Xiong Ao, Jin Ge, Xiong Renping, Lu Hong
Department of Clinical Medicine, Zhengzhou University, Zhengzhou 450001, Henan, China (Xiong A); Department of Biochemistry and Molecular Biology, Basic Medical College of Zhengzhou University, Zhengzhou 450001, Henan, China (Xiong A, Jin G); Molecular Biology Center, the State Key Laboratory of Trauma, Burns and Combined Injury, Research Institute of Surgery and Daping Hospital, Third Military Medical University, Chongqing 400042, China (Xiong RP); Department of Radiology, Chongqing No.7 Hospital, Chongqing 400054, China (Lu H). Corresponding author: Lu Hong, Email:
Zhonghua Wei Zhong Bing Ji Jiu Yi Xue. 2018 Feb;30(2):170-175. doi: 10.3760/cma.j.issn.2095-4352.2018.02.015.
To observe the protein expression related to cognitive and learning memory function, and to investigate the effect of aquaporin 4 (AQP4) silence on learning and memory function in traumatic brain injury (TBI) rats.
Ninety-six healthy adult male Wistar rats were divided into groups according to the random number table. (1) Forty-eight rats were divided into sham operation (sham) group, TBI group (by using modified Feeney method), AQP4 RNA interference (RNAi) negative group [TBI+meaningless small interfering RNA (siRNA)-AQP4 liposome solution 10 μL], and AQP4 RNAi group (TBI+siRNA-AQP4 liposome solution 10 μL). In each group, brain tissues of 4 rats were harvested at 1, 6 and 12 hours respectively. The protein expressions of hippocampus AQP4, general control nonderepressible 2 kinase (GCN2), cyclic adenosine monophosphate response element binding protein (CREB) and phosphorylated CREB (p-CREB) were detected by Western Blot. (2) In addition, 48 rats were divided into normal control group (control group), sham group, TBI group and AQP4 RNAi group, brain water content were measured in 6 of them after 12 hours of injury, and 6 were used in Morris water maze test.
(1) The protein expressions of hippocampus AQP4 and GCN2 in TBI group were significantly higher than those in sham group, and increased gradually with time with statistical difference at 12 hours (AQP4 protein: 5.03±0.09 vs. 1, GCN2 protein: 4.01±0.13 vs. 1, both P < 0.01); the protein expressions of hippocampus CREB and p-CREB were significantly lower than those in sham group, and decreased gradually with time with statistical difference at 12 hours (CREB protein: 0.38±0.03 vs. 1, p-CREB protein: 0.38±0.03 vs. 1, both P < 0.01). Compared with TBI group, the protein expressions of AQP4 in AQP4 RNAi group was significantly decreased (1 hour: 1.02±0.04 vs. 2.23±0.05, 6 hours: 1.23±0.03 vs. 2.59±0.04, 12 hours: 2.20±0.08 vs. 5.03±0.09, all P < 0.01), but there were no significant difference in the expressions of GCN2, CREB or p-CREB. There was no significant difference in the expression of protein between AQP4 RNAi negative group and TBI group. (2) The brain water content in TBI group was significantly higher than that in control group and sham group [(83.7±0.4)% vs. (76.2±0.2)%, (76.2±0.3)%, both P < 0.01]. The brain water content in AQP4 RNAi group [(78.8±0.3)%] was significantly decreased as compared with that in TBI group (P < 0.01). The latency of Morris water maze test was significantly prolonged in the day 11, 13 and 15 after the injury of the TBI group and AQP4 RNAi group, and the exploration time was significantly shortened. Compared with TBI group, the incubation period of AQP4 RNAi group was significantly shortened at 15 days (s: 60.2±11.1 vs. 62.0±11.5, P < 0.05), and the exploration time was significantly prolonged (s: 37.0±8.5 vs. 32.7±9.2, P < 0.05).
The impairment of cognitive and learning memory function in rats after TBI was significantly related to the changes in CREB and GCN2 in cognitive and learning memory function. After RNAi treatment, the cognitive and learning and memory function of rats was not improved obviously, but the brain edema could be alleviated.
观察与认知及学习记忆功能相关的蛋白表达，探讨水通道蛋白4（AQP4）沉默对创伤性脑损伤（TBI）大鼠学习记忆功能的影响。
将96只健康成年雄性Wistar大鼠按随机数字表法分组。（1）48只大鼠分为假手术（sham）组、TBI组（采用改良Feeney法）、AQP4 RNA干扰（RNAi）阴性组[TBI + 无意义小干扰RNA（siRNA）-AQP4脂质体溶液10 μL]和AQP4 RNAi组（TBI + siRNA-AQP4脂质体溶液10 μL）。每组分别于1、6和12小时各取4只大鼠的脑组织，采用蛋白质免疫印迹法检测海马AQP4、一般控制非抑制性2激酶（GCN2）、环磷酸腺苷反应元件结合蛋白（CREB）及磷酸化CREB（p-CREB）的蛋白表达。（2）另外48只大鼠分为正常对照组（对照组）、假手术组、TBI组和AQP4 RNAi组，伤后12小时每组取6只测量脑含水量，另取6只进行Morris水迷宫试验。
（1）TBI组海马AQP4和GCN2的蛋白表达显著高于假手术组，且随时间逐渐升高，12小时时有统计学差异（AQP4蛋白：5.03±0.09 vs. 1，GCN2蛋白：4.01±0.13 vs. 1，均P < 0.01）；海马CREB和p-CREB的蛋白表达显著低于假手术组，且随时间逐渐降低，12小时时有统计学差异（CREB蛋白：0.38±0.03 vs. 1，p-CREB蛋白：0.38±0.03 vs. 1，均P < 0.01）。与TBI组相比，AQP4 RNAi组AQP4的蛋白表达显著降低（1小时：1.02±0.04 vs. 2.23±0.05，6小时：1.23±0.03 vs. 2.59±0.04，12小时：2.20±0.08 vs. 5.03±0.09，均P < 0.01），但GCN2、CREB或p-CREB的表达无显著差异。AQP4 RNAi阴性组与TBI组蛋白表达无显著差异。（2）TBI组脑含水量显著高于对照组和假手术组[（83.7±0.4）% vs. （76.2±0.2）%，（76.2±0.3）%，均P < 0.01]。AQP4 RNAi组脑含水量[（78.8±0.3）%]较TBI组显著降低（P < 0.01）。TBI组和AQP4 RNAi组伤后第11、13和15天Morris水迷宫试验潜伏期显著延长，探索时间显著缩短。与TBI组相比，AQP4 RNAi组15天时潜伏期显著缩短（秒：60.2±11.1 vs. 62.0±11.5，P < 0.05），探索时间显著延长（秒：37.0±8.5 vs. 32.7±9.2，P < 0.05）。
TBI大鼠认知及学习记忆功能障碍与认知及学习记忆功能中CREB和GCN2的变化显著相关。RNAi处理后，大鼠认知及学习记忆功能未明显改善，但可减轻脑水肿。
