Alzheimer's Disease (AD) is a common neurodegenerative disease and has been listed as one of the top five diseases of the 21st century by WHO (World Health Organization) (Hampel et al., 2011). Global pharmaceutical giants have made a great effort to develop the anti-Alzheimer's drugs, but the success rate is very low. Currently, there are only a few anti-AD drugs on the market, including tacrine, donepezil, galantamine, rivastigmine and memantine, etc. The first four are acetylcholinesterase inhibitors, and the last one is a non-competitive NMDA receptor antagonist. In clinical application, it is found that he above drugs have various toxic side effects, and cannot fundamentally change the development of the disease (Keating et al., 2012). Traditional Chinese medicine (TCM), however, has a unique advantage in the treatment of AD because of its complex composition, often acting on multiple targets and multiple pathways, and small side effects.

Kai-Xin-San (KXS) is a classic herbal formula for the treatment of psychiatric disorders such as AD and depression (Cao et al., 2020; Wang et al., 2017a, Wang et al., 2017b; Cheng et al., 2023). KXS was first recorded in Sun Simiao's "Emergency Qianjin Prescription" in Tang Dynasty, and composed of Ginseng radix et rhizoma (short for GR), Polygalae radix (PR), Poria (PO) and Acori tatarinowii rizhoma (AT). Depending on the function and indications, KXS has several compatibility ratios in clinical practice. When used for the treatment of AD, the most common mass ratio is 3:2:3:2 (GR:PR:PO:AT). The results of clinical application analysis of 110 herbs for treating AD showed that the usage frequency of PR, AT, GR and PO ranked 1st, 2nd, 3rd, and 9th, respectively, which was significantly higher than other herbs (May et al., 2013). Modern pharmacological research has shown that KXS can enhance the behavioral memory ability of mice, increase cerebral blood flow, improve brain function, improve hemorheology index, and affect the nervous system (Bi et al., 2019; Shi et al., 2018; Xu et al., 2019). With the deepening of pharmacological research and the continuous renewal of dosage forms, KXS has been widely used in the treatment of senile dementia, neurasthenia, depression, neurosis, children's intellectual disability syndrome, children's epilepsy, and so on (Cao et al., 2012, 2020; Wang et al., 2017a, Wang et al., 2017b). Researchers have used traditional chemistry and pharmacology, serum pharmacochemistry (Wang et al., 2017a, Wang et al., 2017b; Yang et al., 2023), network pharmacology (Bi et al., 2019; Shi et al., 2018), proteomics, prescription metabolomics (Chu et al., 2016; Guo et al., 2020) and other methods to explore the anti-AD effect of KXS, but its material basis and action mechanism still need further experimental verification.

More than 300 years BC, Hippocrates, the father of Western medicine, said, "All diseases come from the gut". Today, when it comes to the pathogenesis of many chronic diseases (such as chronic enteritis, diabetes, Alzheimer's disease, aging, obesity, drug effects, etc.), we are gradually realizing that gut microbes are almost a topic that cannot be sidestep (Goralczyk-Binkowska et al., 2022). Recent studies have shown that gut microbiota communicate in a complex bidirectional manner between gut microbes, the gut, and the central nervous system (CNS) through the microbiota-gut-brain (MGB) axis (Li et al., 2020), and that gut microbiota are not only involved in various physiological processes in the host, but also strongly involved in the pathogenesis of neuropathology (Keogh et al., 2021). The dysbiosis of microbiota ecology may exacerbate neuroinflammation and Aβ aggregation (Liu et al., 2020; Zhuang et al., 2020) Intestinal microbiota can modulate the composition and structure of the microbiota through the changes in community dynamics that alter and influence brain function and behavior, cognitive dysfunction, and neurological disorders such as AD (Liu et al., 2020), cognitive impairment (Saji et al., 2019), and autism (Kotsiliti, 2022). Gut dysfunction and aberrant microbial content may contribute to the pathogenesis of Alzheimer's disease (AD) and potentially other neurodegenerative diseases. The gut microbiome may be an indicator of preclinical Alzheimer's disease and may improve machine learning prediction of AD status (Ferreiro et al., 2023).

Saponins are the most important active ingredients in KXS (Yang et al., 2023; Yu et al., 2019; Yuan et al., 2016). Polygala exerts anti-AD effects mainly through the metabolites (Chu et al., 2016). Ginsenosides are metabolized by Bifidobacterium, Bacteroides and Clostridium in the intestine (Wang et al., 2011). Furthermore, according to TCM theory, KXS may be through regulating the intestines and stomach (intestinal microorganisms), replenishing the deficiency of qi and blood (circulatory system), strengthening Yin and Yang (immune system, nervous system) to "nourish the brain", and achieve the purpose of treatment. Very recently, the pharmacodynamic materials of KXS's anti-depression were explored based on hypothalamic-pituitary-adrenal (HPA) axis, and the composition of intestinal microorganisms in the depression model animals were found to be related with the KXS intervention (Cao et al., 2020; Zhu et al., 2017). In summary, gut microbes may play an important role in KXS's anti-AD effect, and KXS exerts its anti-AD effect mainly through the "microbiota-gut-brain axis" mechanism.

There is interaction between TCM and intestinal microbiota: on the one hand, intestinal microbes have metabolic transformation effect on drugs and produce metabolites with higher activity; On the other hand, drugs may restore and maintain intestinal microecological balance by regulating the structure and function of intestinal microbes. The metabolic activation of KXS has been investigated based on the intestinal content incubation, fecal metabolomics and serum pharmacochemistry in our previous work (Yang et al., 2023; Yu et al., 2019; Yuan et al., 2016; Guo et al., 2020). This work aims at investigating the regulation of KXS on the intestinal microbiota and its mechanism for the improvements of symptoms in AD rats. The whole study process is illustrated in Fig. 1. Firstly, the mulriple animal model that approximates the pathological changes in AD was replicated and evaluated. Secondly, the pharmacodynamic effects of KXS on improving cognitive function, protecting neurons, and reducing inflammation levels in vivo were investigated by Morris water maze test, inflammatory factors, and rat hippocampal and colonic tissue morphometry. Thirdly, the effects of KXS on intestinal microbiota were systematically explored based on 16S rRNA gene sequencing technique, and the composition and differences of intestinal microbiota between KXS group and AD rats were analyzed. Finally, the potential role of gut microbiota in the anti-AD effect of KXS was verified based on the microbial targeting technology-Fecal Microbiota Transplantation (FMT).