Systems Pharmacology Identifies Ajugol-mediated NF-κB/Caspase-3 Inhibition and Isoacteoside-Driven p62/mTOR-mediated Autophagy as Key Mechanisms of Rehmanniae Radix and Its Processed Form in Alzheimer’s Treatment

Xiang Han¹Xianglong Meng¹Yuhui Wu¹Xia Wei²Simin Xue²Xiaoqin Liu¹Chenzi Lyu³Ziang Li¹Xiaoning Yan¹Hyo Won Jung³Shuosheng Zhang^1*

• ¹Shanxi University of Chinese Medicine, Taiyuan, China
• ²Nanjing University of Chinese Medicine, Nanjing, China
• ³Dongguk University, Jung-gu, Republic of Korea

Background: 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 Rehmannia glutinosa (Gaertn.) DC. (Rehmanniae Radix; RR) and its product Rehmanniae Radix Praeparata (RRP) possesses 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. Methods: 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 differences in the medicinal components of RR and RRP as well as in their mechanisms of action in the treatment of AD. The mechanisms of action of two identified critical components, ajugol and isoacteoside, were verified in the D-galactose/AlCl₃-induced 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. Results: Ajugol and isoacteoside were identified as 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, in vivo and in vitro 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 via 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. Conclusion: In this work, we systematically elucidated the differential mechanisms underlying the anti-AD effects of ajugol and isoacteoside. We found that ajugol primarily acts via the TLR/NF-κB/NLRP3 and BCL-2/BAX/cytochrome C/caspase-3 pathways, while isoacteoside acts via the LC3-II/P62/p-mTOR/mTOR pathway. These findings establish a foundation for developing RRP-based complementary medicines and functional foods.