Huang Qi, Zhang Yingli, Huang Li, Guo Zhijun, Wu Deling
Department of Pharmacy, Anhui University of Chinese Medicine, Hefei, 230012, China; Anhui Province Key Laboratory of Traditional Chinese Medicine Decoction Pieces of New Manufacturing Technology, Hefei, 236000, China.
Department of Pharmacy, Anhui University of Chinese Medicine, Hefei, 230012, China.
J Pharmacol Sci. 2025 Feb;157(2):75-87. doi: 10.1016/j.jphs.2024.12.005. Epub 2024 Dec 25.
Alzheimer's disease (AD) is a neurodegenerative disease, and neuroprotection is an important approach to improving AD outcomes. Rhizoma of Anemarrhena asphodeloides (RAA) is a commonly used Traditional Chinese Medicine (TCM) with demonstrated neuroprotective effects, but its anti-AD mechanism requires further exploration.
To elucidate the neuroprotective mechanism of RAA on TMT-induced AD mice.
The AD mice model was established via intraperitoneal injection of TMT. The effect of RAA on ameliorating learning and memory was assessed using the Morris Water Maze (MWM) and Y-maze tests. Haematoxylin-Eosin (HE), Nissl, and TUNEL staining were used to observe the neuroprotective effect of RAA. The components in serum containing RAA (RAA-S) were detected using UPLC-Q-Orbitrap MS. Potential targets were predicted through network pharmacology and molecular docking. Serum levels of reactive oxygen species (ROS), malondialdehyde (MDA), superoxide dismutase (SOD), glutathione (GSH), and glutathione peroxidase (GPx) were measured with ELISA kits. The HT22 hippocampal neuronal cell line injured by l-glutamate (L-Glu) was used to further elucidate the mechanism of RAA. ROS levels in HT22 cells were detected with the 2'-7'-dichlorodihydrofluorescein diacetate (DCFH-DA) fluorescent probe and flow cytometry. Apoptosis in HT22 cells was measured by flow cytometry. The proteins MAP2, GAP-43, Nrf2, Keap1, HO-1, Bax, and Bcl-2 were detected by Western blotting. Immunofluorescence staining was employed to observe Nrf2 nuclear translocation.
RAA significantly increased the residence time of mice in the W zone and enhanced the correct alternation rate in TMT-treated mice. RAA preserved the integrity and orderly arrangement of nerve cells. A total of 12 components were detected in RAA-S. AKT1, PPARG, CASP3, STAT3, HSP90AA1, and NFE2L2 (Nrf2) were involved in the RAA-S target pathway network. Molecular docking revealed that Nrf2 exhibited the highest average binding energy with all components in RAA-S. In vivo, RAA elevated MAP2, GAP-43, Nrf2, and HO-1 levels, along with GPx, GSH, and SOD activity, which had been reduced by TMT. Additionally, RAA reduced serum levels of MDA and ROS, which had been elevated by TMT. In vitro, RAA-S reduced HT22 cell apoptosis and ROS accumulation caused by TMT. Furthermore, RAA-S promoted the expression of N-Nrf2 and HO-1 in L-Glu-injured HT22 cells.
RAA attenuated oxidative stress induced by TMT and L-Glu in AD model mice. The underlying mechanism was associated with the activation of the Nrf2/Keap1-HO-1 pathway.
阿尔茨海默病（AD）是一种神经退行性疾病，神经保护是改善AD预后的重要途径。知母根茎（RAA）是一种常用的具有神经保护作用的传统中药，但其抗AD机制有待进一步探索。
阐明RAA对三甲基锡（TMT）诱导的AD小鼠的神经保护机制。
通过腹腔注射TMT建立AD小鼠模型。采用莫里斯水迷宫（MWM）和Y迷宫试验评估RAA改善学习记忆的效果。采用苏木精-伊红（HE）染色、尼氏染色和TUNEL染色观察RAA的神经保护作用。采用超高效液相色谱-四极杆-轨道阱质谱（UPLC-Q-Orbitrap MS）检测含RAA血清（RAA-S）中的成分。通过网络药理学和分子对接预测潜在靶点。用酶联免疫吸附测定（ELISA）试剂盒检测血清中活性氧（ROS）、丙二醛（MDA）、超氧化物歧化酶（SOD）、谷胱甘肽（GSH）和谷胱甘肽过氧化物酶（GPx）的水平。用L-谷氨酸（L-Glu）损伤的HT22海马神经元细胞系进一步阐明RAA的作用机制。用2',7'-二氯二氢荧光素二乙酸酯（DCFH-DA）荧光探针和流式细胞术检测HT22细胞中的ROS水平。通过流式细胞术检测HT22细胞的凋亡情况。用蛋白质免疫印迹法检测微管相关蛋白2（MAP2）、生长相关蛋白43（GAP-43）、核因子E2相关因子2（Nrf2）、 Kelch样环氧氯丙烷相关蛋白1（Keap1）、血红素氧合酶-1（HO-1）、凋亡相关蛋白Bax和Bcl-2。采用免疫荧光染色观察Nrf2核转位情况。
RAA显著增加了小鼠在W区的停留时间，并提高了TMT处理小鼠的正确交替率。RAA保持了神经细胞的完整性和有序排列。在RAA-S中检测到共12种成分。蛋白激酶B1（AKT1）、过氧化物酶体增殖物激活受体γ（PPARG）、半胱天冬酶3（CASP3）、信号转导和转录激活因子3（STAT3）、热休克蛋白90α家族成员1（HSP90AA1）和核因子E2相关因子2（Nrf2）参与了RAA-S的靶标通路网络。分子对接显示，Nrf2与RAA-S中的所有成分均具有最高的平均结合能。在体内，RAA提高了TMT降低的MAP2、GAP-43、Nrf2和HO-1水平，以及GPx、GSH和SOD活性。此外，RAA降低了TMT升高的血清MDA和ROS水平。在体外，RAA-S减少了TMT诱导的HT22细胞凋亡和ROS积累。此外，RAA-S促进了L-Glu损伤的HT22细胞中N-Nrf2和HO-1的表达。
RAA减轻了AD模型小鼠中TMT和L-Glu诱导的氧化应激。其潜在机制与Nrf2/Keap1-HO-1通路的激活有关。
