组蛋白去乙酰化酶抑制剂丁酸钠对七氟醚麻醉诱发新生大鼠远期认知功能障碍的影响

Effects of histone deacetylase inhibitor sodium butyrate on sevoflurane-induced long-term cognitive dysfunction in neonatal rats

目的观察组蛋白去乙酰化酶（histone deacetylase，HDAC）抑制剂丁酸钠（sodium butyrate，NaB）对七氟醚麻醉诱发新生大鼠远期认知功能障碍的影响。方法24只613龄新生雄性Sprague-Dawley（SD）大鼠按随机数字表法分为3组（每组8只）：O2＋生理盐水组（O2＋NS组）、七氟醚麻醉＋生理盐水组（Sev＋NS组）及七氟醚麻醉＋NaB组（Sev＋NaB组）。Sev＋NS组和Sev＋NaB组分别在出生后6d-8d时，每天接受1次2h的3％七氟醚＋100％O2麻醉；O2＋NS组在相应13龄吸人相同时间100％O：。出生后第9天起至行为学测试结束前分别进行生理盐水（10ml／kg）和NaB（1．2g／kg）腹腔注射，连续32d。第31天行旷场实验，第34～40天行水迷宫实验。行为学测试结束后1h，取海马，Westernblot检测HDAC2、脑源性神经营养因子（brain-derived neurotrophie factor，BDNF）、突触后密度蛋白95（postsynaptic density95，PSD95）及微管相关蛋白2（microtubule associated protein2，MAP2）含量；免疫荧光检测海马分区乙酰化姐蛋白H3赖氨酸9（histone H3 lysine9，H3K9）和乙酰化蛆蛋白H4赖氨酸12（histone H4 lysine12，H4K12）荧光强度。结果旷场实验中，各组探索路程、运动速度及中央格停留时间比较，差异无统计学意义（P〉0．05）。与O2＋NS组比较，Sev＋NS组在水迷宫实验第36、37天的逃避潜伏期明显延长，目标象限停留时间[O2＋NS组（30．2±2．8）S，Sev＋NS组（16．8±2．9）S，Sev＋NaB组（28．3±3．4）S]及穿越原平台次数[O2＋NS组（5．4±0．8）次，Sev＋NS组（2．5±0．8）次，Sev＋NaB组（5．5±0．7）次）]明显减少（P〈0．05），HDAC2含量明显增加，CA1区乙酰化-H3K9和乙酰化-H4K12荧光强度及BDNF、PSD95和MAP2含量明显降低（P〈0．05）；而注射NaB后可改善以上检测指标的异常（P〈0．05）。结论HDAC抑制剂NaB可改善七氟醚麻醉所致新生大鼠远期海马依赖性空间认知功能受损，其机制可能与增加海马内组蛋白乙酰化及突触可塑性有关。

Objective To observe the effects of histone deacetylase （HDAC） inhibitor sodium butyrate （NAB） on sevoflurane-induced long-term cognitive dysfunction in neonatal rats. Methods Twenty-four male Sprague-Dawley （SD） rats at postnatal day 6 were randomly divided into the following three groups （n=8） in accordance with a random number table： O2＋normal saline group （O2＋NS group ）, sevoflurane anesthesia＋normal saline group（Sev＋NS group） and sevoflurane anesthesia＋sodium butyrate group （Sev＋NaB group）. Animals were subjected to 3% sevoflurane plus 100% 02 or 100% O2 2 h daily for 3 consecutive days, respectively. Thereafter, animals in different groups were chronically given intraperitoneal injections of NS（ 10 ml/kg） or NaB （1.2 g/kg） for 32 d, respectively. Open field was performed at postnatal day 31, and the Morris Water Maze test was performed at postnatal day 34-40. The hippocampus was harvested 1 h after the behavioral test to measure the content of HDAC2, brain-derived neurotrophic factor （BDNF）, postsynaptic density 95 （PSD95） and mierotubule associated protein （MAP2）. Immunofluoreseenee was used to confirm the fluorescence intensity of acetyl-histone H3 lysine 9（H3K9） and acetyl-histone H4 lysine 12（H4K12 ） in the hippocampus subfields. Results In the open field test, there was no significant difference in the total travel distance, speed, and the time spent in the center of the arena among the three groups（P〉0.05 ）. In the Morris Water Maze test, the escape latency at postnatal day 36-37 increased significantly, the time spent in the target quadrant [ O2＋NS group： （30.2±2.8） s, Sev＋NS group： （ 16.8±2.9 ） s, Sev＋NaB group： （28.3±3.4） s ] and times across the original platform [O2＋NS group： （5.4±0,8） times, Sev＋NS group ： （2.5±0.8） times, Sev＋NaB group： （5.5±0.7） times] reduced significantly, the content of HDAC2 increased significantly, fluorescence intensity of acetyl-H3K9 and H4K12 in hippocampal CA1 region and the content of BDNF, PSD95 as well as MAP2 decreased significantly in the Sev＋NS group compared with the O2＋NS group （P〈0.05）. However, NaB by intraperitoneal injection could reverse the above measurement indicators to normal levels （P〈0.05）. Conclusions Histone deacetylase inhibitor NaB can alleviate sevoflurane anesthesia-induced hippoeampus-dependent long-term cognitive dysfunction in neonatal rats, which is associated with the up-regulation of histone acetylation and synaptic plasticity in hippocampus.