Neural Regeneration Research ›› 2023, Vol. 18 ›› Issue (6): 1277-1285.doi: 10.4103/1673-5374.357906

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

Xiao-Xue Xu1, 2, #, Rui-Xue Shi1, #, Yu Fu1, #, Jia-Lu Wang2, #, Xin Tong1, Shi-Qi Zhang1, Na Wang3, Mei-Xuan Li1, Yu Tong1, Wei Wang4, Miao He1, Bing-Yang Liu5, *, Gui-Lan Chen3, *, Feng Guo1, *#br#   

  1. 1Department of Pharmaceutical Toxicology, School of Pharmaceutical Science, China Medical University, Shenyang, Liaoning Province, China; 2Department of Neurology, the First Affiliated Hospital of China Medical University, Shenyang, Liaoning Province, China; 3Key Laboratory of Medical Electrophysiology, Ministry of Education & Medical Electrophysiological Key Laboratory of Sichuan Province, Institute of Cardiovascular Research, Southwest Medical University, Luzhou, Sichuan Province, China; 4Department of Endocrinology and Metabolism, the Fourth Affiliated Hospital of China Medical University, Shenyang, Liaoning Province, China; 5Department of Endocrinology, Shengjing Hospital of China Medical University, Shenyang, Liaoning Province, China
  • Online:2023-06-15 Published:2023-01-05
  • Contact: Feng Guo, MD, PhD, blueforest611@hotmail.com; Gui-Lan Chen, PhD, chenguilan@swmu.edu.cn; Bing-Yang Liu, MD, PhD, Liuliuby@sj-hospital.org.
  • Supported by:
    This work was supported by the Natural Science Foundation of China, Nos. 81971212 (to FG), 81601129 (to XXX); the Open Fund of the Key Laboratory of Medical Electrophysiology, Ministry of Education & Medical Electrophysiological Key Laboratory of Sichuan Province, Institute of Cardiovascular Research, Southwest Medical University, No. KeyME-2018-07 (to FG); and Liaoning Province Xingliao Talent Program Project, No. XLYC1907164 (to FG).

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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.

Key words: apoptosis, bioinformatics analysis, cell death, epilepsy, nitric oxide synthase, oxidative stress, pyroptosis, RNA sequencing, Tremor rat, weighted gene co-expression network analysis