COX5A Relieves Traumatic Brain Injury-Induced Neurological Damage in Mice by Correcting Mitochondrial Homeostasis Imbalance
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摘要:
目的 探讨细胞色素c氧化酶亚基5A（cytochrome c oxidase subunit 5A，COX5A）过表达对创伤性脑损伤（traumatic brain injury，TBI）小鼠的神经保护作用及其与线粒体功能的关系。 方法 将4～6周龄C57BL/6J野生型（wild type，WT）小鼠及同龄COX5A过表达（COX5A-UP）转基因小鼠随机分为WT+假手术组、WT+TBI组、COX5A-UP+假手术组和COX5A-UP+TBI组（n = 12，雌雄各半），采用可控皮质冲击法制备TBI模型。术后14 d评估神经行为学表现、脑含水量、海马突触体线粒体氧消耗率、ATP、活性氧、丙二醛、线粒体膜电位及线粒体超微结构。 结果 与WT+假手术组相比，WT+TBI组神经功能和行为学表现受损，脑含水量升高，突触体线粒体能量代谢下降、氧化应激增强，并出现线粒体形态破坏（P < 0.05）。与WT+TBI组相比，COX5A-UP+TBI组上述神经行为学异常、脑水肿及线粒体功能和结构损伤均改善（P < 0.05）。 结论 COX5A过表达可减轻TBI诱导的神经功能损伤，其作用可能与改善突触体线粒体功能障碍和维持线粒体结构稳态有关。 Abstract:Objective To investigate the neuroprotective effect of cytochrome c oxidase subunit 5A （COX5A） overexpression in mice with traumatic brain injury （TBI） and its association with mitochondrial function. Methods C57BL/6J wild-type （WT） mice aged 4–6 weeks and age-matched COX5A-overexpressing （COX5A-UP） transgenic mice were randomly divided into WT+sham operation group, WT+TBI group, COX5A-UP+sham operation group, and COX5A-UP+TBI group （n = 12 per group, male∶female = 1∶1）. The TBI model was established using the controlled cortical impact method. At 14 days after surgery, neurological and behavioral performance, brain water content, oxygen consumption rate of hippocampal synaptosomal mitochondrial , ATP levels, reactive oxygen species, malondialdehyde, mitochondrial membrane potential and mitochondrial ultrastructure were examined. Results Compared with the WT+sham group, WT+TBI group showed impaired neurological and behavioral performance, increased brain water content, reduced mitochondrial energy metabolism, enhanced oxidative stress and mitochondrial morphological damage （P < 0.05）. Compared with the WT+TBI group, the COX5A-UP+TBI group showed improvements in these neurological, behavioral, edema-related and mitochondrial abnormalities （P < 0.05）. Conclusion COX5A overexpression alleviates TBI-induced neurological impairment, and this effect may be associated with amelioration of synaptosomal mitochondrial dysfunction and preservation of mitochondrial structural homeostasis. -
Key words:
- COX5A /
- Traumatic brain injury /
- Mitochondrial dyshomeostasis /
- Neuroprotection /
- Mouse model /
- Behavior
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图 1 各组小鼠脑损伤后14 d神经功能评分及脑含水量测定（mean ± SEM，n = 12）
A：伤后14 d mNSS评分；B：脑组织含水量（%）。采用双因素方差分析及Tukey事后检验。**P < 0.01；****P < 0.0001。
Figure 1. Neurological function scores and brain water content in each group at 14 days after brain injury（mean ± SEM，n = 12）
图 2 各组小鼠脑损伤后14 d神经行为学评估[（mean ± SEM），n = 12]
A：旷场实验中小鼠总移动距离（cm）；B：旷场实验中小鼠中心区域停留时间占比（%）；C：旷场实验中小鼠直立次数；D：新物体识别实验中小鼠识别指数。采用双因素方差分析及Tukey事后检验。*P < 0.05，**P < 0.01，***P < 0.001，****P < 0.0001。
Figure 2. Neurobehavioral evaluation in each group at 14 days after brain injury[（mean ± SEM），n = 12]
图 3 各组小鼠脑损伤后海马突触体线粒体氧耗量检测[（mean ± SEM），n = 12]
A：海马突触体线粒体氧耗曲线；B：基础呼吸；C：最大呼吸；D：备用呼吸容量。采用双因素方差分析及Tukey事后检验。***P < 0.001；****P < 0.0001。
Figure 3. Oxygen consumption rate of hippocampal synaptosomal mitochondria in each group after brain injury[（mean ± SEM），n = 12]
图 4 各组小鼠TBI后海马突触体线粒体功能检测[（mean ± SEM），n = 12]
A：海马突触体线粒体ATP水平；B：海马突触体线粒体ROS相对荧光强度；C：海马突触体线粒体MMP相对荧光强度；D：海马突触体线粒体MDA含量。采用双因素方差分析及Tukey事后检验。*P < 0.05；**P < 0.01；***P < 0.001；****P < 0.0001。
Figure 4. Mitochondrial function assessment of hippocampal synaptosomes in each group after TBI[（mean ± SEM），n = 12]
图 5 各组小鼠TBI后海马神经元线粒体超微结构及形态学定量[（mean ± SEM），n = 5]
A：各组小鼠TEM代表图（×20 000，scale bar = 500 nm）；B：线粒体长径比定量结果。每只动物量化300～400个线粒体后取平均值；采用双因素方差分析及Tukey事后检验。*P < 0.05；**P < 0.01。
Figure 5. Mitochondrial ultrastructure and morphological quantification of hippocampal neurons in each group after TBI [（mean ± SEM），n = 5]
图 6 TBI后不同时间点COX5A表达水平的动态变化（mean ± SEM，n = 12）
A：TBI后海马组织COX5A mRNA表达动态变化；B：TBI后海马组织COX5A蛋白表达定量结果；C：TBI后海马组织COX5A蛋白Western blot条带图。
Figure 6. Dynamic changes in COX5A expression levels at different time points after TBI （mean ± SEM，n = 12）
表 1 引物序列
Table 1. Primer sequences
基因 序列 (5'-3') COX5A F：5'-GGTCAAACCCAACAGAAG-3' COX5A R：5'-TGTCCGTCGCCATCAATAT-3' GAPDH F：5'-GTCCTTGATCACCCGATTC-3' GAPDH R：5'-TCCTGTGTGCTTTCCATTC-3' 
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