Kai-Xin-San alleviates Alzheimer's disease by targeting the DHFR-mediated folate-mitochondrial axis

Kai-Xin-San (KXS) is a classical herbal formula first recorded in the Tang Dynasty and has been used for more than 1000 years for cognitive impairment and dementia.

To investigate whether KXS granules (KXSG) alleviate mitochondrial dysfunction in AD by engaging dihydrofolate reductase (DHFR) -linked folate metabolism.

The pharmacodynamic effects of KXSG were evaluated in APP/PS1 transgenic mice using behavioral testing, neuropathological assessment, and ultrastructural examination of mitochondria. Pathways and candidate targets were first prioritized by brain-tissue DIA proteomics, and were further supported by a network pharmacology analysis based on putative brain-penetrant constituents. Mechanistic validation was performed both in vivo using APP/PS1 transgenic mice and in vitro using an APP-overexpressing HT22 cell model. Mitochondrial function, folate-cycle-related indices, and target protein expression were assessed in both systems, and pharmacological inhibition of DHFR with methotrexate was employed to probe causality.

KXSG treatment improved learning and memory performance, preserved hippocampal neuronal integrity, and reduced Aβ burden in APP/PS1 mice. Proteomic profiling showed that proteins reversed by KXSG were enriched for mitochondrial localization and were closely linked to folate metabolism. DHFR emerged as a key candidate within this network. In cellular assays, KXSG mitigated AD-related mitochondrial impairment while partially normalizing folate-cycle-associated markers and DHFR expression. Notably, methotrexate, a DHFR inhibitor, attenuated the mitochondrial benefits conferred by KXSG.

These data support DHFR-associated folate metabolism as an important mechanistic axis through which KXSG promotes mitochondrial function in AD, providing experimental evidence for a folate-mitochondria link underlying its neuroprotective effects.