were then cut into 7 mm coronal tissue blocks, which were subsequently placed in the fixing solution of the Golgi staining kit (G1069, Servicebio, Wuhan, China). Hippocampal tissue blocks were subsequently transferred to Golgi staining solution and incubated at room temperature. After a 48-h immersion period, a new Golgi staining solution was added, and this process was repeated every 72 h. Following two weeks of immersion in Golgi staining solution, the hippocampal tissue blocks were placed in Golgi tissue treatment solution for one hour, after which they were transferred to a new solution and incubated in the dark at 4 °C for 72 h. The tissue blocks were then cut into 60 μm sections using a vibrating microtome (Leica, Wetzlar, Germany) and washed with double distilled water. The sections were subsequently stained with Golgi developer solution for 30 min and then washed with double distilled water and mounted. A microscope (3DHISTECH, Budapest, Hungary) was used for image acquisition. The analysis of dendritic spine density and the total length of dendrites in hippocampal CA1 neurons was conducted using ImageJ 1.8.0 software. Additionally, Sholl analysis was employed to quantify the number of intersections per neuron [50].

Transmission electron microscopy

In accordance with previously reported methods [47], the brain tissues were removed after the mice were anesthetized, and the CA1 region of the mouse hippocampus was separated using a brain mold. The brain tissues were subsequently cut into 1 mm³ pieces with a scalpel and immediately placed in 2.5% glutaraldehyde for tissue fixation. Brain tissues were post-fixed in 1% osmium tetroxide for 2 h and then rinsed three times with 0.1 M PSB (pH 7.4) for 15 min each. The tissues were permeated with acetone and an embedding agent and subsequently embedded with epoxy resin. Sections of 60–80 nm in size were prepared from the resin blocks using an ultramicrotome (Leica, Germany). The sections were stained with a 2% uranium acetate solution and a 2.6% lead citrate solution, respectively. Neuronal pyroptosis in each group was examined with a HITACHI HT7800 transmission electron microscope (Tokyo, Japan).

Molecular docking

The molecular structure of JC124 (CID: 117,715,670) was obtained from PubChem, and the cryo-electron microscopy structure of human NLRP3 (PDB ID: 8SXN) was retrieved from the Protein Data Bank [51]. The binding site and binding score of JC124 to NLRP3 were analyzed using Maestro 13.5 (Schrödinger, NY, USA).

Statistical analysis

All the data are presented as means ± S.E.M. Statistical analyses were performed using SPSS Statistics 29.0 and Graphpad Prism 10.0. The normality of the data was assessed using the Shapiro‒Wilk test. Data from Figs. 1, 2A, F–H, 3, 4, 5, 6 and 12 were subjected to one-way ANOVA followed by Tukey′s post hoc test. The statistical analyses for Figs. 7, 8A, F-H, 9 and 10 utilized two-way ANOVA with Tukey′s post hoc test. Finally, the data in Figs. 2C-D and 8C-D were evaluated using repeated-measures ANOVA with Tukey′s post hoc test. All tests were two-sided, with statistical significance set at p < 0.05. In the figures, each 'n' corresponds to either an individual animal or a separate sample.

Fig. 1.

JC124 treatment suppresses seizures in KA-induced epileptic mice. A Study design timeline. B Structure of JC124. C Latency to the first convulsive SRS and (D) the number of convulsive SRSs (n = 10). E Representative images of LFP recordings in the post-SE + vehicle, post-SE + JC124, and post-SE + VPA groups. F The number of SLEs, (G) the mean duration of SLE, and (H) the cumulative duration of SLEs in the hippocampal LFP (n = 8). KA-induced epileptic mice treated with JC124 exhibited a marked increase in latency to the first convulsive SRS, accompanied by a reduction in the number of convulsive SRSs, as well as a decrease in the number and cumulative duration of SLEs. Data are represented as mean ± S.E.M. ***p < 0.001. ns indicates no statistical significance

Fig. 2.

JC124 treatment improves depressive-like behavior and cognitive impairment in KA-induced epileptic mice. A Sucrose preference. JC124 treatment significantly improved the consumption of a 1% sucrose solution in KA-induced epileptic mice. B Typical trajectories, (C) escape latency and (D) swimming speed in the spatial acquisition test of the MWM. JC124 treatment notably reduced the escape latency of KA-induced epileptic mice. All the groups exhibited similar swimming speeds, with no significant differences observed. E Typical trajectories, (F) number of platform crossings, (G) percentage of movement distance and (H) percentage of movement time in the platform quadrant during the MWM probe trial. In KA-induced epileptic mice, the JC124 intervention significantly increased the number of platform crossings, as well as the percentage of movement distance and the time spent in the platform quadrant within 60 s. n = 10. Data are represented as mean ± S.E.M. *p < 0.05, **p < 0.01, ***p < 0.001 versus the post-SE + vehicle group. ns indicates no statistical significance

Fig. 3.

JC124 treatment inhibits NLRP3 inflammasome activation in KA-induced epileptic mice. A Representative WB bands. In KA-induced epileptic mice, treatment with JC124 significantly reduced the relative intensities of (B) NLRP3/GAPDH, (C) ASC/GAPDH, (D) pro-Caspase-1/GAPDH, and (E) Caspase-1 p10/GAPDH in the hippocampus (n = 3). F–H Representative immunohistochemical images of NLRP3 (brown), ASC (brown), and Caspase-1 p10 (brown) in the hippocampal CA1 region. Scale bar, 50 μm. The black arrows indicate typical positively stained cells. KA-induced epileptic mice treated with JC124 showed a significant decrease in the mean optical densities of the (I) NLRP3, (J) ASC, and (K) Caspase-1 p10 (n = 5). L-N Representative immunofluorescence images of NLRP3 (green)/GFAP (red), NLRP3 (green)/Iba1 (red), NLRP3 (green)/NeuN (red), ASC (green)/GFAP (red), ASC (green)/Iba1 (red), ASC (green)/NeuN (red), Caspase-1 p10 (green)/GFAP (red), Caspase-1 p10 (green)/Iba1 (red), and Caspase-1 p10 (green)/NeuN (red) in the hippocampal CA1 region (n = 3). Scale bar, 50 μm. The white arrows indicate typical co-labeled cells. Data are represented as mean ± S.E.M. *p < 0.05, **p < 0.01, ***p < 0.001

Fig. 4.

JC124 reduces GSDMD, GSDMD-N, IL-1β and IL-18 levels, and neuronal pyroptosis in KA-induced epileptic mice. A Representative WB bands. KA-induced epileptic mice treated with JC124 showed significantly decreased relative intensities of (B) GSDMD/GAPDH and (C) GSDMD-N/GAPDH in the hippocampus (n = 3). D Representative immunohistochemical images of GSDMD (brown) in the hippocampal CA1 region. Scale bar, 50 μm. The black arrows indicate typical positively stained cells. JC124 treatment significantly reduced the (E) mean optical density of GSDMD in KA-induced epileptic mice (n = 5). F Representative immunofluorescence images of GSDMD (green)/GFAP (red), GSDMD (green)/Iba1 (red) and GSDMD (green)/NeuN (red) in the hippocampal CA1 region (n = 3). Scale bar, 50 μm. The white arrows indicate typical co-labeled cells. G Transmission electron microscopy images showing the pyroptosis morphology of hippocampal CA1 neurons (n = 3). Scale bar, 1 μm. The orange arrows denote membrane pores in the neuronal cell membrane, and asterisks indicate the nucleus. H-I The relative expression levels of IL-1β and IL-18 in the hippocampal homogenates of mice (n = 5). JC124 treatment significantly reduced IL-1β and IL-18 levels in KA-induced epileptic mice. J-K Representative immunohistochemical images of IL-1β (brown) and IL-18 (brown) in the hippocampal CA1 region. Scale bar, 50 μm. The black arrows indicate typical positively stained cells. JC124 treatment in KA-induced epileptic mice significantly reduced the mean optical densities of (L) IL-1β and (M) IL-18 (n = 5). Data are represented as mean ± S.E.M. *p < 0.05, **p < 0.01, ***p < 0.001

Fig. 5.

JC124 treatment attenuates oxidative stress in KA-induced epileptic mice. A-B Representative immunohistochemical images of Nrf2 and HO-1 in the hippocampal CA1 region. Scale bar, 50 μm. The black arrows indicate typical positively stained cells. In KA-induced epileptic mice, the JC124 intervention significantly elevated the mean optical densities of (C) Nrf2 and (D) HO-1 (n = 5). The relative levels of (E) SOD, (F) CAT, (G) 8-OHdG, (H) protein carbonylation, and (I) MDA in the hippocampal homogenates of mice (n = 5). JC124 treatment markedly increased SOD and CAT levels, and reduced 8-OHdG, protein carbonylation, and MDA levels in the hippocampus of KA-induced epileptic mice. Data are presented as mean ± S.E.M. **p < 0.01, ***p < 0.001

Fig. 6.

JC124 treatment attenuates neuronal damage, microgliosis, and astrogliosis in the hippocampus of KA-induced epileptic mice. A-E Representative images of Golgi-stained neurons (scale bar, 10 μm), neuronal dendrites (scale bar, 5 μm), Nissl-stained neurons (scale bar, 100 μm), astrocytes, and microglia (scale bar, 50 μm) in the hippocampal CA1 region. KA-induced epileptic mice treated with JC124 showed a marked increase in (F) total dendritic length, (G) the number of Sholl intersections per neuron, and (H) dendritic spine density in hippocampal neurons (n = 4). Furthermore, the JC124 intervention significantly increased (I) the number of Nissl-stained hippocampal neurons and reduced (J-K) the number of GFAP-positive and Iba1-positive cells (n = 5). Data are represented as mean ± S.E.M. *p < 0.05, ***p < 0.001

Fig. 12.

JC124 inhibits neuroinflammation and oxidative stress in hiPSC-derived neurons treated with LPS + ATP. A Bright-field images of cultured hiPSCs (scale bar, 200 μm), neural stem cells (NSCs; scale bar, 100 μm), and hiPSC-derived neurons (scale bar, 100 μm). B Representative immunofluorescence images of hiPSCs (OcT4, green/SSEA4, red; scale bar, 100 μm), NSCs (Nestin, green/PAX6, red; scale bar, 10 μm), and hiPSC-derived neurons (MAP2, red; scale bar, 50 μm). n = 3. C Effect of JC124 on the cell viability of neurons derived from hiPSCs (n = 3). D Representative WB bands. Treatment with JC124 markedly reduced the relative intensity of (E) Caspase-1 p10/GAPDH in hiPSC-derived neurons induced by LPS and ATP (n = 3). The relative levels of (F) IL-1β, (G) IL-18 (H) SOD, (I) CAT, (J) 8-OHdG, (K) protein carbonylation, and (L) MDA in the neurons derived from hiPSCs (n = 5). The JC124 intervention significantly elevated SOD and CAT levels, while reducing 8-OHdG, protein carbonylation, MDA, IL-1β, and IL-18 levels in hiPSC-derived neurons induced by LPS and ATP. Data are represented as mean ± S.E.M. *p < 0.05, **p < 0.01, ***p < 0.001

Fig. 7.

The suppression of seizures in KA-induced epileptic mice by JC124 is mediated through NLRP3 inhibition. A Latency to the first convulsive SRS and (B) the number of convulsive SRSs (n = 10). C Representative images of LFP recordings in the NLRP3^WT + post-SE + vehicle, NLRP3^WT + post-SE + JC124, NLRP3^−/− + post-SE + vehicle, and NLRP3^−/− + post-SE + JC124 groups. D The number of SLEs, (E) the mean duration of SLE, and (F) the cumulative duration of SLEs in the hippocampal LFP (n = 8). JC124 treatment markedly increased the latency to the first convulsive SRS, decreased the number of convulsive SRSs, and reduced the number and the cumulative duration of SLEs in KA-treated NLRP3^WT mice but not in KA-treated NLRP3^−/− mice. Data are represented as mean ± S.E.M. *p < 0.05, **p < 0.01, ***p < 0.001. ns indicates no statistical significance

Fig. 8.

JC124 attenuates depressive-like behavior and cognitive dysfunction in KA-induced epileptic mice via NLRP3 inhibition. A Sucrose preference. JC124 treatment potently increased the consumption of a 1% sucrose solution in KA-treated NLRP3^WT mice but not in KA-treated NLRP3^−/− mice. B Typical trajectories, (C) escape latency, and (D) swimming speed in the spatial acquisition test of the MWM. JC124 treatment significantly decreased the escape latency in KA-treated NLRP3^WT mice but not in KA-treated NLRP3^−/− mice. All the groups exhibited similar swimming speeds, with no significant differences observed. E Typical trajectories, (F) number of platform crossings, (G) percentage of movement distance, and (H) percentage of movement time in the platform quadrant during the MWM probe trial. JC124 intervention markedly increased the number of platform crossings, as well as the percentage of distance traveled and the time spent in the platform quadrant within 60 s, in KA-treated NLRP3^WT mice but not in KA-treated NLRP3^−/− mice. n = 10. Data are presented as mean ± S.E.M. *p < 0.05, **p < 0.01, ***p < 0.001 versus the NLRP3^WT + post-SE + vehicle group. ns, not statistically significant

Fig. 9.

JC124 inhibits neuroinflammation and oxidative stress in KA-induced epileptic mice through NLRP3 inhibition. A-H Representative immunohistochemical images of NLRP3 (brown), ASC (brown), Caspase-1 p10 (brown), GSDMD (brown), IL-1β (brown), IL-18 (brown), Nrf2 (brown), and HO-1 (brown) in the hippocampal CA1 region (n = 5). Scale bar, 50 μm. The black arrows indicate typical positively stained cells. JC124 treatment markedly decreased the mean optical densities of (I) NLRP3, (J) ASC, (K) Caspase-1 p10, (L) GSDMD, (M) IL-1β, and (N) IL-18, while increasing the mean optical densities of (O) Nrf2 and (P) HO-1 in KA-treated NLRP3^WT mice, but not in KA-treated NLRP3^−/− mice. The relative levels of (Q) IL-1β, (R) IL-18, (S) SOD, (T) CAT, (U) 8-OHdG, (V) protein carbonylation, and (W) MDA in the hippocampal homogenates of mice (n = 5). JC124 treatment notably reduced IL-1β, IL-18, 8-OHdG, protein carbonylation, and MDA levels, while elevating SOD and CAT levels in KA-treated NLRP3^WT mice, but not in KA-treated NLRP3^−/− mice. Data are represented as mean ± S.E.M. **p < 0.01, ***p < 0.001. ns indicates no statistical significance

Fig. 10.

JC124 alleviates neuronal damage, neuronal pyroptosis, and gliosis in the hippocampus of KA-induced epileptic mice through NLRP3 inhibition. A-F Representative images of Golgi-stained neurons (scale bar, 10 μm), neuronal dendrites (scale bar, 5 μm), neuronal pyroptosis (scale bar, 1 μm), Nissl-stained neurons (scale bar, 100 μm), astrocytes, and microglia (scale bar, 50 μm) in the hippocampal CA1 region. The orange arrows denote membrane pores in the neuronal cell membrane, and asterisks indicate the nucleus. JC124 intervention markedly elevated (G) total dendritic length, (H) the number of Sholl intersections per neuron, and (I) dendritic spine density in hippocampal neurons of KA-treated NLRP3^WT mice, but not in KA-treated NLRP3^−/− mice (n = 4). Furthermore, the JC124 intervention significantly increased (J) the number of Nissl-stained hippocampal neurons and reduced (K-L) the number of GFAP-positive and Iba1-positive cells in KA-treated NLRP3^WT mice but not in KA-treated NLRP3^−/− mice (n = 5). Data are presented as mean ± S.E.M. *p < 0.05, **p < 0.01, ***p < 0.001. ns indicates no statistical significance

Results

A positive correlation was identified between NLRP3 gene expression and epilepsy susceptibility through SMR analysis

In the present study, we demonstrate a positive association between the expression of the NLRP3 gene and the susceptibility to epilepsy (odds ratio = 1.28, 95% confidence interval = 1.02–1.57; p = 0.015) through SMR analysis. This finding suggests that inhibition of NLRP3 expression and function could potentially play a protective role in the occurrence and progression of epilepsy.

JC124 treatment suppresses seizures in KA-induced epileptic mice

Preliminary data (Additional file 1: Supplementary Fig. 1) indicated that 50 mg/kg of JC124 effectively suppressed acute seizures. Based on these findings, this dose was selected to assess its impact on seizures in the KA-induced epilepsy model. In addition, VPA, a classical ASM commonly used in clinical practice, was chosen as the positive control drug for this study [52]. After intrahippocampal injection of KA, the mice were divided into JC124, VPA, or vehicle treatment groups and then intraperitoneally injected with JC124, VPA, or vehicle. Compared with those in the post-SE + vehicle group, 4 weeks of continuous treatment with JC124 or VPA significantly prolonged the latency to the first nonconvulsive and the first convulsive SRS, and reduced the number of nonconvulsive and convulsive SRSs (Additional file 1: Supplementary Fig. 5 C-D, and Fig. 1C-D). Compared with the post-SE + vehicle group, the post-SE + JC124 and post-SE + VPA groups showed a notable reduction in both the number and cumulative duration of SLEs (Fig. 1E-F, H).

JC124 treatment alleviates depressive-like behavior and cognitive deficits in KA-induced epileptic mice 

The protective effect of JC124 on depressive-like behavior was assessed using the SPT following LFP recording. The SPT is an effective measure for detecting depression and anhedonia in rodents [36, 53]. In the 24-h SPT test, the consumption of a 1% sucrose solution was significantly lower in the post-SE + vehicle group than in the sham + vehicle and sham + JC124 groups (Fig. 2A). Compared with the post-SE + vehicle group, the post-SE + JC124 and post-SE + VPA groups demonstrated a significantly increased consumption of a 1% sucrose solution (Fig. 2A). These data showed that KA-induced epileptic mice exhibited depressive-like behavior, which was mitigated by JC124 treatment.

During the spatial acquisition test of the MWM test, all groups showed a progressive decrease in escape latency over the course of five days (Fig. 2C). Compared with the sham + vehicle and sham + JC124 groups, the post-SE + vehicle group exhibited a significantly increased time to find a hidden platform (escape latency) in the spatial acquisition test, confirming that KA-induced epileptic mice experienced cognitive dysfunction (Fig. 2C). Compared with vehicle treatment, treatment with JC124 or VPA notably reduced the time required to locate a hidden platform in KA-induced epileptic mice (Fig. 2C). All the groups exhibited similar swimming speeds, with no significant differences observed (Fig. 2D). These findings suggest that intrahippocampal KA injections and drug therapy did not impact the motor function of the mice. In the probe trial, KA-induced epileptic mice treated with JC124 or VPA showed a significant increase in the number of platform crossings, as well as a higher percentage of movement distance and time spent in the platform quadrant within 60 s, than those treated with the vehicle (Fig. 2F-H). These findings indicate that JC124 mitigates cognitive impairment in mice with KA-induced epilepsy. Furthermore, there were no significant differences in the amelioration of depressive-like behavior and cognitive dysfunction between the post-SE + VPA and post-SE + JC124 groups (Fig. 2). The OFT revealed no significant differences in movement time or distance between the groups (Additional file 1: Supplementary Fig. 6), suggesting that JC124 treatment does not affect locomotor or exploratory properties in mice.

JC124 treatment suppresses NLRP3 inflammasome activation in KA-induced epileptic mice

The NLRP3 inflammasome plays a crucial role in neuroinflammation and the development of epilepsy [19]. This study investigated the inhibitory effect of JC124 on NLRP3 inflammasome activation by measuring the expression of NLRP3, apoptosis-associated speck-like protein containing a CARD (ASC), pro-Caspase-1 and Caspase-1 p10 (a marker of activated Caspase-1) in hippocampal homogenates using Western blot analysis. We found that the levels of NLRP3, ASC, pro-Caspase-1, and Caspase-1 p10 in the post-SE + vehicle group were markedly increased compared with those in the sham groups (Fig. 3A-E); however, these alterations were significantly reduced by JC124 treatment (Fig. 3A-E). Consistently, immunohistochemical analysis revealed significantly lower mean optical densities of NLRP3, ASC, and Caspase-1 p10 in the hippocampi of the post-SE + JC124 group than in those of the post-SE + vehicle group (Fig. 3F-K). Immunofluorescence staining of hippocampal sections revealed the colocalization of NLRP3, ASC, and Caspase-1 p10 with the neuron-specific marker NeuN, the microglia-specific marker Iba1, and the astrocyte-specific marker GFAP in the hippocampal CA1 region of the post-SE + vehicle group (Fig. 3L-N). These data suggest that JC124 treatment suppresses NLRP3 inflammasome activation in a KA-induced epilepsy mouse model.

JC124 treatment suppresses the expression of GSDMD, GSDMD-N, IL-1β, and IL-18, as well as mitigates neuronal pyroptosis in KA-induced epileptic mice

Upon activation of the NLRP3 inflammasome, the active form of Caspase-1 cleaves GSDMD to produce pore-forming N-terminal fragments (GSDMD-N), which is one of the key steps in inducing pyroptosis [12]. Our study revealed a significant increase in the expression levels of GSDMD and GSDMD-N in the hippocampal homogenates of the post-SE + vehicle group compared with both the sham + vehicle and sham + JC124 groups (Fig. 4A-C). After JC124 treatment, the levels of GSDMD and GSDMD-N were reduced in the hippocampus of KA-induced epileptic mice (Fig. 4A-C). Consistently, an immunohistochemical assay revealed that treatment with JC124 reduced the mean optical density of GSDMD in the hippocampal CA1 region of KA-induced epileptic mice (Fig. 4D-E). Then, immunofluorescence colabeling was performed to observe the cell types involved in GSDMD-mediated neuroinflammation. GSDMD-immunoreactive cells were co-labeled with GFAP-positive, Iba1-positive, and NeuN-positive cells in the hippocampal CA1 region of the post-SE + vehicle group (Fig. 4F). Notably, KA-induced epileptic mice exhibited numerous membrane pores in neuronal cell membranes in the hippocampal CA1 region, accompanied by rupture of the cell membranes and efflux of cellular contents. Interestingly, JC124 treatment effectively reduced the membrane pores in hippocampal neurons while preserving relatively intact cellular morphology (Fig. 4G).

Furthermore, the enzymatically active form of Caspase-1 facilitates the production of IL-1β and IL-18, which are subsequently secreted via the cell membrane pores, thereby intensifying neuroinflammatory responses [12]. This study confirmed elevated levels of IL-1β and IL-18 in the hippocampus of KA-induced epileptic mice, as determined by ELISA and immunohistochemical analysis (Fig. 4H-M). Notably, JC124 treatment significantly diminished these changes in KA-induced epileptic mice (Fig. 4H-M).

JC124 treatment alleviates oxidative stress in KA-induced epileptic mice

To explore the effects of JC124 treatment on oxidative stress, we detected the protein expression of Nrf2 and HO-1, the activities of the antioxidant enzymes SOD and CAT, and the levels of 8-OHdG (a marker of DNA oxidation), protein carbonylation (an indicator of protein oxidation), and MDA (a marker of lipid peroxidation) [54–56]. Immunohistochemical staining of hippocampal sections indicated that the protein expression of Nrf2 and HO-1 was markedly higher in the post-SE + JC124 group than in the post-SE + vehicle group (Fig. 5A-D). In addition, the post-SE + JC124 group exhibited increased levels of the antioxidant enzymes SOD and CAT, as well as decreased production of 8-OHdG, protein carbonylation, and MDA in hippocampal homogenates compared with those in the post-SE + vehicle group (Fig. 5E-I).

JC124 treatment alleviates hippocampal neuronal damage, microgliosis, and astrogliosis in KA-induced epileptic mice

NLRP3 inflammasome activation drives neuroinflammation and oxidative stress, resulting in neuronal degeneration, neuronal damage, and loss of dendritic spines [5, 6, 24, 57–59]. The morphology of hippocampal neurons was examined using Golgi staining. We found that the dendritic spine density, total dendritic length, and number of Sholl intersections were reduced in the hippocampal CA1 neurons of KA-induced epileptic mice (Fig. 6A-B, F–H). However, these morphological alterations were reversed following treatment with JC124 (Fig. 6A-B, F–H). The Nissl body is a sensitive marker of neuronal degeneration and injury. A reduction in the number or abnormal morphology of Nissl bodies indicates neuronal damage [60]. Our research revealed a notable reduction in Nissl-stained neurons in the hippocampal CA1 region of KA-induced epileptic mice (Fig. 6C, I). KA-induced epileptic mice treated with JC124 had significantly more Nissl-stained hippocampal neurons than those treated with vehicle (Fig. 6C, I). These results indicated that JC124 treatment alleviated hippocampal neuronal damage. In addition, we conducted immunofluorescence staining using the GFAP and Iba1 markers to assess astrogliosis and microgliosis in the CA1 region of the hippocampus following JC124 treatment. The numbers of GFAP-positive and Iba1-positive cells in the post-SE + JC124 group were markedly lower than those observed in the post-SE + vehicle group (Fig. 6D-E, J-K).

The neuroprotective effects of JC124 in KA-induced epileptic mice are mediated by NLRP3 inhibition

A previous study indicated that JC124 directly targets the NLRP3 protein, disrupting the activation of the NLRP3 inflammasome [21]. To clarify whether NLRP3 inhibition contributes to the neuroprotective effects of JC124 in a KA-induced epilepsy mouse model, NLRP3^−/− mice were utilized (Additional file 1: Supplementary Fig. 2). This study revealed that JC124 administration significantly increased the latency to the first nonconvulsive and the first convulsive SRS, decreased the number of nonconvulsive and convulsive SRSs, and reduced the number and the cumulative duration of SLEs in KA-treated NLRP3^WT mice (Additional file 1: Supplementary Fig. 5E-F, and Fig. 7). However, these effects were not significant following the administration of JC124 to KA-treated NLRP3^−/− mice (Fig. 7). Additionally, the ameliorative effects of JC124 on depressive-like behavior and cognitive dysfunction in KA-induced epileptic mice are also mediated by the inhibition of NLRP3. As indicated in Fig. 8, in KA-treated NLRP3^WT mice, the JC124 intervention effectively improved the consumption of a 1% sucrose solution in the SPT, increased the number of platform crossings, enhanced both the percentage of movement distance and the time spent in the platform quadrant during the MWM probe trial, and simultaneously reduced the escape latency in the MWM spatial acquisition test. Nevertheless, these effects were not significant following the administration of JC124 to KA-treated NLRP3^−/− mice (Fig. 8). The OFT revealed no significant differences in movement time or distance across the groups (Additional file 1: Supplementary Fig. 7).

Furthermore, levels of NLRP3, ASC, Caspase-1 p10, GSDMD, IL-1β, IL-18, 8-OHdG, protein carbonylation, and MDA were attenuated, whereas levels of Nrf2, HO-1, SOD, and CAT were elevated in KA-treated NLRP3^WT mice after JC124 administration. However, these effects were not evident when JC124 was administered to KA-treated NLRP3^−/− mice (Fig. 9). JC124 demonstrated neuroprotective properties in the hippocampus of KA-treated NLRP3^WT mice, which was evidenced by increases in dendritic spine density, total dendritic length, Sholl intersections of hippocampal neurons, and the number of Nissl-stained hippocampal neurons, as well as inhibition of neuronal pyroptosis, microgliosis, and astrogliosis, while such effects were not significant in KA-treated NLRP3^−/− mice receiving JC124 treatment (Fig. 10). As anticipated, our findings revealed that NLRP3 knockout in KA-induced epileptic mice had beneficial effects on several key neuropathological processes, including the amelioration of seizures, depressive-like behavior and cognitive dysfunction, as well as the attenuation of hippocampal neuronal damage, neuronal pyroptosis, gliosis, NLRP3 inflammasome activation, and oxidative stress (Figs. 7, 8, 9 and 10). Collectively, these data suggest that JC124 targets NLRP3 and inhibits NLRP3 inflammasome activation, subsequently exerting favorable neuroprotective effects in KA-induced epileptic mice.

The administration of JC124 does not noticeably affect the body weight or histomorphology of the major organs in mice

Throughout the 4-week treatment period with JC124 or vehicle, the body weights of the mice did not significantly differ (Additional file 1: Supplementary Fig. 8 A). Furthermore, treatment with JC124 did not affect the histomorphology of the major organs in mice, including the brain, liver, heart, lung, spleen, kidney, testis, and intestine (Additional file 1: Supplementary Fig. 8B). These findings indicate that JC124 is safe for application in murine models, which provides a promising foundation for future clinical trials and the potential transformation of JC124 into a clinically applicable treatment.

JC124 binds directly to the human NLRP3 protein

The results of the animal experiments in this study demonstrated that JC124 mitigates seizures and associated neurobehavioral comorbidities in KA-induced epileptic mice by targeting NLRP3 and suppressing the activation of the NLRP3 inflammasome. To further investigate the binding capacity of JC124 to the human NLRP3 protein, we employed molecular docking and molecular dynamics (MD) simulations. Molecular docking of JC124 with the human NLRP3 protein revealed that JC124 formed hydrophobic interactions with the residues ALA227, ILE230, LEU371, MET408, PHE410, ILE411, LEU413, VAL414, TYR443, PHE446, PHE525, PHE575, and MET661 of NLRP3; water bridges with the residues GLY226, GLY229, GLU369, ILE370, THR524, ARG578, TYR632, SER658, THR659, and ASP662 of NLRP3; and hydrogen bonds with the residues ALA228, GLY372, and THR439 of NLRP3 (Fig. 11A-B). The docking score of JC124 to the human NLRP3 protein was calculated to be −5.138, indicating favorable binding affinity. Furthermore, MD simulations identified VAL414, THR439, PHE446, PHE525, PHE575, and ARG578 as crucial residues involved in the binding of JC124 to the human NLRP3 protein (Additional file 1: Supplementary Fig. 9D).

Fig. 11.

JC124 binds directly to the human NLRP3 protein. A-B Molecular docking and binding sites of JC124 and the human NLRP3 protein

JC124 inhibits neuroinflammation and oxidative stress in hiPSC-derived neurons treated with LPS + ATP

We differentiated hiPSCs into neurons to further investigate the protective effect of JC124 against inflammatory injury in human neurons (Fig. 12A-B). To activate the NLRP3 inflammasome, a two-step process of priming followed by activation is necessary. LPS enhances the transcription of genes related to the NLRP3 inflammasome, while ATP facilitates the assembly of the NLRP3 inflammasome. Consequently, both LPS and ATP were employed to ensure the complete activation of the NLRP3 inflammasome in neurons derived from hiPSCs. A previous study demonstrated that JC124 inhibits LPS + ATP-induced IL-1β secretion in J774 A.1 cells, with an IC₅₀ value of 3.21 ± 0.49 μM [45]. Furthermore, JC124 at both 3 μM and 10 μM has no significant impact on the viability of hiPSC-derived neurons (Fig. 12C). Consequently, the 3 μM and 10 μM doses of JC124 were selected for subsequent experimental procedures.

LPS and ATP triggered the activation of Caspase-1, a significant effector molecule of the NLRP3 inflammasome, in neurons derived from hiPSCs (Fig. 12D-E). Notably, JC124 treatment markedly reduced the level of Caspase-1 p10, a marker of activated Caspase-1, in hiPSC-derived neurons treated with LPS and ATP (Fig. 12D-E). Furthermore, the JC124 intervention significantly increased the levels of antioxidant enzymes SOD and CAT, as well as decreased the production of 8-OHdG, protein carbonylation, MDA, IL-1β, and IL-18 in hiPSC-derived neurons induced by LPS and ATP (Fig. 12F-L).

Discussion

The present study elucidated a positive association between the expression of the NLRP3 gene and susceptibility to epilepsy. Intervention with JC124, a selective inhibitor of the NLRP3 inflammasome, for 4 weeks in KA-induced epileptic mice mitigated seizures, depressive-like behavior, and cognitive dysfunction. Furthermore, JC124 reduced hippocampal neuronal damage, neuronal pyroptosis, microgliosis, astrogliosis, neuroinflammation, and oxidative stress. Notably, JC124 effectively mitigated neuroinflammation and oxidative stress in hiPSC-derived neurons stimulated by LPS and ATP. Mechanistically, the neuroprotective role of JC124 in epilepsy is mediated through the inhibition of the NLRP3 inflammasome. These findings suggest that JC124 is a promising candidate for mitigating seizures and neurobehavioral comorbidities of epilepsy, although further clinical validation is necessary.

Numerous studies have confirmed that neuroinflammation, particularly that induced by inflammasome activation, is an essential factor in the development of epilepsy and its related neurobehavioral comorbidities [9, 12]. Neuroinflammation is a common pathophysiological mechanism in epilepsy and seizures caused by various etiologies, and it is present throughout epileptogenesis and chronic SRSs [5]. Importantly, neuroinflammation increases neuronal excitability and seizure susceptibility, while also promoting neuronal damage and pathological synaptic remodeling [5, 52, 61, 62]. Moreover, proinflammatory factors disrupt hypothalamic‒pituitary‒adrenal (HPA) axis homeostasis, neurotransmitter function, hippocampal neurogenesis, synaptic plasticity, and long-term potentiation, contributing to depression and cognitive comorbidities in epilepsy [9, 61].

Inflammasome activation is an important step in the formation of neuroinflammatory responses in epilepsy and neuropsychiatric comorbidities [12, 19, 61]. Among the known inflammasomes, the NLRP3 inflammasome is considered a typical member, which includes NLRP3, pro-Caspase-1, and the adaptor protein ASC [11, 12, 63, 64]. Previous studies have reported that the NLRP3 inflammasome is activated in the brains of epilepsy patients, as evidenced by notable increases in the levels of NLRP3, ASC, and Caspase-1 in the epileptogenic zone [65, 66]. ​​Importantly, our use of SMR analysis, a genetic epidemiological approach that minimizes the confounding biases inherent in traditional observational studies, enabled robust causal inference [25]. Through this method, we identified, for the first time, a positive correlation between the NLRP3 gene expression and epilepsy susceptibility, suggesting that NLRP3 plays a critical role in the development and progression of epilepsy. In addition, NLRP3 inflammasome activation promotes seizure susceptibility and neuropsychiatric comorbidities in a variety of animal models of epilepsy [17, 18, 67, 68]. The current study demonstrated that NLRP3^−/− reduces seizures, depressive-like behavior, and cognitive dysfunction in a KA-induced epilepsy mouse model by inhibiting NLRP3 inflammasome activation. Blocking NLRP3 inflammasome-mediated neuroinflammation with chemical compounds (i.e., CY-09, MCC950, curcumin, and amentoflavone) can reduce neuronal damage and seizures, and alleviate neuropsychiatric comorbidities in rodent epilepsy models [17, 19, 68]. Consequently, inhibiting NLRP3 inflammasome activation with anti-inflammatory agents, particularly specific and potent inhibitors of the NLRP3 inflammasome, may serve as an effective therapy to alleviate seizures and improve the neuropsychiatric comorbidities associated with epilepsy.

Glyburide, a glucose-lowering drug approved for clinical use, has been shown to inhibit NLRP3 inflammasome activation. However, the dose of the drug required to achieve this inhibition can lead to hypoglycemia [69]. Through structural optimization of glyburide, JC124, a novel, potent, and BBB-permeable NLRP3 inflammasome inhibitor, was developed [21, 22]. JC124 specifically interacts with the NLRP3 protein, thereby interrupting the activation of the NLRP3 inflammasome [21]. The administration of JC124 inhibited NLRP3 inflammasome activation in animal models of acute myocardial infarction, TBI and AD [21–24]. In this study, we explored the neuroprotective role of JC124 on seizures and neuropsychiatric comorbidities in KA-induced epileptic mice. The KA-induced epilepsy mouse model closely mimics the clinical and pathological features of human epilepsy, and this model is widely used to evaluate seizures and the neurobehavioral comorbidities of epilepsy [70, 71]. The present study revealed that JC124 treatment suppressed seizures, reduced epileptiform discharge, and minimized depressive-like behavior and cognitive impairment in KA-induced epileptic mice by preventing NLRP3 inflammasome activation. Importantly, NLRP3^−/− mice were utilized to further confirm that the neuroprotective role of JC124 in KA-induced epileptic mice is mediated by the inhibition of NLRP3 inflammasome activation. These results provide positive preclinical evidence that blocking NLRP3 inflammasome activation using JC124 represents a promising strategy for mitigating seizures and the neurobehavioral comorbidities associated with epilepsy.

Upon activation of the NLRP3 inflammasome, the enzymatically active form of Caspase-1 cleaves GSDMD to generate GSDMD-N, which subsequently forms pores in the cellular membrane, thereby inducing pyroptosis. Furthermore, IL-1β and IL-18 are released through the cell membrane pores, further exacerbating neuroinflammation [12, 19]. Xia et al. reported that GSDMD and its pore-forming fragment GSDMD-N were significantly upregulated in the mouse hippocampus following hippocampal KA injection and that pharmacological inhibition of GSDMD-mediated pyroptosis relieved seizures [72]. IL-1β and IL-18 levels are increased in the peripheral blood of epilepsy patients and are closely related to the inflammatory cascade [73, 74]. IL-1β, an upstream inflammatory factor, facilitates the nuclear translocation of NF-κB, thereby inducing the transcription of various proinflammatory and prooxidant genes, including NLRP3, IL-1β, IL-18, and NADPH oxidase 2 (NOX2) [19, 75, 76]. IL-1β and IL-18 can induce cellular damage and neurodegeneration, impair synaptic plasticity, and affect neuronal excitability, thereby promoting seizures and the neuropsychiatric comorbidities associated with epilepsy [19, 77, 78]. Importantly, IL-1β blockade significantly reduces seizure frequency and neuronal damage [79, 80]. JC124 has been proven to inhibit neuroinflammation [24]. As expected, this study revealed that the JC124 intervention significantly decreased the levels of IL-1β, IL-18, and GSDMD-N. Additionally, it effectively attenuated membrane pore formation in hippocampal neurons. These data indicate that JC124 can suppress the expression of pyroptosis-associated proteins and neuronal pyroptosis in epilepsy by blocking NLRP3 inflammasome activation, suggesting its potential as a promising ASM.

Neuroinflammation and oxidative stress mutually reinforce each other after epileptogenic brain injury, ultimately contributing to the development of epilepsy and neuropsychiatric comorbidities [5, 6]. Oxidative stress has been shown to persist throughout the process of epileptogenesis and chronic epilepsy, as indicated by decreased antioxidant enzyme activities (such as SOD and CAT) and elevated oxidative damage markers (such as 8-OHdG, protein carbonylation, and MDA) [5, 6, 81, 82]. Reactive oxygen species (ROS) produced during seizures promote NLRP3 inflammasome activation and neuronal excitability [5, 6]. Nrf2, a key factor for antioxidant activity, is also activated by ROS. Upon activation of Nrf2, the expression of HO-1, which has antioxidant and anti-inflammatory properties, is increased [14, 83, 84]. Nrf2 activation reduces seizure severity, neuropsychiatric comorbidities, and NLRP3 inflammasome-driven neuroinflammation [20, 83, 85]. Interestingly, Garstkiewicz et al. reported that NLRP3 inflammasome activation can induce Nrf2 protein degradation; however, this effect is inhibited by NLRP3 deletion [15]. In this study, the inhibition of NLRP3 inflammasome activation through JC124 intervention increased the levels of Nrf2, HO-1, and the antioxidant enzymes SOD and CAT, while also reducing the production of 8-OHdG, protein carbonylation, and MDA in KA-treated NLRP3^WT mice. However, JC124 did not demonstrate additional efficacy in mitigating oxidative stress in KA-treated NLRP3^−/− mice. These data indicate that JC124 facilitates the expression of antioxidant molecules through the inhibition of NLRP3 inflammasome activation, thus alleviating oxidative stress in the context of epilepsy.

Neuroinflammation and oxidative stress promote the activation of glia and induce microgliosis and astrogliosis. The reactive phenotypes of microglia and astrocytes are major sources of proinflammatory factors following NLRP3 inflammasome activation in epilepsy [5, 7, 19]. A recent study has shown that long-term JC124 intervention significantly improved cognitive dysfunction and suppressed microgliosis and astrogliosis in AD transgenic mice [23]. This study revealed that treatment with JC124 for 4 weeks reduced the number of microglia and astrocytes in the hippocampal CA1 region of KA-induced epileptic mice. These findings suggest that JC124 may inhibit gliosis by blocking NLRP3 inflammasome activation in astrocytes and microglia. Moreover, neuroinflammation, oxidative stress, and activated microglia and astrocytes in epilepsy facilitate neuronal damage, synaptic remodeling, and the generation of aberrant dendritic spines [58, 86–88]. Our findings align with those of prior studies [60, 89, 90], which have indicated that KA-induced epileptic mice exhibit a decreased number of Nissl-stained hippocampal neurons, a reduced density of dendritic spines, a shorter total dendritic length, and fewer Sholl intersections of hippocampal neurons. However, these morphological alterations were significantly restored following treatment with JC124. These findings suggest that JC124 plays a neuroprotective role in epilepsy and neuropsychiatric comorbidities by inhibiting neuroinflammation and oxidative stress.

Moreover, the long-term administration of JC124 did not affect locomotor activity, exploratory behavior, body weight, or the histomorphological structure of the major organs in mice. Notably, JC124 demonstrated a favorable binding affinity to the human NLRP3 protein, as indicated by molecular docking and MD simulations. This study effectively triggered activation of the NLRP3 inflammasome in hiPSC-derived neurons using LPS and ATP, and demonstrated that JC124 could mitigate neuroinflammation and neuronal oxidative stress. The presented data establish a foundation for further investigations into the protective role of JC124 in inflammatory diseases of the nervous system, as well as its potential transformation into clinical applications.

Conclusions

The present study demonstrated, for the first time, a positive correlation between the expression of the NLRP3 gene and vulnerability to epilepsy through SMR analysis, implying that NLRP3 is essential for the onset and progression of epilepsy. Our experimental results indicate that JC124, a novel NLRP3 inflammasome inhibitor, exerts neuroprotective effects in KA-induced epileptic mice and in hiPSC-derived neurons stimulated by LPS and ATP. JC124 treatment effectively inhibited seizures; improved depressive-like behavior and cognitive impairment; minimized hippocampal neuronal loss, neuronal pyroptosis, microgliosis, and astrogliosis; and suppressed neuroinflammation and oxidative stress by preventing NLRP3 inflammasome activation. Furthermore, JC124 exhibited a direct binding affinity to the human NLRP3 protein and mitigated neuroinflammation, as well as oxidative stress, in hiPSC-derived neurons subjected to stimulation with LPS and ATP. The current data, together with those of previous studies, strongly indicate that JC124 is a potent and specific NLRP3 inflammasome inhibitor. These findings suggest that JC124 could be developed as a promising ASM. Importantly, the inflammatory injury model of hiPSC-derived neurons provides a new tool for the development of anti-inflammatory drugs and for conducting mechanistic studies. However, this study did not include female mice, as estrogen promotes seizure activity. Future research will explore the potential impact of gender-related variables on the antiseizure efficacy of JC124. Furthermore, future studies are needed to monitor SRS in KA-induced epileptic mice after withdrawal from treatment to assess whether JC124 suppresses epileptogenesis. While JC124 demonstrated neuroprotective effects in preclinical models, further studies are needed to assess its pharmacokinetics, bioavailability, and potential interactions with existing ASMs before clinical trials can be considered.

Abbreviations

AD

Alzheimer’s disease

ANOVA

Analysis of variance

ASC

Apoptosis-associated speck-like protein containing a CARD

ASM

Antiseizure medication

ATP

Adenosine triphosphate

BBB

Blood–brain barrier

CA

Cornus Ammonis

CAT

Catalase

DNA

Deoxyribonucleic acid

ELISA

Enzyme-linked immunosorbent assay

8-OHdG

8-Hydroxy-2’-deoxyguanosine

eQTL

Expression quantitative trait loci

GSDMD

Gasdermin D

GWAS

Genome-wide association study

hiPSC

Human induced pluripotent stem cell

HO-1

Heme oxygenase-1

HPA

Hypothalamic-pituary-adrenal

IL-18

Interleukin-18

IL-1β

Interleukin-1β

KA

Kainic acid

LFP

Local field potential

LPS

Lipopolysaccharide

MD

Molecular dynamics

MDA

Malondialdehyde

MWM

Morris water maze

NLRP3

NOD-, LRR- and pyrin domain-containing protein 3

NOX2

NADPH oxidase 2

Nrf2

Nuclear factor erythroid 2-related factor 2

OD

Optical density

OFT

Open field test

ROS

Reactive oxygen species

S.E.M

Standard error of the mean

SE

Status epilepticus

SLEs

Seizure-like events

SMR

Summary-based Mendelian Randomization

SOD

Superoxide dismutase

SPT

Sucrose preference test

SRS

Spontaneous recurrent seizure

TBI

Traumatic brain injury

VPA

Valproic acid

WB

Western blotting

Authors’ contributions

PZ and HZ designed this study. PZ, HZ, ZL, ML, JY, JZ, JZ, JD, YC, HT, CX, CS, QL, and YL conducted the experiments. PZ, HZ, and JZ wrote the manuscript. LM, JY, and YC revised the manuscript. All the authors reviewed and endorsed the manuscript for submission.

Funding

The China Postdoctoral Science Foundation (grant number: 2024MD764061), the Chongqing Natural Science Foundation (grant number: CSTB2025NSCQ-MSX2082), the Epilepsy Research Foundation of China Association Against Epilepsy (grant number: CS-2025-172) and the National Natural Science Foundation of China (grant number: 82071458) funded this research.

Data availability

No datasets were generated or analysed during the current study.

Declarations

Ethics approval and consent to participate

The Ethics Committee of the Second Affiliated Hospital of Chongqing Medical University authorized the study protocol (ethical approval number: 2022–87). All animal experiments adhered to the ethical standards of Animal Research: Reporting of in Vivo Experiments (ARRIVE) guidelines. The hiPSC studies were conducted in adherence to the ethical standards and regulations of China and the authors' institutions. Participants were given consent to participate in this study.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.