补肾化痰中药复方对3×Tg-AD转基因小鼠脑内未折叠蛋白反应的调节机制研究

Background: β-amyloid deposition and hyperphosphorylated tau are two core pathological changes in Alzheimer's disease (AD), which have been found closely related with the dysregulation of unfolded protein response (UPR). AD can be classified as "Chidai" in Chinese medicine, and its main mechanism is the kidney deficiency and phlegm-turbidity. Jinsiwei, a effective herbal formula developed by our team according to reinforcing kidney and clearing phlegm method, which has been found effectively in improving patient cognition and reducing Aβ deposition in mouse model. Whether Jinsiwei exert such effects via adjusting UPR? And its specific targets are not investigated yet. Aim: Present research aims at observing the influence of Jinsiwei on behavioral, hippocampal and cortical pathological (β-amyloid and hyperphosphorylated tau depositions) changes in AD transgenic mouse and then observing influence of Jinsiwei on changes of UPR and its three classical signaling pathways in AD mouse. Method: Diving platform experiment and Morris water maze experiment will be used to check memory and behavior of AD transgenic mouse; immunohistochemisty and Western blot will be used to test expressions of AD pathological markders β-amyloid and tau, UPR markder GRP78/Bip and key proteins in UPR pathways of PERK/eIF2α pathway, IRE-1/XBP1 pathway, and ATF6 pathway. Application: The whole picture of UPR in AD transgenic mouse will be depicted and pharmaceutical targets in UPR pathways of herbal formula with reinforcing kidney clearing phlegm will be revealed.

背景：β-淀粉样蛋白(Aβ)和过度磷酸化Tau蛋白(P-Tau)堆积是阿尔茨海默病（AD）的主要病理改变，目前认为这些病理蛋白的出现与脑内未折叠蛋白反应(UPR)失调密切相关。AD属于中医痴呆范畴，肾虚痰浊是其主要病机。前期研究发现补肾化痰方-金思维能显著改善患者认知、减少模型Aβ沉积，其是否通过调节UPR起效？具体作用机制如何？目的：在观察金思维对AD转基因小鼠药效学指标的基础上，进一步研究金思维对小鼠脑内UPR及三条通路的影响。方法：应用跳台实验和Morris水迷宫检测小鼠空间记忆学习能力，应用免疫组化和Western blot对AD病理标志Aβ、Tau，UPR标记物GRP78/Bip，以及UPR的PERK/eIF2α途径、IRE-1/XBP1途径、ATF6途径的关键蛋白进行检测。意义：揭示AD转基因小鼠脑内UPR的改变，明确金思维对UPR的调节作用及具体作用靶点。

β-淀粉样蛋白(Aβ)和过度磷酸化Tau蛋白(P-Tau)堆积是阿尔茨海默病（AD）的主要病理改变，目前认为这些病理蛋白的出现与脑内未折叠蛋白反应(UPR)失调密切相关。团队前期研究发现补肾化痰方-金思维能显著改善患者认知、减少模型Aβ沉积，其是否通过调节UPR起效？本研究在观察金思维对AD转基因小鼠药效学指标的基础上，进一步研究金思维对小鼠脑内UPR及其三条通路的影响。本研究应用Morris水迷宫检测小鼠空间记忆学习能力，应用免疫组化和Western blot对AD病理标志Aβ、Tau，UPR标记物GRP78/Bip，以及UPR的PERK/eIF2α途径、IRE-1/XBP1途径、ATF6途径的关键蛋白进行检测。Morris水迷宫检测发现GAPT能显著缩短3×Tg-AD转基因小鼠逃避潜伏期（p＜0.05），改善空间学习能力，此作用与多奈哌齐疗效相似（p＞0.05）。转基因小鼠脑组织Western blot和免疫组化检测发现，GAPT能显著减少海马区Aβ的沉积和Tau蛋白的过度磷酸化（p＜0.05），此作用优于多奈哌齐。进一步探讨作用机制时发现8月龄模型组3×Tg-AD转基因小鼠与正常小鼠相比海马所表达的GRP78/Bip和PERK蛋白均有所升高，但无显著统计学差异（p＞0.05）。GAPT组与模型组相比，GRP78/Bip和XBP1的升高有所降低，但也无明显统计学差异（p＞0.05）。另外，与正常组相比，8月龄模型组小鼠大脑海马所表达的p-eIF2α、IRE1、XBP1、CHOP、ATF4和ATF6α蛋白均未见明显变化（p＞0.05），查找相关文献也证实了这一结果，考虑与模型有关，故未再行这几种蛋白的免疫组化染色。增补观察了电镜下AD转基因小鼠脑内神经元突触的组织形态学变化，发现与对照组相比模型组出现核膜凹陷，染色质浓缩、边集，神经元核固缩等，可见线粒体嵴断裂、肿胀、溶解，粗面内质网扩张、脱颗粒，胞浆内可见脂褐素沉积。与模型组相比，多奈哌齐组和GAPT组神经元变性明显减轻，线粒体仅出现轻度肿胀现象，嵴清晰可见；内质网排列整齐。由此提示GAPT能改善小鼠空间记忆学习能力，减少3×Tg-AD转基因小鼠脑内Aβ的沉积和Tau蛋白的过度磷酸化，在UPR三种通路上可能从GRP78/Bip和PERK途径上起作用，但需要进一步的研究结果进行核实。