Kai-Xin-San Alleviated Neurodegeneration by Reducing Neuroinflammation and Rebalancing Intestinal Flora in APP/PS1 mice

Background : Traditional therapeutics in Alzheimer's disease (AD) are mostly targeted at a single target, such as amyloid-β, tau protein, or APOEε4. Single-target therapy was good pertinence, but AD has many pathogenic factors, thus multi-targeted therapy has its own advantage in treating AD. In the early stage of AD, neuroinflammation and the dysfunction of the blood-brain barrier (BBB) are important factors inducing the development of AD. Additionally, studying AD through intestinal flora was a hotspot in the last decade. Clinical studies have shown that patients in the early stages of AD exhibit gastrointestinal discomfort and imbalance of gut microbiota, which indicates that the imbalance of gut microbiota has an impact on the early onset of AD. Trimethylamine oxide (TMAO) is believed to be associated with the onset of various cardiovascular and cerebrovascular diseases, it has also been confirmed to be a novel AD risk factor. Management in traditional Chinese medicine (TCM) is multi-targeted and multi-mechanism based. Clinical studies have shown that the medication Kai-xin-san (KXS) is helpful in treating dementia by reducing neuroinflammation and enhancing mental impairment. However, the pharmacological mechanism of KXS still needs to be confirmed. Methods : A total of 60 APP/PS1 mice were arbitrarily assigned to the AD, KXS (low dose, middle dose and high dose), and Donepezil categories. As a reference, 12 wild-type mice were used. For 2 months, mice received intragastric medication delivery or saline. To evaluate the cognitive ability, open field test, new object detection, and Morris water maze were performed. Nissl staining and Glogi-cox staining were performed to analyze the structure of the nerve. NLRP3, ASC, Caspase1, TNF-α and IL-1β were perceived in western blot and qPCR to analyze neuroinflammation. ZO-1, Occludin, and MMP9 were detected by qPCR and western blot to analyze the BBB. Meanwhile, we performed IHC to detected the expression and distribution of neuroinflammation and BBB with NLRP3, ZO-1 and MMP9. Fecal were collected and 16S rRNA sequencing was performed to analyze the intestinal flora. Blood samples were collected for determination of TMAO level by LC-MS/MS. Results : KXS improved cognitive degeneration; regulated intestinal flora by abridged ASC, NLRP3, TNF-α, Caspase-1, MMP9 and IL-1β expression, increased ZO-1 and Occludin; improved the structure of neuro cells, regulated Bacteroidetes and Firmicutes in phylum level, reduced the level and production of TMAO. Conclusion : Our study found that KXS could improve cognitive degeneration through reducing neuroinflammation and recovering the BBB function, rebalancing intestinal flora and reducing the expression of TMAO. We provided more powerful experimental proof for the multi-targeted efficacy of KXS in treating AD.