中药解毒消症饮（JXY）治疗胃肠道肿瘤，特别是结直肠癌（CRC）的临床应用已十分成熟，但其治疗结直肠癌的确切生物学机制仍不清楚。本研究致力于揭示JXY调节结直肠癌干细胞的复杂机制，从而阐明其发挥强效抗肿瘤作用的途径。本研究通过网络药理学分析JXY对CRC细胞信号通路和功能的调节影响。使用 HPLC 检测 JXY 的乙酸乙酯提取物的主要化合物，然后处理 HCT-116 细胞进行 RNA 测序 (RNA-Seq)。使用蛋白质印迹分析和基质胶球体测定评估 JXY 提取物处理后 HCT-15 和 HCT-116 细胞的蛋白质表达和干性。此外，使用 TOPflash 报告基因检测在有或没有氯化锂 (LiCl) 刺激的情况下评估 β-连环蛋白转录活性。使用干性维持培养基培养患者来源的 CRC 类器官 (CRC PDO)，并在 JXY 提取物处理后使用 ATP 测定法测量其活力。此外，使用源自HCT-15细胞的异种移植小鼠模型评估了JXY提取物的抗肿瘤功效。网络药理学强调了JXY对癌症干细胞和Wnt信号通路的影响。 HPLC 分析证实，JXY 提取物含有中国药典记载的四种草药中最常见的三种药物化合物（迷迭香酸、槲皮素和山奈酚）。 RNA-Seq 结果进一步阐明了 JXY 提取物的作用，特别是针对癌症干细胞和 Wnt 信号通路的作用。此外，JXY 提取物抑制 CRC 细胞中的球状体形成，并下调 CRC CSC 标记物（CD133、DCLK1 和 C-MYC）。此外，JXY 提取物还抑制 β-catenin 表达和转录活性以及 Wnt 通路靶蛋白，包括 C-MYC 和 Cyclin D1。与细胞系的研究结果一致，JXY 提取物抑制了表现出干性特征的 CRC PDO 的生长。 JXY 提取物对异种移植肿瘤中的肿瘤生长、C-MYC 和 β-catenin 蛋白水平具有显着的抑制作用。这些结果突出了 JXY 提取物通过调节 Wnt 信号通路靶向 CRC CSCs 的新功能，强调了其作为治疗药物的潜力。用于治疗 CRC 的治疗剂。版权所有 © 2024。由 Elsevier B.V. 出版。

The clinical application of the traditional Chinese medicinal formula Jiedu Xiaozheng Yin (JXY) for gastrointestinal tumors, particularly colorectal cancer (CRC), is well-established, yet the precise biological mechanism underlying its efficacy in CRC treatment remains elusive.This study endeavors to unravel the intricate mechanism through which JXY modulates colorectal cancer stem cells, thus elucidating the pathways by which it exerts its potent anti-tumor effects.In this study, the regulatory impact of JXY on the signaling pathway and function of CRC cells was analyzed through Network pharmacology. The ethyl acetate extract of JXY was detected the major compounds using HPLC and then treated the HCT-116 cells for RNA-Sequencing (RNA-Seq). Protein expression and stemness of HCT-15 and HCT-116 cells following JXY extract treatment were assessed using western blot analysis and matrigel spheroid assays. Additionally, the β-catenin transcriptional activity was evaluated using a TOPflash reporter assay with or without Lithium chloride (LiCl) stimulation. Patient-derived organoids of CRC (CRC PDOs) were cultured using a stemness maintenance medium, and their viability was measured using ATP assays after treatment of JXY extract. Furthermore, the anti-tumor efficacy of JXY extract was assessed using a xenograft mice model derived from HCT-15 cells.Network pharmacology emphasized the influence of JXY on cancer stem cells and the Wnt signaling pathway. HPLC analysis confirmed that the JXY extract contained the three most prevalent pharmaceutical compounds among the four herbs documented in the Chinese Pharmacopoeia (rosmarinic acid, quercetin, and kaempferol). RNA-Seq results further elucidated the effect of JXY extract, particularly targeting cancer stem cells and the Wnt signaling pathway. Furthermore, JXY extract inhibited spheroid formation in CRC cells and downregulated CRC CSC markers (CD133, DCLK1, and C-MYC). Additionally, JXY extract suppressed the β-catenin expression and transcriptional activity as well as the Wnt pathway target proteins, including C-MYC and Cyclin D1. Consistent with findings from cell lines, JXY extract suppressed the growth of CRC PDOs exhibiting stemness characteristics. And JXY extract demonstrated a significant inhibitory effect on tumor growth, C-MYC, and β-catenin protein levels in xenograft tumors.These results highlight the novel function of JXY extract in targeting CRC CSCs by regulating Wnt signaling pathway, underscoring its potential as a therapeutic agent for treating CRC.Copyright © 2024. Published by Elsevier B.V.