Chaihu Shugan San (CSS) is a well‐known traditional herbal formula that has the potential to ameliorate hepatocellular carcinoma (HCC); however, its mechanism of action remains unknown. Here, we identified the key targets of CSS against HCC and developed a prognostic model to predict the survival of patients with HCC. The effect of CSS plus sorafenib on HCC cell proliferation was evaluated using the MTT assay. LASSO‐Cox regression was used to establish a three‐gene signature model targeting CSS. Correlations between immune cells, immune checkpoints and risk score were determined to evaluate the immune‐related effects of CSS. The interactions between the components and targets were validated using molecular docking and Surface Plasmon Resonance (SPR) assays. CSS and sorafenib synergistically inhibited HCC cell proliferation. Ten core compounds and 224 targets were identified using a drug compound–target network. The prognostic model of the three CSS targets (AKT1, MAPK3 and CASP3) showed predictive ability. Risk scores positively correlated with cancer‐promoting immune cells and high expression of immune checkpoint proteins. Molecular docking and SPR analyses confirmed the strong binding affinities of the active components and the target genes. Western blot analysis confirmed the synergistic effect of CSS and sorafenib in inhibiting the expression of these three targets. In conclusion, CSS may regulate the activity of immune‐related factors in the tumour microenvironment, reverse immune escape, enhance immune responses through AKT1, MAPK3, and CASP3, and synergistically alleviate HCC. The co‐administration of sorafenib with CSS has a strong clinical outlook against HCC.

中文翻译：

---

综合分析鉴定与柴胡疏肝散方治疗肝细胞癌相关的三基因特征

柴胡疏肝散（CSS）是一种著名的传统草药配方，具有改善肝细胞癌（HCC）的潜力；然而，其作用机制仍不清楚。在这里，我们确定了 CSS 对抗 HCC 的关键靶标，并开发了一个预后模型来预测 HCC 患者的生存率。使用MTT测定评估CSS加索拉非尼对HCC细胞增殖的影响。 LASSO-Cox回归用于建立针对CSS的三基因特征模型。确定免疫细胞、免疫检查点和风险评分之间的相关性，以评估 CSS 的免疫相关作用。使用分子对接和表面等离子共振（SPR）测定验证了成分和靶标之间的相互作用。 CSS和索拉非尼协同抑制HCC细胞增殖。使用药物化合物-靶点网络确定了 10 种核心化合物和 224 个靶点。三个 CSS 目标（AKT1、MAPK3 和 CASP3）的预后模型显示出预测能力。风险评分与促癌免疫细胞和免疫检查点蛋白的高表达呈正相关。分子对接和SPR分析证实了活性成分与靶基因的强结合亲和力。蛋白质印迹分析证实了 CSS 和索拉非尼在抑制这三个靶标表达方面的协同作用。总之，CSS可能调节肿瘤微环境中免疫相关因子的活性，逆转免疫逃逸，通过AKT1、MAPK3和CASP3增强免疫反应，协同缓解HCC。索拉非尼与 ​​CSS 联合用药对于 HCC 具有很强的临床前景。