Supplementary Material for: Effects of Long Noncoding RNA H19 on Isoflurane-Induced Cognitive Dysregulation by Promoting Neuroinflammation

<b><i>Introduction:</i></b> Isoflurane (ISO) may cause neuronal apoptosis and synaptic disorder during development, and damage long-term learning and memory function. This observation aimed to study the function of H19 in vitro and in vivo tests and the further mechanism was identified. <b><i>Methods:</i></b> ISO cell models and rat models were established and reactive oxygen species (ROS) identified. The viability and apoptosis of HT22 cells were detected by the MTT and flow cytometer. Morris water maze test was conducted to analyze the neurotoxicity of ISO on spatial learning and memory ability. Quantitative PCR was the method to verify the expression of H19. The concentration of inflammatory indicators was identified by enzyme-linked immunosorbent assay. <b><i>Results:</i></b> 1.5% and 2% ISO led to the neurotoxicity of HT22 cells and increased expression of H19. Silenced H19 meliorated these adverse impacts of ISO. Interference of H19 exerted neuroprotective roles by repressing modified neurological severity score, inhibiting escape latency, elevating distance and time of target area, and controlling ROS and inflammation. MiR-17-5p might be a promising competing endogenous RNA of H19. The expression of miR-17-5p was reduced in the ISO group and reversed by the absence of H19. <b><i>Conclusion:</i></b> Our results of in vitro and in vivo assay indicated that the absence of HT22 is a neuroprotective regulator of cognition and inflammation by accumulating miR-17-5p.

<b><i>引言：</i></b> 异氟烷（Isoflurane, ISO）可在发育过程中诱发神经元凋亡与突触功能紊乱，并损害长期学习记忆功能。本研究旨在探究H19的生物学功能，通过体外与体内实验明确其潜在分子机制。<b><i>方法：</i></b> 构建异氟烷细胞模型与大鼠模型，并检测活性氧（reactive oxygen species, ROS）水平。采用MTT法与流式细胞仪检测HT22细胞的活力与凋亡情况；通过莫里斯水迷宫实验分析异氟烷对空间学习记忆能力的神经毒性；利用定量PCR（quantitative PCR）验证H19的表达水平；采用酶联免疫吸附实验检测炎症因子的浓度。<b><i>结果：</i></b> 1.5%与2%浓度的异氟烷可诱导HT22细胞产生神经毒性，并上调H19的表达水平；沉默H19可改善异氟烷诱导的上述不良影响。干扰H19可通过降低改良神经功能缺损评分、缩短逃避潜伏期、增加目标象限游泳距离与时间，并调控ROS水平与炎症反应，发挥神经保护作用。MiR-17-5p可能是H19的潜在内源竞争RNA（competing endogenous RNA）；异氟烷处理组中miR-17-5p的表达水平降低，而沉默H19可逆转这一表达变化。<b><i>结论：</i></b> 本研究的体外与体内实验结果表明，沉默H19可通过上调miR-17-5p的表达，作为认知与炎症的神经保护调控因子。