仙莲解毒汤通过抑制Plaur/Hbfegf/EGFR介导的肌动蛋白细胞骨架重塑，抑制癌症肝转移

摘要(中文译文)

结直肠癌肝转移（CRLM）仍是重大的临床挑战。纤连解毒汤（XLJDD）已显示出对结直肠癌的抗肿瘤活性，但其在CRLM中的作用机制尚不明确。为阐明XLJDD在CRLM中的抗转移机制并鉴定其靶向Plaur的活性成分，开展了一项整合体内、体外、转录组学、蛋白质组学和单细胞机制的研究。通过脾内注射CT26细胞建立CRLM小鼠模型。整合转录组学和蛋白质组学分析以鉴定候选靶点。采用单细胞RNA测序（scRNA-seq）表征肿瘤微环境变化。使用划痕实验、Transwell实验、鬼笔环肽染色和体内Plaur过表达回补模型进行功能验证。通过UPLC-MS/MS对生物样本中的XLJDD颗粒及XLJDD来源化合物进行图谱分析，并通过分子对接、表面等离子体共振（SPR）和微量热泳动（MST）检测其与Plaur的结合。进一步通过靶向UHPLC-MRM-MS/MS定量环黄芪皂苷元的肝脏暴露量。XLJDD减轻了转移性肝组织中的肝脏转移负荷。整合转录组学和蛋白质组学分析确定Plaur/Hbegf轴为XLJDD的候选靶点。在机制上，Plaur促进Mmp9相关的Hbegf释放，而XLJDD抑制EGFR/ERK信号传导并降低细胞骨架相关效应分子（包括Fscn1和Cdc42ep1）的表达。scRNA-seq揭示XLJDD减少了Plaur高表达的Car6恶性亚群，增加了Ifit1干扰素反应状态，并削弱了恶性细胞与Thbs1肿瘤相关巨噬细胞之间的Spp1-Cd44信号传导。在体内，Plaur过表达削弱了XLJDD的抗转移效果。环黄芪皂苷元被鉴定为XLJDD的组织可利用成分，可直接结合Plaur并抑制CT26细胞中Plaur的表达。靶向定量证实了口服XLJDD后肝转移组织中存在环黄芪皂苷元。XLJDD通过靶向Plaur、抑制细胞骨架重塑和重塑转移性肿瘤微环境来抑制CRLM。环黄芪皂苷元可能作为直接结合Plaur的成分参与这一活性。

Abstract

Colorectal cancer liver metastasis (CRLM) remains a major clinical challenge. Xian-Lian-Jie-Du Decoction (XLJDD) has shown anti-tumor activity against colorectal cancer, but its mechanism in CRLM remains unclear. To clarify the anti-metastatic mechanism of XLJDD and identify its Plaur-targeting active constituents in CRLM. An integrated in vivo, in vitro, transcriptomic, proteomic, and single-cell mechanistic study. A CRLM mouse model was established by intrasplenic injection of CT26 cells. Transcriptomic and proteomic analyses were integrated to identify candidate targets. Single-cell RNA sequencing (scRNA-seq) was used to characterize tumor microenvironment changes. Wound-healing, Transwell, phalloidin staining, and an in vivo Plaur-overexpression rescue model were used for functional validation. XLJDD granules and XLJDD-derived compounds in biological samples were profiled by UPLC-MS/MS, and their binding to Plaur was examined by molecular docking, surface plasmon resonance (SPR), and microscale thermophoresis (MST). Hepatic exposure of cyclogalegenin was further quantified by targeted UHPLC-MRM-MS/MS. XLJDD reduced hepatic metastatic burden in metastatic liver tissues. Integrated transcriptomic and proteomic analyses identified the Plaur/Hbegf axis as a candidate target of XLJDD. Mechanistically, Plaur promoted Mmp9-associated Hbegf release, whereas XLJDD suppressed EGFR/ERK signaling and reduced the expression of cytoskeleton-related effectors, including Fscn1 and Cdc42ep1. scRNA-seq revealed that XLJDD reduced a Plaur-high Car6 malignant subpopulation, increased an Ifit1 interferon-reactive state, and weakened Spp1-Cd44 signaling between malignant cells and Thbs1 tumor-associated macrophages. In vivo, Plaur overexpression weakened the anti-metastatic effect of XLJDD. Cyclogalegenin was identified as a tissue-available constituent of XLJDD that directly binds Plaur and suppresses Plaur expression in CT26 cells. Targeted quantification confirmed the presence of cyclogalegenin in liver metastatic tissues after oral XLJDD administration. XLJDD suppresses CRLM by targeting Plaur, restraining cytoskeleton remodeling, and remodeling the metastatic tumor microenvironment. Cyclogalegenin may contribute to this activity as a direct Plaur-binding constituent.

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