丁苯酞对急性重度CO中毒大鼠海马区calpain 1和CaMKⅡ蛋白表达的影响

Effects of N-butylphthalide on the expressions of calpain 1 and CaMK Ⅱ in hippocampus in rats with acute severe carbon monoxide poisoning

目的探讨丁苯酞（NBP）对急性重度一氧化碳（CO）中毒大鼠认知功能障碍的治疗作用及其机制。方法按随机数字表法将120只健康雄性SD大鼠分为正常对照组、CO中毒组和NBP治疗组，每组40只。采用高压氧舱法建立急性重度CO中毒动物模型（吸入体积分数为1？000×10-6的CO 40 min，继而吸入3？000×10-6的CO 20 min），并给予高压氧舱治疗1.5 h、每日1次，直至处死。NBP治疗组于染毒后2 h灌胃NBP 60 mg/kg、每日2次，直至处死；正常对照组和CO中毒组灌胃等量纯橄榄油。各组分别于染毒后1、3、7、14、30 d取4只大鼠，采用Morris水迷宫实验进行认知功能评分，透射电镜下观察大鼠海马组织超微结构改变，用免疫荧光染色法检测脑组织海马区钙蛋白酶1（calpain 1）和钙离子/钙调蛋白依赖性蛋白激酶Ⅱ（CaMKⅡ）蛋白表达，用免疫荧光双标染色观察两种蛋白在神经细胞中的定位关系。结果与正常对照组比较，CO中毒组染毒后1 d大鼠逃避潜伏期明显延长（s：55.6±3.2比44.5±3.5，P〈0.05），跨越平台次数明显减少（次：1.3±0.8比6.6±1.2，P〈0.05）；海马区神经细胞超微结构严重损坏；染毒后1 d脑组织calpain 1和CaMKⅡ蛋白表达（A值）即明显增高（calpain 1：41.24±5.21比6.44±1.13，CaMKⅡ：56.19±5.04比9.84±1.53，均P〈0.05）；calpain 1蛋白表达于3 d达峰值（59.34±6.11），CaMKⅡ蛋白表达于1 d达峰值（56.19±5.04）。与CO中毒组比较，NBP治疗组在染毒后期（7~30 d）认知功能明显改善〔逃避潜伏期（s）：7 d为40.3±1.9比49.1±3.1，30 d为30.1±2.9比39.4±3.1；跨越平台次数（次）：14 d为2.8±1.0比1.0±0.9，30 d为3.2±0.8比1.0±0.9，均P〈0.05〕；海马神经元损伤程度相对轻微；染毒后脑组织calpain 1和CaMKⅡ蛋白表达均较CO中毒组明显降低（3 d时calpain 1蛋白为39.63±3.03比59.34±6.11，P〈0.05；1 d时CaMKⅡ蛋白为42.22±3.84比56.19±5.04，P〈0.05）。免疫荧光双标染色提示，calpain 1和CaMKⅡ蛋白不仅可以在同一细胞共存，也可以在不同细胞中单独表达。线性回归分析显示，calpain 1与CaMKⅡ蛋白表达呈明显正相关性（R2=0.852，P=0.002）。结论NBP能够通过修复海马超微结构损伤，恢复其完整性，均衡calpain 1和CaMKⅡ蛋白表达，从而改善急性重度CO中毒大鼠的认知功能，对海马损伤起到积极的保护作用。

ObjectiveTo investigate the effects of N-butylphthalide （NBP） on cognitive function in acute severe carbon monoxide （CO） poisoning rats and its mechanism.Methods120 health Sprague-Dawley （SD） rats were randomly divided into three groups （n = 40）： normal control group （NC group）, CO poisoning group （CO group） and NBP treatment group （NBP group）. The acute severe CO poisoning model was established in a hyperbaric oxygen chamber by intoxicated with 1 000 ×10-6 CO for 40 minutes, followed with 3 000 ×10-6 CO for another 20 minutes, and then received hyperbaric oxygen therapy 1.5 hours once a day until sacrificed. Rats in NBP group were administered orally NBP 60 mg/kg for 2 times daily until death. NC group and CO group were treated with equal amount of pure olive oil. Four rats in each group were taken from 1, 3, 7, 14, 30 days after model setup, respectively. The cognitive function score was assessed by Morris water maze test. The changes in ultrastructure of hippocampus were observed under transmission electron microscope. The expressions of calpain 1 and Ca2＋/calmodulin dependent protein kinase Ⅱ （CaMK Ⅱ） in hippocampus of brain tissue were detected by immunofluorescence staining, and the localization of the two target proteins in neurons was observed by immunofluorescence double staining.ResultsCompared with NC group, the escape latency at 1 day after poisoning in CO group was significantly prolonged （s： 55.6±3.2 vs. 44.5±3.5, P 〈 0.05）, and the times of the platform crossing was significantly decreased （times： 1.3±0.8 vs. 6.6±1.2, P 〈 0.05）; the ultrastructure of hippocampus was obviously injured; the protein expressions of calpain 1 and CaMK Ⅱ in brain tissue were significantly increased at 1 day after CO poisoning [calpain 1 （A value）： 41.24±5.21 vs. 6.44±1.13, CaMK Ⅱ （A value）： 56.19±5.04 vs. 9.84±1.53, both P 〈 0.05], and the protein expression of calpain 1 reached the peak at 3 days （A value： 59.34±6.11）, the protein expression of CaMK Ⅱ reached the peak at 1 day （A value： 56.19±5.04）. Compared with CO group, the cognitive function was significantly improved in NBP group in the late stage of poisoning [7-30 days, escape latency （s）： 40.3±1.9 vs. 49.1±3.1 at 7 days, 30.1±2.9 vs. 39.4±3.1 at 30 days; times of the platform crossing （times）： 2.8±1.0 vs. 1.0±0.9 at 14 days, 3.2±0.8 vs. 1.0±0.9 at 30 days, all P 〈 0.05]; the degree of injury of hippocampal neuron was relatively slight; the protein expression of calpain 1 in brain tissue was significantly decreased from 3 days after CO poisoning （A value： 39.63±3.03 vs. 59.34±6.11, P 〈 0.05）, and the protein expression of CaMK Ⅱ was significantly decreased from 1 day after CO poisoning （A value： 42.22±3.84 vs. 56.19±5.04, P 〈 0.05）. Immunofluorescence double staining suggested that calpain 1 and CaMK Ⅱ protein could not only coexist in the same cell, but also could be expressed separately in different cells. Linear regression analysis showed that the expression of calpain 1 and CaMK Ⅱ was positively correlated （R 2 = 0.852, P = 0.002）.ConclusionsNBP treatment could maintain ultrastructure integrity of hippocampus, balance the expression levels of calpain 1 and CaMK Ⅱ proteins, and significantly improve cognitive impairment induced by CO poisoning, thus play a protective role against hippocampus damage in rats with acute severe CO poisoning.