[1]梁译丹,覃王,黄豪,等.自噬通过降解铁蛋白促进神经元铁死亡参与蛛网膜下腔出血后早期脑损伤[J].第三军医大学学报,2019,41(15):1407-1414.
 LIANG Yidan,QIN Wang,HUANG Hao,et al.Autophagy promotes neuronal ferroptosis by degrading ferritin and induces early brain injury after subarachnoid hemorrhage in rats[J].J Third Mil Med Univ,2019,41(15):1407-1414.
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自噬通过降解铁蛋白促进神经元铁死亡参与蛛网膜下腔出血后早期脑损伤(/HTML )
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《第三军医大学学报》[ISSN:1000-5404/CN:51-1095/R]

卷:
41卷
期数:
2019年第15期
页码:
1407-1414
栏目:
基础医学
出版日期:
2019-08-15

文章信息/Info

Title:
Autophagy promotes neuronal ferroptosis by degrading ferritin and induces early brain injury after subarachnoid hemorrhage in rats
作者:
重庆医科大学附属第一医院神经外科
Author(s):
LIANG Yidan QIN Wang HUANG Hao XIN Yuanjun ZHAO Jun XIANG Xiang WU Xintong HE Zhaohui
Department of Neurosurgery, the First Affiliated Hospital of Chongqing Medical University, Chongqing, 400016, China
关键词:
蛛网膜下腔出血早期脑损伤自噬铁死亡铁蛋白
Keywords:
subarachnoid hemorrhage early brain injury autophagy ferroptosis ferritin
分类号:
R363.21;R742;R743.35
文献标志码:
A
摘要:

目的 观察大鼠蛛网膜下腔出血( subarachnoid hemorrhage,SAH)后抑制自噬对早期脑损伤(early brain injury, EBI)恢复的作用,揭示自噬促进神经元铁死亡从而影响EBI的具体机制。方法 将SD大鼠随机分为对照组、SAH组、SAH+LV-ATG5组和SAH+LV-Scramble组,每组12只。采用血管内穿刺法构建SAH模型,自噬相关基因5 (autophagy-related gene 5, ATG5 )慢病毒(LV-ATG5)抑制自噬,免疫荧光检测SAH后24h ATG5、铁蛋白重链1(ferritin heavy polypeptide 1, FTH1)和谷胱甘肽过氧化物酶4(glutathione peroxidase 4, GPX4)在皮质神经元的表达和定位情况。PCR检测ATG5 mRNA的表达情况,Western blot 检测ATG5、LC3Ⅱ/Ⅰ、FTH1和GPX4的表达变化,尼氏染色(Nissl staining)和神经行为学评分、脑水含量等检测抑制自噬后神经元形态和功能的变化情况,并检测细胞内铁沉积、游离铁含量、脂质过氧化物产物丙二醛( malonaldehyde,MDA) 、GPX4活性等铁死亡指标。结果免疫荧光显示ATG5、FTH1和GPX4广泛表达于SAH后皮质神经元胞质中。PCR和Western blot检测结果显示ATG5 mRNA、ATG5和LC3Ⅱ/I蛋白表达量显著降低(P<0.05),自噬被成功抑制。抑制自噬后,Nissl染色提示神经元数量和形态得到一定程度的改善,神经行为学评分升高(P<0.05),脑水含量减少(P<0.05)。对其可能的具体通路进行探索,Western blot检测结果显示SAH后FTH1和GPX4表达量下降(P<0.05),LV-ATG5抑制自噬后FTH1和GPX4表达量升高(P<0.05),与 SAH 组、空载病毒组相比,慢病毒干预组中细胞内铁沉积减少,铁含量、MDA 减少( P<0.05) ; GSH 含量提高、GPX4 活性增强(P<0.05)。结论 自噬可能通过降解神经元内铁蛋白,导致神经元内游离铁含量增加,促进神经元铁死亡,加重EBI。

Abstract:

Objective To determine the effect of suppressing autophagy on the recovery of early brain injury (EBI) after subarachnoid hemorrhage (SAH) in rats, and to reveal the mechanism in which autophagy promotes neuronal ferroptosis and then affects EBI. MethodsSD rats were randomly divided into control group, SAH group, SAH+LV-ATG5 group and SAH+LV-Scramble group, with 12 animals in each group. Rat model of SAH were established by intravascular puncture. Lentiviral vector LV-ATG5 targeting autophagy-related gene 5 (ATG5) was constructed to inhibit autophagy. Immunofluorescence assay was used to detect the expression and location of ATG5, ferritin heavy polypeptide 1 (FTH1) and glutathione peroxidase 4 (GPX4) in neurons in 24 h after SAH establishment. The mRNA expression of ATG5 was detected by PCR, and the protein expression of ATG5, LC3Ⅱ/Ⅰ, FTH1 and GPX4 were detected by Western blotting. The changes of neuronal morphology and function were detected by Nissl staining, and neurological score and brain water content were also detected. Intracellular iron deposition, iron content, malonaldehyde (MDA) content and GPX4 activity were measured to evaluate the ferroptosis. ResultsImmunofluorescence assay showed that ATG5, FTH1 and GPX4 were widely expressed in the cytoplasm of cortical neurons after SAH. The results of PCR and Western blotting indicated that the expression of ATG5 at mRNA and protein levels, and LC3II/I at protein level was significantly decreased (P<0.05), and autophagy was successfully inhibited. When autophagy was inhibited, Nissl staining showed improvements on neuronal morphology and function, neurological function score was improved (P<0.05), and brain water content was decreased (P<0.05). Western blot assay indicated that the expression levels of FTH1 and GPX4 were decreased after SAH (P<0.05), however, the levels were increased when autophagy was inhibited (P<0.05). Furthermore, autophagy inhibition also reduced iron deposition, and cellular labile iron and MDA contents (P<0.05), and also elevated GSH content and GPX4 activity (P<0.05). ConclusionAutophagy may promote neuronal ferroptosis by degrading ferritin,  result in cellular labile iron increase, and finally early brain injury.

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更新日期/Last Update: 2019-08-13