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

作　　者：张新月 刘晨萌 马瑜徽 孟楠 蒋景英 余小河[3] 王晓莉[1] ZHANG Xin-Yue;LIU Chen-Meng;MA Yu-Hui;Meng Nan;JIANG Jing-Ying;YU Xiao-He;WANG Xiao-Li(School of Medical Imaging,Weifang Medical University,Weifang,Shandong 261053,China)

机构地区：[1]潍坊医学院医学影像学院,山东潍坊261053 [2]北京航空航天大学,北京100191 [3]中南大学湘雅医院,湖南长沙410008

出　　处：《中国当代儿科杂志》2022年第9期1053-1060,共8页Chinese Journal of Contemporary Pediatrics

摘　　要：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

关 键 词：缺氧缺血性脑损伤 铁死亡 海马 TXNIP/Trx-1/GPX4通路 新生大鼠 

分 类 号：R743.3[医药卫生—神经病学与精神病学]