肝细胞癌 (HCC) 是导致癌症相关死亡的一个重要因素。中医 (TCM) 在治疗 HCC 方面具有多种优势。土木宁是土木香花中的关键化合物，已被证明具有抗多种癌症的药理作用，但对其特异性抗HCC作用的研究仍然有限。本研究旨在探讨土木宁的抗HCC作用及其潜在机制。MTT法，克隆采用形成实验和流式细胞术检测Britannin对HCC细胞系的细胞活性、增殖能力和凋亡能力。通过伤口愈合实验和Transwell迁移和侵袭实验评估用Britannin处理的HCC细胞系的细胞迁移和侵袭能力。构建H22异种移植肿瘤小鼠模型并进行不列颠宁治疗，观察不列颠宁对HCC肿瘤的作用。 Elisa检测肝癌生物标志物AFP、AFP-L3、APT、TGF-β的表达水平，HE染色观察肝癌组织病理学变化。利用网络药理学和分子对接方法预测了不列颠宁抗HCC作用可能的信号通路。采用表面等离子共振（SPR）实验验证了britannin与蛋白质之间的相互作用。采用细胞激酶活性函数实验检测不列颠宁对酶活性的影响。采用RT-qPCR和Western-Blot方法验证不列颠宁对小鼠HCC细胞和肿瘤组织中GSK-3β/β-catenin通路相关关键基因mRNA表达和蛋白水平的影响。体外实验表明，不列颠宁抑制HCC细胞的活性、增殖、迁移和侵袭能力，同时促进其凋亡。体内实验表明，英国丹宁对移植性肝癌肿瘤的生长具有抑制作用，降低移植小鼠的炎症浸润以及肝癌标志物AFP、AFP-L3、APT和TGF-β的表达水平。网络药理学和分子对接预测细胞粘附因子和GSK-3β/β-catenin通路可能是相关信号通路，并与关键蛋白具有潜在的对接活性。 SPR 实验阐明了 Britannin 和 GSK-3β 之间的分子相互作用。酶活性测定表明 Britannin 可以调节 GSK-3β 激酶的功能活性。 RT-qPCR 表明 Britannin 可以调节 β-catenin、GSK-3β、E-cadherin 和 Ncadherin 的 mRNA 表达。 Western-Blot进一步证实britannin能够显着上调GSK-3β的表达，下调p-GSK-3β和β-catenin的表达。同时，E-cadherin表达增加，NCadherin表达减少，从而减少EMT的发生，抑制HCC的转移。综上所述，britannin能够抑制HCC的生长、发展和转移，其机制可能与调控 GSK-3β/β-catenin 信号通路抑制 HCC 上皮间质转化。版权所有 © 2024。Elsevier GmbH 出版。

Hepatocellular carcinoma (HCC) stands out as a significant contributor to cancer-related death. Traditional Chinese Medicine (TCM) offers several advantages in the treatment of HCC. Britannin, a pivotal compound in Inulae Flos, has demonstrated pharmacological effects against various cancers, yet research on its specific anti-HCC effects remains limited.This study aims to explore the anti-HCC effects of britannin and its underlying mechanism.MTT assay, clone formation assay and flow cytometry were utilized to detect the cell activity, proliferation ability and apoptosis of britannin against HCC cell lines. Cell migration and invasion abilities of HCC cell lines treated with britannin were evaluated by wound-healing assay and transwell migration and invasion assay. H22 xenografted tumor mouse model was constructed and britannin treatment was performed to observe the effect of britannin on HCC tumors. The expression levels of liver cancer biomarkers AFP, AFP-L3, APT and TGF-β were detected by Elisa, and the histopathology was observed by HE staining. Network pharmacology and molecular docking were used to predict the possible signaling pathway of anti-HCC effect of britannin. The surface plasmon resonance (SPR) experiment was used to verify the interaction between britannin and proteins. The cell kinase activity function experiment was employed to detect the effect of britannin on enzyme activity. RT-qPCR and Western-Blot were used to verify the effect of britannin on the mRNA expressions of key genes and protein levels related to GSK-3β/β-catenin pathway in HCC cells and tumor tissues in mice.In vitro experiments showed that britannin could inhibit the activity, proliferation, migration and invasion abilities of HCC cells, while promoting their apoptosis. In vivo experiments revealed that britannin exerted inhibitory effects on the growth of transplanted liver cancer tumors, reducing the inflammatory infiltration and the expression levels of AFP, AFP-L3, APT and TGF-β of liver cancer markers in transplanted mice. Network pharmacology and molecular docking predicted that cell adhesion factors and GSK-3β/β-catenin pathway might be the related signaling pathway and had potential docking activity with key proteins. The SPR experiments elucidated the molecular interaction between britannin and GSK-3β. Enzyme activity assays indicated that britannin could modulate the functional activity of GSK-3β kinase. RT-qPCR suggested britannin could regulate the mRNA expressions of β-catenin, GSK-3β, E-cadherin and NCadherin. Western-Blot further verified that britannin could significantly up-regulate the expression of GSK-3β and down-regulate the expression of p-GSK-3β and β-catenin. At the same time, the expression of E-cadherin increased and NCadherin decreased, thereby reducing the occurrence of EMT and inhibiting the metastasis of HCC.In conclusion, britannin could inhibit the growth, development and metastasis of HCC, and its mechanism may be related to the regulation of GSK-3β/β-catenin signaling pathway to inhibit epithelial-mesenchymal transition of HCC.Copyright © 2024. Published by Elsevier GmbH.