Neuronal nitric oxide synthase/reactive oxygen species pathway is involved in apoptosis and pyroptosis in epilepsy

Abstract

Dysfunction of neuronal nitric oxide synthase contributes to neurotoxicity, which triggers cell death in various neuropathological diseases, including epilepsy. Studies have shown that inhibition of neuronal nitric oxide synthase activity increases the epilepsy threshold, that is, has an anticonvulsant effect. However, the exact role and potential mechanism of neuronal nitric oxide synthase in seizures are still unclear. In this study, we performed RNA sequencing, functional enrichment analysis, and weighted gene coexpression network analysis of the hippocampus of tremor rats, a rat model of genetic epilepsy. We found damaged hippocampal mitochondria and abnormal succinate dehydrogenase level and Na⁺-K⁺-ATPase activity. In addition, we used a pilocarpine-induced N2a cell model to mimic epileptic injury. After application of neuronal nitric oxide synthase inhibitor 7-nitroindazole, changes in malondialdehyde, lactate dehydrogenase and superoxide dismutase, which are associated with oxidative stress, were reversed, and the increase in reactive oxygen species level was reversed by 7-nitroindazole or reactive oxygen species inhibitor N-acetylcysteine. Application of 7-nitroindazole or N-acetylcysteine downregulated the expression of caspase-3 and cytochrome c and reversed the apoptosis of epileptic cells. Furthermore, 7-nitroindazole or N-acetylcysteine downregulated the abnormally high expression of NLRP3, gasdermin-D, interleukin-1β and interleukin-18. This indicated that 7-nitroindazole and N-acetylcysteine each reversed epileptic cell death. Taken together, our findings suggest that the neuronal nitric oxide synthase/reactive oxygen species pathway is involved in pyroptosis of epileptic cells, and inhibiting neuronal nitric oxide synthase activity or its induced oxidative stress may play a neuroprotective role in epilepsy.

Keywords: RNA sequencing; Tremor rat; apoptosis; bioinformatics analysis; cell death; epilepsy; nitric oxide synthase; oxidative stress; pyroptosis; weighted gene co-expression network analysis.

Figure 1

WGCNA using RNA sequencing of TRM. (A) Determination of the soft thresholding power (β) in WGCNA. (B) The impact of the soft threshold power on the mean connectivity. (C) The dark-red module is the key module related to TRM epileptogenesis with the highest R² and P < 0.05. (D) The gene clustering tree is built by hierarchical clustering of adjacency-based dissimilarity to detect the co-expression clusters with corresponding color assignments. Each color represents a module, and the gray module indicates non-co-expression among the genes. (E) The module-trait relationships. Each row correlates to a module eigengene, each column correlates to a trait. Each cell includes the corresponding correlation and P value. The positive correlation is indicated in red. The red arrow indicates that the dark-red module presents the highest correlation with the TRM. TRM: Tremor rat; WGCNA: weighted gene co-expression network analysis.

Figure 2

The function enrichment assessment highlights the vital role of nitric oxide synthase in TRM epileptogenesis. (A) The correlated heatmap of eigengene adjacency. Light blue represents low adjacency, while red represents high adjacency. (B) The scatterplot of gene significance for the TRM vs. module membership in the dark-red module. (C) The bar blot displays the top ten terms of each GO category, including BP, CC, and MF. The red arrow indicates the BP term “positive regulations of nitric oxide synthase biosynthetic process.” BP: Biological process; CC: cellular component; GO: gene ontology; MF: molecular function; TRM: tremor rat.

Figure 3

Expression and distribution of nNOS in the TRM. (A) Western blot probing of neuronal nNOS. (B) Increased protein expression of nNOS was detected in the TRM hippocampus (n = 3). (C) The nNOS distribution was observed in the hippocampal CA1, CA3, and DG of the TRM and control rats. The relative fluorescence intensity of nNOS localization was elevated in the hippocampal CA1, CA3, and DG regions of the TRMs compared to that in the controls. Arrows point to the immunoreactive cells in which nNOS was labeled with FITC for emitting green fluorescence. Scale bars: 50 μm. (D) Quantification of nNOS fluorescence intensity in CA1 (n = 6). (E) Quantification of nNOS fluorescence intensity in CA3 (n = 6). (F) Quantification of nNOS fluorescence intensity in DG (n = 6). Wistar rats were used as controls. Data are expressed as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001 (unpaired Student’s t-test). CA: Cornu ammonis; DAPI: 4,6-diamino-2-phenyl indole; DG: dentate gyrus; FITC: fluorescein isothiocyanate; nNOS: neuronal nitric oxide synthase; TRM: tremor rat.

Figure 4

Mitochondrial dysfunction and enhanced MDA and LDH in the TRM hippocampus. (A) Transmission electron microscopy revealed damaged mitochondria ultrastructures in the TRM hippocampi compared to that in the controls, which presented with swelling and impaired crista. The yellow arrows point to the representative mitochondria in the animals from different groups. Scale bars: 2 μm. (B) The activity of SDH in the TRM and control hippocampi (n = 6). (C) The activity of Na⁺-K⁺-ATPase in the TRM and the control hippocampi (n = 6). (D). Enhanced MDA levels in the TRM hippocampus (n = 3). (E) Enhanced LDH levels in the TRM hippocampus (n = 3). (F) The SOD level in the TRM hippocampus was reduced compared to the control but without significance (n = 3). Wistar rats were used as controls. Data are expressed as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001 (unpaired Student’s t-test). LDH: Lactate dehydrogenase; MDA: malondialdehyde; SDH: succinate dehydrogenase; SOD: superoxide dismutase; TRM: tremor rat.

Figure 5

nNOS inhibitor 7-NI reverses effect on MDA and LDH levels and ROS generation in PILO-treated N2a cells. (A) The appropriate concentration of 7-NI was determined using a CCK8 assay. ^####P < 0.0001, vs. 0 μM 7-NI-treated cells (unpaired Student’s t-test). (B) Relative MDA content in PILO-treated N2a cells in the presence or absence of 7-NI (n = 3). (C) Relative LDH content in PILO-treated N2a cells in the presence or absence of 7-NI (n = 3). (D) Relative SOD content in PILO-treated N2a cells in the presence or absence of 7-NI (n = 3). (E) ROS level in PILO-treated N2a cells in the presence or absence of 7-NI or NAC. The ROS level was significantly increased in the presence of PILO compared with that in the control group, and this effect was suppressed by blocking nNOS activity with 7-NI. Arrows point to ROS (green). Scale bars: 200 μm. (F) ROS level response to PILO treatment alone and combined with 7-NI or NAC treatment (n = 5). Data are expressed as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001 (one-way analysis of variance followed by Tukey’s post hoc test). 7-NI: 7-Nitroindazole; CCK8: cell counting kit-8; DMSO: dimethyl sulfoxide; LDH: lactate dehydrogenase; MDA: malondialdehyde; NAC: acetylcysteine; nNOS: neuronal nitric oxide synthase; PILO: pilocarpine; ROS: reactive oxygen species; SOD: superoxide dismutase.

Figure 6

nNOS/ROS pathway is involved in PILO-induced apoptosis in N2a cells. (A) Images of apoptotic cells in PILO-treated N2a cells in the presence or absence of 7-NI and/or NAC obtained after TUNEL staining. The enhanced cell apoptosis in PILO-treated cells was significantly reduced after treatment with 7-NI or NAC alone, and after cotreatment with the two inhibitors. Scale bars: 100 μm. (B) PILO treatment led to apoptosis in N2a cells. The 7-NI and/or NAC treatment improved cell survival in the presence of PILO (n = 3) . (C) Western blot analysis of caspase-3 after 7-NI and/or NAC treatment in PILO-treated cells (n = 4). (D) Western blot analysis of cytochrome c after 7-NI and/or NAC in PILO-treated cells (n = 4). Data are expressed as mean ± SEM. *P < 0.05, **P < 0.01 (one-way analysis of variance followed by Tukey’s post hoc test). 7-NI: 7-Nitroindazole; DMSO: dimethyl sulfoxide; NAC: acetylcysteine; nNOS: neuronal nitric oxide synthase; PILO: pilocarpine; ROS: reactive oxygen species; TUNEL: TdT-mediated dUTP nick-end labeling.

Figure 7

nNOS/ROS pathway is involved in PILO-induced apoptosis in N2a cells. (A) Immunofluorescence imaging of NLRP3-positive cells in PILO-treated N2a cells in the presence or absence of 7-NI and/or NAC. The enhanced fluorescence intensity of NLRP3 in the PILO-treated group was significantly reversed by 7-NI, NAC, and 7-NI combined with NAC. Scale bars: 200 μm. (B) Effect of 7-NI and/or NAC on NLRP3 in PILO-treated cells (n = 3), which corresponding to A. (C) The effect of 7-NI and/or NAC on GSDMD in PILO-treated cells (n = 3). (D) Effect of 7-NI and/or NAC on IL-1β in PILO-treated cells (n = 3). (E) Effect of 7-NI and/or NAC on IL-18 in PILO-treated cells (n = 4) Data are expressed as mean ± SEM. *P < 0.05, **P < 0.01, ****P < 0.0001 (one-way analysis of variance followed by Tukey’s post hoc test). 7-NI: 7-Nitroindazole; DMSO: dimethyl sulfoxide; GSDMD: gasdermin-D; NAC: acetylcysteine; NLRP3: nucleotide-binding oligomerization domain-like receptor family pyrin domain-containing 3; nNOS: neuronal nitric oxide synthase; PILO: pilocarpine; ROS: reactive oxygen species.