As one of the classical traditional Chinese medicine (TCM) prescriptions in treating gynecological tumors, Guizhi Fuling Decoction (GFD) has been used to treat breast cancer (BRCA). Nonetheless, the potential molecular mechanism remains unclear so far. Therefore, systems pharmacology was used in combination with high throughput sequencing-based high throughput screening (HTS²) assay and bioinformatic technologies in this study to investigate the molecular mechanisms of GFD in treating BRCA. By computationally analyzing 76 active ingredients in GFD, 38 potential therapeutic targets were predicted and significantly enriched in the “pathways in cancer”. Meanwhile, experimental analysis was carried out to examine changes in the expression levels of 308 genes involved in the “pathways in cancer” in BRCA cells treated by five herbs of GFD utilizing HTS² platform, and 5 key therapeutic targets, including HRAS, EGFR, PTK2, SOS1, and ITGB1, were identified. The binding mode of active compounds to these five targets was analyzed by molecular docking and molecular dynamics simulation. It was found after integrating the computational and experimental data that, GFD possessed the anti-proliferation, pro-apoptosis, and anti-angiogenesis activities mainly through regulating the PI3K and the MAPK signaling pathways to inhibit BRCA. Besides, consistent with the TCM theory about the synergy of Cinnamomi Ramulus (Guizhi) by Cortex Moutan (Mudanpi) in GFD, both of these two herbs acted on the same targets and pathways. Taken together, the combined application of computational systems pharmacology techniques and experimental HTS² platform provides a practical research strategy to investigate the functional and biological mechanisms of the complicated TCM prescriptions.

桂枝茯苓汤（GFD）作为治疗妇科肿瘤的经典中药方剂之一，已用于治疗乳腺癌（BRCA）。尽管如此，潜在的分子机制迄今为止仍不清楚。因此，本研究采用系统药理学结合基于高通量测序的高通量筛选（HTS ² ）分析和生物信息学技术来研究GFD治疗BRCA的分子机制。通过计算分析 GFD 中的 76 种活性成分，预测并显着丰富了“癌症通路”中的 38 个潜在治疗靶点。同时，利用HTS ²平台，检测5种GFD中药治疗BRCA细胞中涉及“癌症通路”的308个基因表达水平的变化，以及5个关键治疗靶点，包括HRAS、EGFR、鉴定出 PTK2、SOS1 和 ITGB1。通过分子对接和分子动力学模拟分析了活性化合物与这五个靶标的结合模式。综合计算和实验数据发现，GFD主要通过调节PI3K和MAPK信号通路抑制BRCA而具有抗增殖、促凋亡和抗血管生成活性。此外，与桂枝和牡丹皮在GFD中协同作用的中医理论一致，这两种草药作用于相同的靶点和途径。 总而言之，计算系统药理学技术和实验HTS ²平台的结合应用为研究复杂中药处方的功能和生物学机制提供了一种实用的研究策略。