生酮饮食对新生期反复惊厥大鼠神经行为的影响及机制

Effect and mechanism of ketogenic diet on neurobehavioral demages induced by recurrent neonatal sei- zures

目的观察生酮饮食（KD）对三氟乙醚所致新生期反复惊厥大鼠神经行为损伤的影响和载脂蛋白E（ApoE）表达的变化。方法48只日龄8d（P8，下同）的sD大鼠依据随机数字表法随机分为对照组（NS组）和惊厥组（Rs组），每组各24只。于P9RS组大鼠吸入三氟乙醚诱导惊厥发作，反复诱导惊厥持续30min，每日1次，同样方法连续诱导8d；NS组同样操作但不吸入三氟乙醚。于P28每组大鼠依据随机数字表法再随机分为两组，即单纯对照组（NS＋ND组）、对照加生酮饮食组（NS＋KD组）、单纯惊厥组（RS＋ND组）、惊厥加生酮饮食组（RS＋KD组），每组各12只。于P42进行神经发育指标监测。于P58每组各随机取6只大鼠断头取大脑皮层组织，采用免疫印迹技术（Westernblot）检测ApoE的表达。结果P42各组大鼠神经发育指标结果显示：平面翻正实验中，NS＋ND组平面翻正时间[（1．04-0．14）s]与RS＋ND组[（0．75±0．32）s]相比差异有统计学意义（P〈0．05），RS＋KD组平面翻正时间与Rs＋ND组相比差异无统计学意义（P〉0．05）；负向趋地反应实验中，NS＋KD组、RS＋ND组负向趋地反应时间[（3．17±0．58）s，（6．754-0．75）s]与NS＋ND组[（1．58±0．52）S]相比差异均有统计学意义护〈0．05），RS＋KD组负向趋地反应时间[（4．58±0．52）s]与Rs＋ND组[（6．75±0．75）s]相比差异有统计学意义（P〈0．05）；悬崖回避实验中，NS＋ND组、RS＋KD组悬崖回避时间[（5．754-2．90）s，（9．50±4．36）S]与RS＋ND组[（14．004-4．79）s]相比差异均有统计学意义（P〈0．05）。Westernblot结果显示：RS＋ND组ApoE在大脑皮层的表达（1．26士0．30）与NS＋ND组（0．78±0．12）相比差异有统计学意义（P〈0．01），且与RS＋KD组（0．894-0．10）相比差异也有统计学意义（P〈0．05）。结论生酮饮食能够改善新生期反复惊厥所致的神经行为损伤，其机制可能与大脑皮层ApoE表达降低有关。

Objective To investigate the intervention effect of ketogenic diet （KD） on neurobehavioral demages after flurothyl-induced recurrent neonatal seizures in rats and on the expression of ApoE. Methods Post- natal day 8 （ P8 ） SD rats （ quantity ： 48 ） were randomly divided into two groups ： the non-seizure group （ NS group, n = 24） and the recurrent-seizure group （RS group, n = 24）. From IX）, rats in RS group were subjected to recurrent seizures induced by volatile flurothyl 30 min each day for consecutive 8 days. While rats in NS group were placed into the container for an equal amount of time to their counterpart without exposuring to flurothyl. At P28 ,each group was divided into two groups again： non-seizure and normal diet group（ NS ＋ ND group, n = 12）, non-seizure and ketogenic diet group（ NS ＋ KD group, n = 12）, recurrent-seizure and normal diet group （RS ＋ ND group, n = 12）, recurrent-seizure and ketogenic diet group（ RS ＋ KD group, n = 12）. At P42, neurodevelopmental indicators were monitored. ApoE protein levels in cerebral cortex were determined by western blot at P58. Results Neurodevelopmental indicators were analyzed at P42：in the plane righting experiment, the rats of group NS ＋ ND （1.0 ＋0.14） about the time of plane righting was significant different comparing with group RS ＋ ND （ （0.75 -＋ O. 32）s）（P 〈 O. 05 ）. There was no significant difference between group RS ＋ KD and group RS ＋ ND about the time of plane righting（P〉 O. 05）. In the negative geotaxis reaction experiment,the rats of groups NS ＋ KD and RS ＋ ND（ （3.17 ~0.58）s, （6.75 ＋ O. 75 ）s） about the time of negative geotaxis reaction were significant different comparing with group NS ＋ ND （ （ 1.58 ＋ O. 52） s） （P 〈 0.05 ）. Compared with group RS ＋ ND, the group RS ＋ KD in the time of negative geotaxis reaction was obviously shortened（P 〈 O. 05）. In the cliff avoidance test, there were significant differences between group NS ＋ ND,RS ＋ KD （ （5.75 ＋2.90） s, （9.50 _＋4.36） s） and group RS ＋ ND（ （14.00 ＋ 4.79）s） about the time of cliff avoidance （P 〈 0.05 ）. In western blot, the expression of ApoE in cere- bral cortex in the RS ＋ ND group （ 1.26 ＋0.30） was obviously increased compared with group NS ＋ ND （0.75 ＋ 0.12） （P 〈 0.05 ）, and there had also significant difference between group RS ＋ KD （0.89 ＋ 0.10） and group RS ＋ ND （P 〈 0.05）. Conclusion The neuroprotective effects of ketogenic diet on recurrent neonatal seizure-induced neurobehavioral demages may be associated with the reduction of ApoE in cerebral cortex.