Table5_The Pharmacological Mechanism of Guchangzhixie Capsule Against Experimental Colitis.DOCX

Ulcerative colitis (UC) is the major type of inflammatory bowel disease (IBD) characterized by an overactive immune response and destruction of colorectal epithelium with intricate pathological factors. Guchangzhixie (GCZX) capsule, included in the Chinese Pharmacopoeia 2020, has been widely utilized against UC. However, the underlying molecular mechanisms have not been elucidated. In the present study, a murine model of experimental colitis was established by orally feeding 4% dextran sodium sulfate (DSS) for 5 days and subsequently subjecting to GCZX treatment for another 15 days. Network pharmacology analysis was performed to predict the pertinent mechanisms of GCZX capsule. Cellular experiments examining the functional changes of intestinal organoids (IOs), macrophages (Mφs), and human colon epithelial cell cells (NCM460 cell line) after GCZX therapy were performed. Sequencing of 16S rRNA was conducted on the stools from the mouse model. Liquid chromatography-mass spectrometry (LC–MS) was utilized to detect serum metabolites. As a result, DSS induced experimental colitis, and this induction was alleviated by GCZX treatment, as evidenced by rescued pathological symptoms in UC mouse models, such as rectal bleeding stopping, decreased levels of albumin, interleukin-17, as well as chemokine (C-X-C motif) ligand 1 (CXCL1), and reduction in colon length. Network pharmacology analysis showed that GCZX-target genes were enriched in pathogen-induced infections, inflammatory pathways, as well as neoplastic processes. DSS treatment decreased microbial diversity and led to the accumulation of pathological bacterial, which was reversed by GCZX capsule. PICRUSt2 (Phylogenetic Investigation of Communities by Reconstruction of Unobserved States) based on profiles of microbiota composition demonstrated a decreased incidence of infectious disease and cancers after GCZX therapy. In full accordance with these data, GCZX administration suppressed Mφ transition to pro-inflammatory phenotype, alleviated tumor necrosis factor-α (TNFα)-compromised IOs functions, and decreased the recruitment of Mφs by epithelial cells. We conclude that GCZX capsule is an effective drug for UC and its pharmacological mechanisms involve re-establishing an anti-inflammatory milieu and favoring mucosal healing.

溃疡性结肠炎（Ulcerative colitis, UC）是炎症性肠病（inflammatory bowel disease, IBD）的主要类型，以免疫应答过度激活及结肠上皮破坏为特征，病理机制复杂。收载于2020年版《中华人民共和国药典》的肠止泻泄（Guchangzhixie, GCZX）胶囊，被广泛用于治疗UC，但其潜在分子机制尚未阐明。本研究通过给小鼠灌胃4%葡聚糖硫酸钠（dextran sodium sulfate, DSS）造模5天，随后给予GCZX干预15天，构建实验性结肠炎小鼠模型。采用网络药理学分析预测GCZX胶囊的相关作用机制，同时开展细胞实验，检测GCZX干预后肠道类器官（intestinal organoids, IOs）、巨噬细胞（macrophages, Mφs）及人结肠上皮细胞系NCM460的功能变化。对小鼠粪便进行16S核糖体RNA（16S rRNA）测序，并通过液相色谱-质谱联用（liquid chromatography-mass spectrometry, LC–MS）检测血清代谢物。结果显示，DSS诱导的实验性结肠炎可被GCZX治疗缓解：UC小鼠模型的病理症状得到改善，包括便血停止、白蛋白水平异常降低的情况得到纠正、白细胞介素-17（interleukin-17）及趋化因子（C-X-C基序）配体1（CXCL1）的异常高表达得到抑制，以及结肠长度缩短的情况得到逆转。网络药理学分析表明，GCZX的靶基因富集于病原体诱导感染、炎症通路及肿瘤发生相关过程。DSS处理会降低微生物多样性并促进病理细菌富集，而GCZX干预可逆转这一现象。基于菌群组成谱的PICRUSt2（通过重建未观察状态对群落进行系统发育研究，Phylogenetic Investigation of Communities by Reconstruction of Unobserved States）分析显示，GCZX治疗后感染性疾病及癌症的发生风险降低。与上述实验结果一致，GCZX给药可抑制巨噬细胞向促炎表型转化，缓解肿瘤坏死因子-α（tumor necrosis factor-α, TNFα）对肠道类器官功能的损伤，并减少上皮细胞对巨噬细胞的招募。综上，GCZX胶囊是治疗UC的有效药物，其药理机制涉及重建抗炎微环境并促进黏膜愈合。