Introduction: This study aimed to investigate the possible role of galectin-3 in epilepsy and further explore its underlying mechanisms. Methods: Sprague-Dawley rats were intraperitoneally injected with 30 mg/kg pilocarpine to induce an animal model of epilepsy. To inhibit galectin-3, the epilepsy model of rats was intraperitoneally injected with TD139. The severity of the seizure was graded according to the Racine score. The pathological changes in hippocampal CA1 regions were observed by hematoxylin and eosin and Nissl staining. Enzyme-linked immunosorbent assay, quantitative real-time polymerase chain reaction, and Western blot were used to detect the levels of cytokines and pyroptosis-related factors. The in vitro effects of galectin-3 were confirmed on BV2 cells and rat primary microglia by transfection with lentivirus vectors carrying Lgals3 shRNA or by treatment with TD139. Results: A higher expression of galectin-3 was observed in the hippocampal CA1 regions of epilepsy rats than in sham rats. Inhibition of galectin-3 by administration of TD139 improved the severity of the seizure, hippocampal damage, and neuron loss. TD139 administration suppressed the expression of NLRP3, ASC, c-caspase-1, and GSDMD-N, and reduced the levels of cytokines. In kainic acid-treated microglia, Lgals3 shRNA or TD139 significantly inhibited Iba1 expression and limited NLRP3/pyroptosis-triggered inflammation. Conclusion: Galectin-3 activates the NLRP3/pyroptosis signaling pathway to promote microglial activation and neuroinflammation during epilepsy disease progression.

Highlights

  • High expression of galectin-3 in microglia of rat model of epilepsy.

  • Inhibition of galectin-3 improves pilocarpine-induced epileptic seizure in rat.

  • Galectin-3 inhibition limits neuroinflammation in epilepsy rat.

  • Galectin-3 inhibition represses microglial activation in KA-treated BV2 cells.

  • Galectin-3 inhibition represses the nod-like receptor (NLR) signaling pathway in KA-treated BV2 cells.

Epilepsy is a chronic disease caused by the sudden abnormal discharge of neurons in the brain, resulting in transient brain dysfunction [1]. Epilepsy occurs in all ages, affects more than 65 million people, and is one of the most common neurological diseases in the world [2]. A core feature of epilepsy is recurrent seizures, which are accompanied by comorbidities like anxiety, depression, and cognitive impairment [3], drastically affecting the quality of life of those affected [4].

Epileptic seizures trigger an inflammatory response in the brain by activating the microglia and releasing pro-inflammatory cytokines [5]. A previous study reported that neuroinflammation is associated with neuron death and gliosis [6]. A drastic increase of tumor necrosis factor-α (TNF-α), interleukin 6 (IL-6), and IL-1β was found in the hippocampus of epileptic mice [7]. Galectin-3 is encoded by the Lgals3 gene and is the only chimeric galectin in the galectin family [8, 9]. Galectin-3 is a centric regulator of key processes in the context of inflammation [9] and plays a significant pro-inflammatory role in the induction of colitis via activating the nod-like receptor family pyrin domain containing 3 (NLRP3) inflammasome and promoting IL-1β production in macrophages [10]. Inhibition of galectin-3 by GB0139 reduces lipopolysaccharide-induced lung inflammation [11]. Importantly, galectin-3 was found to be detectable in the serum of patients with epilepsy [12]. Additionally, an increased galectin-3 level was found in rats that had nonconvulsive status epilepticus [13]. In a mouse model of Alzheimer’s disease, galectin-3 was involved in microglial activation, and deletion of galectin-3 improved the cognitive behavior [14]. Nevertheless, the exact role of galectin-3 in epilepsy remains unclear.

In this study, we used a pilocarpine model of epilepsy in vivo and a kainic acid (KA) model of epilepsy in vitro to investigate the possible role of galectin-3 in epilepsy. Moreover, we further explored the mechanism of galectin-3 in epilepsy. Our findings may provide new targets for the treatment of epilepsy.

Bioinformatic Analysis

The Lgals3 gene expression in the epilepsy mouse model was analyzed using the Gene Expression Omnibus database (GEO; GEO accession: GSE88992) [15]. In the GSE88992 dataset, the mouse model of mesio-temporal lobe epilepsy was established by intrahippocampal microinjection of 1 nmol kainite with saline-injected mice as controls. After 6, 12, or 24 h of injection, mice were decapitated, and the total RNA was extracted from their hippocampi for use in gene chip analysis. To predict the signaling pathways associated with Lgals3 that are involved in epilepsy pathogenesis, Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis was performed to analyze the downstream genes of galectin-3. The downstream genes were enriched to analyze the possible signaling pathways that can be regulated by galectin-3 using the Gene Set Enrichment Analysis software.

Animals

A total of 60 male Sprague-Dawley rats (age: 8–10 weeks; weight 200 ± 20 g; Jinan Pengyue Experimental Animal Breeding Co. Ltd.) were fed in a standard laboratory condition with a 12-h light/dark cycle at 23 ± 1°C with water and food ad libitum. All animal experiments were approved by the Ethics Committee for Animal Research of the Yantai Yuhuangding Hospital (no. 2022-074) and performed in compliance with the Yantai Yuhuangding Hospital guidelines for the care and use of animals.

Rat Model of Epilepsy

All the Sprague-Dawley rats were randomly split into four groups (n = 15 for each group): Sham, EP, EP + Vehicle, and EP + TD139 group. For the epilepsy model, rats were injected with lithium chloride (125 mg/kg; dissolved in normal saline; S24113, Yuanye Bio-Technology) intraperitoneally, followed by an intraperitoneal injection with pilocarpine (30 mg/kg; dissolved in normal saline; B20843, Yuanye Bio-Technology) 18–20 h later. Half an hour before the injection of pilocarpine, atropine (1 mg/kg; dissolved in normal saline; HY-B1205, MedChemExpress, Shanghai, China) was administered to inhibit the peripheral cholinergic effects of pilocarpine. Sham rats were injected with the same volume of normal saline intraperitoneally. The behavior of rats was observed after injection. The EP model was successfully established when the seizure activity reached ≥ grade IV and continued for >30 min. For the EP + TD139 group, EP rats were injected intraperitoneally with TD139 (purity >98%; dissolve in dimethylsulfoxide; S0471, Selleck) at 8 mg/kg/d for 10 days starting 4 h after a seizure. For the EP + Vehicle group, EP rats were injected intraperitoneally with the same amount of dimethylsulfoxide.

Observation of Animal Behavior

The severity of seizure was graded according to the Racine score: grade 0: no response; grade I: facial clonus, including blinking, whiskering, and rhythmic chewing; grade II: rhythmic nodding; grade III: forelimb myoclonus, but no hindlimb erection position; grade IV: hindlimb upright position; and grade V: generalized tonic-clonic seizures and loss of postural control. The experimental animals had seizures of ≥ grade IV for 1 h, which could be judged as successful modeling. All rats in each group were video monitored for spontaneous recurrent seizures after the onset of status epilepticus. The observation period lasted 2 weeks, 24 h/day. The behavioral parameters included seizure frequency and seizure duration. After behavioral observation, all rats were sacrificed under anesthesia for the next experiment. The video recording was analyzed by two independent observers who were blinded to the group allocation.

qRT-PCR

Rats were anesthetized intraperitoneally with 3% pentobarbital sodium (50 mg/kg) and then sacrificed by cervical dislocation. The brain was rapidly removed and placed in cold phosphate buffer saline (PBS, G0002, Servicebio). The hippocampal CA1 regions were microdissected by separating the outer part along the longitudinal axis (long axis region) of the hippocampus and used for qRT-PCR. The total RNA of the hippocampal CA1 region was extracted by TRIzol™ reagent (15596026, Thermo Fisher Scientific). The concentration and purity of RNA were assessed by OD260/OD280 (1.8–2.0). The reverse transcription of RNA to complementary DNA was performed using a BeyoRT™II First Strand complementary DNA Synthesis Kit (D7168, Beyotime), followed by qPCR using a ChamQ SYBR qPCR Master Mix (Q311-02, Vazyme). The PCR reactions were as follows: initial denaturation at 95°C for 60 s, followed by 40 cycles of 8 s at 95°C and 20 s at 60°C, and a melting curve stage at 95°C for 15 s, 60°C for 60 s, and 95°C for 15 s. The expression of Lgals3 (forward 5′-TGG​CCA​TAG​GAA​TCA​GGT​GC-3′ and reverse, 5′-CCG​TCT​GCC​ATT​TTC​CGA​AG-3′) was evaluated using the 2-ΔΔCt method [16]. β-actin (forward 5′-CCC​GCG​AGT​ACA​ACC​TTC​TTG-3′ and reverse 5′-GTC​ATC​CAT​GGC​GAA​CTG​GTG-3′) was used as an internal reference.

Histopathology

The pathological changes of the hippocampal CA1 regions from all five rats in each group were observed by hematoxylin and eosin (H&E) and Nissl staining. The paraffin sections were deparaffinized with xylene and rehydrated with gradient of ethanol and distilled water. For H&E staining, sections were stained with hematoxylin (C0105, Beyotime) for 7 min and counterstained with eosin for 2 min. Subsequently, the sections were dehydrated with 70% ethanol for 2 min, 80% ethanol for 2 min, 90% ethanol for 2 min, and anhydrous ethanol for 2 min. For Nissl staining, sections were stained with Nissl staining solution (G1036 Servicebio, Wuhan, China) at 37°C for 30 min. The sections were dehydrated with 95% ethanol for 2 min and then dehydrated with fresh 95% ethanol for 2 min. Subsequently, sections were transparentized with xylene for 5 min and then transparentized with fresh xylene for 5 min. Finally, sections were sealed using neutral gum. Pictures of H&E and Nissl staining were taken by using a light microscope (Nikon Corporation, Tokyo, Japan).

Preparation of Rat Primary Microglia

Primary microglia were isolated from the cerebral cortices of 1- to 2-day-old Sprague-Dawley rats, as previously reported [17]. After detaching the meninges and dissociating them by trituration, the cerebral cortices were digested in 0.25% trypsin for 30 min at 37°C. The tissue fragments were maintained in Dulbecco’s Modified Eagle Medium (DMEM) (L110KJ, BasalMedia, Shanghai, China) containing 10% FBS (S660JY, BasalMedia), and the single-cell suspension was made by repeated pipetting. After culturing at 37°C for 10–14 days, the microglia were harvested by gentle agitation.

BV2 Cell Culture

The mouse microglia BV2 cells (CL-0493, Procell, Wuhan, China) were cultured in DMEM supplemented with 10% FBS and 1% penicillin/streptomycin (PB180120, Procell). The cells were cultured at 37°C with 5% CO2.

To establish an in vitro model, KA (purity >99%, ab144490, Abcam) was used to stimulate BV2 cells or primary microglia at 100 μm for 24 h [18]. For TD139 treatment, BV2 cells were stimulated with TD139 at 20 μm for 24 h [19].

Cell Transfection

The lentivirus vectors for carrying Lgals3 shRNA (LV-shLgals3) and its negative control were purchased from GeneChem (Shanghai, China). BV2 cells or primary microglia were cultured in serum-free DMEM until they reached 30% confluence. The polybrene (8 μg/mL)- and lentivirus-contained green fluorescent protein was added and incubated for 6 h at 37°C. After replacement with normal medium, cells were cultured continuously for 72 h. The transfection efficiency of the cells was observed by fluorescence microscopy.

ELISA

Blood samples were collected from rats after anesthetization with 3% pentobarbital sodium (50 mg/kg) intraperitoneally and then centrifuged at 500 g for 10 min at 4°C to obtain plasma. The galectin-3 level in plasma was detected by an ELISA kit for galectin-3 (CSB-EL012887RA, CUSABIO, Wuhan, China). The hippocampal CA1 region was homogenized on ice and centrifuged at 300 g for 10 min at 4°C. The levels of inflammatory factors in the rat hippocampal CA1 region were detected by a rat IL-1β ELISA kit (PI303, Beyotime), a rat IL-18 ELISA kit (PI555, Beyotime), a rat TNF-α ELISA kit (E-EL-R2856c, Elabscience, Wuhan, China), a rat IL-6 ELISA kit (E-EL-R0896c, Elabscience), and a rat IL-10 ELISA kit (E-EL-R0016c, Elabscience). The levels of inflammatory factors in the cell supernatant were detected by a mouse IL-1β ELISA kit (PI301, Beyotime), a mouse IL-18 ELISA kit (PI553, Beyotime), a mouse TNF-α ELISA kit (PT512, Beyotime), a mouse IL-6 ELISA kit (PI326, Beyotime), and a mouse IL-10 ELISA kit (PI522, Beyotime).

Cell Immunofluorescence

The expression and localization of Iba1 in BV2 cells or primary microglia were detected by immunofluorescence [20]. Cells were fixed with 4% paraformaldehyde for 15 min at 25°C and then washed thrice in PBS. After permeabilization with 0.25% Triton X-100 (P0096, Beyotime, Shanghai, China) for 15 min and blockade with 5% BSA for 1 h at 37°C, cells were incubated with the primary antibody anti-Iba1 antibody (ab178847, Abcam; 1/100 dilution) at 4°C overnight. Subsequently, the cells were washed with PBS thrice and then incubated with the secondary antibody for 1 h at 37°C, followed by nuclear counterstaining with Hoechst (GDP1025, Servicebio) for 5 min. An image was acquired by immunofluorescence microscopy (Nikon, Japan).

Western Blotting

The total proteins of hippocampal CA1 regions and microglia were extracted using the Radioimmunoprecipitation Assay Lysis Buffer (RIPA, G2002-100 ML, Servicebio, Wuhan, China). The protein concentration was determined using the Bicinchoninic Acid Protein Assay Kit (CW0014S, CWBIO, Jiangsu, China). The protein samples (20 μg) were then separated by 10% SDS-PAGE and transferred onto PVDF membranes. The membranes were blocked with 10% BSA for 2 h at 25°C. Subsequently, the membranes were incubated with primary antibodies against galectin-3 (ab227249, Abcam), Iba1 (ab178846, Abcam), NLRP3 (ab263899, Abcam), apoptosis-associated speck-like protein containing a CARD domain (ASC; ab180799, Abcam), IL-1β (ab254360, Abcam), IL-6 (ab9324, Abcam), c-caspase-1 (PA5-77886, Thermo Fisher Scientific), gasdermin D (GSDMD)-N (#39754, CST), IL-18 (SAB5700754, Sigma-Aldrich), IL-10 (SAB5700775, Sigma-Aldrich), TNF-α (17590-1-AP, Proteintech), and β-actin (20536-1-AP, Proteintech), all at 1/1000 dilution, at 4°C overnight. Anti-rabbit IgG HRP-linked antibody at January 2000 dilution (7074 s, Cell Signaling Technology) was used for 2-h incubation at 25°C. The bands were visualized using a high-sensitivity ECL detection kit (E412-02, Vazyme, Nanjing, China) and analyzed by ImageJ (NIH, Bethesda, MD, USA).

Statistical Analysis

Data were shown as the mean ± standard deviation and analyzed using GraphPad Prism7.0 software (GraphPad Inc., San Diego, CA, USA). The difference between the two groups was analyzed by Student’s t-test. The differences among multiple groups were analyzed by a one-way analysis of variance followed by Tukey’s post hoc test. p < 0.05 was considered statistically significant.

High Expression of Galectin-3 in the Microglia of a Rat Model of Epilepsy

Firstly, we analyzed the differential expression of Lgals3 in the hippocampi of animals based on the GSE88992 dataset. Lgals3 gene mRNA was highly expressed in the hippocampus of epileptic mice compared to sham mice (Fig. 1a). In this study, Sprague-Dawley rats were intraperitoneally injected with pilocarpine to induce epileptic symptoms. Plasma samples were collected at 4 and 24 h after pilocarpine injection to detect galectin-3 levels by ELISA. The data showed that the galectin-3 level was high in the epilepsy group compared to the sham group (Fig. 1b). Lgals3 expression in the hippocampal CA1 regions was higher in rats with epilepsy than in sham rats (Fig. 1c). Additionally, the expression of galectin-3 and Iba1 (a marker of microglia) in the hippocampal CA1 regions was measured by Western blotting and immunofluorescence staining. Both galectin-3 and Iba1 had much higher expression in the hippocampal CA1 regions of rats with epilepsy than in sham rats (Fig. 1d). The high expression of galectin-3 in the hippocampus and plasma of rats with epilepsy suggests that galectin-3 expression may be related to microglial activation during the pathogenesis of epilepsy.

Fig. 1.

High expression of galectin-3 in microglia of the rat model of epilepsy. aLgals3 gene levels in the sham (n = 10) and EP (n = 7) mice from the GSE88992 dataset. b-d Sprague-Dawley rats intraperitoneally injected with pilocarpine were used for animal models of epilepsy. Sham rats were injected with the same volume of normal saline. b The plasma samples were collected 4 and 24 h after pilocarpine injection and the galectin-3 protein level was analyzed using an ELISA kit (n = 15). cLgals3 gene expression in hippocampal CA1 regions was detected by qRT-PCR (n = 15). d The protein expression of galectin-3 and Iba1 in hippocampal CA1 regions was detected by Western blot (n = 5). **p < 0.01, ***p < 0.001.

Fig. 1.

High expression of galectin-3 in microglia of the rat model of epilepsy. aLgals3 gene levels in the sham (n = 10) and EP (n = 7) mice from the GSE88992 dataset. b-d Sprague-Dawley rats intraperitoneally injected with pilocarpine were used for animal models of epilepsy. Sham rats were injected with the same volume of normal saline. b The plasma samples were collected 4 and 24 h after pilocarpine injection and the galectin-3 protein level was analyzed using an ELISA kit (n = 15). cLgals3 gene expression in hippocampal CA1 regions was detected by qRT-PCR (n = 15). d The protein expression of galectin-3 and Iba1 in hippocampal CA1 regions was detected by Western blot (n = 5). **p < 0.01, ***p < 0.001.

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Inhibition of Galectin-3 Improves Pilocarpine-Induced Epileptic Seizures in Rats

To probe the role of galectin-3 in the pathogenesis of epilepsy, the galectin-3 inhibitor, TD139, was administrated. The timeline of pilocarpine and TD139 injections is shown in Figure 2a. The Racine score was used to monitor behavioral changes in rats. The Racine score of the sham rats was 0, while that of the epilepsy model rats reached 4.13 ± 0.74, indicating that the model rats had severe epilepsy (Fig. 2b). Compared to the Racine score of the EP + Vehicle group (4.2 ± 0.68), the Racine score of the EP + TD139 group was 3.27 ± 0.80, indicating that injection of TD139 significantly reduced the Racine score of epilepsy rats (Fig. 2b). The frequency and duration of seizures were also recorded. The frequency of seizures in the epilepsy group was 4.47 ± 1.25 times/day (Fig. 2c), and the mean duration of seizures was 22.23 ± 3.97 s (Fig. 2d). In the EP + TD139 group, the frequency of seizures was 3.6 ± 1.12 times/day, and the mean seizure duration was 16.39 ± 3.94 s. These data showed that TD139 injection significantly reduced the frequency and duration of seizures in rats. In addition, H&E and Nissl staining were performed to assess the pathological changes and neuron survival of the hippocampal CA1 regions. The results showed that neurons were arranged in rows in the hippocampal CA1 regions of rats in the sham group, while the neurons in the epilepsy group were disordered, indicating damage to the hippocampal tissue in rats in the epilepsy group (Fig. 2e). Compared to vehicle injection, TD139 injection alleviated tissue damage in model rats (Fig. 2e). In addition, Nissl staining results showed abundant Nissl bodies in the CA1 regions of sham rats, while fewer Nissl bodies were observed in the epilepsy model rats, suggesting that more neurons were lost in the hippocampus of epilepsy rats (Fig. 2e). Compared to the vehicle injection, the neuronal loss in CA1 regions was obviously alleviated by TD139 injection.

Fig. 2.

Inhibition of galectin-3 improves pilocarpine-induced epileptic seizure in rat. a-d Sprague-Dawley rats intraperitoneally injected with pilocarpine were used for animal models of epilepsy. Sham rats were injected with same volume of normal saline. TD139 at 8 mg/kg/d was intraperitoneally injected into rats for 10 days, starting 4 h after seizure. The same amount of vehicle (DMSO) was injected as a blank control. The Racine score (b), frequency of seizures per day (c), and duration of seizures (d) of rats in each group were recorded after pilocarpine injection (n = 15). e H&E and Nissl staining of hippocampal CA1 regions of rats in each group (n = 5). *p < 0.05, **p < 0.01, ***p < 0.001.

Fig. 2.

Inhibition of galectin-3 improves pilocarpine-induced epileptic seizure in rat. a-d Sprague-Dawley rats intraperitoneally injected with pilocarpine were used for animal models of epilepsy. Sham rats were injected with same volume of normal saline. TD139 at 8 mg/kg/d was intraperitoneally injected into rats for 10 days, starting 4 h after seizure. The same amount of vehicle (DMSO) was injected as a blank control. The Racine score (b), frequency of seizures per day (c), and duration of seizures (d) of rats in each group were recorded after pilocarpine injection (n = 15). e H&E and Nissl staining of hippocampal CA1 regions of rats in each group (n = 5). *p < 0.05, **p < 0.01, ***p < 0.001.

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Inhibition of Galectin-3 Limits NLRP3 Inflammasome-Triggered Neuroinflammation Rats with Epilepsy

To further explore the mechanism of galectin-3 in epilepsy, KEGG analysis based on the GSE88992 dataset was performed to analyze the downstream genes of galectin-3. The downstream genes of galectin-3 are shown in Figure 3a. The enrichment analysis showed that galectin-3 activated the nod-like receptor (NLR) signaling pathway (Fig. 3b). The protein expression of NLRP3, ASC, c-caspase-1, and GSDMD-N was significantly higher in the hippocampal tissues of epilepsy rats compared to sham rats (Fig. 3c), suggesting that pyroptosis mediated by the NLRP3 inflammasome occurred in the hippocampal tissue of rats with epilepsy. Notably, TD139 injection suppressed the expression of those proteins (Fig. 3c). The levels of pro-inflammatory factors IL-1β, IL-18, TNF-α, and IL-6 were markedly higher, while the anti-inflammatory factor IL-10 was lower in the hippocampal tissues of the epilepsy group than in the sham group (Fig. 3d). TD139 injection dramatically inhibited these inflammatory factor changes in hippocampal tissues (Fig. 3d). These results indicate that galectin-3 inhibition attenuates NLRP3/pyroptosis-mediated hippocampal inflammation in rats with epilepsy.

Fig. 3.

Inhibition of galectin-3 limits NLRP3 inflammasome-triggered neuroinflammation in epilepsy rat. a Based on GSE88992 data, KEGG analyzed the downstream genes that can be regulated by galectin-3. b The signaling pathway enrichment analysis showed that galectin-3 activated the nod-like receptor signaling pathway. c-d Sprague-Dawley rats intraperitoneally injected with pilocarpine were used for animal models of epilepsy. Sham rats were injected with the same volume of normal saline. TD139 at 8 mg/kg/d was intraperitoneally injected into rats for 10 days starting 4 h after seizure. The same amount of vehicle (DMSO) was injected as a blank control. c The protein expression of NLRP3, ASC, C-caspase-1, and GSDMD-N in hippocampal tissues was detected by Western blot (n = 5). d The levels IL-1β, IL-18, TNF-α, IL-6, and IL-10 of in the hippocampal tissues were detected using ELISA kits (n = 10). *p < 0.05, **p < 0.01, ***p < 0.001.

Fig. 3.

Inhibition of galectin-3 limits NLRP3 inflammasome-triggered neuroinflammation in epilepsy rat. a Based on GSE88992 data, KEGG analyzed the downstream genes that can be regulated by galectin-3. b The signaling pathway enrichment analysis showed that galectin-3 activated the nod-like receptor signaling pathway. c-d Sprague-Dawley rats intraperitoneally injected with pilocarpine were used for animal models of epilepsy. Sham rats were injected with the same volume of normal saline. TD139 at 8 mg/kg/d was intraperitoneally injected into rats for 10 days starting 4 h after seizure. The same amount of vehicle (DMSO) was injected as a blank control. c The protein expression of NLRP3, ASC, C-caspase-1, and GSDMD-N in hippocampal tissues was detected by Western blot (n = 5). d The levels IL-1β, IL-18, TNF-α, IL-6, and IL-10 of in the hippocampal tissues were detected using ELISA kits (n = 10). *p < 0.05, **p < 0.01, ***p < 0.001.

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Inhibition of Galectin-3 Represses Microglial Activation in KA-Treated Microglia

Studies have demonstrated that KA-stimulated microglia activation exhibits typical postepileptic responses [21, 22]. To verify the effect of galectin-3 on epilepsy, KA-stimulated microglia were used as an in vitro epilepsy model. LV-shLgals3 was transfected into BV2 cells and primary microglia to knockdown Lgals3. Cell immunofluorescence proved that the proportion of Iba1-staining cells was markedly increased and that the cell morphology was more hypertrophic in the KA group compared to the control group (Fig. 4a). LV-shLgals3 transfection significantly inhibited Iba1-staining in KA-stimulated BV2 cells (Fig. 4a). TD139 was employed to inhibit galectin-3 expression in BV2 cells (Fig. 4b). Galectin-3 inhibition markedly suppressed the staining intensity of Iba1 in BV2 cells (Fig. 4b). Similarly, LV-shLgals3 inhibited Iba1-staining in KA-stimulated primary microglia (Fig. 4c). Compared to the control group, the expression of galectin-3 and Ibal was markedly increased in the KA group (Fig. 4d–f. After LV-shLgals3 transfection, the expression of galectin-3 and Iba1 in KA-stimulated BV2 cells and primary microglia was significantly lower than that in nontransfected KA-stimulated cells (Fig. 4d, F). Likewise, protein expression of galectin-3 and Iba1 in the KA + TD139 group was remarkably decreased compared to the KA + Vehicle group (Fig. 4e).

Fig. 4.

Inhibition of galectin-3 represses microglial activation in KA-treated microglia. a-f BV2 cells or primary microglia were treated with 100 μm KA for 24 h. For inhibition of galectin-3, BV2 cells or primary microglia were transfected with lentivirus vectors carrying Lgals3 shRNA or treated with 20 μm TD139 for 24 h. a-c The Iba1 expression was analyzed by immunofluorescence. d-f The protein expression of galectin-3 and Iba1 was analyzed by Western blot (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001.

Fig. 4.

Inhibition of galectin-3 represses microglial activation in KA-treated microglia. a-f BV2 cells or primary microglia were treated with 100 μm KA for 24 h. For inhibition of galectin-3, BV2 cells or primary microglia were transfected with lentivirus vectors carrying Lgals3 shRNA or treated with 20 μm TD139 for 24 h. a-c The Iba1 expression was analyzed by immunofluorescence. d-f The protein expression of galectin-3 and Iba1 was analyzed by Western blot (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001.

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Inhibition of Galectin-3 Represses the Activation of NLRP3 Inflammasome in KA-Treated Microglia

The expression of pyroptosis-related proteins NLRP3, ASC, C-caspase-1, and GSDMD-N in microglia was detected by Western blotting. KA treatment significantly enhanced the expression of these proteins. Notably, the expression of these proteins was significantly reduced by LV-shLgals3 (Fig. 5a). After inhibition of galectin-3 by adding TD139, the expression of these proteins was significantly downregulated in BV2 cells (Fig. 5b). Similarly, LV-shLgals3 significantly reduced the expression of pyroptosis-related proteins in KA-stimulated primary microglia (Fig. 5c). The above data suggest that galectin-3 inhibition alleviates the activation of NLRP3/pyroptosis during epilepsy in vitro.

Fig. 5.

Inhibition of galectin-3 represses the activation of NLRP3 inflammasome in KA-treated microglia. a-c BV2 cells or primary microglia were treated with 100 μm KA for 24 h. For inhibition of galectin-3, BV2 cells or primary microglia were transfected with lentivirus vectors carrying Lgals3 shRNA or treated with 20 μm TD139 for 24 h. The expression of NLRP3, ASC, C-caspase-1, and GSDMD-N was analyzed by Western blot (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001.

Fig. 5.

Inhibition of galectin-3 represses the activation of NLRP3 inflammasome in KA-treated microglia. a-c BV2 cells or primary microglia were treated with 100 μm KA for 24 h. For inhibition of galectin-3, BV2 cells or primary microglia were transfected with lentivirus vectors carrying Lgals3 shRNA or treated with 20 μm TD139 for 24 h. The expression of NLRP3, ASC, C-caspase-1, and GSDMD-N was analyzed by Western blot (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001.

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Inhibition of Galectin-3 Represses Cytokine Levels from Microglia

The changes in inflammatory factor levels in the microglia supernatant were detected by ELISA and Western blotting. KA treatment markedly increased the release of IL-1β, IL-6, IL-18, and TNF-α and decreased the release of IL-10 in BV2 cells and primary microglia (Fig. 6a–c). Transfection of LV-shLgals3 or treatment with TD139 markedly inhibited these changes in inflammatory factors. Similar results were observed on Western blotting. KA treatment significantly decreased IL-10 protein expression and increased the protein levels of IL-1β, IL-6, IL-18, and TNF-α in microglia (Fig. 6d–f. Galectin-3 inhibition obviously suppressed the changes in these inflammatory factors in microglia. These results suggest that inhibition of galectin-3 suppresses the inflammatory levels of KA-stimulated microglia.

Fig. 6.

Inhibition of galectin-3 represses cytokine levels from microglia. a-f BV2 cells or primary microglia were treated with 100 μm KA for 24 h. For inhibition of galectin-3, BV2 cells or primary microglia were transfected with lentivirus vectors carrying Lgals3 shRNA or treated with 20 μm TD139 for 24 h. a-c The concentrations of IL-1β, IL-18, TNF-α, IL-6, and IL-10 in the culture supernatant of cells were analyzed by ELISA kits. d-f The protein levels of IL-1β, IL-18, TNF-α, IL-6, and IL-10 treatment were assessed by Western blot (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001.

Fig. 6.

Inhibition of galectin-3 represses cytokine levels from microglia. a-f BV2 cells or primary microglia were treated with 100 μm KA for 24 h. For inhibition of galectin-3, BV2 cells or primary microglia were transfected with lentivirus vectors carrying Lgals3 shRNA or treated with 20 μm TD139 for 24 h. a-c The concentrations of IL-1β, IL-18, TNF-α, IL-6, and IL-10 in the culture supernatant of cells were analyzed by ELISA kits. d-f The protein levels of IL-1β, IL-18, TNF-α, IL-6, and IL-10 treatment were assessed by Western blot (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001.

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Epilepsy is one of the most common neurological diseases and seriously affects the quality of life of patients and their families [23]. In recent years, there have been many antiepileptic drugs, but their therapeutic effect is not ideal. Still, nearly one-third of patients had poor responses to antiepileptic drugs [24, 25]. A series of pathophysiological changes have been found in the hippocampal CA1 region during epilepsy [26, 27]. The CA1 region is more vulnerable to damage from seizures, ischemia, and head trauma as compared to other areas [28‒30]. Therefore, this study focused on the pathophysiology of the hippocampal CA1 region to investigate the effect of galectin-3 on epileptic rats and microglia. Previous evidence supports that abnormally high galectin-3 expression is related to neuronal degeneration in the hippocampus [31]. Our results suggest that the inhibition of galectin-3 knockdown reduces neuroinflammation and attenuates hippocampal damage in epileptic rats by regulating the NLRP3/pyroptosis pathway.

Abnormal expression of galectin has been found in patients with neuronal degeneration [32]. Galectin-8 and galectin-9 are markedly enhanced in active lesions of multiple sclerosis [33]. Serum galectin-4 levels are quite higher in patients with Parkinson’s disease than in healthy subjects [32]. Galectin-3 levels are significantly increased in patients with neurological diseases, such as Parkinson’s disease, amyotrophic lateral sclerosis, Huntington’s disease, and epilepsy [31, 34‒36]. In our study, galectin-3 was observed to be highly expressed in the hippocampal CA1 region of epileptic rats. At present, the expression of galectin-3 in the central nervous system (CNS) remains controversial. Mangano et al. [37] mentioned that galectin-3 was significantly highly expressed in microglia, astrocytes, and oligodendrocytes isolated from the spinal cord of EAE mice, as well as in human MS-related white matter lesions. However, using fluorescence localization assays, Bischoff et al. [38] and Lalancette-Hébert et al. [39] reported that galectin-3 is exclusively expressed by microglia rather than by astrocytes, neurons, and other cellular types in CNS as well. In addition, the role of galectin-3 in microglia, rather than other cellular types, has been widely studied. As its pivotal role in modulating microglial activation, it has been recently considered a potential target for neuroinflammatory diseases [40, 41]. We cannot be so sure that galectin-3 is only expressed by microglia, but we can conclude that microglia are the major cellular type for producing galectin-3.

Microglia are the resident immunomodulatory cells within the CNS and can be classified into two phenotypes: M1 and M2. M1-polarized cells are pro-inflammatory cells that express iNOS, CD16/32, and cytokines, like TNF-α and IL-1β. M2-polarized cells are anti-inflammatory cells that express anti-inflammatory cytokines, like IL-10, to exert reparative and neuroprotective capacities [42]. The number of M1/M2 microglia varies in CNS diseases like epilepsy [42]. Taniwaki et al. [43] did not observe activated microglia 4 h after seizure induction in rats but detected activated microglia 8 and 24 h later. In addition, activated microglia are typically located in the hippocampal CA1 area, which is the major location for hyperexcitability and seizures [44, 45]. Microglial activation has been recognized as one of the major pathological hallmarks of epilepsy [42]. Iba1, also known as allograft inflammatory factor 1, is a cellular marker of microglia and is upregulated during microglial activation [46, 47]. Our results showed that Iba1 was highly expressed in the hippocampal CA1 area of epileptic rats, indicating microglial activation in epileptic rats. In addition, fluorescence localization found that galectin-3 was mainly expressed in microglia, both in the cytoplasm and nucleus [38, 39]. These results suggest that galectin-3 is highly expressed in epileptic rats and that the high expression of galectin-3 may be related to the activation of microglia during the pathogenesis of epilepsy.

The inflammatory process in the CNS is commonly referred to as neuroinflammation [14]. Clinical studies have shown that the levels of pro-inflammatory factors in the cerebrospinal fluid of epilepsy patients are significantly increased [48]. Increased pro-inflammatory factors have also been observed in the brain tissue of an animal model of epilepsy [49]. In our study, the levels of pro-inflammatory factors, including IL-1β, TNF-α, IL-18, and IL-6, were significantly increased in the hippocampal CA1 area of epileptic rats. Microglia are the main source of pro-inflammatory factors within the CNS [50]. We used a KA model of epilepsy in vitro to investigate the possible role of galectin-3 in epilepsy. Elevated levels of pro-inflammatory factors, including IL-1β, TNF-α, IL-18, and IL-6, and decreased levels of the anti-inflammatory factor IL-10 were observed in KA-induced microglia. Additionally, M1 polarization of microglia was observed in KA-induced microglia. Galectin-3, a key regulator of microglial activity, plays a key role as an inflammatory mediator in neurodegeneration [40, 51]. In the present study, galectin-3 expression was suppressed by using TD139 or Lgals3 shRNA. We found that galectin-3 inhibition significantly reduced the levels of pro-inflammatory factors and raised the level of the anti-inflammatory factor in microglia. Moreover, galectin-3 inhibition reduced the frequency and duration of seizures, as well as hippocampal damage in epileptic rats, indicating that galectin-3 inhibition may alleviate epilepsy-induced hippocampal damage by reducing neuroinflammation.

NLRs are a family of proteins containing nucleotide-binding oligomerization domains, which are widely present in the human cytoplasm [52]. NLRs, together with the apoptosis-associated speck-like protein containing ASC and caspase-1, constitute the inflammasome [53]. Activation of the inflammasome can promote the cleavage and activation of caspase-1 [54]. Caspase-1 activation promotes the maturation and secretion of IL-1β and IL-18, as well as the cleavage of GSDMD, which eventually leads to pyroptosis [55]. In the present study, bioinformatics analysis indicates that galectin-3 activated the NLR signaling pathway. The levels of pyroptosis-related proteins were markedly increased in rats with epilepsy and KA-stimulated microglia. After galectin-3 expression was inhibited by TD139 or galectin-3 shRNA, the expression of these pyroptosis-related proteins was significantly suppressed in both rats with epilepsy and KA-stimulated microglia. These findings suggest that galectin-3 inhibition alleviates NLRP3/pyroptosis activation during epilepsy.

In conclusion, galectin-3 is highly expressed during the pathogenesis of epilepsy. High-level expression of galectin-3 promotes microglial activation and neuroinflammation, thereby participating in the progression of epilepsy by activating NLRP3/pyroptosis.

All animal experiments were approved by the Ethics Committee for Animal Research of the Yantai Yuhuangding Hospital (no. 2022-074) and performed in compliance with the Yantai Yuhuangding Hospital guidelines for the care and use of animals.

The authors declare that they have no conflicts of interest.

Funding is not included in this study.

Conception, design, and manuscript writing: W.W.S.; collection and assembly of data: W.W.S., Y.H., and C.X.L.; data analysis and interpretation: Y.Y.Z., H.S.Y., and L.W.; and final approval of manuscript: all authors.

Additional Information

Weiwei Sun and Ying Hao contributed equally to this work.

All data generated or analyzed during this study are included in this article. Further inquiries can be directed to the corresponding author.

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