TXNIP/Trx-1/GPX4通路促进新生大鼠缺氧缺血后海马神经元铁死亡的作用机制

The TXNIP/Trx-1/GPX4 pathway promotes ferroptosis in hippocampal neurons after hypoxia-ischemia in neonatal rats

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。

Objective 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 shamoperation 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.