金钗石斛总生物碱对双侧侧脑室注射链脲佐菌素所致大鼠学习记忆减退的影响

发布时间：2018-04-28 12:24

本文选题：金钗石斛总生物碱 + 散发性老年痴呆　；参考：《遵义医学院》2017年硕士论文

【摘要】：目的:探索金钗石斛总生物碱(DNLA)对链脲佐菌素(STZ)诱导的大鼠学习记忆减退模型的作用。方法:将50只雄性SD大鼠随机分成5组:空白对照组(Control)、空白给药组(Control+DNLA40)、模型组(STZ)、低剂量组(STZ+DNLA20)和高剂量组(STZ+DNLA40)。每组10只大鼠。对大鼠进行麻醉,后将其固定于立体定位仪上,定位注射时的坐标位置:前囟后0.8 mm,左右旁开1.5 mm,颅骨下3.6mm。使用微量进样器对侧脑室缓慢注STZ溶液(70.5μg/μL),以1μL/min速度注入,5 min注完,留针5 min,然后对另一侧侧脑室进行相同操作,最终使每只用于模型的大鼠接受STZ剂量为3 mg/kg,制备散发性老年痴呆(sporadic Alzheimer’s Disease,SAD)动物模型。模型制备完成后,对需要给药的实验组每天定时给予20 mg/kg或40 mg/kg的DNLA,连续28天;而模型组和空白对照组则给予等体积的溶媒处理。本研究使用Morris水迷宫法检测单次双侧侧脑室注射链脲佐菌素(ICV-STZ)对大鼠的空间学习记忆功能的影响,从ICV-STZ后第23天开始连续进行5天,并在该测试结束后第二天,进行空间探索实验。经苏木素-伊红染色及尼氏染色,光镜观察DNLA对大鼠海马神经元密度和形态的影响。采用Western blot法检测大鼠海马内Tau蛋白,胰岛素受体(IR),胰岛素底物-1(IRS-1),蛋白激酶B(Akt)和糖原合成激酶-3β(GSK-3β)的蛋白表达水平和磷酸化程度,并检测了蛋白磷酸酶2-α(PP2A-α)的蛋白表达水平。结果:行为学实验中,通过Morris水迷宫实验测试发现,模型组的大鼠的逃逸潜伏期较空白对照组的从第4天开始表现为的显著升高;给药DNLA后,高剂量组的大鼠逃逸潜伏期较模型组在第五天出现显著降低;而低剂量组的大鼠逃逸潜伏期较模型组无显著差异;另外,对大鼠的游泳速度测试,各组大鼠未见显著差异。组织切片实验中,经HE和Nissl染色发现,模型组的大鼠海马CA3区的神经元数量显著减少,细胞排列层次减少,部分细胞出现深染、核固缩;给药DNLA后,高剂量组的大鼠海马CA3区正常神经元数量较模型组明显增多,排列层次增加,且未见明显的细胞深染及核固缩现象;低剂量组的大鼠海马CA3区亦可观察到正常神经元数量较模型组增多,深染及核固缩的细胞减少;免疫印迹实验中,经Western blot法检测发现,在模型组的大鼠海马内,与正常组比较,IR在酪氨酸1361位点和GSK-3β在丝氨酸9位点的磷酸化程度降低,GSK-3β在酪氨酸216位点的磷酸化水平以及PP2A-α蛋白水平无显著改变;IRS-1在丝氨酸616位点,Akt在丝氨酸473位点,Tau蛋白在丝氨酸199、396、404、422位点、苏氨酸616位点的磷酸化水平升高。给药DNLA后,在高剂量组的大鼠海马内,与模型组的大鼠比较,IR在酪氨酸1361位点和GSK-3β在丝氨酸9位点的磷酸化程度与模型组比较增加,GSK-3β在酪氨酸216位点的磷酸化水平以及PP2A-α蛋白水平无显著改变;IRS-1在丝氨酸616位点,Akt在丝氨酸473位点,Tau蛋白在丝氨酸199、396、404、422位点、苏氨酸616位点的磷酸化水平降低;而在低剂量组的大鼠海马内,与模型组相比较,可见Tau蛋白在丝氨酸404、苏氨酸231位点的磷酸化水平较模型组降低,Akt在丝氨酸473位点和GSK-3β在丝氨酸9位点磷酸化水平降低。结论:DNLA可减轻STZ诱导的大鼠学习记忆减退及神经元损伤,该作用与DNLA改善STZ导致的大鼠海马中的Tau蛋白过度磷酸化及胰岛素信号通路障碍有关。
[Abstract]:Objective: To explore the effect of Dendrobium nobile total alkaloid (DNLA) on the learning and memory impairment model induced by streptozotocin (STZ) in rats. Methods: 50 male SD rats were randomly divided into 5 groups: blank control group (Control), blank administration group (Control+DNLA40), model group (STZ), low dose group (STZ+DNLA20) and high dose group (STZ+DNLA40). 10 rats in each group were large. Rats were anesthetized, then fixed to the stereotaxis, positioning the coordinates of the coordinates: 0.8 mm in the anterior Fontane, 1.5 mm beside the side of the skull, and the slow injection of STZ solution (70.5 mu g/ mu L) to the lateral ventricle using a micro injector for the lower 3.6mm. of the skull, 5 Min injection, 5 min, and the same exercise on the other side of the ventricle. As a result, each rat used for the model was eventually accepted STZ 3 mg/kg to prepare sporadic Alzheimer's (sporadic Alzheimer 's Disease, SAD) animal model. After the model preparation was completed, the experimental group needed to be given a dose of 20 mg/kg or 40 mg/kg DNLA for even 28 days, while the model group and the blank control group were given equal volume. The effect of single lateral lateral ventricle injection of streptozotocin (ICV-STZ) on the spatial learning and memory function of rats was detected by Morris water maze. The space exploration was carried out for 5 days from twenty-third days after ICV-STZ, and the space exploration experiment was carried out at the end of the test after the end of the test. The effects of DNLA on the density and morphology of hippocampal neurons in rats were observed. The protein expression level and phosphorylation level of Tau protein, insulin receptor (IR), insulin substrate -1 (IRS-1), protein kinase B (Akt) and glycogen kinase -3 beta (GSK-3 beta) were detected by Western blot method, and the protein phosphatase 2- alpha protein was detected. Results: in the behavior test, the Morris water maze test showed that the escape latency of the rats in the model group was significantly higher than that in the blank control group from fourth days. After DNLA, the escape latency of the rats in the high dose group was significantly lower than that of the model group at fifth days, while the rats in the low dose group fled. There was no significant difference between the model group and the model group. In addition, there was no significant difference in the swimming speed test of rats. In the tissue section experiment, HE and Nissl staining showed that the number of neurons in the hippocampus CA3 area of the rat model group decreased significantly, the level of cell arrangement decreased, some cells appeared deep dyed and nuclear pyknosis; after the administration of DNLA, a high dose of drug was used. The number of normal neurons in the hippocampal CA3 area of rats in the dose group increased significantly, the arrangement level increased, and there was no obvious cell deep staining and nuclear condensation. The number of normal neurons in the hippocampus CA3 area of the low dose group could also be observed to be more than the model group, and the deep staining and nuclear pyknosis cells decreased; in Western blot experiments, Western blo T assay found that in the rat hippocampus of the model group, the phosphorylation of IR at the tyrosine 1361 site and GSK-3 beta at the serine 9 site was lower than that in the normal group. The phosphorylation level of GSK-3 beta at the tyrosine 216 site and the level of PP2A- alpha protein were not significantly changed; IRS-1 was at the 616 site of serine, Akt at the serine 473 site, and Tau protein in silk The phosphorylation level of the threonine 616 site was increased at the 199396404422 site of the ammonia acid site. After DNLA, the phosphorylation of IR at the tyrosine 1361 site and the GSK-3 beta at the serine 9 site increased in the hippocampus of the high dose group, compared with the model group. The phosphorylation level of GSK-3 beta at the tyrosine 216 site and PP2A were higher than that of the model group. There was no significant change in alpha protein level; IRS-1 at the serine 616 site, Akt at the serine 473 site, Tau protein at the serine 199396404422 site, and the phosphorylation level of the threonine 616 loci, while in the hippocampus of the low dose group, the phosphorylation level of Tau protein at the serine 404 and the threonine 231 loci was compared with the model group. The model group decreased. Akt decreased the phosphorylation level of serine 473 and GSK-3 beta at the serine 9 site. Conclusion: DNLA can reduce the learning and memory impairment and neuronal damage induced by STZ in rats. This effect is related to the DNLA improvement of STZ induced overphosphorylation of Tau protein in the hippocampus and the obstruction of the isdin signaling pathway in the hippocampus of rats.

【学位授予单位】：遵义医学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R285.5

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