摘要
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 protein25-35,Aβ25-35)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Aβ1-42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Aβ1-42、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 withβ-amyloid protein 25-35(Aβ25-35)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;Aβexpression in hippocampal tissue of mice was observed by sulfur-S staining;Immunohistochemistry was used to detect the expressions of Aβ1-42 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,Aβ1-42 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.
作者
宋冲
占伟
谌静
李心亮
SONG Chong;ZHAN Wei;CHEN Jing;LI Xinliang(School of Medical,Changsha Social Work College,Changsha 410004,China;National Clinical Research Center for Geriatric Diseases(Xiangya)Institute of Health Care,Changsha 410004,China;Xiangya School of Nursing,Central South University,Changsha 410013,China;Department of Imaging,Reproductive&Genetic Hospital of Citic-Xiangya,Changsha 410008,China;Stomatological Center,Xiangya Hospital of Central South University,Changsha 410008,China;Institute of Clinical Pharmacology of Traditional Chinese Medicine,Hunan Academy of Chinese Medicine,Changsha 410017,China)
出处
《中草药》
北大核心
2025年第22期8234-8244,共11页
Chinese Traditional and Herbal Drugs
基金
湖南省自然科学基金资助项目(2023JJ60263)。
