Supplementary file 1_Wuji Pill and Akkermansia muciniphila alleviates intestinal dysfunction and depression-like behavior in irritable bowel syndrome through the microbiota-gut-brain axis.docx

IntroductionIrritable bowel syndrome (IBS) is a typical disorder of gut-brain interaction (DGBI). The microbiota-gut-brain (MGB) axis is pivotal in preventing and treating IBS. Wuji Pill is a traditional Chinese medicine commonly used to treat IBS. This study aimed to investigate the mechanism by which Wuji Pill improves IBS via the MGB axis.
MethodsThe visceral sensitivity and colonic motor function were evaluated using the abdominal wall withdrawal reflex test and the colonic motility curve. Depression-like behavior were evaluated using sucrose preference test, open field test, novelty-suppressed feeding test, and forced swimming tests. The intestinal mucus secretion and the activation status of microglia was detected using AB-PAS staining and immunofluorescence staining, respectively. The species composition and abundance of gut microbiota were detected through 16S rRNA sequencing and RT-qPCR. Targeted metabonomics and RT-qPCR were used for metabolites and metabolic enzymes analysis.
ResultsIn this study, Wuji Pill improved the symptoms of IBS rats and increased the relative abundance of Akkermansia muciniphila in feces. Additionally, antibiotics affected the repair of intestinal mucus secretion and significantly reduced the level of short-chain fatty acids. Subsequently, fecal microbiota transplantation and A. muciniphila transplantation can improve the symptoms of IBS rat by increasing intestinal mucus secretion, elevating the levels of acetic acid and butyric acid in feces. Additionally, the microglia in the cortex were suppressed, and the tryptophan-kynurenine pathway in the hippocampus was inhibited, leading to the conversion of tryptophan into 5-HT.
DiscussionThis study highlights the Wuji Pill may alleviate IBS symptoms by modulating A. muciniphila and regulating the tryptophan metabolism pathway through MGB axis.

引言
肠易激综合征（Irritable bowel syndrome, IBS）是典型的脑肠互动紊乱（disorder of gut-brain interaction, DGBI）。微生物群-肠-脑（microbiota-gut-brain, MGB）轴在肠易激综合征的防治中发挥关键作用。无极丸是临床常用于治疗肠易激综合征的中药。本研究旨在探究无极丸通过微生物群-肠-脑轴改善肠易激综合征的作用机制。

方法
采用腹壁撤回反射试验与结肠动力曲线评估内脏敏感性与结肠运动功能。通过蔗糖偏好试验、旷场试验、新奇抑制摄食试验及强迫游泳试验评估大鼠的抑郁样行为。分别运用AB-PAS染色与免疫荧光染色检测肠道黏液分泌情况及小胶质细胞的活化状态。通过16S rRNA测序与实时定量聚合酶链反应（RT-qPCR）检测肠道菌群的物种组成与丰度。采用靶向代谢组学与RT-qPCR开展代谢物及代谢酶相关分析。

结果
本研究中，无极丸可改善肠易激综合征模型大鼠的症状，并提升粪便中嗜黏蛋白阿克曼菌（Akkermansia muciniphila）的相对丰度。此外，抗生素可干扰肠道黏液分泌的修复过程，并显著降低短链脂肪酸水平。后续粪便菌群移植与嗜黏蛋白阿克曼菌移植可通过提升肠道黏液分泌、升高粪便中乙酸与丁酸水平，改善肠易激综合征模型大鼠的症状。同时，皮层内小胶质细胞的活化受到抑制，海马体内的色氨酸-犬尿氨酸通路被阻滞，促使色氨酸转化为5-羟色胺（5-HT）。

讨论
本研究表明，无极丸可能通过调控嗜黏蛋白阿克曼菌，并经微生物群-肠-脑轴调节色氨酸代谢通路，从而缓解肠易激综合征的症状。