Objectives: To observe the change in ferroptosis in hippocampal neurons after hypoxia-ischemia (HI) in neonatal rats and investigate the related mechanism based on the TXNIP/Trx-1/GPX4 signaling pathway.
Methods: Healthy neonatal Sprague-Dawley rats, aged 7 days, were randomly divided into three groups: sham-operation (n=30), hypoxic-ischemic brain damage (HIBD) (n=30) and siRNA (TXNIP siRNA) (n=12). The classic Rice-Vannucci method was used to establish a neonatal rat model of HIBD. At 6 hours, 24 hours, 72 hours, and 7 days after modeling, Western blot was used to measure the protein expression of GPX4 in the hippocampal tissue at the injured side; at 24 hours after modeling, laser speckle imaging combined with hematoxylin-eosin staining was used to determine whether the model was established successfully; NeuN/GPX4 and GFAP/GPX4 immunofluorescence staining combined with Western blot and other methods was used to measure the protein expression of GPX4 and the signal molecules TXNIP and Trx-1 in the hippocampal tissue at the injured side; the kits for determining the content of serum iron and tissue iron were used to measure the change in iron content; quantitative real-time PCR was used to measure the mRNA expression of TXNIP, Trx-1, and GPX4.
Results: At 6 hours, 24 hours, 72 hours, and 7 days after modeling, the HIBD group had a significantly lower protein expression level of GPX4 than the sham-operation group (P<0.05). At 24 hours after modeling, the HIBD group had a significantly lower cerebral blood flow of the injured side than the sham-operation group (P<0.05), with loose and disordered arrangement and irregular morphology of hippocampal CA1 neurons at the injured side. Compared with the sham-operation group, the HIBD group had a significantly higher number of TXNIP+ cells and significantly lower numbers of Trx-1+ cells and NeuN+GPX4+/NeuN+ cells in the hippocampal CA1 region at the injured side (P<0.05), with almost no GFAP+GPX4+ cells in the hippocampal CA1 region. Compared with the sham-operation group, the HIBD group and the siRNA group had significantly higher levels of serum iron and tissue iron in the hippocampus at the injured side (P<0.05). Compared with the HIBD group, the siRNA group had significantly lower levels of serum iron and tissue iron in the hippocampus at the injured side (P<0.05). The HIBD group and the siRNA group had significantly higher mRNA and protein expression levels of TXNIP than the sham-operation group (P<0.05), and the siRNA group had significantly lower expression levels than the HIBD group (P<0.05). The HIBD group and the siRNA group had significantly lower mRNA and protein expression levels of Trx-1 and GPX4 in the hippocampus at the injured side than the sham-operation group (P<0.05), and the siRNA group had significantly higher expression levels than the HIBD group (P<0.05).
Conclusions: HI induces ferroptosis of hippocampal neurons in neonatal rats by activating the TXNIP/Trx-1/GPX4 pathway, thereby resulting in HIBD.
目的: 观察新生大鼠缺氧缺血后海马神经细胞铁死亡的变化，并从TXNIP/Trx-1/GPX4信号通路探讨其机制。方法: 将健康7日龄Sprague-Dawley新生大鼠随机分为假手术组（n=30）、缺氧缺血性脑损伤（hypoxic-ischemic brain damage，HIBD）组（n=30）及siRNA（TXNIP siRNA）组（n=12）。采用经典Rice-Vannucci法建立新生大鼠HIBD模型。造模后6 h、24 h、72 h、7 d，Western blot法检测新生大鼠损伤侧海马组织铁死亡蛋白GPX4蛋白表达水平变化；造模后24 h，采用激光散斑成像结合苏木精-伊红染色法鉴定模型；采用NeuN/GPX4、GFAP/GPX4免疫荧光双标染色结合Western blot等方法检测各组新生大鼠损伤侧海马组织GPX4、TXNIP、Trx-1蛋白表达水平；检测新生大鼠血清铁和损伤侧海马组织铁含量的变化；采用实时荧光定量聚合酶链式反应法检测各组新生大鼠TXNIP、Trx-1、GPX4 mRNA的表达水平。结果: 造模后6 h、24 h、72 h、7 d，HIBD组GPX4蛋白表达水平低于假手术组（P<0.05）。造模后24 h，HIBD组损伤侧脑血流量低于假手术组（P<0.05），HIBD组损伤侧海马CA1区神经细胞排列疏松紊乱，形态不规则。HIBD组损伤侧海马CA1区TXNIP+细胞数高于假手术组，Trx-1+细胞数、NeuN+GPX4+/NeuN+低于假手术组（P<0.05），海马CA1区几乎未见GFAP+GPX4+细胞。HIBD组和siRNA组血清铁、损伤侧海马组织铁含量高于假手术组，且siRNA组血清铁、损伤侧海马组织铁含量低于HIBD组（P<0.05）。HIBD组和siRNA组TXNIP mRNA及蛋白表达水平均高于假手术组，且siRNA组TXNIP mRNA及蛋白表达水平低于HIBD组（均P<0.05）；HIBD组和siRNA组损伤侧海马组织Trx-1、GPX4 mRNA及蛋白表达水平均低于假手术组，且siRNA组损伤侧海马组织Trx-1、GPX4 mRNA及蛋白表达水平高于HIBD组（均P<0.05）。结论: 新生大鼠缺氧缺血通过激活TXNIP/Trx-1/GPX4通路，导致新生大鼠海马神经元铁死亡，进而导致HIBD。.
Keywords: Ferroptosis; Hippocampus; Hypoxic-ischemic brain damage; Neonatal rat; TXNIP/Trx-1/GPX4 pathway.