Clinic Medical College, Chengdu Medical College, No. 783 Xindu Avenue, Xindu District, Chengdu, Sichuan Province 610500, PR China; Department of Pediatrics, The First Affiliated Hospital of Chengdu Medical College, No. 278, Middle Section of Baoguang Avenue, Xindu District, Chengdu, Sichuan Province 610500, PR China.
Department of Pediatrics, The First Affiliated Hospital of Chengdu Medical College, No. 278, Middle Section of Baoguang Avenue, Xindu District, Chengdu, Sichuan Province 610500, PR China.
Brain Res Bull. 2022 Oct 15;189:139-150. doi: 10.1016/j.brainresbull.2022.08.014. Epub 2022 Aug 17.
BACKGROUND AND RESEARCH QUESTION/HYPOTHESIS: Excessive oligodendrocyte precursor cell (OPC) apoptosis occurs during intrauterine infection-induced white matter injury (WMI) in premature infants, preventing excessive apoptosis of OPCs is one of the mechanisms protecting WMI. Micro-RNA-21-5p (miR-21-5p) mediating anti-apoptotic activity was observed in other diseases. Therefore, the aim of this study was to determine whether miR-21-5p protects against WMI by modulating phosphatase and tensin homologue deleted on chromosome 10/phosphatidylinositol-3-kinase/protein kinase B (PTEN/PI3K/Akt) signalling pathway.
A lipopolysaccharide (LPS)-induced neonatal Sprague-Dawley (SD) rat model of preterm WMI was established. To explore the effect of miR-21-5p on WMI, we intraventricularly injected miR-21-5p agomir and miR-21-5p antagomir to activate or inhibit endogenous miR-21-5p. Immunofluorescent labelling of myelin basic protein, immunohistochemical labelling of 2',3'-cyclic-nucleotide 3'-phosphodiesterase (CNPase), and terminal deoxynucleotidyl transferase dUTP nick end labelling assays were conducted to observe pathological white matter changes. The antibody of anti-oligodendrocyte marker 4 (O4) was used to specifically recognise OPCs. The expressions of miR-21-5p and PTEN mRNA in the brain were detected with quantitative real-time polymerase chain reaction (qRT-PCR). PTEN, Akt, and phosphorylated Akt (p-Akt) protein levels were assayed with western blotting, and apoptotic proteins associated with PI3K/Akt signalling were quantified.
Intense white matter dysplasia and excessive OPC apoptosis were observed in the brains of rats with WMI. When the miR-21-5p agonist miR-21-5p agomir was used in the WMI group, apoptosis of OPCs was significantly reduced, and myelin maturation increased. MiR-21-5p agomir relieved WMI. MiR-21-5p agomir inhibited the mRNA and protein expression of PTEN, increased p-Akt phosphorylation, and decreased the expression and activation of related apoptotic proteins.On the other hand, the administration of miR-21-5p specific blocker, miR-21-5p antagomir, reduced the level of p-AKT, increased OPC apoptosis, and worsened WMI.
Our findings revealed that miR-21-5p agomir had anti-OPC over-apoptotic effects and enhanced myelin development in WMI by modulating the PTEN/Akt signalling pathway.
背景和研究问题/假设：在早产儿宫内感染诱导的白质损伤（WMI）中，过度的少突胶质前体细胞（OPC）凋亡发生，防止 OPC 过度凋亡是保护 WMI 的机制之一。在其他疾病中观察到 micro-RNA-21-5p（miR-21-5p）介导的抗凋亡活性。因此，本研究旨在确定 miR-21-5p 是否通过调节磷酸酶和张力蛋白同源物缺失于染色体 10/磷脂酰肌醇-3-激酶/蛋白激酶 B（PTEN/PI3K/Akt）信号通路来保护 WMI。
建立脂多糖（LPS）诱导的早产 Sprague-Dawley（SD）大鼠 WMI 模型。为了探讨 miR-21-5p 对 WMI 的影响，我们通过脑室注射 miR-21-5p 激动剂和 miR-21-5p 拮抗剂来激活或抑制内源性 miR-21-5p。通过免疫荧光标记髓鞘碱性蛋白、2',3'-环核苷酸 3'-磷酸二酯酶（CNPase）免疫组织化学标记和末端脱氧核苷酸转移酶 dUTP 缺口末端标记法观察病理性白质变化。用抗少突胶质细胞标记物 4（O4）抗体特异性识别 OPC。采用实时定量聚合酶链反应（qRT-PCR）检测脑内 miR-21-5p 和 PTEN mRNA 的表达。采用 Western blot 法检测 PTEN、Akt 和磷酸化 Akt（p-Akt）蛋白水平，并定量分析与 PI3K/Akt 信号相关的凋亡蛋白。
WMI 大鼠脑内白质严重发育不良，OPC 凋亡明显增加。当 WMI 组使用 miR-21-5p 激动剂 miR-21-5p agomir 时，OPC 凋亡明显减少，髓鞘成熟增加。miR-21-5p agomir 缓解了 WMI。miR-21-5p agomir 抑制了 PTEN、p-Akt 磷酸化和相关凋亡蛋白的表达和激活。另一方面，给予 miR-21-5p 特异性阻滞剂 miR-21-5p antagomir 降低了 p-AKT 水平，增加了 OPC 凋亡，加重了 WMI。
我们的研究结果表明，miR-21-5p agomir 通过调节 PTEN/Akt 信号通路对 OPC 过度凋亡具有抗凋亡作用，并增强了 WMI 中的髓鞘发育。
