Han Xiang, Meng Xianglong, Wu Yuhui, Xia Wei, Xue Simin, Liu Xiaoqin, Lyu Chenzi, Li Ziang, Yan Xiaoning, Won Jung Hyo, Zhang Shuosheng
College of Chinese Materia Medica and Food Engineering, Shanxi University of Chinese Medicine, Jinzhong, Shanxi, China.
Shanxi Key Laboratory of Traditional Herbal Medicines Processing, Shanxi University of Chinese Medicine, Jinzhong, Shanxi, China.
Front Pharmacol. 2025 Aug 29;16:1644847. doi: 10.3389/fphar.2025.1644847. eCollection 2025.
Alzheimer's disease (AD) is a progressive neurodegenerative disorder characterized by the deposition of senile plaques, neurofibrillary tangles, and neuronal dysfunction, resulting in severe cognitive and memory decline. The root of the Scrophulariaceae plant (Gaertn.) DC. (Rehmanniae radix; RR) and its product Rehmanniae radix praeparata (RRP) possess high nutritional and medicinal value. Both show therapeutic potential for AD in traditional medical settings. However, the differences in their bioactive components and the mechanisms of action underlying their anti-AD effects remain unclear.
In this study, APP/PS1 mice were used as the animal model of AD. Ultra-high-performance liquid chromatography coupled with Q-Exactive tandem mass spectrometry (MS/MS) (UPLC-QE-MS/MS), network pharmacology, proteomics, molecular docking, and 16S rRNA sequencing were used to investigate the differences in the medicinal components of RR and RRP and their mechanisms of action in the treatment of AD. The mechanisms of action of two identified critical components, ajugol and isoacteoside, were further verified in the D-galactose/AlCl-induced Institute of Cancer Research (ICR) mouse model of AD-with cognitive function evaluated using the Morris water maze and open-field tests-and the amyloid-beta (Aβ)-induced BV2 cell model of inflammation.
Ajugol and isoacteoside were identified as the key anti-AD bioactive compounds in RR and RRP, respectively, through UPLC-QE-MS/MS. Integrated network pharmacology, proteomics, and 16S rRNA sequencing implicated neuroinflammation, apoptosis, and autophagy as critical pathways for their anti-AD effects. Subsequently, and experiments demonstrated that ajugol exerted its effects mainly by modulating the TLR/NF-κB/NLRP3 and BCL-2/BAX/cytochrome C/caspase-3 pathways, while isoacteoside primarily acted the LC3-Ⅱ/P62/p-mTOR/mTOR pathway. Ajugol and isoacteoside mitigated cognitive impairment in AD models, decreased Aβ plaque accumulation in hippocampal tissues, and attenuated inflammatory injury-induced cytotoxicity in BV2 microglia, thereby suppressing AD progression.
In this work, we systematically elucidated the differential mechanisms underlying the anti-AD effects of ajugol and isoacteoside. We found that ajugol primarily acts the TLR/NF-κB/NLRP3 and BCL-2/BAX/cytochrome C/caspase-3 pathways, while isoacteoside acts the LC3-II/P62/p-mTOR/mTOR pathway. These findings establish a foundation for developing RRP-based complementary medicines and functional foods.
阿尔茨海默病（AD）是一种进行性神经退行性疾病，其特征为老年斑沉积、神经原纤维缠结和神经元功能障碍，导致严重的认知和记忆衰退。玄参科植物地黄（Gaertn.）DC.的根（地黄；RR）及其制品熟地黄（RRP）具有很高的营养和药用价值。在传统医学中，二者均显示出对AD的治疗潜力。然而，它们生物活性成分的差异以及抗AD作用的潜在机制仍不清楚。
本研究中，将APP/PS1小鼠用作AD动物模型。采用超高效液相色谱联用Q-Exactive串联质谱（MS/MS）（UPLC-QE-MS/MS）、网络药理学、蛋白质组学、分子对接和16S rRNA测序，研究RR和RRP药用成分的差异及其治疗AD的作用机制。在D-半乳糖/氯化铝诱导的AD ICR小鼠模型（通过莫里斯水迷宫和旷场试验评估认知功能）和淀粉样β（Aβ）诱导的BV2细胞炎症模型中，进一步验证了两种已鉴定的关键成分——筋骨草醇和异麦角甾苷的作用机制。
通过UPLC-QE-MS/MS鉴定出筋骨草醇和异麦角甾苷分别是RR和RRP中关键的抗AD生物活性化合物。综合网络药理学、蛋白质组学和16S rRNA测序表明，神经炎症、细胞凋亡和自噬是它们抗AD作用的关键途径。随后的实验证明，筋骨草醇主要通过调节TLR/NF-κB/NLRP3和BCL-2/BAX/细胞色素C/半胱天冬酶-3途径发挥作用，而异麦角甾苷主要作用于LC3-Ⅱ/P62/p-mTOR/mTOR途径。筋骨草醇和异麦角甾苷减轻了AD模型中的认知障碍，减少了海马组织中Aβ斑块的积累，并减轻了BV2小胶质细胞中炎症损伤诱导的细胞毒性，从而抑制了AD的进展。
在本研究中，我们系统地阐明了筋骨草醇和异麦角甾苷抗AD作用的差异机制。我们发现，筋骨草醇主要作用于TLR/NF-κB/NLRP3和BCL-2/BAX/细胞色素C/半胱天冬酶-3途径，而异麦角甾苷作用于LC3-II/P62/p-mTOR/mTOR途径。这些发现为开发基于RRP的补充药物和功能性食品奠定了基础。
