Pyroptosis was involved in SE but not in absence seizures

Pilocarpine (PILO) is a widely-used drug that induces spontaneous seizures and partially mimics temporal lobe epilepsy [7]. Here, we injected PILO to C57BL/6 J mice to establish an in vivo model of status epilepticus (SE), according to the previous study [29].

We first conducted RNA sequencing on hippocampus from PILO-treated and vehicle-treated C57BL/6 J mice. In Fig. 1A, we identified 165 differentially expressed genes (DEGs) between PILO-treated mice and control mice, with 153 up-regulated genes and 12 down-regulated genes. Gene Ontology (GO) enrichment analysis (Fig. 1B) of the upregulated genes suggested that cell death processes were significantly activated in the PILO-induced SE model, potentially initiating epileptogenesis.

Fig. 1

Pyroptosis was involved in SE but not in absence seizures A, B The volcano plot and GO enrichment analysis of the DEGs in mice treated with PILO compared with healthy C57BL/6 J controls (n = 5). The red dots represent up-regulated genes, while the blue dots represent down-regulated ones. C, D ELISA analysis of IL-18 (C) and IL-1β (D) in the serum of idiopathic epilepsy patients (n = 11) and healthy controls (n = 9). E–G Immunofluorescence analysis of NLRP3 (green) and NeuN (red) expression in hippocampus of PILO-treated C57BL/6 J mice (60 × lens), including the CA1, CA3 and DG regions (n = 9). Scale bar: 100 μm. DAPI (blue) was used to label nucleus. H, I The representative protein bands of NLRP3, GSDMD-N, and caspase-1 p20 in hippocampus of PILO-treated C57BL/6 J mice (n = 6) and cultured neurons treated with PILO for 24 h (n = 6). J Immunofluorescence analysis of NLRP3 (green) expression in cultured neurons (20 × lens) treated with PILO for 24 h (n = 6). Scale bar: 100 μm. DAPI (blue) was used to label nucleus. K The representative protein bands of NLRP3, GSDMD, and caspase-1 p20 of control Wistar and TRM rats (n = 6). Full scans of all the blots are in the Supplementary Note. *P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001

Pyroptosis is a programmed cell death that has been not commonly associated with the occurrence of SE, we then investigated whether pyroptosis associated factors were altered in patients with idiopathic epilepsy and the PILO-treated SE models. Serum levels of free IL-18 and IL-1β were significantly higher in idiopathic epilepsy patients compared to healthy controls, as measured by ELISA (Fig. 1, C and D).

Additionally, the protein expression levels of NLRP3, GSDMD, and caspase-1 p20 (Fig. 1H) were enhanced in hippocampal tissues of PILO-treated SE mice, as confirmed by western blot. Moreover, we detected the expression of NLRP3 and neuronal nuclei (NeuN), a neuron marker, in hippocampus of PILO-treated SE mice by immunofluorescence. PILO-treated epileptic mice exhibited higher relative fluorescence intensity of NLRP3 in cornu ammonis 1 (CA1), cornu ammonis 3 (CA3), and dentate gyrus (DG) regions (Fig. 1, E and F). Meanwhile, the number of neurons were decreased in CA1, CA3, and DG regions (Fig. 1, E and G) of PILO induced SE mice.

Then, hippocampal neurons treated with 10 μM PILO for 24 h showed significantly increased protein levels of NLRP3, GSDMD, and caspase-1 p20 (Fig. 1I). For investigate the location and expression level of NLRP3 protein, immunofluorescence staining was performed in cultured hippocampal neurons after 10 μM PILO treatment for 24 h. 10 μM PILO treatment significantly enhanced the relative fluorescence intensity of NLRP3 (Fig. 1J).

The tremor rat (TRM) is capable to mimic human absence-like seizures and demonstrate spontaneous spike and wave discharges in electroencephalogram (EEG) recordings [30]. However, compared with the Wistar controls, the changes of pyroptosis associated factors including NLRP3, GSDMD, and caspase-1 p20 were unaltered (Fig. 1K) in TRM group, suggesting that pyroptosis was involved in SE but not in absence seizures.

To further explore the effects of PILO, we treated N2a cells with various concentrations of PILO and analyzed cell viability after 48 h using the CCK-8 assay. Cell vitality decreased in a dose-dependent manner, with a half maximal inhibitory concentration (IC50) of 12 mM in N2a cells (Supplemental Fig. 1A). After treating N2a cells with 12 mM PILO for different time intervals 0 h, 12 h, 24 h, 36 h, and 48 h, we tested the changes of pyroptosis associated factors by western blot. Compared with the response at 0 h, protein levels of NLRP3, GSDMD, and caspase-1 p20 (Supplemental Fig. 1B) in 24 h group were significantly enhanced. As a positive control for caspase-1-dependent pyroptosis [31], N2a cells were pretreated with 1 μg/mL lipopolysaccharide (LPS) for 4 h and then were treated with 15 μM Nigericin for 1 h to measure the changes of pyroptosis associated factors. Compared with the control group, the protein levels of NLRP3, GSDMD, caspase-1 p20 (Supplemental Fig. 1C), IL-18 (Supplemental Fig. 1D), and IL-1β (Supplemental Fig. 1E) were significantly increased in the N2a cells of LPS group. Meanwhile, compared with the control group, the protein levels of pyroptosis associated factors (Supplemental Fig. 1, F–H) in the C57BL/6 J mice injected intraperitoneally with LPS (20 mg/kg) were also significantly increased. Taken together, PILO treatment can promote pyroptosis in SE models. Thus, pyroptosis was involved in SE but not in absence seizures.

TRPM7 was increased in SE models and participated in pyroptosis in vitro

TRPM7 has been involved in several neurological diseases including ischemic injury, Alzheimer’s disease, and Parkinson’s disease [32]. However, the relationship between TRPM7 and SE is currently unknown. We analyzed six transcriptome datasets of the hippocampus of normal mice (Supplemental Fig. 2A) and normal human (Supplemental Fig. 2B) brains, and found that TRPM7 was in the top three levels of TRPM subfamily gene expression in both healthy human and healthy mice hippocampal samples in each dataset. To investigate the involvement of TRPM7 in SE, we detected the change of TRPM7 in SE models in vivo and in vitro. TRPM7 protein expression in C57BL/6 J mice hippocampal tissues was enhanced in PILO-treated SE mice by western blot (Fig. 2, A, B). Moreover, we detected TRPM7 protein expression and the colocalization with NeuN in hippocampus of PILO-treated SE mice by immunofluorescence. The expression of TRPM7 was increased in CA1, CA3, and DG regions (Fig. 2, E and F) of PILO-treated SE mice. Additionally, PILO-treated SE mice exhibited higher amount of TRPM7+ cells in NeuN+ neurons in CA1, CA3, and DG regions (Fig. 2, E and G). Similarly, TRPM7 protein expression in Mg2+-free treatment group (Fig. 2, C and D) was also increased. Consistently, PILO treatment significantly enhanced the expression of TRPM7 protein in PILO-treated hippocampal neurons (Fig. 2, C and D). Additionally, the protein expression of TRPM7 in PILO-treated N2a cells was significantly increased in PILO group compared with the control group (Fig. 2, H, I).

Fig. 2

TRPM7 was increased in SE models and participated in pyroptosis in vitro. A, B The representative protein bands and analysis of TRPM7 in hippocampus of PILO-treated C57BL/6 J mice (n = 6). C, D The representative protein bands and analysis of TRPM7 of cultured neurons treated with PILO for 24 h or cultured in Mg2+-free extracellular fluid for 3 h before cultured in the original medium for 21 h (n = 6).E–G Immunofluorescence analysis of TRPM7 (green) and NeuN (red) expression in hippocampus of PILO-treated C57BL/6 J mice, including the CA1, CA3 and DG regions (20 × lens or 60 × lens). The arrows indicated positive co-localization neurons (n = 9). Scale bar: 100 μm. DAPI (blue) was used to label nucleus. H, I The representative protein bands and analysis of TRPM7 in PILO-treated N2a cells (n = 6). J, K The representative protein bands and analysis of NLRP3, GSDMD, and caspase-1 p20 in TRPM7-overexpressed N2a cells (n = 5 or 6). Full scans of all the blots are in the Supplementary Note. *P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001

The relationship between TRPM7 and pyroptosis remained unclear and we hypothesized that TRPM7 could contribute to pyroptosis. Therefore, we transfected TRPM7 siRNA and TRPM7 (ion channel segment) overexpression plasmid into N2a cells to explore the relationship between TRPM7 and pyroptosis. The knockdown (Supplemental Fig. 2C) and overexpression (Supplemental Fig. 2D) efficiency were examined by real-time qRT-PCR. TRPM7 overexpression significantly enhanced the protein levels of NLRP3, GSDMD, and caspase 1 p20 (Fig. 2, J and K). Meanwhile, in empty vector-transfected N2a cells, PILO treatment elevated the protein levels of NLRP3, GSDMD, and caspase 1 p20 (Supplemental Fig. 2E). PILO treatment in TRPM7-overexpressed N2a cells further increased the protein levels of NLRP3, GSDMD, and caspase 1 p20 (Supplemental Fig. 2E).

Additionally, PILO treatment significantly enhanced the protein levels of NLRP3, GSDMD, and caspase 1 p20 (Supplemental Fig. 2F). Following PILO treatment, the protein levels of NLRP3, GSDMD, and caspase 1 p20 (Supplemental Fig. 2F) were decreased in si-TRPM7-1 transfected N2a cells. In addition, the reduction in levels of NLRP3, GSDMD, and caspase 1 p20 (Supplemental Fig. 2F) protein were observed in si-TRPM7-2 transfected N2a cells.

Thus, TRPM7 was increased in SE models in vivo and in vitro and participated in pyroptosis.

TRPM7 contributed to pyroptosis in SE models in vivo

To further elucidate whether TRPM7 gene knockdown reduces pyroptosis and hyperexcitability in SE mice, AAV‐sh-TRPM7 containing with a green fluorescent (EGFP) tag was delivered to C57BL/6 J mice via stereotaxic injection. As shown in Supplemental Fig. 2G, EGFP (green) was expressed in the hippocampus, indicating that the AAV was successfully injected. Protein expression levels of TRPM7 in the hippocampus of AAV-sh-TRPM7-EGFP and AAV-sh-NC-EGFP transfection SE mice was evaluated by western blot (Fig. 3A) and immunofluorescence staining analysis (Supplemental Fig. 3A). TRPM7 levels in the hippocampus of AAV-sh-TRPM7-EGFP transfected mice were reduced compared with AAV-sh-NC-EGFP group (Fig. 3A), where the immunostaining intensity of TRPM7 (Supplemental Fig. 3, A and B) was decreased as well, suggesting that AAV-sh-TRPM7-EGFP transfection reduced PILO‐induced TRPM7 expression.

Fig. 3

TRPM7 contributed to pyroptosis in SE models in vivo. A The representative protein bands of TRPM7 in the hippocampus of PILO-treated C57BL/6 J mice after AAV-sh-TRPM7-EGFP or AAV-sh-NC-EGFP transfection (n = 6). B The representative protein bands of NLRP3, GSDMD, and caspase-1 p20 of PILO-treated C57BL/6 J mice after AAV-sh-TRPM7-EGFP or AAV-sh-NC-EGFP transfection (n = 5). C, D ELISA analysis of IL-18 (C) and IL-1β (D) in hippocampus homogenates of PILO-treated C57BL/6 J mice after AAV-sh-TRPM7-EGFP or AAV-sh-NC-EGFP transfection (n = 6). E The representative protein bands of NLRP3, GSDMD, and caspase-1 p20 expression in hippocampus of PILO-treated C57BL/6 J mice after NS8593 pretreatment (n = 6). F, G Immunofluorescence assessment of NLRP3 (red) expression in the hippocampus (120 × lens) of PILO-treated C57BL/6 J mice after AAV-sh-TRPM7-EGFP (green) or AAV-sh-NC-EGFP (green) transfection, including the CA1, CA3 and DG regions (n = 9). Scale bar: 200 μm. DAPI (blue) is used to label nucleus. H, I Immunofluorescence analysis of NLRP3 (green) expression in hippocampus (40 × lens) of PILO-treated C57BL/6 J mice after NS8593 pretreatment, including the CA1, CA3, and DG regions (n = 9). Scale bar: 500 μm. DAPI (blue) was used to label nucleus. Full scans of all the blots are in the Supplementary Note. *P < 0.05; **P < 0.01; ***P < 0.001; **** P < 0.0001

To assess whether AAV-sh-TRPM7-EGFP transfection could affect PILO‐induced pyroptosis, the protein expression levels of pyroptosis factors were evaluated by western blot and ELISA. The transfection of AAV-sh-TRPM7-EGFP reduced NLRP3, GSDMD, caspase-1 p20 (Fig. 3B), IL-18 (Fig. 3C), and IL-1β (Fig. 3D) levels compared with AAV-sh-NC-EGFP transfected mice. The immunostaining intensity of NLRP3 was decreased in the hippocampus including CA1, CA3, and DG regions (Fig. 3, F and G) of AAV-sh-TRPM7-EGFP transfected mice compared with that in the AAV-sh-NC-EGFP transfection group.

To assess the effect of NS8593, a non-specific inhibitor of TRPM7[33], in neuronal damage and pyroptosis in PILO-treated mice, 5 mg/kg NS8593 was applied intravenously 30 min before PILO administration. The PILO group revealed significant up-regulation of NLRP3, GSDMD, and caspase-1 p20 (Fig. 3E) compared with the control group by western blot. Compared with the PILO group, pretreatment with NS8593 group reduced the expression of NLRP3, GSDMD, and caspase-1 p20 ((Fig. 3E). Additionally, NLRP3 was up-regulated in the CA1, CA3, and DG (Fig. 3, H and I) regions. NS8593 pretreatment in PILO-treated group caused a significant reduction of NLRP3 in the CA1, CA3, and DG (Fig. 3, H and I) regions compared with PILO-treated group. These data indicate that silencing TRPM7 and TRPM7 inhibition by NS8593 reduced pyroptosis in PILO-treated SE mice.

TRPM7-mediated Zn 2+contributed to pyroptosis

TRPM7 is a high Zn2+ permeable ion channel[34]. The abnormal accumulation of intracellular Zn2+ can lead to neuron death and brain disorders including epileptic seizures and stroke[35]. In order to evaluate whether the intracellular Zn2+ levels were changed in the hippocampus of PILO-treated SE mice, we used the Zn2+-specific stain the N-(6-methoxy-8-quinolyl)-para-toluenesulfonamide (TSQ) to detect the change of intracellular Zn2+ accumulation. TSQ fluorescence was observed in the hippocampus CA1, CA3, and DG regions (Fig. 4A). Compared with the control group, higher TSQ fluorescence intensity was detected in the hippocampus CA1, CA3, and DG regions (Fig. 4, A and B) of PILO-treated SE mice. Thus, intracellular Zn2+ accumulation was enhanced in the hippocampus of PILO-treated mice.

Fig. 4

TRPM7-mediated Zn2+ contributed to pyroptosis. A The TSQ staining detected in hippocampus (4 × lens) of PILO-treated C57BL/6 J mice, including the enlarged CA1, CA3 and DG regions (10 × lens). Scale bar: 500 μm. B Statistical analysis of TSQ intensity (n = 9). C The representative protein bands of NLRP3, GSDMD, and caspase-1 p20 in N2a cells treated with ZnCl2 (30 μM and 100 μM) (n = 6). D The representative protein bands of NLRP3, GSDMD, and caspase-1 p20 in PILO-treated N2a cells after TRPM7 overexpression and TPEN (1 μM) pretreatment (n = 5 or 6). E The representative protein bands of NLRP3, GSDMD, and caspase-1 p20 in TRPM7-overexpressed N2a cells after TPEN(1 μM), DFX(1 μM), or TTM(1 μM) pretreatment (n = 6). Full scans of all the blots are in the Supplementary Note. *P < 0.05; ** P < 0.01

Next, we checked whether Zn2+ can induce pyroptosis in vitro. Here, we treated N2a cells with 30 μM and 100 μM ZnCl2 for 24 h to allow Zn2+ influx to the cytoplasm. In western blot experiments, the activation of pyroptosis by Zn2+ was dose-dependent (Fig. 4C).

To further investigate the involvement of TRPM7-mediated Zn2+ in pyroptosis, we measured the changes of pyroptosis associated factors in empty vector-transfected N2a cells, TRPM7-overexpressed cells after PILO treatment, and TRPM7-overexpressed cells after exposed to PILO combined with TPEN, a membrane-permeable Zn2+ chelator. We used 1 μM TPEN to chelate intracellular Zn2+ (Kd Zn = 0.7 fM) following the same treatment regimen as previously shown[36]. After PILO treatment, the protein levels of NLRP3, GSDMD, and caspase-1 p20 (Fig. 4D) were significantly enhanced in TRPM7-overexpressed cells. In TRPM7-overexpressed N2a cells, PILO combined with TPEN treatment remarkably down-regulated the protein levels of NLRP3, GSDMD, and caspase-1 p20 (Fig. 4D). Thus, the application of TPEN reversed pyroptosis via TRPM7.

TPEN also has high affinity to Fe2+(Kd Fe = 2.4 fM) and Cu2+ (Kd Cu = 17 zM)[37]. To verify whether TRPM7-mediated Fe2+ or Cu2+ participates in pyroptosis, we applied the iron chelator deferoxamine (DFX)[38] and the specific Cu2+ chelator tetrathiomolybdate (TTM)[39]. In TRPM7-overexpressed cells, treatment with 1 μM TPEN down-regulated the protein levels of NLRP3, GSDMD, and caspase-1 p20 (Fig. 4E). Nevertheless, exposure to 2 μM DFX and 1 μM TTM failed to diminish the expression of these pyroptosis associated factors in TRPM7-overexpressed cells (Fig. 4E), suggesting that Zn2+ rather than Fe2+ or Cu2+ was involved in TRPM7-mediated pyroptosis.

P-STAT3 promoted both Trpm7 and Nlrp3 transcription in PILO -treated N2a cells

STAT3 is a member of the signal transducer and activator of transcription (STAT) family, and it is activated by tyrosine phosphorylation, translocating from the cytoplasm to the nucleus to mediate the transcription of target genes[40]. The protein levels of STAT3, p-STAT3 and p-STAT3/STAT3 were increased in PILO-treated animals[41,42,43]. Here, we used RNA sequencing to explore the DEGs associated with PILO treatment. KEGG analysis disclosed that the up-regulated genes were mainly enriched in members of the JAK-STAT signaling pathway, which is the TOP 5 items of KEGG (Fig. 5A). In PILO-treated N2a cells, we found that STAT3 phosphorylation inhibitor Stattic reversed PILO-treated up-regulation of NLRP3 (Fig. 5, B and C), indicating that NLRP3 was up-regulated in PILO-treated SE model via p-STAT3 signaling.

Fig. 5

P-STAT3 promoted the transcription of Trpm7 and Nlrp3 in PILO-treated N2a cells. KEGG pathway analysis revealed that the up-regulated genes in DEGs between control C57BL/6mice and PILO-treated mice were involved in many signaling pathways, including JAK-STAT signaling pathway, which is the TOP 5 items of KEGG (n = 5). B, C The representative protein bands (B) and data analysis of NLRP3 (C) in N2a cells treated with PILO alone or combined with Stattic (0.5 μM) pretreatment (n = 6). D Top: P-STAT3 was recruited to the promoter regions of Trpm7 and Nlrp3. The sequence logo of potential STAT3 binding sites in the Trpm7 or Nlrp3 sequence in JASPAR. Bottom: ChIP assays performed on the promoter regions of Trpm7 or Nlrp3 with the p-STAT3 antibodies in N2a cells with or without PILO (10 μM) treatment (n = 5). TSS indicates transcription start site. Full scans of all the blots are in the Supplementary Note. * P < 0.05; ** P < 0.01; NS not significant

We then hypothesized that p-STAT3 could promote the transcription of Nlrp3 and Trpm7. ChIP assay was applied to verify the interaction of p-STAT3 protein with the Nlrp3 and Trpm7 genes. We performed JASPAR database analysis to predict binding sites between STAT3 and promoters of Trpm7 and Nlrp3, and designed three pairs of primers for ChIP-qPCR assay (Fig. 5D and Supplemental Fig. 4A). Here, p-STAT3 was specifically recruited to the GTGCGGGAAG region of Trpm7 after PILO treatment (Fig. 5D). Additionally, STAT3 recruitment to the TTACAGTAAA region of Trpm7 was augmented in the the PILO-treated group specifically (Supplemental Fig. 4A). Similarly, the recruitment of p-STAT3 to the TTCTGGGATG region of Nlrp3 was enhanced in PILO treated N2a cells (Fig. 5D) compared with the normal N2a cells. There was no recruitment of p-STAT3 to the other two regions (Fig. 5D). STAT3 was also recruited to the Nlrp3 promoter region of CTTCCTGTCT in the absence (Supplemental Fig. 4A) of PILO. Collectively, our results suggest that STAT3 or p-STAT3 was recruited to the promoter region of Trpm7 and Nlrp3, thereby activating Trpm7 and Nlrp3 transcription in PILO-treated cells.

TRPM7 contributed to pyroptosis via the Zn 2+/ROS/JAK2/STAT3 pathway in SE

To explore the function of STAT3 in SE, we evaluated STAT3 activation by measuring the relative level of STAT3 Tyr705 phosphorylation (pY705-STAT3) using western blot analysis. Here, levels of pY705-STAT3/STAT3 were enhanced in the total, cytosolic, and nuclear fractions (Fig. 6, A and B) of PILO-treated N2a cells, suggesting that p-STAT3 upregulation was apparent in the nucleus and cytoplasm.

Fig. 6

TRPM7 participated in pyroptosis via the Zn2+/ROS/JAK2/STAT3 pathway in SE. A, B The representative protein bands (A) and data analysis of STAT3 and pY705-STAT3/STAT3 (B) in the total fractions (TF), cytosolic fractions (CF), and nuclear fractions (NF) of PILO-treated N2a cells (n = 6). C, D The representative protein bands (C) and data analysis of STAT3 and pY705-STAT3/STAT3 (D) in PILO-treated N2a cells after TRPM7 overexpression and TPEN (1 μM) pretreatment (n = 6). E, F The representative protein bands (E) and data analysis of STAT3 and pY705-STAT3/STAT3 (F) in TRPM7-overexpressed N2a cells after TPEN(1 μM), DFX(1 μM), or TTM(1 μM) pretreatment (n = 6). G, H The representative protein bands (G) and data analysis of STAT3 and pY705-STAT3/STAT3 (H) in N2a cells treated with ZnCl2 (30 μM and 100 μM) (n = 6). I-L ROS and MMP production were measured by DCFH-DA dying or JC-1 in PILO-treated N2a cells after TRPM7 knockdown or TPEN (1 μM) pretreatment (n = 6). Scale bar: 100 μm. M, N The representative protein bands (M) and data analysis of NLRP3, GSDMD, caspase-1 p20, pY705-STAT3/STAT3, p-JAK2/JAK2, STAT3, and JAK2 (N) in PILO-treated N2a cells after NAC (5 mM), AG490 (50 μM) or Stattic (0.5 μM) pretreatment (n = 6). Full scans of all the blots are in the Supplementary Note. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001; NS not significant

To understand the relationship between TRPM7-mediated Zn2+ and STAT3, we detected the relative levels of pY705-STAT3/STAT3 in empty vector-transfected N2a cells, TRPM7-overexpressed cells after PILO treatment, and TRPM7-overexpressed cells after exposure to PILO and 1 μM TPEN. The results indicated that after PILO treatment, the protein expression of pY705-STAT3/STAT3 (Fig. 6, C and D) were significantly enhanced in TRPM7-overexpressed cells, while TPEN treatment diminished the expression of pY705-STAT3/STAT3 (Fig. 6, C and D). In TRPM7-overexpressed N2a cells, exposure to 2 μM DFX and 1 μM TTM did not attenuate the elevated protein levels of pY705-STAT3/STAT3 (Fig. 6, E and F). Meanwhile, the influx of Zn2+ into the cytoplasm also enhanced the protein expressions of pY705-STAT3/STAT3 (Fig. 6, G and H). Thus, TRPM7-mediated Zn2+ contributed to STAT3 activation in PILO-treated N2a cells.

Disturbances in cellular Zn2+ levels are toxic to N2a cells and Zn2+ is related to the generation of reactive oxygen species (ROS)[44]. To assess oxidative stress of TRPM7-Zn2+-induced pyroptosis, mitochondrial membrane potential (MMP) and ROS production were examined in N2a cells treated with PILO, PILO combined with TRPM7 siRNA transfection, or PILO combined with 1 μM TPEN with the JC-1 and DCFH-DA dyes (fluorescent probe 2,7-Dichlorodi-hydrofluorescein diacetate (DCFH-DA)). Here, the intracellular ROS was up-regulated in N2a cells treated with PILO for 24 h (Fig. 6, I and K). Additionally, the si-TRPM7 and TPEN treatment significantly reduced the up-regulation of ROS (Fig. 6, I and K). Similarly, the treatment of si-TRPM7 or TPEN inhibited the decreased mitochondrial potential in N2A cells treated with PILO compared with the control group (Fig. 6, J and L), indicating that knockdown of TRPM7 or TPEN treatment inhibited oxidative stress responses in PILO-treated N2a cells.

Moreover, when N2a cells were pre-treated with the ROS scavenger NAC[45] and JAK2 inhibitor AG490[46] and then treated with PILO, pyroptosis associated protein expression, as well as the activation of JAK2/STAT3 were suppressed (Fig. 6M). In addition, the STAT3 phosphorylation inhibitor Stattic reversed PILO-treated up-regulation of the relative levels of pyroptosis indicators (Fig. 6M) but did not affect the protein level of p-JAK2/JAK2 (Fig. 6, M and N). Additionally, the protein levels of pY705-STAT3/STAT3 in PILO-treated cells were significantly higher than control group. This effect was inhibited by NAC, AG490, and Stattic treatment (Fig. 6, M and N). Thus, TRPM7 contributed to pyroptosis via the Zn2+/ROS/JAK2/STAT3 pathway in SE.

Sliencing TRPM7 and TRPM7 inhibition by NS8593 alleviated neuronal damage in PILO -treated SE mice

We determined the effect of AAV-sh-TRPM7-EGFP transfection on PILO‐induced neuronal damage. Astrocytes respond to neuronal damage by proliferating and modulating their activity[47]. Here, we investigated the distribution of neurons labeled by NeuN and that of astrocytes labeled by glial fibrillary acidic protein (GFAP) in the hippocampus. AAV-sh-TRPM7-EGFP transfection markedly reduced the immunostaining intensity of GFAP (Supplemental Fig. 3, C and D) and increased the number of neurons (Fig. 7 A, B) compared with those of the AAV-sh-NC-EGFP transfected mice, indicating the critical role of TRPM7 in mediating PILO‐induced neuronal damage.

Fig. 7

Silencing TRPM7 and TRPM7 inhibition by NS8593 mitigated neuronal damage and pyroptosis in SE mice. A, B Immunofluorescence analysis of NeuN (red) expression in the hippocampus (120 × lens) of PILO-treated C57BL/6 J mice after AAV-sh-TRPM7-EGFP (green) or AAV-sh-NC-EGFP (green) transfection, including the CA1, CA3 and DG regions (n = 9). Scale bar: 200 μm. DAPI (blue) is used to label nucleus. C The representative protein bands of NeuN and GFAP in hippocampus of PILO-treated C57BL/6 J mice after NS8593 pretreatment (n = 6). D-F Immunofluorescence analysis of NeuN (red) and GFAP (green) expression in hippocampus (10 × lens) of PILO-treated C57BL/6 J mice after NS8593 pretreatment, including the enlarged CA1, CA3, and DG regions (40 × lens) (n = 9). Scale bar: 100 μm and 500 μm. DAPI (blue) was used to label nucleus. Full scans of all the blots are in the Supplementary Note. *P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001

Meanwhile, we assessed the effect of NS8593 in neuronal damage in PILO-treated mice (Fig. 7F). Compared with the control group, the number of neurons was reduced in CA1, CA3, and DG regions (Fig. 7, D, F) of hippocampus in PILO-treated SE mice. When compared with PILO-treated mice, NS8593 pretreatment with PILO (PILO + NS8593) showed the increasing number of neurons in CA1, CA3, and DG regions (Fig. 7 D, F). In addition, compared with the control group, GFAP intensity changes were confirmed in CA1, CA3, and DG regions (Fig. 7 E, F) in the hippocampus of PILO-treated mice. NS8593 pretreatment remarkably reduced the intensity of GFAP in CA1, CA3, and DG regions (Fig. 7 E, F) of hippocampus compared with those of the PILO-treated group.

Additionally, NeuN protein levels were decreased (Fig. 7C) and GFAP protein levels were significantly increased (Fig. 7C) in PILO-treated SE mice. The PILO + NS8593 group showed decreased GFAP protein levels (Fig. 7C) and increased levels of NeuN (Fig. 7C). Thus, silencing TRPM7 and TRPM7 inhibition by NS8593 suppressed neuronal damage and neuronal death in PILO-treated SE mice.

Silencing TRPM7 and TRPM7 inhibition by NS8593 reversed neuronal hyperexcitability in PILO -treated SE models

We evaluated the impact of AAV-sh-TRPM7-EGFP transfection in an in vivo SE model by EEG. Flowcharts of the injection protocol is shown in Supplemental Fig. 5A. Representative EEG recordings are shown in Fig. 8A. Based on EEG recordings, we found the mean total duration of seizure activity was decreased in the group pretreated with AAV-sh-TRPM7-EGFP (median score on the Racine scale was 2) compared with the group using vector and PILO (median score on the Racine scale was 4.5) (Fig. 8, A and B), implying that TRPM7 has a crucial role in regulating PILO‐induced hyperexcitability. Therefore, TRPM7 knockdown lessened neuronal damage and pyroptosis and reversed disease associated neuronal hyperexcitability.

Fig. 8