银杏叶提取物对2型糖尿病大鼠学习记忆及海马胶质纤维酸性蛋白表达的影响

背景:研究表明,银杏叶提取物具有促进脑损伤后功能修复、改善老年人学习与记忆功能障碍以及促进中枢神经系统可塑性的作用,但其对2型糖尿病大鼠学习记忆功能的影响又如何呢?目的:观察银杏叶提取物对2型糖尿病大鼠学习记忆功能障碍的改善作用和对海马内星形胶质细胞特异性蛋白胶质纤维酸性蛋白表达的影响。设计:完全随机分组设计,对照实验。单位:广西医科大学药学院药理学教研室。材料:选用84只雄性清洁级Wistar大鼠,8周龄,体质量180～220g。银杏叶提取物粉,广西桂林思特新技术公司提供,含黄酮24.8%,萜内酯6.2%,生产批号:200405。方法:实验于2003-06/2005-03在广西医科大学中心实验室(省重点实验室)药理室完成。①随机抽取70只大鼠,禁食24h,按55mg/kg腹腔注射链脲佐菌素(pH4.4柠檬酸缓冲液配制),注射后第4天,空腹血糖>15mmol/L的56只为2型糖尿病大鼠。②将56只糖尿病大鼠按随机数字表法分为4组:模型组,胰岛素治疗组,银杏叶提取物高、低剂量组,每组14只。4组糖尿病大鼠的空腹血糖差异不明显(P>0.05)。另取未注射链脲佐菌素的大鼠14只作正常对照组。胰岛素治疗组:皮下注射10U/(kg·d);银杏叶提取物高、低剂量治疗组:灌胃银杏叶提取物混悬液100和500mg/(kg·d);正常对照组和模型组:每天灌胃等量生理盐水,连续6个月。③Morris水迷宫实验观察记忆能力改变:圆形水池直径为100cm,高60cm,平台高40cm,水深42cm。大鼠每天上、下午各训练2次,连续4d。第5和8天上午测定每只大鼠学习能力[90s内找到平台的时间(逃避潜伏期);搜寻平台的策略直线式,记分4分;趋向式,记分3分;随机式,记分2分;圆圈式,记分1分)]。④测试结束后,各组取大鼠分别进行反转录聚合酶链反应(n=8)及免疫组织化学(n=6)实验,观察大鼠海马星形胶质细胞胶质纤维酸性蛋白mRNA及其蛋白的表达,分别以目的基因胶质纤维酸性蛋白区带的灰度值与内参β-actin区带的灰度值之比和胶质纤维酸性蛋白反应阳性细胞的体密度比表示。⑤组间差异显著性比较采用方差分析和q检验。主要观察指标:银杏叶提取物对2型糖尿病大鼠Morris水迷宫实验结果及海马星形胶质细胞胶质纤维酸性蛋白mRNA及其蛋白表达的影响。结果:造模失败脱失14只,其余70只大鼠进入结果分析。①Morris水迷宫实验结果:模型组大鼠第5,8天的逃避潜伏期明显长于正常对照组,搜寻平台策略得分明显低于正常对照组(P<0.01)。胰岛素治疗组及银杏叶提取物高、低剂量组的第5,8天逃避潜伏期均明显短于模型组,搜寻平台策略得分明显高于模型组(P<0.05～0.01)。胰岛素治疗组和银杏叶提取物高、低剂量组Morris水迷宫实验结果差异不明显(P>0.05)。②海马星形胶质细胞胶质纤维酸性蛋白mRNA及其蛋白表达:模型组大鼠海马的胶质纤维酸性蛋白mRNA表达水平明显高于其他3组(P<0.01)。免疫组化结果显示,与正常对照组比较,模型组大鼠海马星形胶质细胞胞体明显增大、数量增加、突起明显增粗,胶质纤维酸性蛋白阳性表达细胞的体密度比明显增高(P<0.01)。与模型组比较,胰岛素治疗组和银杏叶提取物高、低剂量组海马星形胶质细胞胞体缩小、数量减少、突起明显变短,胶质纤维酸性蛋白表达的体密度比明显下降(P<0.01)。胰岛素治疗组和银杏叶提取物高、低剂量组胶质纤维酸性蛋白mRNA表达水平和胶质纤维酸性蛋白表达的体密度比比较,差异不明显(P>0.05)。结论:2型糖尿病大鼠存在学习记忆能力障碍,反应性星形胶质细胞可能参与了该障碍发生过程,银杏叶提取物可能通过抑制星形胶质细胞反应性增生对糖尿病大鼠学习记忆损伤起到保护作用。

Effects of Gingko biloba leaf extract on the learning and memory and expression of glial fibrillary acidic protein in hippocampal astrocytes of type 2 diabetic rats

BACKGROUND: Studies have shown that Gingko biloba leaf extract (GbE) is effective in promoting the functions recovery of the brain that follows traumatic injury, in improving the dysfunctions of learning and memory of the elderly, and it is also effective in improving the plasticity in central nervous system (CNS). However, what is the effect on learning and memory functions of rats with type 2 diabetes mellitus?OBJECTIVE: To study the effects of GbE on the learning and memory dysfunction and glial fibrillary acidic protein (GFAP) expressions in hippocampus of diabetic rats.DESIGN: Complete-random design, controlled experimental study.SETTING: Department of Pharmacology, Guangxi Medical University.MATERIALS: A total of 84 male Wistar rats (180-220 g), 8 weeks old,SPF, were used in this study. GbE (containing 24.8% flavone glycosides and 6.2% diterpene lactone) was purchased from Guilin Xintejia Natural plants Pharmaceutical Factory, Guangxi Province, Lot No. 200405.METHODS: The experiment was completed at the Pharmacology Lab (Provincial Lab) of the Experimental Center of Guangxi Medical University from June 2004 to March 2005. ① A total of 70 rats were rendered diabetic by a single intraperitoneal injection of streptozotocin at a dose of 55 rmg/kg dissolved in citrate buffer (pH4.4) after 24 hours fasting. Tail vein blood glucose concentration was determined 4 days later using ONE TOUCH glucose meter. A total of 56 streptozotocin-treated rats with a blood glucose concentration of ＞ 15 mmol/L were recognized as type 2 diabetic rats. ② These diabetic rats were randomly divided into model group, insulin group, high-dose GbE group, and low-dose GbE group. There were 14 rats in each group. There was no difference in the blood glucose concentration among the groups. Another 14 male rats with an intraperitoneal injection of citrate buffer solution were served as control group. After division, drugs were given. Insulin 10 μ/kg was injected subcutaneously every day for 6 months. GbE 100 mg/kg and GbE 50 mg/kg were administered through intra-gastric method every day for 6 months.The diabetic group and control group were administered normal solution through intra-gastric method every day for 6 months. ③ Six months later,Morris water maze was operated on each group of rats. The Morris water maze consisted of a large circular pool [100 cmdimension, 60 cm height,filled to a depth of 42 cm with water at (25±1) ℃]. Within the pool a submerged platform (round, black, 8 cm diameter, 2 cm below the water surface) was hidden on a fixed location, 20 cm from the edge of the pool,in which milk powder was dissolved to obscure the platform. The rat could climb on the platform to escape from the necessity of swimming. The rats were trained to locate the hidden platform. The animals were received 4 trials (2 in the morning, and 2 in the afternoon) per day on 4 consecutive days. The rat was given a maximum of 90 s to find the hidden platform.On the 5th day, the rat's learning ability was examined by observing the time to find the hidden platform (escape latency) in 90 s and the platformfinding strategy (prompt and straight way, marked 4 scores; hesitating first and then straight way, marked 3 scores; random way, marked 2 scores;aimless way and around the pool border, marked 1 score). On the 8th day,the escape latency and the platform-finding strategy were observed to examine the rat's memory level. ④ After the Morris water maze test, 8 rats of each group were sacrificed by decapitation for RT-PCR of GFAP mRNA expression, and 6 rats of each group were sacrificed for immunohistochemistry of GFAP protein expression. GFAP mRNA expression level was analyzed by the expression ratio of the interest GFAP to the control β-actin according to the computer image analysis. The GFAP protein expression was analyzed by the volume density of GFAP in hippocampus. ⑤ Data were analyzed with one-way ANOVA and q-test.MAIN OUTCOME MESURES: The effects of GbE-on the performances of the water maze Morris of type 2 diabetic rats and both GFAP mRNA and protein expressions in hippocampus.RESULTS: A total of 14 streptozotocin-treated rats with a blood glucose concentration of ＜ 15 mmol/L were rejected from the study. ① The performance of diabetic rats in the Morris water maze was significantly impaired compared to control group, the results on the 5th day and the 8th day showed that both escape latency and platform-finding strategy scores were decreased (P ＜ 0.01). The escape latency of both insulin treatment and GbE treatments on the 5th day and the 8th day was shorter than that of diabetic group, the platform-finding strategy scores was higher than that of diabetic group (P ＜ 0.05-0.01). There was no marked difference among the insulin treatment and GbE treatments groups in performance of the water maze Morris (P ＞ 0.05). ② The levels of both GFAP mRNA and protein expressions in hippocampus: Statistical analyses indicated that the level of GFAP mRNA expression of diabetic rats was significantly higher than that of the 3 other groups (P ＜ 0.01). Compared to control group, the diabetic rats showed a high immunoreactivity, the GFAP body was enlarged markedly, apophysis was obviously longer, the expressed numbers were increased, and the volume density of GFAP was also increased significantly (P ＜ 0.01). Compared to the diabetic group, the insulin treatment and GbE treatments groups showed a low immunoreactivity, the GFAP body was markedly smaller, apophysis was obviously shorter, the expressed numbers were decreased, and the volume density of GFAP was also decreased significantly (P ＜ 0.01). There were no marked differences in both GFAP mRNA and protein expressions among the insulin treatment and GbE treatments groups (P ＞ 0.05).CONCLUSION: There is cognition impairment in type 2 diabetic rats, the responsive GFAP may take a part in the progress of the learning and memory dysfunction. GbE can decrease markedly the reactive hypertrophy of astrocytes of diabetic hippocampus and improve the learning and memory dysfunction in diabetic rats.