Ze-qi decoction inhibits neutrophil extracellular trap formation to suppress the growthand metastasis of NSCLC

This study aimed to explore the material basis for anit-NSCLC efficacy of Ze-qi decoction (ZQD) and elucidate the molecular mechanisms. UPLC-HRMS was used to identify the primary chemical components. The potent molecular mechanisms were analyzed using network pharmacology. A subcutaneous tumor mouse model was established. The subcutaneous tumor was stripped for transcriptomic analysis. 253 DEGs were identified in the ZQD-H versus Model, including 167 upregulated and 86 downregulated genes. The heatmap demonstrated that the gene expression patterns differed significantly between the two groups. Functional exploration was conducted using GO and KEGG analyses. Most entries were immune-associated, such as the regulation of tumor necrosis factor and cytokine production, as well as immune receptor activity. From the KEGG enrichment analysis, we identified 29 significant KEGG pathways mostly associated with cancer, immunology, and metabolism, which corresponded to the results of the GO analysis. The neutrophil extracellular traps (NETs) formation was uncovered as the anti-NSCLC target of ZQD by pharmacological research and transcriptomic profile. The molecular docking analysis was conducted to predict the interaction of 16 core components with proteins HIF1A, CD18, and MPO that were jointed to neutrophil recruitment and NETs formation.The underlying molecular mechanisms of ZQD against NSCLC were investigated by western blot, immunofluorescence, immunohistochemistry, and Elisa assays. ZQD hindered the neutrophils recruitment and activation by decreasing HIF-1α, CD18, and ICAM-1 levels and suppressed NETs formation shown as the downregulated biomarkers (CitH3, MPO, and NE). Finally, the lung metastasis mouse model was constructed dor further validation. Fewer nodules were observed on the surface of the lungs in mice treated with ZQD. NETs-related proteins CitH3 and Ly6G, as well as MPO were less expressed in the lungs after ZQD intervened in comparison of model group.本研究旨在探讨泽漆汤（Ze-qi decoction, ZQD）抗非小细胞肺癌（non-small cell lung cancer, NSCLC）疗效的物质基础，并阐明其分子作用机制。采用超高效液相色谱-高分辨质谱（UPLC-HRMS）鉴定其主要化学成分，通过网络药理学分析潜在分子机制。构建皮下移植瘤小鼠模型，剥离皮下肿瘤组织进行转录组学分析。在ZQD高剂量组与模型组中，共鉴定出253个差异表达基因（differentially expressed genes, DEGs），其中167个为上调基因，86个为下调基因。热图分析显示，两组间基因表达模式存在显著差异。通过基因本体（Gene Ontology, GO）与京都基因与基因组百科全书（Kyoto Encyclopedia of Genes and Genomes, KEGG）富集分析开展功能注释与探索，结果显示多数富集条目与免疫过程相关，包括肿瘤坏死因子调控、细胞因子生成及免疫受体活性等。KEGG富集分析共筛选出29条显著富集通路，主要涉及癌症、免疫学与代谢通路，与GO分析结果高度一致。结合药理学研究与转录组学特征，发现中性粒细胞胞外陷阱（neutrophil extracellular traps, NETs）形成是泽漆汤抗非小细胞肺癌的核心作用靶点。采用分子对接技术，预测16种核心活性成分与中性粒细胞招募及NETs形成相关蛋白HIF1A、CD18和MPO的相互作用。通过蛋白质印迹、免疫荧光、免疫组化及酶联免疫吸附测定（Enzyme-Linked Immunosorbent Assay, ELISA）实验，进一步探究泽漆汤抗非小细胞肺癌的潜在分子机制。结果表明，泽漆汤可通过降低HIF-1α、CD18及细胞间黏附分子-1（ICAM-1）的表达水平，抑制中性粒细胞的招募与活化，并下调中性粒细胞胞外陷阱相关标志物CitH3、MPO及NE的表达，从而阻滞NETs形成。最后构建肺转移小鼠模型进行验证：泽漆汤给药组小鼠肺表面肿瘤结节数量显著少于模型组；与模型组相比，泽漆汤干预后小鼠肺组织中NETs相关蛋白CitH3、Ly6G及MPO的表达水平显著降低。