背景:颞叶癫痫(TLE),一种普遍的神经系统疾病,与海马氧化应激和炎症有关。最近的一项研究表明,在TLE患者的海马中,长链非编码RNAILF3差异转录本(ILF3-AS1)水平升高;然而,ILF3-AS1在TLE中的功能作用和潜在机制值得进一步研究。因此,本研究旨在阐明ILF3-AS1是否通过调节氧化应激和炎症参与TLE的发病,并探讨其在体外的作用机制。
方法:将人海马神经元置于无镁(无Mg2+)溶液中,以建立TLE的体外模型。通过TargetScan/Starbase预测ILF3-AS1和miRNA之间的潜在结合位点,并通过双荧光素酶报告基因测定确认。通过细胞计数试剂盒-8和乳酸脱氢酶测定试剂盒评估细胞活力和损伤,分别。活性氧的水平,丙二醛,和超氧化物歧化酶通过商业试剂盒测定。白细胞介素-6(IL-6)的水平,IL-1β,和肿瘤坏死因子-α通过酶联免疫吸附试验进行定量。通过实时定量聚合酶链反应和Westernblot分析确定基因和蛋白质的表达。
结果:在无Mg2+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敲低改善了无Mg2+诱导的细胞损伤,氧化应激,和炎症反应。生物信息学分析显示,miR-504-3p是ILF3-AS1的靶标,受到ILF3-AS1的负调控。MiR-504-3p抑制剂阻断ILF3-AS1敲低对无Mg2+诱导的神经元损伤的保护作用。进一步分析显示ILF3-AS1通过海绵作用miR-504-3p调节HMGB1表达。此外,HMGB1过表达逆转了ILF3-AS1敲低的保护功能。
结论:我们的研究结果表明,ILF3-AS1有助于无Mg2+诱导的海马神经元损伤,氧化应激,和炎症通过靶向miR-504-3p/HMGB1轴。这些结果提供了对TLE中ILF3-AS1的新的机制理解,并提出了治疗癫痫的潜在治疗目标。
BACKGROUND: 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.
METHODS: Human hippocampal neurons were subjected to a magnesium-free (Mg2+-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.
RESULTS: In Mg2+-free-treated hippocampal neurons, both ILF3-AS1 and HMGB1 were highly up-regulated, whereas miR-504-3p was down-regulated. ILF3-AS1 knockdown ameliorated Mg2+-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 Mg2+-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.
CONCLUSIONS: Our findings indicate that ILF3-AS1 contributes to Mg2+-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.