Department of Pediatrics, Cangzhou Central Hospital, Cangzhou, Hebei Province, People's Republic of China.
Brain Behav. 2024 Aug;14(8):e3615. doi: 10.1002/brb3.3615.
Temporal lobe epilepsy (TLE), a prevalent neurological disorder, is associated with hippocampal oxidative stress and inflammation. A recent study reveals that the long noncoding RNA ILF3 divergent transcript (ILF3-AS1) level is elevated in the hippocampus of TLE patients; however, the functional roles of ILF3-AS1 in TLE and underlying mechanisms deserve further investigation. Hence, this study aimed to elucidate whether ILF3-AS1 is involved in the pathogenesis of TLE by regulating oxidative stress and inflammation and to explore its underlying mechanism in vitro.
Human hippocampal neurons were subjected to a magnesium-free (Mg-free) solution to establish an in vitro model of TLE. The potential binding sites between ILF3-AS1 and miRNA were predicted by TargetScan/Starbase and confirmed by dual luciferase reporter assay. Cell viability and damage were assessed by cell counting kit-8 and lactate dehydrogenase assay kits, respectively. Levels of reactive oxygen species, malondialdehyde, and superoxide dismutase were determined by commercial kits. Levels of Interleukin-6 (IL-6), IL-1β, and tumor necrosis factor-alpha were quantified by enzyme-linked immunosorbent assay. The expressions of gene and protein were determined by quantitative real-time polymerase chain reaction and Western blot analysis.
In Mg-free-treated hippocampal neurons, both ILF3-AS1 and HMGB1 were highly up-regulated, whereas miR-504-3p was down-regulated. ILF3-AS1 knockdown ameliorated Mg-free-induced cellular damage, oxidative stress, and inflammatory response. Bioinformatics analysis revealed that miR-504-3p was a target of ILF3-AS1 and was negatively regulated by ILF3-AS1. MiR-504-3p inhibitor blocked the protection of ILF3-AS1 knockdown against Mg-free-induced neuronal injury. Further analysis presented that ILF3-AS1 regulated HMGB1 expression by sponging miR-504-3p. Moreover, HMGB1 overexpression reversed the protective functions of ILF3-AS1 knockdown.
Our findings indicate that ILF3-AS1 contributes to Mg-free-induced hippocampal neuron injuries, oxidative stress, and inflammation by targeting the miR-504-3p/HMGB1 axis. These results provide a novel mechanistic understanding of ILF3-AS1 in TLE and suggest potential therapeutic targets for the treatment of epilepsy.
颞叶癫痫（TLE）是一种常见的神经疾病，与海马氧化应激和炎症有关。最近的一项研究表明，长非编码 RNA ILF3 发散转录本（ILF3-AS1）在 TLE 患者的海马体中升高；然而，ILF3-AS1 在 TLE 中的功能作用及其潜在机制仍有待进一步研究。因此，本研究旨在通过调节氧化应激和炎症来阐明 ILF3-AS1 是否参与 TLE 的发病机制，并在体外探讨其潜在机制。
用无镁（Mg-free）溶液处理人海马神经元，建立 TLE 的体外模型。通过 TargetScan/Starbase 预测 ILF3-AS1 与 miRNA 的潜在结合位点，并通过双荧光素酶报告基因检测进行验证。分别用细胞计数试剂盒-8 和乳酸脱氢酶试剂盒评估细胞活力和损伤。通过商业试剂盒测定活性氧、丙二醛和超氧化物歧化酶的水平。用酶联免疫吸附试验定量测定白细胞介素-6（IL-6）、白细胞介素-1β和肿瘤坏死因子-α的水平。通过实时定量聚合酶链反应和 Western blot 分析测定基因和蛋白的表达。
在 Mg-free 处理的海马神经元中，ILF3-AS1 和 HMGB1 均高度上调，而 miR-504-3p 下调。ILF3-AS1 敲低可改善 Mg-free 诱导的细胞损伤、氧化应激和炎症反应。生物信息学分析表明，miR-504-3p 是 ILF3-AS1 的靶点，受 ILF3-AS1 负调控。miR-504-3p 抑制剂阻断了 ILF3-AS1 敲低对 Mg-free 诱导的神经元损伤的保护作用。进一步分析表明，ILF3-AS1 通过海绵吸附 miR-504-3p 调节 HMGB1 表达。此外，HMGB1 过表达逆转了 ILF3-AS1 敲低的保护作用。
我们的研究结果表明，ILF3-AS1 通过靶向 miR-504-3p/HMGB1 轴，促进 Mg-free 诱导的海马神经元损伤、氧化应激和炎症。这些结果为 ILF3-AS1 在 TLE 中的作用提供了新的机制理解，并为癫痫的治疗提供了潜在的治疗靶点。
