基于转录组学探讨EI24参与电针调控自噬减轻大鼠脑缺血再灌注损伤的机制研究

关键词：针灸  脑缺血再灌注损伤  依托泊苷诱导蛋白24  自噬

Keywords:acupuncture  cerebral ischemia-reperfusion injury  etoposide-induced gene 24  autophagy

摘要：

      摘要 目的：利用转录组测序技术，探讨EI24参与电针调控自噬减轻脑缺血再灌注损伤的分子机制。 方法：33只SD雄性大鼠随机分为假手术组、模型组、电针组。建立大脑中动脉闭塞/再灌注模型，电针刺曲池、百会、足三里，20min/d，持续3天，末次治疗后进行神经功能评分，高架十字实验，TTC染色，HE染色，电镜观察自噬小体，转录组学测序分析差异基因并进行生物信息学分析，Western Blot检测EI24及LC3、p62的蛋白表达水平。 结果：与假手术组相比，模型组神经功能评分明显升高（P<0.01），高架十字实验开臂时间显著减少（P<0.01），脑梗死体积显著增加（P<0.01），自噬小体增多；与模型组比较，电针组神经功能损伤明显改善（P<0.01），高架实验开臂停留时间显著增加（P<0.01），脑梗死体积减小（P<0.01），自噬小体减少。转录组测序结果显示，假手术组vs模型组、模型组vs电针组之间的差异基因GO功能和KEGG通路富集分析，均表明这些差异基因与自噬过程密切相关。此外，通过EI24与自噬基因的相关系数矩阵分析，结果显示它们之间存在正相关或负相关关系。Western Blot显示，与假手术组相比，模型组LC3-II及EI24显著增加（P<0.01），而p62蛋白显著下调（P<0.01）；与模型组比较，电针组LC3-II和EI24显著降低（P<0.01），p62蛋白显著上调（P<0.01）。 结论：EI24可能参与电针刺抑制细胞过度自噬，改善大鼠脑缺血再灌注损伤。

Abstract:

      Abstract Objective： To explore the molecular mechanism of etoposide-induced gene 2.4（EI24） involved in the regulation of autophagy by electroacupuncture to attenuate cerebral ischemia-reperfusion injury using transcriptome sequencing technology. Method： Thirty-three SD male rats were randomly divided into the sham，model，and electroacupuncture groups. A middle cerebral artery occlusion/reperfusion model was established，followed by electroacupuncture at the “Quchi”（LI11），“Baihui”（DU20），and “Zhusanli”（ST36） points for 20 minutes daily over the course of three days. Assessments included neurological function scoring，elevated plus maze testing，TTC staining，HE staining，electron microscopy for observing autophagic vesicles，transcriptomic sequencing for differential gene analysis and bioinformatics，and Western Blot to evaluate the protein expression levels of EI24，LC3，and p62 after the final treatment. Result： Compared to the sham group， the model group exhibited significantly higher neurological function scores （P<0.01）， reduced open-arm dwell time in the elevated plus maze （P<0.01）， increased cerebral infarct volume （P<0.01）， and a higher number of autophagic vesicles. In contrast， the electroacupuncture group demonstrated improved functional recovery （P<0.01）， significantly increased open-arm dwell time （P<0.01）， reduced cerebral infarct volume （P<0.01）， and decreased numbers of autophagic vesicles compared to the model group. Transcriptome sequencing results revealed that GO function and KEGG pathway enrichment analyses of differential genes between the sham and model groups， as well as between the model and electroacupuncture groups， indicated a strong association with the autophagy process. In addition， the correlation coefficient matrix analysis of EI24 and autophagy genes showed that there was a positive or negative correlation between them. Western Blot analysis showed that LC3-II and EI24 were significantly increased （P<0.01）， while p62 expression was notably down-regulated in the model group compared to the sham-operated group （P<0.01）； additionally， LC3-II and EI24 levels were significantly decreased, whereas p62 expression was notably upregulated in the electroacupuncture group relative to the model group（P<0.01）. Conclusion： EI24 may play a pivotal role in electroacupuncture to inhibit autophagy to ameliorate cerebral ischemia-reperfusion injury.