Data Sheet 1_Therapeutic effects of traditional Chinese medicine Hua-Feng-Dan in a rat model of ischemic stroke involve renormalization of gut microbiota.docx

Hua-Feng-Dan is a traditional Chinese medicine used to treat ischemic stroke, but little is known about its therapeutic mechanism. This study explored whether and how the mechanism involves readjustment of gut microbiota. Rats were subjected to middle cerebral artery occlusion as a model of ischemic stroke or to sham surgery, then treated or not with Hua-Feng-Dan. The different groups of animals were compared in terms of neurological score, cerebral infarct volume, brain edema, brain and gut histopathology to assess stroke severity. They were also compared in terms of indices of intestinal barrier permeability, inflammation and oxidative stress, brain metabolites as well as composition of the gut microbiota and their metabolites. Hua-Feng-Dan significantly reduced cerebral infarct volume and brain water content and improved neurological score, ischemic brain histopathology, and gut histopathology. It partially reversed stroke-induced intestinal barrier disruption and leakage, inflammation, dyslipidemia and oxidative stress, as well as the stroke-induced increase in pathogenic gut microbiota (e.g., Escherichia-Shigella, Enterococcus, Clostridium_innocuum_group) and decrease in beneficial microbiota (e.g., Lachnospiraceae, unclassified__f__Lachnospiracea and Ruminococcus_torques_group). The treatment altered levels of 39 and 38 metabolites produced during gut microbial and brain tissue metabolism respectively, mainly of amino acids, nucleosides, short-chain fatty acids, and essential fatty acids. Levels of factors related to inflammation and intestinal barrier permeability correlated positively with relative abundance of Escherichia-Shigella and Clostridium_innocuum_group, and negatively with 4-(glutamylamino) butanoate, 2-hydroxy-3-methylbutyric acid, dihomo-α-linolenic acid, dihomolinoleic acid, and 10-nitrolinoleic acid. Conversely, levels of 4-(glutamylamino) butanoate, 2-hydroxy-3-methylbutyric acid, and 10-nitrolinoleic acid correlated positively with relative abundance of unclassified__f__Lachnospiracea. Our results suggest that Hua-Feng-Dan may mitigate ischemic stroke injury by renormalizing gut microbiota and restoring gut barrier function, gut metabolism, thereby helping to alleviate inflammatory, neurological damage, and brain metabolic disorders.

华风丹（Hua-Feng-Dan）是一款用于治疗缺血性脑卒中的传统中药，但其具体治疗机制尚未阐明。本研究探讨了华风丹的治疗机制是否涉及肠道菌群的重塑，以及其具体作用路径。实验采用大鼠大脑中动脉闭塞（middle cerebral artery occlusion）模型模拟缺血性脑卒中，同时设置假手术对照组；造模完成后，将大鼠分为给药组与非给药组，分别给予华风丹或安慰剂处理。随后通过神经功能评分、脑梗死体积、脑水肿程度、脑与肠道组织病理学检查评估各组大鼠的脑卒中严重程度；同时检测肠道屏障通透性相关指标、炎症与氧化应激水平、脑代谢物水平，以及肠道菌群组成及其代谢产物。结果显示，华风丹可显著降低脑梗死体积与脑含水量，改善神经功能评分、缺血性脑组织病理学及肠道组织病理学表现。其可部分逆转脑卒中诱导的肠道屏障破坏与渗漏、炎症反应、血脂异常与氧化应激，同时扭转脑卒中引发的致病肠道菌群（如埃希氏菌-志贺氏菌属（Escherichia-Shigella）、肠球菌属（Enterococcus）、无害梭菌群（Clostridium_innocuum_group））丰度升高，以及有益菌群（如毛螺菌科（Lachnospiraceae）、未分类毛螺菌科（unclassified__f__Lachnospiracea）、Torques瘤胃球菌群（Ruminococcus_torques_group））丰度降低的异常变化。该干预分别改变了肠道菌群代谢与脑组织代谢产生的39种和38种代谢物，主要涉及氨基酸、核苷、短链脂肪酸与必需脂肪酸。炎症与肠道屏障通透性相关因子的水平与埃希氏菌-志贺氏菌属、无害梭菌群的相对丰度呈正相关，而与4-（谷氨酰胺基）丁酸（4-(glutamylamino) butanoate）、2-羟基-3-甲基丁酸（2-hydroxy-3-methylbutyric acid）、双高-α-亚麻酸（dihomo-α-linolenic acid）、双高亚油酸（dihomolinoleic acid）及10-硝基亚油酸（10-nitrolinoleic acid）的水平呈负相关。反之，4-（谷氨酰胺基）丁酸、2-羟基-3-甲基丁酸与10-硝基亚油酸的水平与未分类毛螺菌科的相对丰度呈正相关。本研究结果表明，华风丹或可通过重塑肠道菌群稳态、恢复肠道屏障功能与肠道代谢，进而减轻炎症反应、神经损伤与脑代谢紊乱，从而缓解缺血性脑卒中损伤。