目的 基于核因子E2相关因子2（nuclear factor erythroid 2-related factor 2，Nrf2）/p62/Kelch样ECH相关蛋白1（Kelch-like ECH-associated protein 1，Keap1）信号通路探讨葛根素联合雷帕霉素对阿尔茨海默病（Alzheimer’s disease，AD）模型小鼠的神经保护作用。方法 BALB/c小鼠随机分为对照组、模型组、葛根素组、雷帕霉素组、葛根素＋雷帕霉素组、葛根素＋雷帕霉素＋Nrf2抑制剂ML385组，除对照组外，其余小鼠均通过脑内注射β-淀粉样蛋白25-35（β-amyloid protein 25-35，Aβ_25-35）构建AD模型，给予药物干预42 d。采用Y迷宫交替实验及新物体识别实验检测小鼠学习记忆能力；ELISA检测海马组织氧化应激水平；苏木素-伊红（hematoxylin-eosin，HE）染色及尼氏染色观察海马组织病理损伤情况；硫磺素-S染色观察小鼠海马组织中Aβ表达；免疫组化检测海马组织Aβ_1-42和磷酸化tau蛋白（phosphorylated tau，p-Tau）的表达；TUNEL染色观察海马组织神经元凋亡情况；Western blotting检测海马组织凋亡、自噬及Nrf2/p62/Keap1通路相关蛋白表达。结果 与模型组比较，葛根素组、雷帕霉素、葛根素＋雷帕霉素组小鼠自主交替率、偏好系数、辨别系数显著升高（P＜0.05）；海马组织中超氧化物歧化酶（superoxide dismutase，SOD）活性及谷胱甘肽（glutathione，GSH）水平显著升高（P＜0.05），丙二醛（malondialdehyde，MDA）水平显著降低（P＜0.05）；海马组织病理损伤表现出不同程度的改善，神经元凋亡率显著降低（P＜0.05）；海马组织Aβ及Aβ_1-42、p-Tau表达降低（P＜0.05）；海马组织半胱氨酸天冬氨酸蛋白酶-3（cystein-asparate protease-3，Caspase-3）、B细胞淋巴瘤-2（B-cell lymphoma-2，Bcl-2）相关X蛋白（Bcl-2 associated X protein，Bax）、Keap1、p62蛋白表达水平显著降低（P＜0.05），Beclin-1、微管相关蛋白轻链3-II（microtubule-associated protein light chain 3-II，LC3-II）/LC3-I、Nrf2蛋白表达水平显著升高（P＜0.05）；与单独给药比较，联合给药的变化最为显著（P＜0.05）。与葛根素＋雷帕霉素组比较，葛根素＋雷帕霉素＋ML385组可部分逆转上述指标及病理的变化趋势（P＜0.05）。结论 葛根素联合雷帕霉素对AD小鼠发挥神经保护作用，其作用机制与激活Nrf2/p62/Keap1信号通路有关。

Objective To explore the neuroprotective effects of puerarin combined with rapamycin in Alzheimer’s disease (AD) mice model based on nuclear factor E2-related factor 2 (Nrf2)/p62/Kelch-like ECH associated protein 1 (Keap1) signaling pathway. Methods BALB/c mice were randomly divided into control group, model group, puerarin group, rapamycin group, puerarin + rapamycin group and puerarin + rapamycin + Nrf2 inhibitor ML385 group. Except for the control group, all other mice were injected with β-amyloid protein 25-35 (Aβ_25-35) into the brain to establish an AD model and received drug intervention for 42 d. Y-maze alternation experiment and new object recognition experiment were used to detect the learning and memory ability of mice; ELISA was used to detecte oxidative stress levels in hippocampal tissue; Hematoxylin-eosin (HE) staining and Nissl staining were used to observe the pathological damage of hippocampal tissue; Aβ expression in hippocampal tissue of mice was observed by sulfur-S staining; Immunohistochemistry was used to detect the expressions of Aβ_1-42 and phosphorylated tau protein (p-Tau) in hippocampal tissue; TUNEL staining was used to observe neuronal apoptosis in hippocampal tissue; Western blotting was used to detect apoptosis, autophagy and Nrf2/p62/Keap1 pathway related protein expressions in hippocampal tissue. Results Compared with model group, the spontaneous alternation rate, preference coefficient and discrimination coefficient of mice in puerarin group, rapamycin group and puerarin + rapamycin group were significantly increased (P < 0.05); The activity of superoxide dismutase (SOD) and level of glutathione (GSH) in hippocampal tissue were significantly increased (P < 0.05), while the level of malondialdehyde (MDA) was significantly decreased (P < 0.05); The pathological damage of hippocampal tissue showed varying degrees of improvement, with a significant decrease in neuronal apoptosis rate (P < 0.05); The expressions of Aβ, Aβ_1-42 and p-Tau in hippocampal tissue were decreased (P < 0.05); The expression levels of cystein-asparate protease-3 (Caspase-3), B-cell lymphoma-2 associated X protein (Bax), Keap1 and p62 proteins in hippocampal tissue were significantly reduced (P < 0.05), while the expression levels of Beclin-1, microtubule associated protein light chain 3-II (LC3-II)/LC3-I, and Nrf2 proteins were significantly increased (P < 0.05); Compared with individual administration, the changes in combination administration were the most significant (P < 0.05). Compared with puerarin + rapamycin group, puerarin + rapamycin + ML385 group partially reversed the above indicators and pathological changes (P < 0.05). Conclusion The combination of puerarin and rapamycin exerts neuroprotective effects in AD mice, and its mechanism is related to the activation of Nrf2/p62/Keap1 signaling pathway.

R285.5

湖南省自然科学基金资助项目(2023JJ60263)