节点文献
基于“毒损脑络”病机理论的阿尔茨海默病机制研究
【作者】 刘洋；
【作者基本信息】 北京中医药大学 ， 中西医结合基础， 2013， 博士
【摘要】 1.背景阿尔茨海默病(Alzeimer disease, AD)是老年痴呆症最常见的一种,以认知功能障碍为主要临床表现。现代医学研究表明,AD病因主要与β淀粉样蛋白(Amyloid β, Aβ)在脑组织异常沉积、Aβ清除障碍、胆碱能神经元受损、氧化应激反应、脑内炎症级联反应或基因突变等因素有关,为此研究者虽然针对改善胆碱能神经元功能、抗氧化应激等多方面研制了各种药物,但是目前尚未找到有效的治疗措施,尤其是仍未使脑损伤得以根本解决。然而,在中医理论知道下,本研究团队前期研究表明,AD隶属于中医“络病”范畴,“毒损脑络”是其病机关键,在临床中运用针对该病机关键的解毒通络法进行辨证论治取得了显著疗效。以脑微血管内皮细胞为主要物质基础的中医络脉系统具有渗灌气血、贯通营卫之功效,是经脉气血进行物质交换的场所。络脉瘀滞引发气血渗灌失常,营卫失和,卫气壅滞,郁而化热,进而损伤络脉。若发于脑部络脉则形成“毒损脑络”之病机。随着对该疾病认识的逐渐深入,研究人员日益认识到以脑微血管内皮细胞为主要物质基础的脑部络脉系统对脑损伤的保护作用,除了能够调节血管细胞生长迁移、基质降解、活化多种细胞受体、激活细胞内信号转导通路外,还能调节脑组织内特定物质的脑外转运,对维持脑组织内环境的平衡稳定具有重要的调节作用。2.目的本研究在中医络脉理论指导下,以脑部络脉系统的主要物质基础-脑微血管内皮细胞为切入点,以具有解毒通络功效的通络救脑注射液(TLJN)为干预药物,重点探讨在拟AD损伤的大鼠脑微血管内皮细胞上特定β淀粉样蛋白转运体的动态改变以及由此转运体介导的细胞内特定信号转导通路的变化,在揭示该转运体改变与内皮细胞损伤程度的同时,阐明TLJN与血管内皮保护生物效应之间的关系,从分子水平对该注射液作用内皮细胞的可能靶点进行科学诠释,为“毒损脑络”病机假说在阿尔茨海默病的确立以及TLJN的临床应用提供体外实验依据。3.方法本研究共分为四部分：第一部分：观察解毒通络药物对拟AD损伤大鼠脑组织的保护作用。第二部分：探讨拟AD损伤大鼠脑微血管内皮细胞与神经元损伤的时程变化及TLJN干预作用。第三部分：探讨Aβ1-42对β淀粉样蛋白转运体的影响以及TLJN干预作用。第四部分：探讨拟AD损伤的脑微血管内皮细胞内PI3K/Akt/Caspase-3信号转导通路变化及TLJN干预作用。具体实验方案为：第一部分：大鼠双侧海马注射凝聚态Aβ1-42拟建立AD损伤动物模型,动物实验分为五组,分别是control组(正常对照组)、sham组(假手术组),model组(模型组)、TLJN组(通络救脑注射液组)和DNP组(阳性药组),观察各组大鼠在药物干预7天、14天和21天后的Morris水迷宫行为学改变,阿尔茨海默病脑组织特征性病理改变(ELISA (?)去检测脑组织A β1-42含量、Western Blot法检测P-tau表达,HE染色观察神经元损伤程度)。第二部分：Western Blot法和ELISA法分别观察拟AD损伤大鼠在7天、14天和21天脑组织内皮细胞特异性损伤因子vWF和神经元特异性损伤因子NSE的蛋白表达以及血液中上述蛋白含量的变化。第三部分：通过体外培养拟AD损伤大鼠脑微血管内皮细胞,运用Western Blot和Real-time PCR实验技术分别从蛋白水平和mRNA水平考察拟AD损伤的大鼠脑微血管内皮细胞在不同时段中特定β淀粉样蛋白转运体LRP-1和RAGE表达变化、分光光度法检测氧化应激反应的变化(MDA含量、SOD活性、Cat活性、GSH-Px活性)、MTT法检测内皮细胞的损伤程度以及TLJN液的干预作用。第四部分：通过培养拟AD损伤大鼠脑微血管内皮细胞,运用Western Blot实验技术检测A β1-42通过LRP-1转运体介导的脑微血管内皮细胞内PI3K/Akt/Caspase-3信号转导通路中主要信号分子的蛋白表达变化(LRP-1、T-tau、 P-tau、Caspase-3)以及TLJN对该信号转导通路的调节作用。4.结果①在本次动物实验观察范围内,通过morris水迷宫实验,我们发现造模后散发型AD大鼠行为学发生显著改变。体现在其逃避潜伏期延长、游泳总路程距离增加、目标象限停留时间缩短以及穿台次数减少。此外,AD大鼠形态学亦发生显著变化,表现在海马神经元出现不同程度的细胞皱缩、核固缩、核溶解以及核碎裂等现象；整个脑组织Tau蛋白过度磷酸化；脑组织中A β1-42含量显著增加。在通络就脑注射液干预后,AD大鼠行为学、脑部组织结构和功能均得到了不同程度的改善。②在本实验中,AD大鼠脑组织和血液中的vWF蛋白量与假手术组比较在检测的三个时程中均显著升高,而AD大鼠脑组织和血液中的NSE蛋白量从检测的14天时间点开始才明显升高。在TLJN干预后,与模型组比较,vWF和NSE蛋白量在14天后明显降低,显示出对脑组织的改善作用。③在本次细胞实验观察范围内,LRP-1和RAGE蛋白转运体在拟AD损伤的内皮细胞中,呈现动态改变,表现在0h、3h、6h和12h四个时间点LRP-1蛋白表达升高,在24h、36h、48h和72h四个时间点蛋白表达降；而其mRNA表达在0h、3h、6h三个时间点增加,在后续时间点降低。RAGE蛋白和mRNA表达在全部8个时间点逐渐增加；在拟AD损伤的内皮细胞中,LRP-1/RAGE蛋白比值在0h、3h、6h和12h四个时间点未见统计学差异,而在后续四个时间点显著下降;然而,LRP-1/RAGE的mRNA比值在检测的8个时间点未见规律性改变。经TLJN干预后,RAGE蛋白和mRNA表达在全部8个时间点均有不同程度地降低；而对LRP-1的干预作用,仅体现在其对LRP-1蛋白表达的下调作用,未见对mRNA的调节作用;LRP-1/RAGE蛋白比值在0h、3h、6h和12h四个时间点未见组间及组内比较的统计学差异,而在后续四个时间点其蛋白比值与A β1-42损伤组比较明显升高；在拟AD损伤的内皮细胞中,细胞内氧化应激反应(MDA含量)在8个时间点逐渐增加,抗氧化酶系统(SOD、Cat、GSH-Px活性)活性在全程逐渐下降,经TLJN干预后,细胞内氧化应激反应减弱,抗氧化酶系统活性增加。④在细胞实验中,A β1-42诱导拟AD损伤内皮细胞中LRP-1受体蛋白表达下降,P-tau蛋白表达下降,Caspase-3蛋白表达升高,诱导细胞凋亡；在TLJN干预A β1-42诱导的内皮细胞后,LRP-1受体蛋白表达增加,P-tau蛋白表达增加,Caspase-3蛋白表达降低,减缓细胞凋亡；在TLJN药物和阻滞剂LY200942共同作用于A β1-42诱导的内皮细胞后,LRP-1受体蛋白表达增加,P-tau蛋白表达降低,而Caspase-3蛋白表达与Aβ1-42损伤的内皮细胞比较,明显升高,减缓细胞凋亡。5.结论①以通络救脑注射液为代表的解毒通络方药能够减轻拟AD损伤大鼠认知功能障碍,改善大鼠脑部组织AD病变的特征性病理损伤。②拟AD损伤的大鼠动物模型和脑微血管内皮细胞模型中,脑微血管病变先于神经元的损伤,由于脑微血管内皮细胞上LRP-1和RAGE两个p淀粉样蛋白转运体的动态平衡被打破,引起A β脑外转运减少,脑内聚集增加,通过LRP-1转运体介导的PI3K/Akt/Caspase-3信号转导通路,使Caspase-3蛋白表达增加,促进内皮细胞凋亡,加重拟AD损伤模型中神经元的损伤,最终形成了以认知功能障碍为主要表现的脑功能紊乱。③解毒通络药物-TLJN在干预拟AD损伤模型后,恢复了脑微血管内皮细胞上LRP-1和RAGE两个β淀粉样蛋白转运体的动态平衡,可能通过TLJN中药复方多靶点的作用途径抑制内皮细胞内Caspase-3蛋白表达,减少内皮细胞凋亡,通过调节脑微血管的功能,最终达到了改善受损神经元目的,在阐明脑微血管在神经退行性病变中重要作用的同时,一定程度上为阿尔茨海默病“毒损脑络”病机假说的确立提供了体外实验依据。
【Abstract】 1. BackgroundAlzeimer’s disease (AD) is the most common one of dementia, regarding cognitive dysfunction as the main mlinical manifestations. Modern medical research in this field demonstrates that the pathogenesis of AD is related to the accumulation of amyloid beta (Aβ) in brains, disorder of Aβ clearance, cholinergic neuron damage, oxidative stress, brain inflammatory cascade, gene mutation, and so forth. Although researchers have produced a variety of drugs aimed at improving cholinergic neuronal function, oxidative stress and other aspects of AD, there are not effective ways to treat AD, especially in the aspect of brain injury recovery. However, in the direction of TCM theory, our previous research showed that AD belongs to the category of ’collateral disease’, and ’toxin hurts brain collaterals’ is the key point in its pathogenesis. It is also realized that brain microvascular endothe lial cells (BMECs) are not only the substance exchange barrier of blood and tissue, but also have important incretion function. Our researches pay more attention to BMECs rather than neurons, which is a meaningful probe to guide Traditional Chinese Medicine mordernation in brain diseases.2. PurposeIn this study, with the guidance of collateral disease theory, we take the main material basis of brain collaterals system-BMECs as the breakthrough point of our research, choose Tongluojiunao injection(TLJN)withthe function of detoxification and smoothing collaterals as intervening drugs, mainly explore the dynamic changes of Aβprotein transportersin ADimitated BMECs andchanges in thetransportermediatedspecific intracellular signal pathway, elucidate both the transporter change and endothelial cells injury and the biological effect between TLJN and vascular endothelial protection and explain the targets of TLJN to BMECs from the molecular level. At last, provide an in vitro experimental basis for "establishingthe toxin hurtingbraincollaterals"pathogenesis hypothesis in Alzheimer’s disease and the clinical application of TLJN.3. MethodsThe experiments are divided into4parts, the first part is to observe the protective effect of Jiedu Tongluo drugs on AD imitated rat brain; The second part is to investigate time course changes between cerebral vessels and neuronal injury in the AD imitated rat and the intervention effect of TLJN; The third part is to investigate the effect of Aβ1-42on the amyloid beta protein transporter and the intervention effect of TLJN; The fourth part is to investigate the AD imitated brain microvascular endothelial cells in PI3K/Akt/Caspase-3 intracellular signal pathway and the intervention effect of TLJN.The experimental plans as followed:the first part:condensed Aβ1-42was injected into both hippocampus of rats to establish AD imitated animal model, animal experiments were divided into five groups, respectively, control group (normal control group), sham group (sham operation group), model group (model group), TLJN group (TLJN injection group) and DNP group (positive drug group), behavior of rats were detected by Morris water maze after drug administration for7days,14days and21days, characteristic pathological changes were observed in Alzheimer’s disease brains (ELISA method for detecting the content of Aβ1-42in brain tissue, Western Blot method to detect the expression of P-tau, HE staining was chosen for neuron injury). The second part:Western Blot method and ELISA method were used to observe the expressions of both vWF and NSE in protein levels in cerebral tissues and blood in AD imitated rats of3detecting period of time. The third part:in vitro culture of AD imitated rat brain micro vascular endothelial cell, from the level of protein and mRNAto study brain damage in AD rat, the expression changes of specific amyloid beta protein transporters LRP-1and RAGE in AD imitated micro vascular endothelial cells in different period of time, detection of oxidative stress reaction by spectrophotometry (MDA content, SOD activity, Cat activity, GSH-Px activity), the viability of endothelial cells were detected by MTT method. The fourth part:through the cultivation of the AD imitated rat brain micro vascular endothelial cells to detect the main signal molecules change of LRP-1mediated PI3K/Akt/Caspase-3signal pathway induced by Abetal-42with the method of Western Blot and the regulating effect of TLJN on the signal pathway.4. Results①In the observation of the animal experimental range, by the Morris water maze test, we found that the behavior of AD rats changed significantly, demonstrated by the prolonged escape latency, increased swimming total distance, increased target quadrant stay time and reduced number of wear stage. In addition, the morphology of AD rats also changed significantly, showing different degrees of cell shrinkage, nuclear condensation in hippocampal neurons, karyolysis and nuclear fragmentation phenomenon:Tau protein hyperphosphorylation and increased Aβ1-42content in the whole brain tissue. After the administration of TLJN. behavior of AD rats, brain tissue structure and function were improved to some extent.②In this experiment, the vWF protein level in blood and brain in AD rats compared with sham operation group in three period of time were increased significantly, while the NSE protein in brain tissue and the blood in AD rats increased significantly after14days. In TLJN group, compared with model group, vWF and NSE protein decreased significantly in14days, showing the beneficial effects on AD imitated rats.③In the observation of cells experimental range, LRP-1and RAGE protein expressions in the AD imitated endothelial cells showed the dynamic change, presenting the elevated expression of LRP-1in Oh,3h,6h and12h, four period of time, but decreased in24h,36h,48h and72h four period of time. As for mRNA expression of LRP-1, increased at Oh,3h and6h three time points, decreased in the subsequent time points. Expression of RAGE in both protein and mRNA levels increased gradually in all8time points; LRP-1/RAGE protein ratio showed no statistical differences in Oh,3h,6h and12h four time points, but significantly decreased in the following four period of time; however, the ratio of LRP-1/RAGE in mRNA level showed no change. After TLJN administration, the expression of RAGE both in protein and mRNA levels were decreased in all8time points; as for the regulation of LRP-1expression, TLJN could down regulate the protein expression of LRP-1, but showed no effect on the expression of LRP-1in mRNA level. In AD imitated endothelial cells, intracellular oxidative stress (MDA content) increased at all detected8time points, antioxidant enzymes (SOD, Cat, GSH-Px activity) activity decreased gradually in the whole process, after the administration of TLJN, intracellular oxidative stress was decreased, the activities of antioxidant enzymes were increased.④In cellular experiments, in Aβ1-42induced AD imitated RMECs, LRP-1protein expression decreased, P-tau protein expression decreased, Caspase-3protein expression increased, inducing endothelial cell apoptosis; with the administration of TLJN, the expression of LRP-1increased, the expression of P-tau protein increased, the expression of Caspase-3decreased, reducing cell apoptosis; after the interaction of both TLJN and inhibitor-LY200942in Aβ1-42induced endothelial cells, the expression of LRP-1increased, the expression of P-tau protein reduced, and Caspase-3protein expression decreased, alleviating endothelial cell apoptosis.5. Conclusion①TLJN as the representative of the decoction with the function detoxification and smoothing collaterals can reduce t cognitive impairment, improve the characteristic pathological injury of brain in AD imitated rats.②In the AD imitated rats and rats micro vascular endothelial cell models, the cerebral microvessels are prior to the injury of neurons, due to break of the dynamic balance between LRP-1and RAGE, Aβ1-42were transported decreased and increased aggregation in brains, via LRP-1mediated PI3K/Akt/Caspase-3signal pathway, Caspase-3expression was increased, promoting the apoptosis of endothelial cells and increasing the damage to the neurons in AD imitated rats, eventually led to the cognitive dysfunction. ③After TLJN was administrated, it could recover the dynamic balance between LRP-1and RAGE, with the help of compound target in decoction, inhibited the expression of Caspase-3, reduced the apoptosis of endothelial cells, with the regulation to microvascular endothelial cells, achieve the purpose of improving the damaged neurons, to a certain extent for the Alzheimer’s disease"toxin damaging brain collaterals" pathogenesis hypothesis establishment provides experimental evidence in vitro.