Supporting data for “Effects of Angong Niuhuang Pill on ischemic stroke via the regulation of gut microbiota targeting TMA/TMAO metabolic pathway”

BackgroundIschemic stroke is a cerebrovascular event with underlying pathology in which gut microbiota and their metabolites participate via the gut-brain axis. The traditional Chinese medicine Angong Niuhuang Pill (AGNHP) is widely used in treatment of stroke, but its mechanisms via gut microbiota modulation remain obscure. ResultsIn this study, we established a middle cerebral artery occlusion (MCAO) mouse model and administered AGNHP at different timepoints after stroke. Multi-omics approaches including 16S rRNA sequencing and LC-MS/MS-based targeted metabolomics were conducted to analyze gut microbiota composition and TMA/TMAO metabolic pathways dynamically. The results showed that AGNHP treatment significantly reduced infarct volume, improved neurological function, relieve inflammatory conditions and restored gut and blood-brain barrier (BBB) integrity. Mechanistically, AGNHP altered gut microbiota composition. Through integrating abundance profiling, Lefse analysis, and correlation analysis related to metabolites, further study revealed AGNHP exclusively enriched the probiotic Akkermansia muciniphila, which suppressed TMAproducing-related genes (CntA/B, CutC/D, YeaW/X), thereby reducing systemic and cerebral TMA/TMAO accumulation. Complementary in vitro studies demonstrated that TMA may directly disrupted tight junction proteins in BBB and intestinal barrier. ConclusionsThis study elucidates the neuroprotective mechanism of AGNHP on ischemic stroke through modulation of the A. muciniphila-TMA/TMAO. By establishing a novel “Complex Drugs-Core Strains-Key Metabolites” paradigm, our study elucidates how AGNHP protects against ischemic stroke through time-dependent regulation of the gut microbiota-TMA/TMAO axis, with A. muciniphila playing a pivotal role. These findings not only advance our understanding of stroke pathophysiology, but also provide a methodological framework for investigating complex traditional medicines in modern biomedical research.背景：缺血性脑卒中是一类存在潜在病理机制的脑血管事件，肠道菌群及其代谢产物可通过肠-脑轴参与该病的发生发展。中药安宫牛黄丸（Angong Niuhuang Pill, AGNHP）已被广泛应用于脑卒中的临床治疗，但其通过调控肠道菌群发挥治疗作用的具体机制仍未明确。 结果：本研究构建了大脑中动脉闭塞（middle cerebral artery occlusion, MCAO）小鼠模型，并在脑卒中发生后的不同时间点给予安宫牛黄丸干预。采用16S rRNA测序、基于液相色谱-串联质谱（LC-MS/MS）的靶向代谢组学等多组学技术手段，动态分析了肠道菌群组成以及三甲胺（trimethylamine, TMA）/三甲胺N-氧化物（trimethylamine N-oxide, TMAO）代谢通路的变化。结果显示，安宫牛黄丸干预可显著缩小脑梗死体积、改善神经功能缺损、缓解机体炎症状态，并恢复肠道屏障与血脑屏障（blood-brain barrier, BBB）的完整性。 机制分析：安宫牛黄丸可重塑肠道菌群组成。通过整合菌群丰度谱、线性判别分析效应大小（linear discriminant analysis effect size, Lefse）以及代谢物相关性分析，本研究进一步发现，安宫牛黄丸可特异性富集有益菌嗜黏蛋白阿克曼菌（Akkermansia muciniphila），该菌可抑制产三甲胺相关基因（CntA/B、CutC/D、YeaW/X）的表达，从而降低全身及脑组织内三甲胺/三甲胺N-氧化物的蓄积水平。补充体外实验结果表明，三甲胺可直接破坏血脑屏障与肠道屏障的紧密连接蛋白。 结论：本研究阐明了安宫牛黄丸通过调控嗜黏蛋白阿克曼菌-三甲胺/三甲胺N-氧化物轴发挥缺血性脑卒中神经保护作用的具体机制。本研究构建了全新的"复杂药物-核心菌株-关键代谢物"研究范式，明确了安宫牛黄丸通过时间依赖性调控肠道菌群-三甲胺/三甲胺N-氧化物轴发挥抗脑卒中作用的机制，其中嗜黏蛋白阿克曼菌发挥了关键核心作用。本研究结果不仅加深了我们对脑卒中病理生理机制的认知，同时也为现代生物医学研究中探究复杂中药的作用机制提供了一套标准化的方法学框架。