NMDA受体2A亚基磷酸化对幼鼠肝缺血再灌注后脑损伤及远期认知功能障碍的影响

目的 评价肝缺血再灌注 （HIR） 对幼鼠海马以及远期认知功能的影响， 并探讨N-甲基-D-天冬氨酸受体 2A亚基 （NR2A） 磷酸化在其中发挥的作用。方法 健康C57小鼠54只， 2周龄， 体质量6～9 g， 采用随机数字表法分为3组 （n=18）： 假手术组 （S）、 HIR组 （I） 和NR2A抑制剂NVP-AAM077处理组 （N）。建立70% HIR模型， N组于术后连续3 d每天予NVP-AAM077 （10 mg/kg） 腹腔注射。S组和I组以相同方式腹腔注射等体积生理盐水。每组随机取 10只小鼠于术后3 d处死， 分离血清并取脑组织， 酶联免疫吸附测定 （ELISA） 检测血清脑损伤标志物S100β和神经元特异性烯醇化酶 （NSE） 水平， HE染色观察海马组织病理改变， Western blot检测cleaved caspase-3、 NR2A、 p-NR2A Y1325蛋白水平。其余小鼠于术后30 d进行Morris水迷宫实验， 评价远期认知功能。结果 与S组比较， I组与N组血清S100β和NSE水平明显升高， 海马组织水肿， 细胞排列紊乱， cleaved caspase-3、 p-NR2A Y1325蛋白水平升高， 平台所在象限滞留时间缩短 （P＜0.05）； 与I组比较， N组血清S100β和NSE水平降低， 海马神经元病理损伤减轻， cleaved caspase-3、 p-NR2A Y1325蛋白水平下降， 平台所在象限滞留时间明显延长 （P＜0.05）。3组之间NR2A表达水平、 逃逸潜伏期差异无统计学意义 （P＞0.05）。结论 幼鼠HIR可导致脑损伤和远期认知功能障碍， 其机制可能与 NR2A Y1325过度磷酸化介导的兴奋性毒性和细胞凋亡有关。

The role of NMDA receptor subunit 2A phosphorylation in hepatic ischemia reperfusion induced brain injury and long-term cognitive dysfunction in young mice

Objective To evaluate the effect of hepatic ischemia reperfusion on hippocampus and long-term cognitive function in young mice, and investigate the mechanisms related to N-methyl-D-aspartic acid receptor subunit 2A (NR2A) phosphorylation. Methods Fifty-four healthy C57 mice, 2-week old, with body mass 6-9 g, were randomly divided into three groups (n=18 / group): sham-operated group (S), hepatic ischemia reperfusion group (I) and NR2A inhibitor NVP- AAM077 treatment group (N). The mice in group N were given the intraperitoneal injection of NVP-AAM077 at the dose of 10 mg/kg for 3 days after the surgery. The mice in group S and group I were given equal amount of normal saline in the same way. Ten mice from each group were sacrificed 3 d after the surgery. Serum concentrations of S100β and neuron-specific enolase of enzyme (NSE) were detected by ELISA. Histopathological changes of hippocampus were determined by HE staining. Western blot assay was used to detect the expression levels of cleaved caspase-3 and NR2A, p-NR2A Y1325. Long-term cognitive function was evaluated by Morris water maze 30 d after surgery. Results Compared with group S, the serum concentrations of S100β and NSE increased significantly, hippocampal tissue was edematous, the cell arrangement was disorder and sparse, the expression levels of cleaved caspase-3 and p-NR2A Y1325 were up-regulated, and the time spent in the target quadrant decreased in group I and group N (P＜0.05). Compared with group I, the serum concentrations of S100β and NSE decreased significantly, morphological changes of hippocampal tissue were ameliorated, the expression levels of cleaved caspase-3 and p-NR2A Y1325 were down-regulated, and the time spent in the target quadrant increased in group N (P＜0.05). There were no significant differences in the expression level of NR2A and escape latency between the three groups (P＞0.05). Conclusion Hepatic ischemia reperfusion could induce brain injury and long-term cognitive dysfunction in young mice, and the mechanism may be associated with the excitotoxicity mediated by excessive phosphorylation of NR2A Y1325.