Li Yuanchao, Zhou Lingwei, Sun Kang, Guo Ran, Li Zehua, Wen Qingqing, Fu Guifeng, Yang Shuohui
Department of Radiology, Shanghai Municipal Hospital of Traditional Chinese Medicine, Shanghai University of Traditional Chinese Medicine, 274 Middle Zhi-Jiang Road, Shanghai, 200071, People's Republic of China.
Department of Laboratory, Shanghai Municipal Hospital of Traditional Chinese Medicine, Shanghai University of Traditional Chinese Medicine, 274 Middle Zhi-Jiang Road, Shanghai, 200071, People's Republic of China.
J Ethnopharmacol. 2025 Apr 9;345:119589. doi: 10.1016/j.jep.2025.119589. Epub 2025 Mar 6.
Bufalin is a potent bioactive compound extracted from the venom of toads such as Bufo gargarizans. It has rich pharmacological effects, and its traditional applications mainly include anti-cancer, anti-inflammatory and analgesic, especially in cancer treatment, which has been a hot topic of research. Prior research has suggested that bufalin may have anti-tumor angiogenic effects. However, the efficacy and mechanism of bufalin inhibiting hepatocellular carcinoma (HCC) angiogenesis have yet to be further investigated.
An extensive detailed strategy via network pharmacology, proteomics, histopathological analysis, molecular docking, in vitro experiments, and in vivo magnetic resonance imaging (MRI) examinations were adopted to investigate the efficacy and mechanisms of bufalin against HCC angiogenesis.
Micro-vessel density (MVD) and intravoxel incoherent motion (IVIM) perfusion-related parameters based on magnetic resonance diffusion-weighted imaging were used to identify the effect of bufalin against HCC angiogenesis. Potential bufalin and HCC targets were gathered from appropriate databases. The STRING database was used to construct the target protein interaction networks. The "clusterprofiler" package (version 4.2.2) in R was applied to conduct the target-related Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment and Gene Ontology (GO) analysis. Network pharmacology and proteomics were integrated to identify key targets and pathways related to bufalin against HCC angiogenesis. Molecular docking and Western Blot were utilized to validate the findings.
Analysis through IVIM and MVD showed that bufalin could inhibit HCC angiogenesis in nude mice models. A total of 159 common targets of bufalin and HCC were identified by network pharmacology. GO analysis revealed that these targets focused on multiple angiogenesis-related biological processes, including endothelial cell proliferation and migration, sprouting angiogenesis, and regulation of angiogenesis. The KEGG enrichment results suggested that bufalin could regulate multiple signaling pathways to inhibit HCC angiogenesis, including VEGF, MAPK, PI3K-Akt, mTOR, and HIF-1 signaling pathways. MAPK1, MAPK14, PRKCA, EIF4E, and APEX1 might be critical targets in regulating the above pathways. The molecular docking and Western blot analysis verified the effects of bufalin on target proteins.
This study demonstrated that bufalin might inhibit HCC angiogenesis by regulating multiple targets and pathways. These findings offer theoretical insights and experimental foundations for the clinical application and commercial development of bufalin in the treatment of HCC.
蟾毒灵是一种从中华大蟾蜍等蟾蜍毒液中提取的强效生物活性化合物。它具有丰富的药理作用，其传统应用主要包括抗癌、抗炎和镇痛，尤其是在癌症治疗方面，一直是研究的热点。先前的研究表明，蟾毒灵可能具有抗肿瘤血管生成作用。然而，蟾毒灵抑制肝细胞癌（HCC）血管生成的疗效和机制尚有待进一步研究。
采用网络药理学、蛋白质组学、组织病理学分析、分子对接、体外实验和体内磁共振成像（MRI）检查等广泛详细的策略，研究蟾毒灵抗HCC血管生成的疗效和机制。
基于磁共振扩散加权成像的微血管密度（MVD）和体素内不相干运动（IVIM）灌注相关参数用于确定蟾毒灵对HCC血管生成的影响。从适当的数据库中收集潜在的蟾毒灵和HCC靶点。使用STRING数据库构建目标蛋白相互作用网络。应用R语言中的“clusterprofiler”软件包（版本4.2.2）进行与目标相关的京都基因与基因组百科全书（KEGG）通路富集和基因本体论（GO）分析。整合网络药理学和蛋白质组学以确定与蟾毒灵抗HCC血管生成相关的关键靶点和通路。利用分子对接和蛋白质免疫印迹法验证研究结果。
通过IVIM和MVD分析表明，蟾毒灵可抑制裸鼠模型中的HCC血管生成。通过网络药理学确定了蟾毒灵和HCC的159个共同靶点。GO分析显示，这些靶点集中在多个与血管生成相关的生物学过程，包括内皮细胞增殖和迁移、芽生血管生成以及血管生成调节。KEGG富集结果表明，蟾毒灵可调节多种信号通路以抑制HCC血管生成，包括VEGF、MAPK、PI3K-Akt、mTOR和HIF-1信号通路。MAPK1、MAPK14、PRKCA、EIF4E和APEX1可能是调节上述通路的关键靶点。分子对接和蛋白质免疫印迹分析验证了蟾毒灵对目标蛋白的作用。
本研究表明，蟾毒灵可能通过调节多个靶点和通路来抑制HCC血管生成。这些发现为蟾毒灵在HCC治疗中的临床应用和商业开发提供了理论见解和实验基础。
