A study on the effect of HIE treatment by umbilical cord blood and EPO using mouse microarray

Neonatal hypoxic–ischemic encephalopathy (HIE) results in neurological impairments; cell-based therapy has been suggested as a therapeutic avenue. Previous research has demonstrated the synergistically potentiated therapeutic efficacy of human umbilical cord blood (UCB) by combining recombinant human erythropoietin (EPO) treatment for recovery from HIE. However, its molecular mechanism is not entirely understood. In the present study, we analyzed the mechanisms underlying the effect of combination treatment with EPO and UCB by transcriptomic analysis, followed by gene enrichment analysis. The HIE model of the neonate was prepared and randomly divided into five groups: sham, HIE, and UCB, EPO, and UCB+EPO treatments after HIE. A total of 376 genes were differentially expressed when |log2FC| ≥ 1-fold change expression values were considered to be differentially expressed between UCB+EPO and HIE. Further assessment through qRT-PCR and gene enrichment analysis confirmed the expression and correlation of its potential target Gene N as an essential gene involved in the synergistic effect of the UCB+EPO combination. At 7 days post-injury, the operated pups were randomly assigned into 5 groups (each group, n=8 to 10). UCB (3 ×10^7 /kg, intraperitoneal injection once at 7d post-injury), EPO (500 IU/kg, intraperitoneal injection for five consecutive days from 7d HIE), UCB+EPO (at the same dose and schedule as the other groups) were intraperitoneally administered, and PBS (five consecutive days from 7 d HIE) was injected as a control (HIE). Five array data were obtained for a total of five samples, one each. A total of 5 samples were named 1) sham, 2) HIE, 3) HIE+EPO, 4) HIE+UCB, and 5) HIE+EPO+UCB according to the treatment method.

新生儿缺氧缺血性脑病（Neonatal hypoxic–ischemic encephalopathy, HIE）可引发神经系统损伤，细胞疗法被视为潜在的治疗途径。既往研究证实，将重组人促红细胞生成素（recombinant human erythropoietin, EPO）与人脐带血（human umbilical cord blood, UCB）联合应用，可协同增强其用于HIE康复的治疗效果，但该联合疗法的分子机制尚未完全阐明。本研究通过转录组分析结合基因富集分析，探究EPO与UCB联合治疗的潜在作用机制。研究人员构建了新生儿HIE模型，并将其随机分为5组：假手术组、HIE模型组、HIE后UCB治疗组、HIE后EPO治疗组以及HIE后UCB+EPO联合治疗组。以|log₂FC|≥1作为差异表达基因筛选阈值时，UCB+EPO联合治疗组与HIE模型组间共鉴定出376个差异表达基因。后续通过实时荧光定量聚合酶链反应（qRT-PCR）与基因富集分析进一步验证，潜在靶基因N的表达及其相关性，证实其为介导UCB+EPO联合治疗协同效应的关键基因。造模后第7天，将构建HIE模型的幼鼠随机分为5组（每组n=8~10），各组分别给予如下干预：UCB组（3×10⁷/kg，造模后第7天腹腔注射1次）、EPO组（500 IU/kg，自造模后第7天起连续5天腹腔注射）、UCB+EPO联合组（给药剂量与方案同前两组），对照组（HIE组）则给予磷酸盐缓冲液（PBS，自造模后第7天起连续5天腹腔注射）。本研究共获取5份样本的基因芯片测序数据，根据干预方案，5份样本分别命名为：1）假手术组、2）HIE模型组、3）HIE+EPO治疗组、4）HIE+UCB治疗组、5）HIE+EPO+UCB联合治疗组。