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线粒体自噬对新生大鼠缺氧缺血脑损伤的影响
Role of mitophagy in neonatal rats with hypoxic-ischemic brain damage
目的 了解缺氧缺血脑损伤的动物模型中线粒体自噬情况，探讨其在缺氧缺血脑损伤中的作用。方法 将120只新生7日龄Sprague-Dawley大鼠分为假手术组、缺氧缺血脑损伤 （HIBD）组、自噬抑制剂干预组 （3MA组）。HIBD组行右侧颈总动脉结扎后置于低氧仓 （8%氧气和92%氮气）2.5h，3MA组腹腔注射2μL 3MA 30min后再进行结扎和低氧处理，假手术组不予结扎和低氧处理。在造模后提取各组单细胞悬液，用Mitotracker标记线粒体、Lysotracker标记自噬小体、LC3标记自噬进行免疫荧光共定位观察线粒体自噬情况；荧光探针JC-1染色后，采用流式细胞仪检测线粒体膜电位。TTC染色法检测脑梗死灶大小。提取皮质神经元中的细胞浆蛋白，Western blot法检测线粒体自噬相关蛋白的表达情况。结果 与假手术组相比，HIBD组线粒体膜电位明显降低 （P < 0.05），线粒体自噬增加 （P < 0.05），线粒体分裂相关蛋白Drp1、Fis1的表达升高，线粒体外膜蛋白受体Tom20与内膜蛋白受体Tim23的表达降低 （P < 0.05）；3MA组膜电位比HIBD组降低更为明显 （P < 0.05），但线粒体自噬程度明显减少 （P < 0.05）。3MA组大鼠脑组织梗死程度较HIBD组明显增加 （P < 0.05）。结论 HIBD能够增加线粒体自噬的发生，抑制线粒体自噬会加重新生大鼠HIBD的损伤程度。
Objective To investigate mitophagy in an animal model of hypoxic-ischemic brain damage (HIBD) and its role in HIBD. Methods A total of 120 neonatal Sprague-Dawley rats aged 7 days were divided into three groups: sham-operation, HIBD, and autophagy inhibitor intervention (3MA group). The rats in the HIBD group were treated with right common carotid artery ligation and then put in a hypoxic chamber (8% oxygen and 92% nitrogen) for 2.5 hours. Those in the 3MA group were given ligation and hypoxic treatment at 30 minutes after intraperitoneal injection of 2 μL 3MA. Those in the sham-operation group were not given ligation or hypoxic treatment. Single cell suspension was obtained from all groups after model establishment. Immunofluorescence localization was performed for mitochondria labeled with MitoTracker, autophagosomes labeled with LysoTracker, and autophagy labeled with LC3 to observe mitophagy. After staining with the fluorescent probe JC-1, flow cytometry was used to measure mitochondrial membrane potential. TTC staining was used to measure infarct volume. Cytoplasmic proteins in cortical neurons were extracted, and Western blot was used to measure the expression of mitophagy-related proteins. Results Compared with the shamoperation group, the HIBD group had a significant reduction in mitochondrial membrane potential (P < 0.05), a significant increase in mitophagy (P < 0.05), a significant increase in the expression of the proteins associated with the division of the mitochondrial Drp1 and Fis1 (P < 0.05), and a significant reduction in the expression of the mitochondrial outer membrane protein Tom20 and the mitochondrial inner membrane protein Tim23 (P < 0.05). Compared with the HIBD group, the 3MA group had a significantly greater reduction in mitochondrial membrane potential (P < 0.05), but showed significantly reduced mitophagy (P < 0.05). In addition, the 3MA group had a significantly increased degree of cerebral infarction compared with the HIBD group (P < 0.05). Conclusions HIBD can increase the degree of mitophagy, and the inhibition of mitophagy can aggravate HIBD in neonatal rats.
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国家自然科学基金（81330016；81630038；81300526）；国家重点基础发展计划（973计划）（2013CB967404）；四川省科技计划项目（2014SZ0149）；临床重点专科计划项目（1311200003303）。
