Li_2025_J.Ethnopharmacol_343_119484

ETHNOPHARMACOLOGICAL RELEVANCE: Kai-Xin-San (KXS) has a significant effect therapeutic on Alzheimer's disease (AD) in clinical practice. According to the compatibility theory of traditional Chinese medicine, Acori Tatarinowii Rhizoma (ATR) serves as the guiding drug in the KXS formulation and is believed to enhance the bioavailability and brain tissue distribution of the other drugs. However, the mechanism underlying the "guiding medicine upwards" effect of ATR in KXS remains unexplored. AIM OF THE STUDY: The aim of this study is to investigate the role of ATR in the efficacy of KXS on amyloid precursor protein/presenilin 1 (APP/PS1) mice, as well as its impact on the brain tissue distribution of other active ingredients in the KXS formula, and to elucidate the mechanism of ATR's "guiding medicine upwards" effect in KXS. MATERIALS AND METHODS: The pharmacodynamic effects of ATR in KXS were assessed through behavioral tests, immunohistochemical staining, and Nissl staining. Additionally, the levels of inflammatory factors, as well as the activities of malondialdehyde, superoxide dismutase, and acetylcholinesterase, were measured using enzyme-linked immunosorbent assay kits. Subsequently, the effect of ATR on the ultrastructure of the blood-brain barrier (BBB) in APP/PS1 mice was observed using transmission electron microscopy (TEM), and the pharmacodynamic components of KXS in cerebrospinal fluid were quantified by ultra-high-performance liquid chromatography-triple quadrupole mass spectrometry (UPLC-MS/MS). Furthermore, Western blot (WB) analysis was used to quantitatively assess the expression of tight junction proteins (Claudin-5, Occludin, and ZO-1) and transporters (OCT3, OATP2, P-gp, and MRP1) in the BBB. Finally, bEND.3 cells and astrocyte cells were co-cultured to validate the effect of ATR on KXS. The expressions of OCT3/OATP2 and P-gp/MRP1 in BBB cell model were determined by WB and the content of pharmacodynamic components in the lower chamber of the transwell were also analyzed by UPLC-MS/MS. RESULTS: Behavioral test results suggest that KXS significantly improved the learning and memory capacities of APP/PS1 mice compared to the ATR-free KXS group. Furthermore, KXS was more effective in reducing amyloid-beta protein deposition in the brain and repairing damaged neurons in the CA1 and CA3 regions than ATR-free KXS. Notably, KXS significantly reversed the pathological biochemical indices compared to the ATR-free KXS group. These results indicate that ATR has a positive effect on the pharmacodynamics of KXS in treating AD. Most importantly, TEM results revealed that KXS repaired the damaged BBB in AD mice, and ATR contributed to the improvement of BBB integrity. Furthermore, KXS and ATR increased the expression levels of Claudin-5, Occludin, and ZO-1 proteins in AD mice. Meanwhile, the levels of ginsenoside Rg1, ginsenoside Rb1, and polygalaxanthone III in the cerebrospinal fluid of the KXS group were 1.47, 1.39, and 2.02 times higher than those in the ATR-free KXS group, respectively. WB results showed that ATR and KXS significantly upregulated the expression of OCT3/OATP2 uptake transporters and downregulated the expression of P-gp/MRP1 efflux transporters compared to ATR-free KXS. Concurrently, in vitro BBB cell experimental results suggest that ATR promoted the transport of ginsenoside Rg1, ginsenoside Rb1, and polygalaxanthone III across BBB cells in KXS, and the regulation of OCT3/OATP2 and P-gp/MRP1 expression was consistent with the in vivo trends observed in AD mice. CONCLUSIONS: ATR plays a critical role in enhancing the efficacy of KXS in treating AD and facilitates the entry of other pharmacodynamic components into the brain. The mechanism underlying the "guiding medicine upwards" effect of ATR may involve the regulation of OCT3/OATP2 and P-gp/MRP1 transporters.