【作者】 李宝龙；

【导师】 周忠光；

【作者基本信息】 黑龙江中医药大学 ， 中西医结合基础， 2012， 博士

【摘要】 目的：研究通天草提取物对老年痴呆(AD)大鼠的保护作用及机制。方法：通天草提取物由本实验室提取并制成水提物和醇提物。将初筛后的大鼠随机分成以下8组：阴性对照组、假手术组、AD模型组、西药治疗组、通天草醇提物低剂量组和高剂量组、水提物低剂量组和高剂量组。采用脑内注射Aβ1-40(10μg/鼠),建立大鼠老年痴呆模型,然后给予通天草提取物治疗28d。采用Morris水迷宫实验检测大鼠的学习记忆等行为学能力；采用HE染色和透射电镜对海马组织进行病理学检测；血清中GSH-Px和T-SOD的活力以及MDA含量采用分光光度法进行测定；采用ELISA法检测血清中IL-1、IL-6、TNF-α的水平；采用免疫组织化学方法检测nNOS、iNOS、bcl-2、bax、GFAP蛋白的表达；采用RT-PCR法检测海马组织中NF-κB、IκB、H SP70、APP和BACE1mRNA的表达。结果1、通过Morris水迷宫实验和病理学检测结果,可以肯定本实验成功建立了Aβ诱导的AD大鼠模型。主要表现为逃避潜伏期明显延长AD模型组为(45.5±6.25)s,明显高于阴性对照组(24.5±5.75)s。AD模型组的穿越跳台次数(1.7±0.89)明显低于阴性对照组(3.2±1.27)。透射电镜结果显示,AD模型组海马神经元细胞间隔增大,突触明显减少,突触小泡融合,肿胀明显；神经元细胞皱缩明显,核膜皱缩并出现阶段性融合,核形异常,异染色质聚集,细胞器如线粒体、核糖体、溶酶体损伤明显,脂褐素异常增多2、通天草提取物能明显改善AD大鼠的学习记忆能力。低、高剂量的醇提物投予动物后,能以剂量依赖方式明显缩短大鼠的逃避潜伏期,分别为(34.5±7.25)s和(24.0±6.5)s,与AD模型组(45.5±7.3)比较,差异有统计学意义；高剂量醇提物的降低作用与西药治疗组相似。同时通天草提取物各组均在不同程度上增加了大鼠穿越跳台的次数,高剂量醇提物的作用尤为明显3、病理学HE染色结果可见,高剂量醇提物作用组大鼠海马区神经元细胞数量较AD模型组明显增多,神经元细胞核形态及排列紧密度上均明显改善,类似于西药治疗组。透射电镜可见,与AD模型组大鼠海马神经元细胞(细胞膜、细胞器等)的明显损伤相比,西药治疗组神经元细胞间界限清晰,连接较紧密,核形基本正常,核糖体、线粒体结构基本正常,脂褐素增多不明显,但仍可见毛细血管及星形胶质细胞肿胀；高剂量的通天草水提物和醇提物组海马神经元细胞皱缩不明显,突触数量有一定的增加,神经元细胞核尚欠规则,低剂量醇提物组可见大量的自噬体存在。4、血清学检测结果发现,通天草提取物各组能明显抑制血清中Aβ1-40水平,与AD模型组(316.8±28.2pg/mL)相比,差异有显著性(p<0.05,p<0.01),醇提物的作用更明显。通天草醇提物(高、低剂量)和水提物高剂量组能明显降低MDA水平,同时增加GSH-Px的活性；通天草提取物各组均能增加T-SOD的活性,与AD模型组比较,差异显著(p<0.01,p<0.05)。血清学炎症因子(IL-1、IL-6、TNF-α)检测结果显示,通天草提取物各组均能在一定程度上降低Aβ诱发的炎症因子分泌,特别是高剂量醇提物对各炎症因子的抑制作用更加明显。5、大鼠海马组织的免疫组化结果显示,通天草提取物能抑制Aβ诱导的海马GFAP蛋白的表达,诱导抗凋亡蛋白bcl-2同时抑制促凋亡蛋白bax蛋白的表达,使bcl-2/bax的比值增加。此外,高剂量的通天草提取物(醇提物和水提物)能明显抑制诱导型一氧化氮合酶(iNOS)的表达,同时促进神经元型一氧化氮合酶(nNOS)的表达6、RT-PCR结果显示,通天草提取物能明显降低Aβ前体APP mRNA的表达,同时降低APP的β-裂解酶BACE1mRNA表达,高剂量醇提物的抑制作用尤为明显。此外,通天草提取物(特别是醇提物)能明显降低NF-κB mRNA的表达,同时增强其抑制因子IκB mRNA的表达。HSP70的表达在AD模型组并无显著变化(与阴性对照组比较,p>0.05)。结论1、采用脑内注射Aβ蛋白,成功建立了Aβ诱导的AD大鼠模型。2、通天草提取物能明显改善AD大鼠的学习记忆能力,高剂量醇提物的作用与西药治疗组相似。通天草提取物能部分改善Aβ对大鼠海马神经元细胞的病理学表现。通天草提取物能明显抑制血清中Aβ1-40水平,该作用与抑制Aβ前体物APP mRNA的表达、降低APP裂解酶BACE1mRNA的表达有关。3、通天草提取物保护AD大鼠的可能分子机制如下(1)通过诱导GSH-Px和T-SOD的活性、抑制MDA生成而增强抗氧化能力;(2)通过抑制炎症因子IL-1、IL-6和TNF-α的分泌,降低Aβ引起的炎性损伤,该作用与NF-κB/IκB调节通路及iNOS有关。(3)通过诱导bcl-2蛋白同时抑制bax蛋白的表达,抑制海马神经细胞凋亡；通过抑制海马星形胶质细胞中GFAP蛋白的表达来增加神经突触的传递能力；通过诱导nNOS的表达来发挥神经保护作用。更多还原

【Abstract】 Objective:To study the protective effects of extracts from waternut Herb on Alzheimer’s disease(AD)and its molecular mechanisms in rats.Methods:Waternut Herb extracts were prepared into aqueous extract and alcohol extract in our lab. The screened rats were randomly divided into eight groups:negative group, sham operated group, Alzheimer’s disease (AD) model group, positive therapy group, two alcohol extract groups with high or low dose and two aqueous extract groups with high or low dose. Alzheimer’s disease (AD) model in rats was established by subdural injection with β-amyloid protein (Aβ,10μg/rat). Morris water maze was used to test the learning and memory abilities of rats; Pathological changes were observed under light microscope (HE staining) and transmission electron microscope; The activities of GSH-Px, T-SOD and the content of MDA in the serum were individually determined by spectrophotometric method. Also ELISA assays were performed to measure the levels of IL-1, IL-6and TNF-α in the serum. The expressions of NOS (including nNOS and iNOS), bcl-2/bax and GFAP proteins in the tissue of cormu ammon (CA) were detected by immunohistochemistry technique. And the transcriptional expressions of NF-κB/IκB, HSP70, APP and BACE1genes in CA tissue were measured by reverse-transcription PCR (RT-PCR) method.Results1. AD model in rats was successfully established and qualified by the results of Morris water maze test and pathological evidences. Escape latency period was significantly prolonged in the AD group (45.5±6.25s), compared with negative group (24.5±5.75s). Similarly, numbers of times across platform were largely decreased in the AD group (1.7±0.89) comparing with (3.2±1.27) in the negative group. Under the transmission electron microscope, slides of AD group showed that cell compartment among the nerve cells were extremely broadened with much less synapse and confluence of synaptic vesicle, as well as severe swelling. And also cell membrane and nuclear membrane were shrinked and partly confluenced with abnormal karyomorphism, heterochromatin aggregation as well as the evident damages of organelles such as mitochondria, ribosome, lysoome and lipofuscin accumulation.2. Waternut Herb extracts obviously improved the capabilities of learning and memory in AD rats. Low and high alcohol extract extremely shortened the escape latency period in dose-dependent way (34.5±7.25s and24.0±6.5s respectively), significant with AD model (45.5±7.3s). Especially, similar effect was observed in both high alcohol extract group and positive therapy group. Meanwhile, the numbers of times crossing platform were increased by the individual Waternut Herb extracts in the different extent with the most evident effect in high alcolhol extract group.3. Pathological examinations by HE staining showed that the quantities of nerve cells in the tissue of CA were significantly increased by the treatment with high alcohol extract, with improved karyomorphism and tight arrange. Also the evidence from transmission electron microscope exhibited that clear cell limit, tighter conjunction and relative normal of nuclear karyomorphism, ribosome, mitochrondria and liposuscin, accompanying with swelling micrangium and astrocyte (AS) in the positive therapy group. High aqueous and alcohol extracts improved, in some extent, pathological appearances such as cell shrinkage, numbers of synapses, nuclear karyomorphism. It should be noted that a large number of autophagosome were observed in the low dose of alcohol extract.4. All Waternut Herb extracts significantly inhibited Aβ1-40level compared with its level (316.8±28.2pg/mL) in AD model (p<0.05, p<0.01), with the most evident in alcohol extracts. Alcohol extracts (both low and high doses) and high aqueous extract decreased the contents of MDA, meanwhile increased the activity of GSH-Px in the serum of rats. All the extracts enhanced the activities of T-SOD compared with AD model (p<0.01,p<0.05). The secretion of inflammatory factors such as IL-1, IL-6and TNF-α in the serum were also obviously suppressed by all extracts, with the most inhibitory effect in the high alcohol extract group.5. Waternut Herb extracts inhibited the expression of GFAP protein, induced the anti-apoptotic protein bcl-2expression, and meanwhile reduced bax apoptotic protein expression which lead to the increase of bcl-2/bax ratio in CA. In addition, both aqueous and alcohol extracts with high doses lessened the expression of induced-NOS(iNOS), while enhanced the expression of neron-type NOS(nNOS).6. Waternut Herb extracts down-regulated APP mRNA expression, the precursor of Aβ and BACE1mRNA, β-site App cleaving enzyme of APP. Additionally, all extracts (especially for alcohol extract) largely inhibited the expression of NF-κB mRNA, while increased IκB mRNA expression, an inhibitor of NF-κB. There is no observed difference in the expression of HSP70between negative group and AD model group (p>0.05).Conclusions1. Alzheimer’s disease model in rat was successfully by subdural injection with β-amyloid protein (Aβ,10μg/rat).2. Waternut Herb extracts present the therapeutic effect on AD rats.3. The molecular mechanisms were involved in the inhibition of neuron cell apoptosis and inflammatory factors secretion, as well as in the improvement of anti-oxidant capabilities.更多还原