调肠消瘤方抑制CXCR2介导的中性粒细胞募集并重塑肠道菌群代谢微环境干预肠道肿瘤发生

Tiaochang Xiaoliu Decoction Inhibits CXCR2-Mediated Neutrophil Recruitment and Reshapes Gut Microbiota Metabolic Microenvironment to Intervene in Intestinal Tumor Development

    摘要
  • 摘要:
    目的 探讨调肠消瘤方（Tiaochang Xiaoliu Decoction，TXD）治疗结直肠腺瘤的疗效及其相关机制。
    方法 采用液相色谱-质谱（LC-MS）对TXD化学成分进行鉴定，并通过网络药理学分析筛选其治疗结直肠腺瘤的潜在作用靶点。采用低剂量或高剂量TXD治疗ApcMin/+小鼠，并以瑞戈非尼作为对照药物。通过统计各组小鼠肿瘤负荷，并通过组织病理学染色评估肠上皮细胞增殖情况。采用免疫荧光法和流式细胞术评估中性粒细胞及其亚群CXCR2⁺中性粒细胞在肿瘤组织的浸润情况。通过RNA转录组测序解析TXD对ApcMin/+小鼠基因表达的影响。利用组织病理学染色、16S rRNA测序及非靶向代谢组学技术评估肠道屏障完整性、肠道菌群组成及粪便代谢变化。
    结果 LC-MS分析揭示了TXD在正离子和负离子模式下的复杂化学成分；网络药理学分析发现，TXD的多种成分可作用于结直肠腺瘤相关的核心靶点，包括CXCR2、IL-6、TNF和MMP9，以及炎症和癌症相关信号通路。TXD治疗显著降低了ApcMin/+小鼠的肿瘤负荷及上皮细胞增殖。免疫荧光结果显示，TXD治疗显著减少了结肠及肿瘤组织中的Ly6G⁺MPO⁺细胞的浸润（P<0.001）。流式细胞术分析发现TXD治疗明显降低了CD45⁺Ly6G⁺CD11b⁺中性粒细胞及其亚群CXCR2⁺中性粒细胞的比例（P<0.01，P<0.001），同时CXCL1/2/5及IL-6的浓度显著下降（P<0.01）。转录组学分析结果表明TXD治疗可抑制中性粒细胞趋化性和活化特征相关基因的表达，TXD治疗后CXCR2、Ly6G和MPO基因表达显著下调。同时，TXD治疗显著改善了ApcMin/+小鼠结直肠上皮屏障的完整性，并改善了ApcMin/+小鼠肠道微生物群组成以及脂质和胆汁酸代谢。
    结论 TXD治疗可通过抑制CXCR2介导的中性粒细胞募集与活化通路，并重塑重塑肠道菌群代谢微环境，从而抑制ApcMin/+小鼠结直肠腺瘤的发生。

     

    Abstract:
    OBJECTIVE To investigate the therapeutic efficacy of Tiaochang Xiaoliu Decoction (TXD) in colorectal adenomas and to elucidate its underlying mechanisms.
    METHODS The chemical constituents of TXD were characterized using liquid chromatography-mass spectrometry (LC-MS), and potential therapeutic targets were identified through network pharmacology analysis. The ApcMin/+ mice were treated with low- or high-dose TXD, with regorafenib serving as a positive control. Tumor burden was quantified, and epithelial proliferation was assessed by histopathological staining. Neutrophil infiltration, including the CXCR2⁺ neutrophil subset, was evaluated by immunofluorescence and flow cytometry. Transcriptomic profiling (RNA-seq) was performed to determine gene expression changes induced by TXD. Intestinal barrier integrity, gut microbiota composition (16S rRNA sequencing), and fecal metabolomic profiles (untargeted metabolomics) were comprehensively analyzed.
    RESULTS LC-MS analysis revealed the complex chemical composition of TXD under both positive and negative ion modes. Network pharmacology identified multiple active components targeting key molecules implicated in colorectal adenoma progression, including CXCR2, IL-6, TNF, and MMP9, as well as inflammation- and cancer-related signaling pathways. TXD treatment significantly reduced tumor burden and suppressed epithelial proliferation in ApcMin/+ mice. Immunofluorescence analysis demonstrated a marked decrease in Ly6G⁺MPO⁺ neutrophil infiltration in colonic and tumor tissues (P < 0.001). Flow cytometry further showed that TXD significantly reduced the proportions of CD45⁺Ly6G⁺CD11b⁺ neutrophils and CXCR2⁺ neutrophil subsets (P < 0.001, P < 0.01), accompanied by decreased levels of CXCL1/2/5 and IL-6 (all P < 0.01). Transcriptomic analysis revealed that TXD downregulated genes associated with neutrophil chemotaxis and activation, including CXCR2, Ly6G, and MPO. In addition, TXD significantly restored intestinal epithelial barrier integrity and reshaped gut microbiota composition, along with lipid and bile acid metabolic profiles.
    CONCLUSION TXD suppresses colorectal adenoma development in ApcMin/+ mice by inhibiting CXCR2-mediated neutrophil recruitment and activation, while concurrently remodeling the gut microbiota-metabolite microenvironment.

     

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