目的 研究调心补肾方（Tiao Xin Bu Shen Fang, TXBSF）对Presenilin1/2条件性双基因敲除（presenilin1/2 conditional double knockout mice,PS cDKO）的阿尔茨海默病（Alzheimer s disease，AD）模式小鼠记忆障碍的影响。方法 实验动物采用3-3.5月龄小鼠，雌雄各半，随机双盲分组法分3组：同窝野生型组（Wildtype,WT）、PS cDKO组（cDKO）、调心补肾方组（cDKO + TXBSF），每组20只。利用PS cDKO小鼠作为AD模式小鼠，cDKO+TXBSF组为PS cDKO小鼠给予含有TXBSF（17913 ppm）饲料治疗60 d，其余2组食用普通饲料，治疗结束后进行行为学检测。分别采用旷场实验、高架十字迷宫实验、Y迷宫实验和条件性恐惧实验观察TXBSF对PS cDKO小鼠学习记忆的影响；并采用Western blot方法检测TXBSF对PS cDKO小鼠海马突触相关蛋白和促炎症因子表达的影响；采用免疫组化观察TXBSF对PS cDKO小鼠海马CA3区小胶质细胞激活的影响。结果 旷场实验结果显示，TXBSF没有改变PS cDKO小鼠的活动时间及活动距离（P > 0.05），说明TXBSF并不会影响PS cDKO小鼠的自发运动能力。高架十字迷宫实验结果显示，PS cDKO小鼠较WT小鼠在开放臂逗留的频率降低（P < 0.01），时间相比则增高（P < 0.001）；而TXBSF治疗后增加PS cDKO小鼠在开放臂逗留的频率（P < 0.01），但减少逗留的时间（P < 0.05），说明TXBSF能改善PS cDKO小鼠的焦虑样行为。Y迷宫结果显示，PS cDKO小鼠较WT小鼠在闭合臂逗留的频率和时间均降低（P < 0.01），而cDKO + TXBSF小鼠在闭合臂逗留的频率和时间较PS cDKO小鼠均增加（P < 0.01），说明TXBSF能改善PS cDKO转基因小鼠空间学习记忆能力。条件性恐惧实验结果显示，在Contextual阶段与Cued阶段，PS cDKO小鼠的僵直时间百分明显低于WT小鼠（P < 0.001），而cDKO + TXBSF小鼠的僵直时间百分比较PS cDKO小鼠显著增高（P < 0.05），说明TXBSF可以改善PS cDKO小鼠的联合记忆障碍。Western blot检测结果发现，TXBSF治疗可降低PS cDKO小鼠海马组织中促炎症因子iNOS和 COX-2表达的增加，表明TXBSF可以抑制PS cDKO小鼠的神经炎症反应；并且TXBSF治疗提高PS cDKO小鼠海马组织中MAP2和PSD95蛋白表达的下降，改善神经元和突触损伤。另外，免疫组化结果表明TXBSF对PS cDKO小鼠海马CA3区的小胶质细胞激活有抑制作用。结论 TXBSF对PS cDKO小鼠学习记忆障碍具有一定程度的改善作用。TXBSF对PS cDKO小鼠具有神经保护作用可能是通过抑制神经炎症反应实现的。
Objective To study the effect of Tiaoxin Bushen Fang (TXBSF) on memory impairment in Alzheimer s disease (AD) model mice with presenilin 1/2 conditional double knockout (PS cDKO). Methods 3-3.5-month-old mice with half of male and female, were randomly divided into three groups: wild type group (Wildtype, WT), PS cDKO group (cDKO), and Tiaoxin Bushen Fang group (cDKO + TXBSF), 20 per group. PScDKO mice were used as AD mode mice, and TXBSF group were fed standard chow containing 17913 ppm TXBSF for 60 days. After the treatment, behavioral tests were performed. The effects of TXBSF on learning and memory in PS cDKO mice were observed by open field experiment, elevated plus maze test, Y maze test and conditional fear test. Effect of TXBSF on the expression of pro-inflammatory factors and hippocampal synapse-related proteins in PS cDOK mice were detected by Western blot in hippocampal tissue. In addition, immunohistochemistry staining was used to observe the effect of TXBSF on the activation of microglia in CA3 region of hippocampus of PS cDKO mice. Results The results of open field experiments showed that TXBSF did not change the activity time and activity distance of PS cDKO mice (P > 0.05), indicating that TXBSF did not affect the spontaneous exercise ability of PS cDKO mice. The results of the elevated plus maze test showed that PS cDKO mice had a lower frequency of staying in the open arms than WT mice (P < 0.01), and the time was increased (P < 0.001). Meanwhile, PS cDKO mice had a lower frequency of staying in the open arms after TXBSF treatment (P < 0.01), but the duration of stay reduced (P < 0.05), indicating that TXBSF can improve anxiety-like behavior in PS cDKO mice. Y-maze results showed that frequency and time of staying in closed arms decreased in PScDKO mice compared with WT mice (P < 0.01), while DKO + TXBSF mice stayed in closed arms more frequently than PS cDKO mice (P < 0.01). This indicates that TXBSF can improve the spatial learning and memory ability of PS cDKO mice. The conditional fear test showed that the percentage of immediate freezing in PS cDKO mice was significantly lower than that in WT mice in the Contextual and Cued stages (P < 0.001). The percentage of immediate freezing in cDKO + TXBSF mice was significantly higher than that in PS cDKO mice (P < 0.05), indicating that TXBSF can improve the combined memory ability of mice. Western blot analysis showed that TXBSF treatment reduced proinflammatory cytokines iNOS and COX-2 in hippocampus of PS cDKO mice, which indicates that TXBSF can inhibit the neuroinflammatory response in PS cDKO mice. In addition, TXBSF treatment increased the expression of MAP2 and PSD95 protein in hippocampus of PS cDKO mice, which improved neuronal and synaptic damage. In addition, immunostaining showed that TXBSF inhibited microglial activation in hippocampus CA3 of PS cDKO mice.Conclusion TXBSF has a potential of improving memory impairment in PS cDKO mice. The neuroprotective effect of TXBSF on PS cDKO mice may be achieved by inhibiting neuroinflammatory responses.
＊ 上海中医药大学第十一批大学生创新活动计划项目 2018KCZ14＊ 上海中医药大学第十一批大学生创新活动计划项目（2018KCZ14）：调心补肾方对PS1/2双敲除AD模型小鼠记忆障碍的影响，负责人：王迪霖。