Objective: To study the methylation level and dynamic change of 5-hydroxymethylcytosine (5hmC) in mitochondrial DNA (mtDNA) in the cerebral cortex of neonatal rats with hypoxic-ischemic brain damage.
Methods: A total of 24 male Sprague-Dawley rats aged 7 days were randomly divided into control group, 24-hour model group and 48-hour model group (n=8 each). Common carotid artery ligation combined with hypoxic treatment was performed to establish an animal model of hypoxic-ischemic brain damage. The rats in the control group were not given ligation or hypoxic treatment. Oxidative bisulfite sequencing was used to measure the level of 5hmC in the cerebral cortex. Western blot was used to measure the expression of 5hmC-related enzymes TET1, TET2 and DNMT1.
Results: The 24- and 48-hour model groups had a significantly higher level of 5hmC than the control group (P<0.05). Western blot showed a significant increase in the expression of DNMT1 in the 24- and 48-hour model groups (P<0.05). Compared with the control group, the 24- and 48-hour model groups had significant differences in the 5hmC level at multiple mitochondrial genetic loci (P<0.05).
Conclusions: The level of DNMT1, a key enzyme for 5hmC modification in mtDNA, in the cerebral cortex increases in neonatal rats with hypoxic-ischemic brain damage, suggesting that there is an abnormal methylation level of 5hmC after hypoxic-ischemic brain damage, which might be associated with the regulation of hypoxic-ischemic brain damage.
目的: 检测缺氧缺血脑损伤新生大鼠大脑皮质组织线粒体DNA（mtDNA）中5-羟甲基胞嘧啶（5hmC）的甲基化含量及动态变化。
方法: 将24只7日龄雄性Sprague-Dawley大鼠随机分为对照组、24 h模型组和48 h模型组，每组8只。采用结扎颈总动脉结合缺氧法构建新生大鼠缺氧缺血性脑损伤动物模型，对照组不接受结扎及缺氧处理。采用化学氧化法结合重亚硫酸盐转化测序技术（oxBS-Seq）检测大鼠大脑皮质组织mtDNA的5hmC水平。使用Western blot验证5hmC相关酶TET1、TET2、DNMT1的表达水平。
结果: 模型组24 h、模型组48 h的5hmC含量显著高于对照组（P < 0.05）。Western blot显示mtDNA中5hmC相关酶DNMT1的表达在24 h和48 h模型组中显著升高（P < 0.05）。与对照组相比，多个线粒体基因位点在模型组大鼠中均呈现5hmC水平差异（P < 0.05）。
结论: 缺氧缺血脑损伤新生大鼠大脑皮质组织mtDNA中5hmC相关酶DNMT1的表达水平增高，提示大鼠在缺氧缺血脑损伤后5hmC甲基化水平存在异常，可能与缺氧缺血大脑损伤的调节有关。