目的 探究砂生槐总生物碱（TA-SM）体内、体外抗肝细胞癌的作用及作用机制。方法 体外实验以HepG2和Huh-7细胞为研究对象，采用CCK-8法、克隆形成实验、细胞划痕实验、侵袭实验和流式细胞术评价TA-SM对2种肝癌细胞增殖、迁移、侵袭、凋亡和细胞周期进程的影响；随后运用蛋白质组学数据依赖采集（DIA）检测技术和分子对接技术等生物信息学分析手段，探究TA-SM对HepG2细胞内蛋白表达水平的影响并探讨可能的信号通路；应用Western blotting和实时荧光定量PCR（qRT-PCR）实验验证TA-SM对Hippo通路关键靶点蛋白——含WW域转录调节蛋白1（WWTR1）、转录增强因子4（TEAD4）、丝氨酸/苏氨酸激酶25（STK25）、丝氨酸/苏氨酸-蛋白激酶38（STK38L）的影响。采用裸鼠建立HepG2细胞的人源肿瘤细胞系异种移植（CDX）模型，血常规、生化指标、主要脏器病理检测评估TA-SM（50 mg·kg^-1）体内抗肿瘤的安全性；记录裸鼠体质量、肿瘤体积、肿瘤质量变化； Western blotting、qRT-PCR、免疫组化、免疫荧光法检测肿瘤组织WWTR1、TEAD4、STK25、STK38L表达变化；免疫组化和免疫荧光检测肿瘤组织CD31、CD34、血管内皮生长因子（VEGF）表达变化；免疫荧光检测细胞增殖抗原（Ki67）表达变化。结果 TA-SM通过浓度和时间相关方式抑制HepG2和Huh-7细胞的增殖、迁移及侵袭，还可以通过阻滞HepG2的G₁、G₂期和Huh-7细胞的G₂期，诱导细胞凋亡（P＜0.05、0.01、0.001）。蛋白质组学结果显示，TA-SM作用于HepG2细胞与TNF信号通路、Hippo信号通路、p53信号通路相关。分子对接结果显示TA-SM中4种主要生物碱（苦参碱、氧化苦参碱、槐果碱和槐定碱）与Hippo通路关键靶点（TK25、STK38L、WWTR1和TEAD4）的亲和力较好，具有很强的结合活性。与对照组比较，TA-SM在体外显著上调STK25、STK38L、WWTR1和TEAD4蛋白和基因表达（P＜0.05、0.01、0.001）。在裸鼠CDX模型中，TA-SM抗肿瘤安全性良好；与对照组比较，肿瘤体积显著降低（P＜0.001），STK25、STK38L、WWTR1和TEAD4表达显著升高，CD31、CD34、VEGF、Ki67表达显著降低（P＜0.05、0.01、0.001）。结论 TA-SM通过激活Hippo信号通路，抑制HepG2和Huh-7细胞的增殖、迁移和侵袭，并诱导细胞凋亡。此外，TA-SM还能协同阻滞HepG2和Huh-7细胞周期，抑制新生血管生成相关因子以及肿瘤增殖因子的表达，从多途径发挥其抗肝癌作用。

Objective To investigate the in vivo and in vitro anti-hepatocellular carcinoma effects and mechanisms of total alkaloids from Sophora moorcroftiana (TA-SM). Methods In vitro experiments were conducted using HepG2 and Huh-7 cells as the research subjects. The effects of TA-SM on the proliferation, migration, invasion, apoptosis and cell cycle progression of the two liver cancer cell lines were evaluated by CCK8 assay, colony formation assay, cell scratch assay, invasion assay and flow cytometry. Subsequently, proteomics data-dependent acquisition (DIA) detection technology and molecular docking technology and other bioinformatics analysis methods were used to explore the effects of TA-SM on the protein expression levels in HepG2 cells and to discuss the possible signaling pathways. Western blotting and real-time fluorescence quantitative PCR (qRT-PCR) experiments were used to verify the effects of TA-SM on the key target proteins of the Hippo pathway - WWTR1, TEAD4, STK25, and STK38L. A human tumor cell line xenograft (CDX) model of HepG2 cells was established in nude mice. The safety of TA-SM (50 mg·kg^-1) in vivo was evaluated by blood routine, biochemical indicators, and pathological examination of major organs. The changes in body weight, tumor volume, and tumor mass of nude mice were recorded. Western blotting, qRT-PCR, immunohistochemistry, and immunofluorescence were used to detect the expression changes of WWTR1, TEAD4, STK25, and STK38L in tumor tissues. Immunohistochemistry and immunofluorescence were used to detect the expression changes of CD31, CD34, vascular endothelial growth factor (VEGF), and tumor proliferation antigen (Ki67) in tumor tissues. Results TA-SM inhibited the proliferation, migration, and invasion of HepG2 and Huh-7 cells in a concentration- and time-dependent manner. It also induced cell apoptosis by blocking the G1 and G2 phases of HepG2 cells and the G2 phase of Huh-7 cells (P<0.05, 0.01, 0.001). Proteomics results showed that TA-SM acted on HepG2 cells and was related to the TNF signaling pathway, Hippo signaling pathway, and p53 signaling pathway. Molecular docking results showed that the four main alkaloids in TA-SM (matrine, oxymatrine, sophoridine, and sophoramine) had good affinity with the key targets of the Hippo pathway (STK25, STK38L, WWTR1, and TEAD4) and had strong binding activity. Compared with the control group, TASM significantly upregulated the protein and gene expression of STK25, STK38L, WWTR1, and TEAD4 in vitro (P<0.05, 0.01, 0.001). In the nude mouse CDX model, TA-SM had good anti-tumor safety. Compared with the control group, the tumor volume was significantly reduced (P<0.001), and the expression of STK25, STK38L, WWTR1, and TEAD4 was significantly increased, while the expression of CD31, CD34, VEGF, and Ki67 was significantly decreased (P<0.05, 0.01, 0.001). Conclusion TA-SM inhibits the proliferation, migration, and invasion of HepG2 and Huh-7 cells by activating the Hippo signaling pathway and inducing apoptosis. It also synergistically disrupts the cell cycle in both cell lines and suppresses the expression of tumor proliferation and angiogenesisrelated factors. These findings suggest that TA-SM exerts anti-hepatocellular carcinoma effects through multiple, complementary mechanisms.

R285.5

青海省科学技术厅资助（2025-ZJ-965J）