穴位埋线对慢性缺血性认知障碍模型大鼠空间学习记忆能力及海马兴奋性氨基酸毒性相关蛋白的影响

Effect of Catgut Implantation on Spatial Learning-memory Ability,Expression of Hippocampal Protein Kinase C Interacting Protein 1 and GluR 2 and Ca^(2+) Content in Rats with Chronic Ischemic Cognitive Impairment

目的:观察穴位埋线对慢性缺血性认知障碍大鼠空间学习记忆能力及海马蛋白激酶C相互作用蛋白1(PICK 1)、谷氨酸受体2(GluR 2)及Ca^(2+)水平的影响,探讨穴位埋线改善缺血性认知障碍的可能途径。方法:雄性SD大鼠随机分为假手术组、模型组、埋线组、药物组,每组14只。采用双侧颈总动脉永久性结扎法复制慢性缺血性认知障碍模型。埋线组取"百会""大椎"、双侧"肾俞""悬钟"穴埋线,每周1次,共埋线4次;药物组予以单唾液酸四己糖神经节苷脂钠注射液腹腔注射(0.33mg/kg),每天1次,共4周。采用Morris水迷宫进行学习记忆能力测试,尼氏染色法观察大鼠海马组织内尼氏小体病理变化,蛋白免疫印迹法检测大鼠海马组织内PICK 1、GluR 2蛋白相对表达量,BCA浓度测定法检测大鼠海马组织内Ca^(2+)浓度。结果:与假手术组比较,模型组大鼠定位航行实验逃避潜伏期延长,空间探索实验穿越原平台次数减少(P<0.01);海马组织尼氏染色仅显示少量尼氏小体,排列分散,胞核呈深染固缩、体积变小,细胞大量丢失,结构不清晰;海马组织内PICK 1蛋白相对表达量增加,GluR 2蛋白相对表达量显著减少,Ca^(2+)浓度明显增高(均P<0.01)。与模型组比较,埋线组、药物组大鼠逃避潜伏期明显缩短(P<0.01),穿越原平台次数增加(P<0.05),尼氏体较丰富,仅见少量细胞核深染固缩,细胞丢失少;海马组织内PICK 1蛋白相对表达量减少(P<0.01,P<0.05),GluR 2蛋白相对表达量增加(P<0.05,P<0.01),Ca^(2+)浓度降低(P<0.01)。结论:穴位埋线疗法能抑制大鼠海马内PICK 1蛋白与A-氨基-3-羟基-5-甲基-4-异恶唑丙酸(AMPA)受体的相互作用,使AMPA受体亚基GluR 2含量增加,Ca^(2+)浓度降低,从而降低兴奋性氨基酸毒性,这可能是其改善大脑学习记忆等认知功能障碍的机制之一。

Objective To observe the effect of catgut embedment at ＂Baihui＂ （GV 20）, ＂Dazhui＂ （GV 14）, etc. on learning-memory ability, expression of hippocampal protein kinase O interacting protein 1 （PICK 1 ） and glutamate receptor 2 （GluR 2） proteins and level of calcium ions, so as to explore its mechanism underlying improvement of vascular cognitive impairment. Methods A total of 56 male SD rats were randomly divided into sham operation, model, catgut embedment and medica- tion groups （n = 14 in each）. The chronic ischemic cognitive impairment model was established by permanent occlusion of bilate- ral common carotid arteries. The catgut embedment was applied to GV 20, GV 14, ＂Shenshu＂（BL 23） and ＂Xuanzhong＂（GB 39）, once a week, for 4 weeks. Rats of the medication group received intraperitoneal injection of monosialate tetrahexose ganglioside sodium （GM-1, 0.33 mg/kg）, once daily for 4 weeks. The rats＇ learning-memory ability was detected by Morris water mazetasks, pathological changes of hippocampal Nissl＇s bodies were tested by Nissl staining. The expression levels of PICK 1 and GluR 2 proteins in the hippocampus were detected by Western bolt （WB）, and the concentration of calcium ions in the hippocam- pus tissue was measured by Bicinchoninic acid （BOA） assay. Results After modeling, the mean escape latencies of place navi- gation test were significantly increased while the crossing times of target platform quadrant of space probing test notably decreased in the model, catgut embedment and medication groups compared with their own individual pre-modeling （P 〈0.01 ）. Following the treatment, the increased mean escape latencies and decreased crossing times were markedly reversed in both catgut embedment and medication groups relevant to the model group （P〈0.01, P〈0.05）. Nissl staining showed that after mode- ling, a smaller amount of Nissl bodies with dispersing arrangement, reduction in cellular volume, and loss of large amount of cells with vague structure, and hyperchromatic nuclear pyknosis were found in the hippocampus tissue, which was relatively milder in both catgut embedment and medication groups. The hippocampal PICK 1 protein expression and the calcium ion concentration were obviously higher in the model group than in the sham operation group （P〈0.01 ）, and significantly lower in both embedment and medication groups than in the model group （P〈0.01, P〈0.05）, while hippocampal GluR 2 protein expression was obviously lower in the model group than in the sham operation group （P〈0.01）, and markedly higher in both embedment and medication groups than in the model group （ P〈0.05, P〈0.01 ）. No significant differences were found between the embedment and medication groups in the abovementioned indexes （P〈0.05）. Cenclusion Catgut implantation at GV 20 etc. can effectively improve the learning-memory ability in rats with chronic ischemic cognitive impairment, which may be related to its effects in down-regula- ting the expression of PICK 1 and calcium ion concentration and up-regulating the expression of AMPA receptor subunit GluR 2 pro- tein in the hippocampus.