[Role of the nuclear factor-kappa B signaling pathway in the repair of white matter injury in neonatal rats through human umbilical cord mesenchymal stem cell transplantation]

Abstract

Objectives: To observe the reparative effects of human umbilical cord mesenchymal stem cell (hUC-MSC) transplantation on white matter injury (WMI) in neonatal rats and explore its mechanism through the nuclear factor-kappa B (NF-κB) signaling pathway mediated by microglial cells.

Methods: Sprague-Dawley rats, aged 2 days, were randomly divided into three groups: sham-operation,WMI, and hUC-MSC (n=18 each). Fourteen days after modeling, hematoxylin-eosin staining was used to observe pathological changes in the white matter, and immunofluorescence staining was used to measure the expression level of ionized calcium-binding adapter molecule 1 (Iba1). Western blotting was used to measure the protein expression levels of inhibitory subunit of nuclear factor-kappa B alpha (IκBα), phosphorylated IκBα (p-IκBα), phosphorylated NF-κB p65 (p-NF-κB p65), myelin basic protein (MBP), and neuron-specific nuclear protein (NeuN). Quantitative real-time PCR was used to assess the mRNA expression levels of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), MBP, and NeuN. Immunohistochemistry was used to measure the protein expression levels of MBP and NeuN. On day 28, the Morris water maze test was used to evaluate spatial cognitive ability.

Results: Fourteen days after modeling, the sham-operation group exhibited intact white matter structure with normal cell morphology and orderly nerve fiber arrangement. In the WMI group, large-scale cell degeneration and necrosis were observed, and nerve fiber arrangement was disordered. The hUC-MSC group showed relatively normal cell morphology and more orderly nerve fibers. Compared with the sham-operation group, the WMI group had significantly higher proportions of Iba1-positive cells, increased protein levels of p-IκBα and p-NF-κB p65, and higher mRNA levels of TNF-α and IL-1β. The protein expression of IκBα and the positive expression of MBP and NeuN, as well as their protein and mRNA levels, were significantly reduced in the WMI group (P<0.05). Compared with the WMI group, the hUC-MSC group showed reduced proportions of Iba1-positive cells, decreased protein levels of p-IκBα and p-NF-κB p65, and lower mRNA levels of TNF-α and IL-1β. Furthermore, IκBα protein expression and MBP and NeuN expression (both at the protein and mRNA levels) were significantly increased in the hUC-MSC group (P<0.05). On day 28, the Morris water maze results showed that compared with the sham-operation group, the WMI group had significantly longer escape latency and fewer platform crossings (P<0.05). In contrast, the hUC-MSC group had significantly shorter escape latency and more platform crossings than the WMI group (P<0.05).

Conclusions: hUC-MSC transplantation can repair WMI in neonatal rats, promote the maturation of oligodendrocytes, and support neuronal survival, likely by inhibiting activation of the NF-κB signaling pathway mediated by microglial cells.

Keywords: Human umbilical cord mesenchymal stem cell; Neonatal rat; Nuclear factor-kappa B pathway; White matter injury.

图1. 各组新生大鼠脑白质区域组织病理学变化（苏木精-伊红染色，×400） 假手术组组织结构完整，细胞形态正常，神经纤维排列规则；WMI组组织结构破坏，大量细胞水肿、细胞核固缩坏死，神经纤维排列疏松、紊乱；hUC-MSC组组织结构较完整，细胞形态相对正常，神经纤维排列较为规整。图中箭头所指为细胞核固缩。

图2. 各组新生大鼠Iba1和NF-κB信号通路相关蛋白表达水平比较 A：各组新生大鼠脑室旁区Iba1阳性细胞比较（免疫荧光染色，×400），图中白色箭头示Iba1阳性细胞。Iba1标记细胞染色为红色，DAPI标记细胞核染色为蓝色。WMI组Iba1阳性细胞占比高于假手术组，hUC-MSC组Iba1阳性细胞占比低于WMI组。B：各组新生大鼠IκBα、p-IκBα、p-NF-κB p65蛋白表达电泳图。

图3. 各组新生大鼠白质区域MBP、NeuN表达水平比较 A：各组新生大鼠MBP、NeuN阳性表达比较（免疫组化，×400）。图中棕黄色为阳性表达，WMI组中MBP、NeuN阳性表达低于假手术组，hUC-MSC组MBP、NeuN阳性表达高于WMI组。B：各组新生大鼠MBP、NeuN蛋白表达电泳图。

图4. 各组新生大鼠Morris水迷宫实验行径路线比较 A：各组新生大鼠定向航行训练阶段（第5天）行径路线图。第4象限内小圆圈为平台所在位置，WMI组逃避潜伏期较假手术组延长，hUC-MSC组逃避潜伏期较WMI组缩短。B：各组新生大鼠空间探索测试阶段行径路线图。WMI组穿越平台次数较假手术组减少，hUC-MSC组穿越平台次数较WMI组增加。