TRPV4 is involved in NLRP3 and NLRP1 inflammasomes activation after pilocarpine-induced status epilepticus in mice

Abstract

The nucleotide-binding oligomerization domain (NOD)-like receptor family pyrin domain-containing 1 (NLRP1) and NLRP3 inflammasomes are activated in temporal lobe epilepsy (TLE), leading to neuroinflammation, pyroptosis, and neuronal injury. Protein phosphatase 2 A (PP2A) and the toll-like receptor 4 (TLR4)–p38 MAPK signaling pathway participate in regulating these inflammasomes. Although blocking transient receptor potential vanilloid 4 (TRPV4) alleviates inflammation and neuronal damage after pilocarpine-induced status epilepticus (PISE), the underlying mechanisms remain unclear. Here, we found that NLRP1 and NLRP3 expression and activation were markedly increased in the hippocampus during the acute phase post-PISE, accompanied by enhanced pyroptosis and inflammatory responses. Both PP2A activity and TLR4–p38 MAPK signaling were upregulated. Pharmacological inhibition revealed that PP2A primarily drove NLRP3 activation, whereas TLR4–p38 MAPK signaling promoted NLRP1 activation, collectively contributing to pyroptosis and neuronal injury. TRPV4 blockade simultaneously suppressed PP2A activity and TLR4–p38 MAPK signaling, thereby inhibiting NLRP1/NLRP3 inflammasome activation and exerting neuroprotective effects. Conversely, TRPV4 activation with GSK1016790A promoted inflammasome activation, pyroptosis, and activation of PP2A and TLR4–p38 MAPK signaling; these effects were attenuated by inhibiting PP2A or TLR4–p38 MAPK. In HT-22 cells, TRPV4 activation activated the TLR4–p38 MAPK–NLRP1 axis, and NLRP1 knockdown alleviated pyroptosis. Finally, inhibition of TRPV4, PP2A, NLRP3, or NLRP1 markedly reduced seizure frequency and duration in PISE mice. Collectively, these findings suggest that TRPV4 blockade suppresses the PP2A–NLRP3 and TLR4–p38 MAPK–NLRP1 pathways, attenuating neuroinflammation and pyroptosis, and thereby reducing seizure activity during the acute phase following PISE.

Supplementary Information

The online version contains supplementary material available at 10.1007/s00018-026-06185-2.

Introduction

Epilepsy is a common neurological disorder characterized by recurrent and unprovoked seizures [1]. Temporal lobe epilepsy (TLE), one of the most prevalent and drug-resistant forms of epilepsy, is primarily associated with pathological changes in the temporal cortex and hippocampus [2]. Status epilepticus (SE) can induce immune inflammatory responses, resulting in functional and structural damage to neurons and synapses. Studies have shown that the inflammatory response is involved in the pathogenesis of epilepsy. For instance, inflammatory cytokines can increase neuronal excitability and provoke neuronal damage, thereby exacerbating epileptic seizures [3]. Additionally, the overactivation of glial cells and heightened immune inflammatory responses have been observed in brain tissues of patients with epilepsy and rodent model of experimental epilepsy [4]. Meanwhile, anti-inflammatory therapy has been demonstrated to reduce neuronal damage during epilepsy [3, 5].

The inflammasome is a protein complex mainly composed of receptor proteins (e.g., nucleotide-binding oligomerization domain (NOD)-like receptor family, pyrin domain containing 1 (NLRP1), nucleotide-binding oligomerization domain (NOD)-like receptor family, pyrin domain containing 3 (NLRP3), absent in melanoma 2 (AIM2)), the adaptor protein apoptosis-associated Speck-like protein containing a CARD (caspase recruitment domain) (ASC), and the effector protein cysteine-dependent aspartate-directed protease 1 (caspase-1, cas-1). Inflammasomes can be activated by signals released by pathogens and injured neurons, following which they participate in immune inflammatory responses [6, 7]. Pyroptosis is a form of programmed cell death highly related to the inflammatory response. Activated cas-1 can promote the release of mature inflammatory cytokines and cleave gasdermin-D (GSDMD), generating an N-terminal fragment (N-GSDMD) that triggers pyroptosis [6]. Patients with medial TLE exhibit elevated levels of NLRP1 and NLRP3 in the hippocampus and increased cas-1 and interleukin-1 beta (IL-1β) levels in plasma [8]. Additionally, mice with pilocarpine-induced epilepsy display neuronal damage in the hippocampus, accompanied by elevated expression of NLRP3 [5]. Bioinformatics analysis also indicates that the levels of NLRP1, NLRP3, AIM2, GSDMD, toll-like receptor 4 (TLR4), and IL-1β are increased after SE [9].

Protein phosphatase 2 A (PP2A) is a heterotrimeric protein phosphatase composed of catalytic subunit C, scaffold subunit A, and regulatory subunit B. The B subunit, of which there are multiple subtypes, is responsible for regulating PP2A enzymatic activity, substrate specificity, and subcellular localization [10, 11]. The B subunit containing the PR72 subtype is expressed in the brain and can be directly activated by calcium ion (Ca²⁺) [10]. When activated, PP2A facilitates the interaction between the pyrin domains (PYDs) of ASC and NLRP3 [12], thus influencing the assembly and activation of the NLRP3 inflammasomes. The application of okadaic acid (OA), an inhibitor of PP2A, suppresses adenosine triphosphate-induced NLRP3 inflammasome activation in microglia and reduces the production of IL-1β [13]. However, it is unclear whether PP2A is involved in the assembly and activation of the NLRP3 inflammasome in TLE.

TLR4 is an innate immune system receptor widely expressed in microglia, astrocytes, and neurons [14]. The level of TLR4 protein is significantly increased in serum and lesional brain tissue of patients with epilepsy, as well as in the hippocampi of TLE model mice [15, 16]. p38 mitogen-activated protein kinase (MAPK), a member of the MAPK family, plays a role in inflammatory response, cell proliferation, cell differentiation, and apoptosis via the activation of transcription factors and proteases [17]. Downregulating TLR4 or inhibiting p38 MAPK decreases IL-1β and tumor necrosis factor-alpha (TNF-α) protein levels [18]. Moreover, the suppression of p38 MAPK activity was shown to markedly reduce NLRP1 protein levels and attenuate NLRP1-dependent neuronal pyroptosis following cerebral ischemia–reperfusion injury [19]. Nevertheless, whether the TLR4-p38 MAPK signaling pathway is involved in regulating inflammasome expression during TLE remains unknown.

Transient receptor potential vanilloid 4 (TRPV4), a member of the transient receptor potential (TRP) superfamily of ion channels, is expressed in neurons and glial cells in brain regions such as the hippocampus, cortex, and thalamus [20]. TRPV4 is a Ca²⁺ channel, and its activation can increase intracellular Ca²⁺ concentration ([Ca²⁺]_i) [21]. TRPV4 has been identified as a genetic risk factor for epilepsy, and it has been observed to be upregulated in surgical brain specimens from patients with epilepsy and hippocampal tissue of mice with pilocarpine-induced epilepsy [22]. Furthermore, the inhibition of TRPV4 was shown to inhibit TLR4-nuclear factor-κB (NF-κB) signaling, reduce inflammatory cytokine levels, and attenuate neuronal injury in the hippocampus of mice with pilocarpine-induced status epilepticus (PISE) [5, 23]. These observations indicate that TRPV4 is involved in the inflammatory response during epilepsy. The activation of transient receptor potential vanilloid 1 (TRPV1) mediates Ca²⁺ influx and promotes PP2A-dependent NLRP3 inflammasome activation in microglia [13]. Additionally, TRPV4 activation has been reported to increase p38 MAPK activity in the hippocampus in mice [24]. However, during epilepsy, it remains unclear whether TRPV4 activation enhances the inflammatory response through the regulation of inflammasomes, and whether this effect is related to PP2A or the TLR4-p38 MAPK signaling pathway.

In the present study, we examined whether TRPV4 blockade affected the activation of NLRP3 and NLRP1 inflammasomes in PISE mice, and whether these effects were mediated through the regulation of PP2A activity and/or the TLR4–p38 MAPK signaling pathway. Early epileptic seizures after SE have been implicated in the progression of epilepsy [25–27]; however, the impact of TRPV4 inhibition on seizure behavior has not been clarified. Therefore, we also assessed whether TRPV4 blockade altered seizure activity during the acute phase following PISE and determined whether any observed behavioral effects were associated with changes in NLRP3 and NLRP1 inflammasome activation.

Materials and methods

Animals

Male ICR mice (Oriental Bio Service Inc., Nanjing, China), aged 6 weeks and weighing between 25 and 30 g, were used in this study. The mice were housed in the Animal Core Facility of Nanjing Medical University under controlled environmental conditions (temperature: 23 ± 2 °C; relative humidity: 55 ± 5%) with a 12-hour light/dark cycle and were provided ad libitum access to food and water. This study was approved by the Ethics Committee of Nanjing Medical University (No. IACUC2009007) and all animal experiments were performed in accordance with the university’s guidelines for Laboratory Animal Research. Sample sizes for all experimental groups were provided in the Supplementary Methods.

Preparation for PISE mice

Mice were intraperitoneally injected with pilocarpine (300 mg/kg) to induce SE [23]. Methylscopolamine (1 mg/kg) was intraperitoneally injected 20 min before pilocarpine injection to inhibit peripheral muscarinic activity. Seizure severity was rated using the Racine scale, which is defined as follows: category 1: facial clonus, including mouth and facial movements; category 2: head nodding, often accompanied by more pronounced facial clonus; category 3: unilateral forelimb clonus; category 4: bilateral forelimb clonus with rearing; category 5: generalized tonic-clonic seizures with rearing and falling. SE was defined as the onset of category 4–5 seizures. After 1 h, SE was terminated using diazepam (10 mg/kg). Animals that did not develop category 4–5 seizures within 30 min after pilocarpine injection were excluded from the study. Control mice were injected with the same volume of saline.

Cell culture and treatment

The HT-22 hippocampal cell line (cat. no. ZQ0476, purchased from Shanghai Zhong Qiao Xin Zhou Biotechnology Co., Ltd, Shanghai, China, originally from MilliporeSigma, Burlington, MA, USA, cat. no. SCC129, RRID: CVCL_0321) was used in this study. The cells were cultured in DMEM supplemented with 10% fetal bovine serum (FBS) and 1% penicillin–streptomycin (cat. no. ZM0476, Shanghai Zhong Qiao Xin Zhou Biotechnology Co., Ltd, Shanghai, China) and were incubated in a humidified incubator with 5% CO₂ at 37 °C. For treatments, HT-22 cells were cultured in 6-well plates, and, at 70%–80% confluence, they were either transfected with shRNA or incubated with drugs. GSK1016790A (0.1 µM), SB203580 (10 µM), or vehicle were added to the DMEM and the cells were subsequently incubated for 24 h. For the knockdown of NLRP1, the following three oligonucleotide sequences were designed: (shRNA-NLRP1-A) 5′-GGAGCTGGGATTGCAAAGA-3′, (shRNA-NLRP1-B) 5′-CCTCCAAATTGGTGGAAAT-3′, (shRNA-NLRP1-C) 5′-CAGCTAGAGAGGAACTTGAAGCTAA-3′. The negative control sequence was 5′-TTCTCCGAACGTGTCACGTAA-3′. All shRNAs were constructed by Zebrafish Biotech (Nanjing, China). HT-22 cells were transfected with shRNA-NLRP1 or shRNA-control for 12 h and then allowed to grow for another 12 h. To identify the optimal sequence for subsequent cell and animal experiments, the NLRP1 knockdown efficiency was determined by examining NLRP1 protein levels (see Supplementary Methods). HT-22 cells that exhibited good growth after lentivirus (shRNA-NLRP1-B) infection were selected, treated with GSK1016790A (0.1 µM) for 24 h and collected for subsequent experiments.

Drug treatment

The TRPV4 agonist GSK1016790A, the TRPV4 antagonist HC-067047, the PP2A inhibitor OA, and the p38 MAPK inhibitor SB203580 were administered by intracerebroventricular (icv.) injection, while the TLR4 antagonist TAK-242 and the NLRP3 inhibitor MCC950 were administered by intraperitoneal (ip.) injection. Mice were anesthetized with ketamine (100 mg/kg)/xylazine (10 mg/kg; ip.) and then placed on a stereotaxic device (Kopf Instruments, Tujunga, CA, USA). A guide cannula comprising 23-gauge stainless steel tubing was implanted into the right lateral ventricle (0.3 mm posterior, 1.0 mm lateral, and 2.5 mm ventral to bregma) and anchored to the skull with stainless steel screws and dental cement. Drugs were injected with a 26-gauge stainless steel needle (Plastics One, Roanoke, VA, USA) at the rate of 0.2 µL/min. GSK1016790A (1 µM/mouse) was injected once daily for 3 consecutive days (GSK mice). To block TRPV4 in PISE mice, HC-067047 (10 µM/mouse) was injected 1 h after the termination of SE, and subsequently once daily for 3 days. OA (2 µg/kg), MCC950 (20 mg/kg), TAK-242 (3 mg/kg), and SB203580 (1 mM/mouse) were injected 30 min before GSK1016790A injection or 1 h after the termination of SE, and then once daily for 3 days. The doses of the above drugs were chosen based on previous reports [5, 23, 28–30].

Injection of adenovirus-associated vector (AAV)

To knock down NLRP1 in vivo, an adeno-associated virus (AAV) vector containing shRNA-NLRP1-B or shRNA-control (Zebrafish Biotech, Nanjing, China) was employed (see Supplementary Methods). In brief, shRNA-NLRP1-B and shRNA-control were separately cloned into sc-AAV-U6-CAG-EGFP-WPRE-SV40pA (AAV9, 1 × 10¹² TU/mL) and confirmed by sequencing. The recombinant plasmids were packaged using a triple-transfection, helper virus-free method, and purified. For virus infusion, mice were anesthetized with ketamine (100 mg/kg)/xylazine (10 mg/kg; ip.), and 1 µL of shRNA-control or shRNA-NLRP1-B were injected into the hippocampus (− 1.6 mm anteroposterior, ± 1.8 mm mediolateral, − 1.6 mm dorsoventral relative to bregma) at a rate of 0.2 µL/min using a 5-µL Hamilton syringe connected to a 30-gauge stainless steel needle. Transfection efficiency was assessed by measuring NLRP1 protein levels in the hippocampus of mice 4 weeks after virus infusion.

Western blot analysis

Hippocampal samples were obtained 8 h after the last drug injection or 3 days after SE induction. Total protein was extracted using the Whole Cell Lysis Assay Kit (cat. no. KGP250, Nanjing KeyGen Biotech Co., Ltd, Nanjing, China) according to the manufacturer’s protocol. Protein concentrations were determined using a BCA Protein Assay Kit (cat. no. P0012, Beyotime Institute of Biotechnology, Shanghai, China). Equal amounts of protein (20 µg) were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride (PVDF) membranes. After blocking using 5% nonfat milk in Tris-buffered saline (TBS)/Tween 20, the membranes were incubated at 4 °C overnight with primary antibodies against NLRP1, NLRP3, ASC, cleaved caspase-1 (c-cas-1), IL-1β, GSDMD, PP2A, phospho-PP2A (p-PP2A), TLR4, p38 MAPK, phospho-p38 MAPK (p-p38 MAPK), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). After washing with TBST, the membranes were incubated with an HRP-conjugated secondary antibody, developed using an ECL Detection Kit (Amersham Biosciences, Piscataway, NJ, USA). Protein bands were quantified using a Tanon Digital Gel Imaging Analysis System (Tanon-5200, Shanghai Tanon Science & Technology, Shanghai, China), and analyzed using ImageJ software (National Institutes of Health). The primary antibodies were chosen according to previous reports (see Supplementary Methods). The protein levels in PISE mice were normalized to those of control mice. The protein levels in drug-treated PISE mice were normalized to those of vehicle-treated PISE mice. The protein levels in drug-injected mice were normalized to those of vehicle-injected mice.

Histological examination

After anesthesia, mice were transcardially perfused first with ice-cold phosphate-buffered saline (PBS) and then with 4% paraformaldehyde 8 h after the last drug injection or 3 days after SE onset. For Toluidine blue staining, excised brains were fixed at 4 °C overnight and paraffin-embedded. Coronal sections (5 μm) were cut at the level of the dorsal hippocampus and stained with Toluidine blue. Pyramidal cells in the hippocampal CA1 and CA2/3 regions were identified using a light microscope (Olympus DP70, Olympus Corporation, Tokyo, Japan). The surviving neurons in the hippocampal CA1 and CA2/3 subregions were counted in six sections per mouse as previously reported [31]. The density of surviving neurons in PISE mice was expressed as a percentage of that in control mice injected with methylscopolamine and saline. In drug- or AAV-shRNA-NLRP1-treated PISE mice, the surviving neuron density was expressed as a percentage of that in vehicle- or AAV-shRNA-control-treated PISE mice, respectively. In GSK mice, the surviving neuron density was expressed as a percentage of that in vehicle-treated normal controls. In OA- or AAV-shRNA-NLRP1-treated GSK mice, the surviving neuron density was expressed as a percentage of that in vehicle- or AAV-shRNA-control-treated GSK mice, respectively [31]. For ASC and GSDMD staining, brains were coronally sectioned at 30 μm, and free-floating sections were incubated overnight at 4 °C with primary antibodies against ASC (cat. no. AL177, 1:200, AdipoGen, San Diego, CA, USA) or GSDMD (cat. no. AF-4012, 1:100, Affinity, Melbourne, Australia). The sections were then incubated with a fluorescent secondary antibody (Alexa Fluor 594, cat. no. A21207, 1:500, RRID: AB_141637, Invitrogen, Carlsbad, CA, USA) for 1.5 h at room temperature, followed by mounting with antifade medium containing DAPI (Life Technologies Corporation, Gaithersburg, MD, USA). ASC-immunopositive (ASC⁺) or GSDMD-immunopositive (GSDMD⁺) cells in the hippocampus were visualized using a light microscope (Olympus DP70, Olympus Corporation, Tokyo, Japan) and analyzed using ImageJ software. ASC⁺ or GSDMD⁺ cells were quantified in the hippocampal region, and the number of ASC⁺ or GSDMD⁺ cells was expressed as the number of cells per square millimeter. For HT-22 cells cultured on chamber slides, cells were fixed with 4% paraformaldehyde and permeabilized with PBS containing 0.1% Triton X-100. The cells were then incubated overnight at 4 °C with primary antibodies against NLRP1 (cat. no. sc-390133, 1:200, Santa Cruz Biotechnology, Dallas, TX, USA) or GSDMD (cat. no. AF-4012, 1:100, Affinity, Melbourne, Australia). After washing, cells were incubated for 2 h at 37 °C with the corresponding biotin-conjugated secondary antibodies (goat anti-mouse IgG, cat. no. ab6788, 1:2000, RRID: AB_954885; or rabbit anti-goat IgG, cat. no. ab6740, 1:100, RRID: AB_954844; both from Abcam, Cambridge, UK). Nuclei were counterstained with DAPI (Life Technologies, Gaithersburg, MD, USA). Fluorescence signals were captured using an Olympus DP70 microscope (Olympus Corporation, Tokyo, Japan) and quantified using ImageJ software (see Supplementary Methods).

Caspase-1 activity assay

Cas-1 activity was measured using a cas-1 activity assay kit (cat. no. C1101, Beyotime Biotechnology, Shanghai, China) as instructed by the manufacturer. Absorbance was measured at a wavelength of 405 nm.

Video monitoring of seizures

Epileptic seizure activity in mice following PISE was continuously monitored for 72 h after diazepam injection using a video recording system (Cat. CS-H6c, EZVIZ Network Co., Ltd, Hangzhou, China). Seizures occurring on days 2 and 3 post-PISE were evaluated using the Racine scale. The frequency and duration of seizures were recorded by the observer who was blinded to the experimental groups.

Chemicals

HC-067047 (cat. no. HY-100208), TAK-242 (cat. no. HY-11109), adezmapimod (SB203580, cat. no. HY-152121), and MCC950 (cat. no. HY-12815A) were obtained from MedChemExpress (Shanghai, China); OA (cat. no. S1786) was obtained from Beyotime Biotechnology (Shanghai, China); pilocarpine (cat. no. 14487), methylscopolamine (cat. no. 23862), ketamine (cat. no. 11630), and xylazine (cat. no. 14113) were obtained from Cayman Chemical Company (Ann Arbor, MI, USA). Unless otherwise stated, all other chemicals, including GSK1016790A (cat. no. G0798) and diazepam (cat. no. D0899), were obtained from Sigma Chemical Company (St Louis, MO, USA).

Statistical analysis

Data were analyzed using the Statistical Package for the Social Sciences, version 27.0 (SPSS Inc., Chicago, IL, USA). Normality was assessed with the Shapiro–Wilk test, and homogeneity of variance was evaluated using Levene’s test. For normally distributed data with homogeneous variances, independent-samples two-tailed Student’s t-tests were performed, and results were presented as mean ± standard deviation. For data that did not meet assumptions of normality or variance homogeneity, the nonparametric Mann–Whitney U test was applied, and results are reported as median with interquartile range (25th–75th percentile). P value < 0.05 was considered statistically significant (see Supplementary Table 1, 2 and 3). The experimenters were blinded to the control and experimental groups when collecting data.

Results

1. Changes in PP2A-NLRP3 signaling pathway in the hippocampus following PISE

Studies have shown that inhibiting or knocking down PP2A significantly inhibits NLRP3 inflammasome activation [12, 32]. PP2A becomes inactive when its Y307 residue is phosphorylated [33]. In this study, we evaluated the activity of PP2A by measuring the levels of PP2A protein phosphorylated at Y307 and total PP2A protein in the hippocampus of PISE mice, as well as examining the ratio of p-PP2A to total PP2A protein level (p-PP2A/PP2A ratio). As shown in Fig. 1A, compared with control animals, the total PP2A protein levels in the hippocampi of PISE mice were increased, whereas the p-PP2A protein levels and the p-PP2A/PP2A ratio were decreased, indicating that PP2A activity was increased in the hippocampus of mice after PISE.

Fig. 1.

PP2A-NLRP3 pathway is activated in the hippocampus of PISE mice. A. The protein level of p-PP2A was decreased and that of NLRP3 was increased in the hippocampus of PISE mice. B. The protein levels of ASC-D, ASC-M, c-cas-1, IL-1β, and N-GSDMD were increased in the hippocampus of PISE mice. C. The administration of the PP2A inhibitor OA increased p-PP2A protein level in the hippocampus of PISE mice. D. OA treatment decreased the protein levels of ASC-D, ASC-M, c-cas-1, IL-1β, and N-GSDMD in the hippocampus of PISE mice. E. The administration of the NLRP3 inflammasome inhibitor MCC950 decreased the protein levels of ASC-D, ASC-M, c-cas-1, IL-1β, and N-GSDMD in the hippocampus of PISE mice. F. Cas-1 activity was increased in the hippocampus of PISE mice; however, treatment with OA or MCC950 suppressed this effect. G. The numbers of ASC⁺cells and GSDMD⁺ cells were increased in the hippocampal area of PISE mice (G-i, G-iii), whereas the administration of OA or MCC950 reversed these effects (G-ii, G-iii). H. The numbers of surviving pyramidal neurons in the hippocampal CA1 and CA2/3 subregions were decreased in PISE mice (H-i, H-ii); treatment with OA (H-i, H-iii) or MCC950 (H-i, H-iv) mitigated the changes. Scale bar = 50 µm. ×10 objective for panels G-i-DAPI, G-i-ASC, G-i-GSDMD, G-i-merge, G-ii-DAPI, G-ii-ASC, G-ii-merge, H-ii-middle, H-iii-middle, and H-iv-middle. ×40 objective for panels G-i-enlarge, G-ii-enlarge, H-ii-left, H-ii-right, H-iii-left, H-iii-right, H-iv-left, and H-iv-right.**P < 0.01 vs. control; ^#P < 0.05,^##P < 0.01 vs. PISE+vehicle (icv.);^&&P < 0.01 vs. PISE+vehicle (ip.). Sample sizes were as follows: for Western blot analyses in panels A–E, n = 9 per group; for cas-1 activity assays in panel F, n = 4 per group; and for immunofluorescence and Toluidine blue staining experiments in panels G and I, n = 5 per group (see Supplementary Methods)

In this study, the protein levels of NLRP3 and monomeric ASC (ASC-M) and the number of ASC⁺ cells were examined to determine the expression levels of the NLRP3 inflammasome; the protein levels of dimeric ASC (ASC-D) were measured to detect the extent of NLRP3 inflammasome assembly; and the protein levels of c-cas-1, IL-1β, and N-GSDMD, as well the activity of cas-1, were evaluated to detect NLRP3 inflammasome activation. As shown in Fig. 1A and B, and 1G-i, compared with control animals, the protein levels of NLRP3, ASC-M, and ASC-D were markedly increased in the hippocampi of PISE mice, as was the number of ASC⁺ cells. Moreover, ASC⁺/Iba-1⁺, ASC⁺/GFAP⁺ and ASC⁺/NeuN⁺ cells were observed in the hippocampal region following PISE (Supplementary Fig. 7 A). Similarly, the c-cas-1 protein levels and cas-1 activity were higher in the hippocampi of PISE mice than in those of control animals (Fig. 1A and F), with similar trends being observed for the protein levels of IL-1β and N-GSDMD and the number of GSDMD⁺ cells (Fig. 1A and G-i). These results indicate that the expression, assembly, and activation of the NLRP3 inflammasome in the hippocampus are enhanced following PISE.

Next, OA, an inhibitor of PP2A, and MCC950, a specific inhibitor of the NLRP3 inflammasome, were applied to examine the effect of PP2A on the NLRP3 inflammasome following PISE. As shown in Fig. 1C and D, compared to vehicle-treated controls, PISE mice treated with OA showed a marked increase in the p-PP2A/PP2A ratio and a decrease in both ASC-M and ASC-D protein levels and ASC⁺ cell numbers in the hippocampus, including ASC⁺/Iba-1⁺, ASC⁺/GFAP⁺ and ASC⁺/NeuN⁺ cells (Fig. 1G-ii and Supplementary Fig. 7B). OA administration significantly reduced c-cas-1 protein expression and cas-1 activity in the hippocampi of PISE mice (Fig. 1D and F), accompanied by a notable reduction in the protein levels of IL-1β and N-GSDMD (Fig. 1D). Subsequently, we found that the administration of MCC950 significantly decreased the protein levels of ASC-M, ASC-D, and c-cas-1, and reduced the activity of cas-1 in the hippocampi of PISE mice compared with that seen in the control condition (Fig. 1E and F). Compared with the vehicle treatment, MCC950 administration resulted in fewer ASC⁺ cells in the hippocampal area of PISE mice, including ASC⁺/Iba-1⁺, ASC⁺/GFAP⁺ and ASC⁺/NeuN⁺ cells (Fig. 1G-ii and Supplementary Fig. 7 C), and also significantly reduced the protein levels of IL-1β and N-GSDMD in the hippocampus (Fig. 1E and Supplementary Fig. 1). Moreover, we observed a reduced number of surviving neurons in the hippocampal CA1 and CA2/3 region of PISE mice compared to control mice, and this change was markedly attenuated by OA or MCC950 administration (Fig. 1H). The above results suggest that during PISE, PP2A activity promotes NLRP3 inflammasome activation, thereby enhancing the inflammatory response, and mediates cell pyroptosis in the hippocampal region.

• 2. Changes in TLR4-p38 MAPK-NLRP1 signaling pathway in the hippocampus following PISE

TLR4 is an important pattern recognition receptor in the immune system and its expression has been reported to be upregulated in epilepsy [15]. The activation of this receptor can enhance p38 MAPK activity, thus modulating the NLRP1 inflammasome and the inflammatory response. As shown in Fig. 2A, the protein levels of TLR4, p-p38 MAPK, and NLRP1 in the hippocampi of PISE mice were significantly increased compared with those of control animals. Additionally, compared to vehicle treatment, the administration of TAK-242, a TLR4 inhibitor, markedly decreased the protein levels of p-p38 MAPK, NLRP1, ASC-M, and ASC-D in the hippocampi of PISE mice (Fig. 2C and D). Moreover, the administration of TAK-242 resulted in fewer ASC⁺ cells in the hippocampus of PISE mice, including ASC⁺/Iba-1⁺, ASC⁺/GFAP⁺ and ASC⁺/NeuN⁺ cells (Fig. 2G and Supplementary Fig. 8 A), while also decreasing c-cas-1 protein levels and inhibiting cas-1 activity (Fig. 2D and B). As shown in Fig. 2D, the administration of TAK-242 also significantly reduced the hippocampal protein levels of IL-1β and N-GSDMD in PISE mice. Meanwhile, we found that treatment with SB203580, a p38 MAPK inhibitor, significantly reduced the protein levels of p-p38 MAPK, NLRP1, and c-cas-1 and the activity of cas-1 in the hippocampi of PISE mice (Fig. 2B and E, and 2F). Compared with vehicle-treated PISE mice, SB203580-treated PISE mice exhibited reduced ASC-D protein levels and a decreased number of ASC-positive cells, including ASC⁺/Iba-1⁺, ASC⁺/GFAP⁺ and ASC⁺/NeuN⁺ cells, in the hippocampus (Fig. 2F and G and Supplementary Fig. 8B). The administration of SB203580 also markedly decreased the protein levels of IL-1β and N-GSDMD in the hippocampi of PISE mice (Fig. 2F and Supplementary Fig. 2).

Fig. 2.

TLR4-p38 MAPK-NLRP1 pathway is activated in the hippocampus of PISE mice. A. The protein levels of TLR4, p-p38 MAPK, and NLRP1 were increased in the hippocampus of PISE mice. B. The administration of a TLR4 antagonist, TAK-242 (left), or a p38 MAPK inhibitor, SB203580 (right), decreased cas-1 activity in the hippocampus of PISE mice. C and E. The administration of TAK-242 (C) or SB203580 (E) decreased p-p38 MAPK and NLRP1 protein levels in the hippocampus of PISE mice. D and F. The administration of TAK-242 (D) or SB203580 (F) decreased the protein levels of ASC-D, ASC-M, c-cas-1, IL-1β, and N-GSDMD in the hippocampus of PISE mice. G. The administration of TAK-242 or SB203580 reduced ASC⁺ cell numbers in the hippocampal region of PISE mice. H. The protocol for AAV and pilocarpine injection. Fluorescence images showing the expression of AAV-shRNA-NLRP1-EGFP in the hippocampus 28 days after infusion. I–K. AAV-shRNA-NLRP1 markedly decreased NLRP1, c-cas-1, IL-1β, and N-GSDMD protein levels (I), pyramidal neuron survival (J), and cas-1 activity (K) in the hippocampus of PISE mice. Scale bar = 50 µm. ×10 objective for panels G-DAPI, G-ASC, G-merge, H-left, and J-middle and ×40 objective for panels G-enlarge, H-right, J-left, and J-right. **P < 0.01 vs. control; ^#P < 0.05, ^##P < 0.01 vs. PISE+vehicle (icv.); ^&&P < 0.01 vs. PISE+vehicle (ip.); ^%%P < 0.01 vs. AAV-shRNA-control. Sample sizes were as follows: for Western blot analyses in panels A, C‒F and I, n = 9 per group; for cas-1 activity assays in panels B and K, n = 4 per group; and for immunofluorescence and Toluidine blue staining experiments in panels G and J, n = 5 per group (see Supplementary Methods)

To further verify the role of the NLRP1 inflammasome in the inflammatory response and neuronal damage observed in PISE mice, AAV-shRNA-NLRP1 was injected into the hippocampus to knock down NLRP1. Compared with PISE mice treated with AAV-shRNA-control, those treated with AAV-shRNA-NLRP1 exhibited significantly reduced NLRP1 protein levels in the hippocampus (Fig. 2I). Knocking down NLRP1 markedly reduced c-cas-1 levels and cas-1 activity in the hippocampi of PISE mice, accompanied by a decrease in IL-1 β and N-GSDMD protein levels (Fig. 2I and K). Finally, knocking down NLRP1 increased neuronal survival in the hippocampal CA1 and CA2/3 subregions of PISE mice (Fig. 2J). These results suggest that during PISE, elevated TLR4-p38 MAPK pathway activity increases NLRP1 expression and inflammatory responses, inducing cell pyroptosis in the hippocampus. The inhibition of the NLRP1 inflammasome could suppress the inflammatory response and reduce neuronal damage in PISE mice.

• 3. Blocking TRPV4 inhibits the PP2A-NLRP3 and TLR4-p38 MAPK-NLRP1 pathways in the hippocampus following PISE

Blocking TRPV4 has been reported to alleviate the inflammatory response during PISE [5]. In this study, we explored whether this effect was related to the PP2A-NLRP3 and TLR4-p38 MAPK-NLRP1 signaling pathways. As shown in Fig. 3A and B, the administration of HC-067047, a specific antagonist of TRPV4, markedly increased the p-PP2A/PP2A ratio and decreased the protein levels of TLR4 and p-p38 MAPK in the hippocampus of PISE mice, compared to that observed with vehicle treatment. Additionally, the administration of HC-067047 significantly reduced the protein levels of NLRP3, NLRP1, ASC-M, ASC-D, and c-cas-1, along with the activity of cas-1, in the hippocampi of PISE mice. A similar trend was observed in the protein levels of IL-1 β and N-GSDMD (Fig. 3A and D and Supplementary Fig. 3) as well as in the numbers of ASC⁺ cells, including ASC⁺/Iba-1⁺, ASC⁺/GFAP⁺ and ASC⁺/NeuN⁺ cells, and GSDMD⁺ cells (Fig. 3E and Supplementary Fig. 9). In contrast, the number of surviving neurons in the hippocampal CA1 and CA2/3 subregions of PISE mice was markedly increased with HC-067047 administration (Fig. 3F). The above results indicate that during PISE, blocking TRPV4 alleviates the inflammatory response and exerts neuroprotective effects by inhibiting the PP2A-NLRP3 and TLR4-p38 MAPK-NLRP1 signaling pathways.

Fig. 3.

Blockade of TRPV4 inhibits PP2A-NLRP3 and TLR4-p38 MPAK-NLRP1 pathways in the hippocampus of PISE mice. A–D. The administration of a TRPV4 antagonist, HC-067047, markedly decreased p-PP2A, NLRP3 (A), TLR4, p-p38 MAPK, NLRP1 (B), ASC-D, ASC-M, c-cas-1, IL-1β, and N-GSDMD (C) protein levels and cas-1 activity (D) in the hippocampus of PISE mice. E and F. The administration of HC-067047 decreased ASC⁺ and GSDMD⁺ cell numbers (E) and increased the survival of pyramidal neurons (F) in the hippocampal region of PISE mice. Scale bar = 50 µm.×10 objective for panels E-DAPI, E-ASC, E-GSDMD, E-merge, and F-middle. ×40 objective for panels E-enlarge one, F-left, and F-right. ^##P < 0.01 vs. PISE+vehicle (icv.). Sample sizes were as follows: for Western blot analyses in panels A‒C, n = 9 per group; for cas-1 activity assays in panel D, n = 4 per group; and for immunofluorescence and Toluidine blue staining experiments in panels E and F, n = 5 per group (see Supplementary Methods)

• 4. TRPV4 activation enhances the PP2A-NLRP3 signaling pathway in the hippocampus

To further verify the regulatory effect of TRPV4 on the PP2A-NLRP3 signaling pathway, we examined the levels of proteins related to this signaling pathway in the hippocampi of GSK mice. As shown in Fig. 4A, compared with control animals, the p-PP2A/PP2A ratio in the hippocampus was significantly decreased in GSK mice, whereas the protein levels of NLRP3 were notably increased. The protein levels of ASC-D and ASC-M, along with the number of ASC⁺ cells, including ASC⁺/Iba-1⁺, ASC⁺/GFAP⁺ and ASC⁺/NeuN⁺ cells, were markedly elevated in the hippocampi of GSK mice (Fig. 4B and G and Supplementary Fig. 10). Similarly, increased levels of c-cas-1 protein levels and enhanced cas-1 activity were also observed (Fig. 4B and F). We also found that the protein levels of IL-1β and N-GSDMD and the number of GSDMD⁺ cells in the hippocampus were significantly higher in GSK mice than those in control mice (Fig. 4B and G).

Fig. 4.

PP2A-NLRP3 pathway is activated in the hippocampus of GSK mice. A and B. The protein level of p-PP2A was decreased (A) and the protein levels of NLRP3 (A), ASC-D, ASC-M, c-cas-1, IL-1β, and N-GSDMD (B) were increased in the hippocampus of GSK mice. C. The administration of OA increased p-PP2A protein level in the hippocampus of GSK mice. D and E. The administration of OA (D) or MCC950 (E) decreased the protein levels of ASC-D, ASC-M, c-cas-1, IL-1β, and N-GSDMD in the hippocampus of GSK mice. F. Cas-1 activity was increased in the hippocampus of GSK mice; the administration of OA or MCC950 reversed this effect. G. ASC⁺ and GSDMD⁺ cell numbers were increased in the hippocampal region of GSK mice. H. Treatment with OA or MCC950 reduced ASC⁺ cell numbers in the hippocampal region of GSK mice. I. Pyramidal neuron survival was decreased in the hippocampal CA1 and CA2/3 subregions of GSK mice; this change was attenuated by OA or MCC950 administration. Scale bar = 50 µm. ×10 objective for panels G-DAPI, G-ASC, G-GSDMD, G-merge, H-DAPI, H-ASC, H-merge, and I-middle. ×40 objective for panels G-enlarge, H-enlarged one, I-left, and I-right. ^^P < 0.01 vs. control; ^$$P < 0.01 vs. GSK1016790A+vehicle (icv.); ^~~P < 0.01 vs. PISE+vehicle (ip.). Sample sizes were as follows: for Western blot analyses in panels A‒E, n = 9 per group; for cas-1 activity assays in panel F, n = 4 per group; and for immunofluorescence and Toluidine blue staining experiments in panels G‒I, n = 5 per group (see Supplementary Methods)

We further noted that the administration of the PP2A inhibitor OA markedly increased the p-PP2A/PP2A ratio in the hippocampi of GSK mice compared with that detected with the control (vehicle) treatment (Fig. 4C). Additionally, after OA administration, the protein levels of ASC-D and ASC-M and the number of ASC⁺ cells were both decreased in the hippocampus in GSK mice (Fig. 4D and H). Similarly, both c-cas-1 protein level and cas-1 activity in the hippocampus were decreased in GSK mice relative to those in control animals (Fig. 4D and F), as were the protein levels of IL-1β and N-GSDMD (Fig. 4D).

We also found that treatment with the NLRP3 inhibitor MCC950 markedly decreased the protein levels of ASC-D and ASC-M, as well as the number of ASC⁺ cells in the hippocampal area of GSK mice (Fig. 4E and H). Compared with vehicle-treated GSK mice, both c-cas-1 protein level and cas-1 activity were significantly reduced in the hippocampus of GSK mice following OA administration, concomitant with a decrease in the protein levels of IL-1β and N-GSDMD (Fig. 4E and F and Supplementary Fig. 4).

Finally, after treatment with either OA or MCC950, the number of surviving neurons in the hippocampal CA1 and CA2/3 subregions of GSK mice was significantly increased (Fig. 4I). The above results demonstrate that TRPV4 activation upregulates the PP2A-NLRP3 signaling pathway, which promotes an inflammatory response and mediates cell pyroptosis, ultimately leading to neuronal damage in the hippocampus.

• 5. TRPV4 activation enhances TLR4-p38 MAPK-NLRP1 signaling in the hippocampus

We also examined the changes in the hippocampal levels of proteins related to the TLR4-p38 MAPK-NLRP1 signaling pathway in GSK mice. As shown in Fig. 5A, compared with control mice, the protein levels of TLR4, p-p38 MAPK, and NLRP1 in the hippocampus were markedly increased in mice treated with GSK. Subsequently, GSK mice were administered an inhibitor of TLR4 (TAK-242), or an inhibitor of p38 MAPK (SB203580). Compared with vehicle-treated GSK mice, the administration of TAK-242 resulted in a significant decrease in the protein levels of p-p38 MAPK and NLRP1 in the hippocampus of GSK mice (Fig. 5B). The protein levels of ASC-D and ASC-M and the number of ASC⁺ cells were also greatly decreased in the hippocampus of GSK mice (Fig. 5C and G), as were the protein level of c-cas-1 and the activity of cas-1 (Fig. 5C and F), along with the protein levels of IL-1β and N-GSDMD (Fig. 5C).

Fig. 5.

TLR4-p38 MPAK-NLRP1 pathway is activated in the hippocampus of GSK mice.  A. The protein levels of TLR4, p-p38 MAPK, and NLRP1 were increased in the hippocampus of GSK mice. B and C. The administration of TAK-242 decreased the protein levels of p-p38 MAPK, NLRP1 (B), ASC-D, ASC-M, c-cas-1, IL-1β, and N-GSDMD (C) in the hippocampus of GSK mice. D and E. The administration of SB203580 decreased the protein levels of p-p38 MAPK, NLRP1 (D), ASC-D, ASC-M, c-cas-1, IL-1β, and N-GSDMD (E) in the hippocampus of GSK mice. F. The administration of TAK-242 or SB203580 decreased cas-1 activity in the hippocampus of GSK mice. G. The administration of TAK-242 or SB203580 decreased ASC⁺ cell numbers in the hippocampus of GSK mice. Scale bar = 50 µm. ×10 objective for panels G-DAPI, G-ASC, and G-merge. ×40 objective for panel G-enlarge. ^^P < 0.01 vs. control; ^$$P < 0.01 vs. GSK1016790A+vehicle (icv.); ^~~P < 0.01 vs. PISE+vehicle (ip.). Sample sizes were as follows: for Western blot analyses in panels A‒E, n = 9 per group; for cas-1 activity assays in panel F, n = 4 per group; and for immunofluorescence and Toluidine blue staining experiments in panel G, n = 5 per group (see Supplementary Methods)

As shown in Fig. 5D, compared with vehicle-treated GSK mice, the protein levels of p-p38 MAPK and NLRP1 in the hippocampus were significantly reduced in GSK mice treated with SB203580. Furthermore, the protein levels of ASC-D and ASC-M were decreased and there were fewer ASC⁺ cells in the hippocampal region in GSK mice (Fig. 5E and G). As observed with TAK-242 treatment, c-cas-1 protein level and cas-1 activity in the hippocampus were markedly reduced in GSK mice following SB203580 administration, accompanied by a decrease in IL-1β and N-GSDMD protein levels (Fig. 5E and F and Supplementary Fig. 5). The above results suggest that the activation of TRPV4 could upregulate the TLR4-p38 MAPK-NLRP1 signaling pathway, thereby enhancing the inflammatory response and mediating cell pyroptosis.

• 6. Knockdown of NLRP1 attenuates pyroptosis following TRPV4 activation

To explore the role of NLRP1 in TRPV4 activation-induced pyroptosis, AAV-shRNA-NLRP1 was injected into the mouse hippocampus to knock down Nlrp1. Knocking down NLRP1 significantly reduced NLRP1, c-cas-1, IL-1β, and N-GSDMD protein levels; decreased cas-1 activity (Fig. 6A and B); and significantly increased the number of surviving neurons in the hippocampus of GSK mice (Fig. 6C). As NLRP1 is highly expressed in neurons, we further validated the role of the TLR4-p38 MAPK-NLRP1 signaling pathway in TRPV4-induced neuronal pyroptosis using the HT-22 mouse hippocampal neuronal cell line. As shown in Fig. 6D, following incubation with GSK1016790A (GSK-HT-22 cells), the protein levels of TLR4, p-p38MAPK, NLRP1, c-cas-1, and N-GSDMD were all markedly increased in HT-22 cells compared with vehicle-treated HT-22 cells. The fluorescence intensity of NLRP1 and GSDMD was also significantly increased in GSK-HT-22 cells (Fig. 6G-iii and 6G-iv). In contrast, after administering SB203580, the protein levels of p-p38 MAPK, NLRP1, c-cas-1, and N-GSDMD were greatly reduced in GSK-HT-22 cells (Fig. 6E and Supplementary Fig. 6), as was the fluorescence intensity of NLRP1 (Fig. 6G-v). These results suggest that the activation of TRPV4 in HT-22 cells enhances TLR4-p38 MAPK-NLRP1 signaling, thereby activating cas-1 and promoting GSDMD cleavage, ultimately leading to cell pyroptosis. These findings were consistent with the in vivo results (Fig. 2). As shown in Fig. 6F and G-vi, knocking down NLRP1 significantly reduced its protein level and fluorescence intensity in GSK-HT-22 cells, accompanied by a decrease in c-cas-1 and N-GSDMD protein levels. These results indicate that knocking down NLRP1 reduces cas-1 activity and alleviates neuronal pyroptosis caused by TRPV4 activation.

Fig. 6.

Knockdown of NLRP1 attenuates pyroptosis following TRPV4 activation. A–C. The injection of AAV-shRNA-NLRP1 decreased NLRP1, c-cas-1, IL-1β, and N-GSDMD protein levels (A) as well as cas-1 activity (B) in the hippocampus, and increased the number of surviving pyramidal neurons in the hippocampal CA1 and CA2/3 subregions of GSK mice (C). D. The protein levels of TLR4, p-p38 MAPK, NLRP1, ASC-D, ASC-M, c-cas-1, and N-GSDMD were increased in GSK-HT-22 cells. E. The administration of SB203580 decreased the protein levels of p-p38 MAPK, NLRP1, ASC-D, ASC-M, c-cas-1, and N-GSDMD in GSK-HT-22 cells. F. The protein levels of NLRP1, c-cas-1, and N-GSDMD were decreased in GSK-HT-22 cells transfected with shRNA-NLRP1. G. The fluorescence intensity of NLRP1 (G-i, G-iii) and GSDMD (G-i, G-iv) was increased in GSK-HT-22 cells. Treatment with SB203580 or transfection with shRNA-NLRP1 decreased the fluorescence intensity of NLRP1 (G-ii, G-v, G-vi) in GSK-HT-22 cells. ×10 objective for C-middle. ×40 objective for C-left, C-right, G-iii, G-iv, G-v, and G-vi. Scale bar = 50 µm (C) and 200 µm (G).^▲▲P < 0.01 vs. GSK1016790A+AAV-shRNA-control; ^∆∆P < 0.01 vs. GSK1016790A; ^▼▼P < 0.01 vs. GSK1016790A+vehicle; ^>>P < 0.01 vs. GSK1016790A+shRNA-control. Sample sizes were as follows: for Western blot analyses in panel A, n= 9 per group; for cas-1 activity assays in panel B, n = 4 per group; for Toluidine blue staining experiments in panel C, n = 5 per group; for Western blot analyses in panels D‒F, n = 3 per group; and for immunofluorescence staining experiments in panel G, n = 3 per group. Data shown in panels D–G were expressed as mean ± standard error of the mean (SEM) (see Supplementary Methods)

• 7.Inhibition of TRPV4, PP2A, or NLRP3, or knocking down NLRP1 attenuates seizures in the acute phase following PISE

Finally, we investigated the role of TRPV4-induced activation of NLRP3 and NLRP1 inflammasomes in seizure activity in acute phase post-PISE. As shown in Table 1, seizures at Racine stages 1–3 and 4–5 occurred on days 2 and 3 post-PISE. Compared with the vehicle-treated group, administration of the TRPV4 antagonist HC-067047 significantly reduced both the frequency and duration of seizures with Racine stages 1–3 and 4–5. Similarly, treatment with OA or MCC950 markedly decreased seizure frequency and duration at both seizure stages. Furthermore, knockdown of NLRP1 also led to a significant reduction in the frequency and duration of seizures in PISE mice. These findings suggest that TRPV4 blockade suppresses seizures in the acute-phase following PISE, likely through the inhibition of NLRP3 and NLRP1 inflammasome activation.

Table 1.

Early seizure behaviors in PISE mice and drug-treated PISE mice

Experimental groups	Seizure numbers		Seizure duration (s)
	Racine 1–3	Racine 4–5	Racine 1–3	Racine 4–5
control (n = 8)	0.00 (0.00 ~ 0.00)	0.00 (0.00 ~ 0.00)	0.00 (0.00 ~ 0.00)	0.00 (0.00 ~ 0.00)
PISE (n = 6)	21.00(16.75 ~ 22.75) **	3.00 (0.75 ~ 5.75) **	44.53 (40.37 ~ 45.51) **	34.20 (23.75 ~ 38.16) **
PISE+vehicle† (n = 7)	21.86 ± 3.33	3.00 (1.00 ~ 4.00)	43.54 (41.21 ~ 45.60)	32.33 (30.50 ~ 35.50)
PISE + HC-067047 (n = 6)	11.50 ± 2.25##	0.00 (0.00 ~ 0.00) ##	39.19(35.79 ~ 40.50) #	0.00 (0.00 ~ 0.00) ##
PISE+vehicle† (n = 7)	21.86 ± 3.33	3.00 (1.00 ~ 4.00)	43.35 ± 3.09	32.33 (30.50 ~ 35.50)
PISE + OA (n = 6)	10.00 ± 4.04##	0.50 (0.00 ~ 1.00) #	35.77 ± 6.11#	7.00 (0.00 ~ 19.25) #
PISE+vehicle‡ (n = 7)	18.00 ± 2.70	3.00 (3.00 ~ 5.00)	40.38 ± 4.36	28.33 (27.00 ~ 31.50)
PISE+MCC950 (n = 8)	9.50 ± 2.56&&	0.00 (0.00 ~ 0.00) &&	31.63 ± 2.58&&	0.00 (0.00 ~ 0.00) &&
PISE + AAV-control (n = 7)	24.50 (15.00 ~ 26.00)	5.00 (2.50 ~ 8.25)	45.51 ± 3.53	29.31 (27.91 ~ 32.37)
PISE + AAV-NLRP1 (n = 8)	14.00 (12.25 ~ 15.75) %	0.00 (0.00 ~ 1.75) %%	33.47 ± 3.43%%	0.00 (0.00 ~ 28.50) %

**P < 0.01 vs. control, ^#P < 0.05, ^##P < 0.01 vs. PISE+vehicle†, ^&&P < 0.01 vs. PISE+vehicle‡, ^%P < 0.05, ^%%P < 0.01 vs. PISE + AAV-control 

The data were analyzed using either the Mann-Whitney U test or the independent samples t-test, and are presented as medians with interquartile ranges or means ± standard deviations, respectively

†: icv. injection of vehicle

‡: ip. injection of vehicle

Discussion

In the present study, male ICR mice were used to establish the epilepsy model for the following reasons. First, hormonal fluctuations caused by the estrous cycle in female animals may increase the variability of phenotypes in the pilocarpine-induced epilepsy model. Additionally, female animals have enhanced anti-epileptic capabilities through metabolic adaptation and blood-brain barrier protection. In contrast, pilocarpine-induced epilepsy models in male mice exhibit more stable phenotypes, along with prominent neuroinflammation, gliosis, and neuronal injury in brain regions such as the hippocampus [34, 35]. Previous studies investigating pilocarpine-induced epilepsy have predominantly used male animals [5, 23, 34–37]. Second, ICR mice are more sensitive to pilocarpine, with higher SE induction efficiency and a shorter latency period before SE onset. The temporal lobe epilepsy model established in ICR mice is well-defined, showing not only typical neuronal loss in the hippocampus and mossy fiber sprouting but also a significant inflammatory response, making them suitable for studying inflammation-related pathways [5, 23, 34–38]. Studies have shown that inflammasome (e.g., NLRP3, NLRP1) activity is upregulated and inflammatory cytokines levels are increased in the hippocampus of TLE model rodents and patients with TLE [8, 39]. After inflammasome activation, c-cas-1 promotes the production of inflammatory cytokines (such as IL-1β), thus mediating the inflammatory response. Inflammasome cytokines can also enhance inflammasome expression, which further promotes the release of inflammatory cytokines, thereby creating a positive feedback loop [40, 41]. Meanwhile, c-cas-1 cleaves GSDMD, yielding the functional fragment N-GSDMD, which induces pyroptosis [6]. In this study, we observed ASC⁺/Iba-1⁺, ASC⁺/GFAP⁺, and ASC⁺/NeuN⁺ cells in the hippocampal region of PISE mice, with ASC⁺ cells predominantly expressed in glial cells (Iba-1⁺ microglia and GFAP⁺ astrocytes). This finding is consistent with the previous study that ASC-associated inflammasomes, such as NLRP3, are mainly expressed in glial cells [4]. The pyroptosis of glial cells results in the release of inflammatory cytokines, exacerbating the inflammatory response, while neuronal pyroptosis directly leads to further neuronal damage. Inflammatory cytokines can stimulate neurons and increase their excitability [42]. Through their deleterious effects on neurons, the inflammatory response and pyroptosis lead to structural and functional remodeling of neural networks in the hippocampus. These observations indicate that excessive or sustained inflammasome activation contributes to the inflammatory response and the neuronal damage that occur during epilepsy. In this study, NLRP3 and NLRP1 inflammasome expression (NLRP3, NLRP1, and ASC-M protein levels and ASC⁺ cell numbers), assembly (ASC-D protein levels), and activation (cas-1 activity and c-cas-1, IL-1β, and N-GSDMD protein levels) increased markedly in the acute phase of PISE mice (3 days post-PISE) (Figs. 1 and 2). The colocalization of ASC and GSDMD with glial and neuronal markers indicates the dual roles of inflammasome activation in driving both neuroinflammation (via inflammatory factors release and glial pyroptosis) and direct neuronal injury (via neuronal pyroptosis), thereby amplifying the pathogenic cascade in epilepsy [43]. Given these results, we next sought to identify the mechanisms underlying the upregulation of the NLRP1 and NLRP3 inflammasomes during PISE.

Activated TRPV4 participates in the modulation of a variety of biological processes through its promotive effect on intracellular calcium elevation [21]. TRPV4 has been identified as a susceptibility gene for epilepsy [22], and its expression is markedly increased in cortical lesions of patients with focal cortical dysplasia as well as in hippocampal lesions of patients with TLE [44, 45]. Consistent with these clinical observations, TRPV4 expression in the hippocampus was also reported to be robustly upregulated from 3 h to 30 days after SE onset in PISE mice [5]. Functionally, blocking TRPV4 in mice not only reduced the incidence of pilocarpine-induced SE but also prolonged the latency of its onset [5]. Moreover, TRPV4 inhibition has been shown to attenuate hippocampal injury by suppressing inflammatory responses [5], and pharmacological or genetic blockade of TRPV4 effectively ameliorated lipopolysaccharide (LPS)-induced cognitive impairment by reducing inflammatory cytokines and pyroptosis-related proteins [46]. In addition to TRPV4, other members of the TRP channel family, such as TRPV1 and TRPM2, have also been implicated in neuroinflammatory regulation. TRPV1 activation promotes microglial reactivity and facilitates NLRP3 inflammasome assembly, thereby exacerbating neuroinflammation in various neurological conditions [13, 47, 48]. Likewise, TRPM2, a redox-sensitive and reactive oxygen species-responsive channel, enhances oxidative stress-driven signaling cascades that contribute to NLRP3 inflammasome activation [49, 50]. These findings indicate that multiple TRP channels converge on inflammasome-related inflammatory pathways and may participate in seizure-associated neuroinflammation. However, most of these TRP members predominantly modulate the NLRP3 inflammasome. In contrast, our study demonstrates a distinct role of TRPV4: blocking TRPV4 inhibited the expression, assembly, and activation of both NLRP3 and NLRP1 inflammasomes in the hippocampus of PISE mice (Fig. 3), whereas TRPV4 activation exerted the opposite effects (Fig. 4). TRPV4 blockade also attenuated neuronal pyroptosis, thereby exerting significant neuroprotective effects (Fig. 3). This regulatory effect distinguishes TRPV4 from other TRP channels, suggesting that TRPV4 may act as an upstream regulator of seizure-related neuroinflammation. Given its pivotal role in modulating both NLRP3 and NLRP1 inflammasomes, TRPV4 is proposed as a promising therapeutic target for epilepsy. Targeting TRPV4, especially with selective small-molecule inhibitors, could reduce neuroinflammation and protect neurons from pyroptosis, both of which play crucial roles in the pathophysiology of epilepsy. Further studies are needed to evaluate the safety, efficacy, and optimal delivery methods for TRPV4-targeted therapies in preclinical models and clinical settings. These investigations could contribute to more targeted treatment strategies for epilepsy, potentially improving options for patients with seizures. To further explore the therapeutic potential of TRPV4, it is essential to investigate the mechanisms through which TRPV4 drives the activation of both NLRP3 and NLRP1 inflammasomes.

The expression, assembly, and activation of inflammasomes are strictly regulated. The NLRP3 inflammasome, highly expressed in microglia and astrocytes, is composed of NLRP3, cas-1, and ASC [7]. In the resting state, the PYD of NLRP3 undergoes phosphorylation, which hinders the interaction between it and the PYD of ASC through electrostatic repulsion, which prevents accidental assembly [12]. ASC is primarily localized to the nucleus under resting conditions. When NLRP3 on the cell membrane is activated by damage-associated or pathogen-associated molecular patterns, ASC oligomerizes through PYD-PYD interactions, which triggers ASC translocation from the nucleus to the perinuclear space [51]. However, at this time, ASC is still under the control of inhibitor of kappa B kinase α (IKKα) and cannot participate in NLRP3 inflammasome assembly [32]. PP2A is a serine and threonine protein phosphatase that can dephosphorylate IKKα, thereby inactivating it, which allows interaction between ASC-PYD and NLRP3-PYD, and, consequently, the formation of ASC-D [32, 52]. ASC-D then further oligomerizes, forming ASC specks, which participate in NLRP3 inflammasome assembly [53]. Moreover, PP2A dephosphorylates NLRP3-PYD (at S3 in mice and S5 in humans), which removes the electrostatic repulsion between NLRP3-PYD and ASC-PYD, and thus promotes inflammasome assembly [12]. After the formation of ASC-D and ASC specks, the CARD of ASC interacts with the CARD of cas-1, which leads to the production of cleaved (active) cas-1 [54, 55]. Studies have shown that reducing PP2A activity markedly inhibits the assembly and activation of the NLRP3 inflammasomes and reduces the production of IL-1β in LPS-treated macrophages and microglia [13, 56]. In this study, PP2A activity was increased in the hippocampi of PISE mice, but this activity was decreased with the administration of OA, a PP2A inhibitor. The decline in PP2A activity inhibited NLRP3 inflammasome assembly and activation, reduced the inflammatory response, thereby exerting neuroprotective effects. Moreover, the blockade of the NLRP3 inflammasome via MCC950 administration inhibited the inflammatory response and mitigated neuronal damage following PISE (Fig. 1). The above results suggest that during TLE, enhanced PP2A activity promotes NLRP3 inflammasome assembly and activation, which triggers an inflammatory response and results in neuronal damage. Nevertheless, no study to date has investigated the mechanism underlying PP2A activation during TLE.

It has been reported that TRPV1 can activate PP2A by increasing [Ca²⁺]_i [13]. Both TRPV1 and TRPV4 are members of the TRPV subfamily within the TRP superfamily, and the activation of either of these ion channels can increase [Ca²⁺]_i [57]. In this study, blocking TRPV4 markedly reduced hippocampal PP2A activity in PISE mice (Fig. 3), whereas activating TRPV4 resulted in the opposite effect (Fig. 4). Moreover, blocking PP2A markedly suppressed NLRP3 inflammasome assembly and activation in the hippocampus in GSK mice (Fig. 4). In summary, TRPV4 activation accounted for the increase in PP2A activity detected during TLE, which led to the assembly and activation of NLRP3 inflammasomes.

The NLRP1 inflammasome is mainly expressed in neurons, and there are differences in NLRP1 protein structure among mammals [58, 59]. Given that both the CARD and the PYD are present in human NLRP1 protein, ASC is not an essential component of the NLRP1 inflammasome in humans [60], but it can enhance NLRP1 inflammasome activation [61]. Mouse NLRP1 protein lacks a PYD, which has been replaced by a unique N-terminal domain. Like in humans, ASC is not necessary for NLRP1 inflammasome assembly and activation in mice [62, 63]. Concerning the effect of PP2A on ASC, PP2A may be involved in the assembly and activation of the NLRP1 inflammasome in humans, but not mice. RNA-seq analysis of hippocampal tissues revealed that NLRP1 is a candidate causative gene for drug-refractory epilepsy in patients with mesial temporal lobe epilepsy with hippocampal sclerosis [64]. Additionally, NLRP1 protein levels were noted to be significantly increased in the hippocampi of rats with amygdala kindling-induced TLE [65]. However, the mechanism underlying the modulation of NLRP1 protein expression during TLE is not fully understood. NLRP1 protein levels were reported to be significantly elevated in both a cultured cortical neuron oxygen–glucose deprivation model and middle cerebral artery occlusion model rats. Notably, curcumin displayed neuroprotective effects against cerebral ischemia–reperfusion injury by inhibiting NLRP1-dependent neuronal pyroptosis via the suppression of the p38 MAPK pathway [19]. TLR4 can activate the p38 MAPK signaling cascade through either a MyD88-dependent mechanism or the TAK1 pathway [66]. In this study, both TLR4 expression and p38 MAPK activity were elevated in the hippocampus of PISE mice (Fig. 2). Inhibition of TLR4 reduced p38 MAPK activity, and blockade of either TLR4 or p38 MAPK significantly decreased NLRP1 expression, suppressed cas-1 activation, and attenuated pyroptosis in the hippocampus of PISE mice (Fig. 2B and G). Previous studies have shown that TRPV4 activation upregulates the TLR4/NF-κB and p38 MAPK signaling pathways [23, 24]. Here, TRPV4 blockade reduced TLR4 expression and p38 MAPK activity in the hippocampus of PISE mice (Fig. 3). Moreover, TLR4 inhibition diminished p38 MAPK activity, and blocking either TLR4 or p38 MAPK reduced NLRP1 expression, cas-1 activity, and pyroptosis in GSK-treated mice (Fig. 5). Collectively, these findings indicate that during TLE, TRPV4 activation increases TLR4-p38 MAPK signaling, with TLR4 functioning upstream of p38 MAPK to promote NLRP1 inflammasome expression. Besides the NLRP1 inflammasome, TLR4 can also regulate the expression and activation of the NLRP3 and AIM2 inflammasomes by activating the NF-κB, JNK or p38 MAPK or increasing reactive oxygen species production [67, 68]. Here, we found that TLR4 activation was responsible for the increase in NLRP3 inflammasome expression following PISE (Supplementary Fig. 11). However, whether the TRPV4-TLR4 pathway also influences the expression of other inflammasomes during TLE remains unknown.

To further clarify the role of NLRP1 in neuronal pyroptosis during TLE, we knocked down NLRP1 in GSK-HT-22 cells, as well as in the hippocampal region in GSK mice and PISE mice. We observed that TLR4-p38 MPAK pathway activity was increased in GSK-HT-22 cells, concomitant with an increase in NLRP1 expression, cas-1 activity, and pyroptosis (Fig. 6). Importantly, knocking down NLRP1 reversed the effects of GSK treatment (Fig. 6). These results indicate that the activation of TRPV4 upregulates the TLR4-p38 MAPK pathway, thus inducing NLRP1-dependent neuronal pyroptosis. Moreover, the knockdown of NLRP1 in GSK mice or PISE mice significantly reduced NLRP1 expression in the hippocampus, accompanied by a decline in cas-1 activity and the inhibition of pyroptosis (Fig. 6). Collectively, our findings suggest that the TRPV4-TLR4-p38 MPAK pathway is likely responsible for the increased expression and activation of the NLRP1 inflammasome. Notably, NLRP1 inflammasome inhibition attenuated neuronal pyroptosis in the hippocampus during TLE, thus displaying neuroprotective effects.

While previous studies have demonstrated that TRPV4 blockade suppresses epileptiform discharges [22, 45, 69], its effect on seizure behaviors remained unclear. Our findings revealed that TRPV4 blockade markedly decreased seizure frequency and duration in the acute phase of PISE mice (Table 1), providing the first evidence that TRPV4 blockade can effectively attenuate seizure behaviors. Seizures occurred in this phase was different from the spontaneous recurrent seizures (SRS) observed in the chronic phase of TLE [26]. Notably, pharmacokinetic analyses of pilocarpine indicate that pilocarpine is no longer detectable in blood or brain at 24 h after its injection [26]. In this study, seizure behaviors were observed on days 2 and 3 post-PISE, indicating that the early seizures were not caused by residual exposure to pilocarpine. Previous studies have shown that epileptiform discharges and seizures can occur as early as 1–5 days after SE [25, 26], and that the frequency of seizures during this period may be positively correlated with the progression in the severity and number of seizures and the hippocampal pathology, such as neuronal loss and gliosis [26]. These findings suggest that early seizures may serve as a driving factor in disease progression, promoting the development of chronic epilepsy. Furthermore, our study revealed that, in addition to TRPV4 antagonist, inhibition of PP2A, NLRP3, or knockdown of NLRP1 also significantly reduced early seizures in PISE mice (Table 1), suggesting that TRPV4 may exert its pro-epileptic effects through activation of both NLRP3 and NLRP1 inflammasomes. That is, TRPV4 activation may enhance PP2A-NLRP3 or TLR4-p38 MAPK-NLRP1 pathways, leading to inflammasomes activation and the subsequent release of pro-inflammatory cytokines such as IL-1β. IL-1β has been shown to exacerbate neuronal hyperexcitability by enhancing glutamatergic transmission and suppressing GABAergic inhibition [42], which may be one of the important mechanisms underlying early seizures. Thus, TRPV4 inhibition may disrupt the vicious cycle of seizures and inflammation by suppressing neuroinflammatory cascades. Besides its role in inflammation, the effect of TRPV4 activation on blood-brain barrier (BBB) integrity is also noteworthy. Previous studies have reported that TRPV4 activation can upregulate matrix metalloproteinase-9, thereby degrading tight junction proteins (e.g., ZO-1, occludin) and exacerbating cerebral edema in cerebral ischemic mice [70]. Moreover, BBB-protective agents such as β-theanine have been shown to alleviate early seizures [25], implying that BBB disruption may be another trigger of early seizures. Our study did not assess the protective effects of TRPV4 blockade on BBB integrity in PISE mice, this hypothesis needs to be investigated in future research.

Several limitations should be acknowledged in this study. First, although male animals were selected to minimize phenotypic variability associated with estrous-cycle–related hormonal fluctuations and to ensure the stability of the pilocarpine-induced epilepsy model, this design inherently restricts the generalizability of our findings. Estrogenic hormones such as estradiol and progesterone are known to modulate neuroinflammation and inflammasome activation [71], and may also influence TRPV4 channel activity [72]. These hormone-dependent regulatory mechanisms raise the possibility that TRPV4 signaling and NLRP1/NLRP3 inflammasome responses could differ substantially between sexes. Future studies including female mice and directly assessing estrogen/progesterone-dependent modulation of TRPV4–inflammasome pathways will therefore be necessary to determine sex-specific effects on seizure susceptibility and the therapeutic potential of TRPV4 inhibition. Second, the present study focused on the effects of TRPV4 blockade on inflammation and seizure behaviors during the acute phase following PISE. Future research should investigate whether TRPV4 blockade also influences SRS during the chronic phase of PISE and whether these potential effects are associated with the suppression of early seizures. Third, this study explored the effects of TRPV4 blockade on early seizure behavior, further validation and clarification of TRPV4’s role in early epileptic seizures is needed through electroencephalographic analysis in future studies.

Conclusion

This study demonstrates that TRPV4 activation during PISE promotes the expression and activation of the NLRP3 and NLRP1 inflammasomes, likely through the activation of PP2A and the TLR4-p38 MAPK signaling pathway (Fig. 7). Notably, this is the first report showing that inhibition of TRPV4 can attenuate early seizures in PISE mice, an effect associated with the suppression of NLRP3 and NLRP1 inflammasomes expression and activation. These findings provide new insights into the molecular mechanisms by which TRPV4 contributes to the pathogenesis of temporal lobe epilepsy.

Fig. 7.

Proposed mechanisms underlying TRPV4-mediated activation of NLRP3 and NLRP1 inflammasomes following PISE. Activation of TRPV4 enhances PP2A activity, leading to NLRP3 inflammasome activation, while simultaneously facilitating activation of the TLR4–p38 MAPK signaling pathway, resulting in NLRP1 inflammasome activation. Activation of both NLRP3 and NLRP1 inflammasomes promotes caspase-1 activation, thereby increasing the production of pro-inflammatory mediators and inducing pyroptosis, which together contribute to neuronal damage and the exacerbation of epileptic seizures. In contrast, pharmacological blockade of TRPV4 suppresses these signaling cascades, leading to reduced inflammasome activation and neuronal injury, and contributing to the alleviation of epileptic seizures

Supplementary Information

Below is the link to the electronic supplementary material.

Authors’ contributions

Lei Chen designed the study and wrote the manuscript. Lihan Liu performed the material preparation, data collection and analysis, and wrote the first draft of the manuscript. Guowen Zhang and Xiaolin Wang performed data analysis. Yu Xu, Sha Sha, Wei Li, and Chunfeng Wu helped to revise the manuscript. All authors read and approved the final manuscript.

Funding

This work was supported by grants from the National Natural Science Foundation of China (81971274, Lei Chen), the Jiangsu Provincial Health Commission Key Medical Research Project (ZD2022053, Chunfeng Wu), the Clinical New Technology Project of the Children’s Hospital of Nanjing Medical University (XJS2024-203, Wei Li), and the Nanjing Medical Science and Technology Development Project (YKK24164, Wei Li).

Data availability

All data and materials are presented in this published article or the supplementary information or are available from the corresponding author upon reasonable request.

Declarations

Ethics approval

This study was approved by the Ethics Committee of Nanjing Medical University (No. IACUC2009007).

Consent for publication

Not applicable. This study does not contain any individual person’s data.

Competing interests

The authors declare no competing interests.