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

Abstract

ETHNOPHARMACOLOGICAL RELEVANCE: 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. AIM OF THE STUDY: To investigate whether KXS granules (KXSG) alleviate mitochondrial dysfunction in AD by engaging dihydrofolate reductase (DHFR) -linked folate metabolism. MATERIALS AND METHODS: 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. RESULTS: 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. CONCLUSION: 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.