基于GPX4/SLC7A11表达变化探讨半枝莲诱导肝癌细胞铁死亡的实验研究

Experimental study of Scutellaria barbata D. Don-induced ferroptosis in hepatocellular carcinoma cells based on changes in GPX4/SLC7A11 expression

摘要

目的 探讨半枝莲调控肝癌细胞铁死亡的作用机制。方法 通过网络药理学筛选半枝莲与肝癌及调控铁死亡的交集靶点，并将半枝莲的主要活性成分与筛选出的关键靶点进行分子对接验证。体外培养 Huh7 细胞，采用细胞计数试剂盒-8（CCK-8）法检测半枝莲（0、3.15、6.3、12.5、25、50 mg/mL）处理 24 h 后的细胞增殖能力，筛选最佳药物浓度。将 Huh7 细胞分为对照组和半枝莲组（20 mg/mL），测定乳酸脱氢酶（LDH）释放量、Fe²⁺含量、活性氧（ROS）水平及线粒体膜电位；采用定量聚合酶链反应（qPCR）和蛋白免疫印迹法检测细胞中铁死亡相关指标[谷胱甘肽过氧化物酶 4（GPX4）、溶质载体家族 7 成员 11（SLC7A11）、铁反应元件结合蛋白 2（IREB2）、长链酰基辅酶 A 合成酶 4（ACSL4）及关键靶点[丝裂原活化蛋白激酶 14（MAPK14）、肿瘤蛋白 p53（TP53）、低氧诱导因子-1α（HIF-1α）、NF-κB 亚单位（p65）基因（RELA）、MAPK1]的 mRNA 与蛋白表达水平。构建雄性 BALB/c 裸鼠 Huh7 皮下移植瘤模型，随机分为对照组和半枝莲组。对照组给予生理盐水灌胃，半枝莲组给予半枝莲颗粒 140 mg/kg 灌胃，定期监测移植瘤体积与质量，取材后行苏木精-伊红（HE）染色评估肿瘤病理变化。结果 网络药理学筛选得到半枝莲排名前五位的活性成分分别为槲皮素、木犀草素、汉黄芩素、黄芩素和圣草酚，其与核心靶点 MAPK14、TP53、HIF-1α、RELA、MAPK1 均具有较强的结合力；基因本体论（GO）和京都基因与基因组百科全书（KEGG）富集分析显示，半枝莲诱导肝癌细胞铁死亡与 HIF-1 信号通路、肿瘤相关信号通路、IL-17 信号通路等有关。体外实验证实，半枝莲呈浓度依赖性抑制 Huh7 细胞增殖；与对照组相比，半枝莲组细胞内 ROS 及 Fe²⁺水平升高，线粒体膜电位降低；铁死亡相关指标中，GPX4 与 SLC7A11 的 mRNA 及蛋白表达水平下调，IREB2 与 ACSL4 表达上调；网络药理学筛选靶点 MAPK1、TP53、MAPK14、HIF1A 的 mRNA 及蛋白表达水平升高，而 RELA 表达水平降低（均 P＜0.05）。动物体内实验结果显示，半枝莲组小鼠肿瘤体积与质量均低于对照组（均 P＜0.05），且肿瘤组织细胞排列紊乱，可见明显坏死灶。结论 半枝莲在体内外均可有效抑制肝癌恶性进展，其机制可能与诱导肝癌细胞铁死亡有关。

Abstract

Objective To investigate the mechanism by which Scutellaria barbata D. Don regulates ferroptosis in hepatocellular carcinoma (HCC) cells. Methods Network pharmacology was used to screen the intersecting targets of Scutellaria barbata D. Don, HCC, and ferroptosis regulation. Molecular docking was then performed to validate the binding between the main active components of Scutellaria barbata D. Don and the screened key targets. Huh7 cells were cultured in vitro, and the cell counting kit-8 (CCK-8) assay was used to detect cell proliferation after treatment with Scutellaria barbata D. Don at different concentrations (0, 3.15, 6.3, 12.5, 25, and 50 mg/mL) for 24 h, so as to determine the optimal drug concentration. Huh7 cells were divided into a control group and a Scutellaria barbata D. Don group (20 mg/mL). Lactate dehydrogenase (LDH) release, Fe ²⁺ content, reactive oxygen species (ROS) levels, and mitochondrial membrane potential were measured. Quantitative polymerase chain reaction (qPCR) and Western blot were used to detect the mRNA and protein expression levels of ferroptosis-related indicators, including glutathione peroxidase 4 (GPX4), solute carrier family 7 member 11 (SLC7A11), iron-responsive element-binding protein 2 (IREB2), and long-chain acyl-CoA synthetase 4 (ACSL4), as well as key targets, including mitogen-activated protein kinase 14 (MAPK14), tumor protein p53 (TP53), hypoxia-inducible factor-1α, NF-κB subunit (p65) gene (RELA), and MAPK1. A Huh7 subcutaneous xenograft model was established in male BALB/c nude mice, which were randomly divided into a control group and a Scutellaria barbata D. Don group. The control group was given normal saline by gavage, while the Scutellaria barbata D. Don group was given Scutellaria barbata D. Don granules at 140 mg/kg by gavage. Tumor volume and weight were monitored regularly. After tissue collection, hematoxylin-eosin (HE) staining was performed to evaluate pathological changes in tumor tissues. Results Network pharmacology identified the top five active components of Scutellaria barbata D. Don as quercetin, luteolin, wogonin, baicalein, and eriodictyol. These components showed strong binding affinities with the core targets MAPK14, TP53, HIF-1α, RELA, and MAPK1. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses showed that Scutellaria barbata D. Don-induced ferroptosis in HCC cells was associated with the HIF-1 signaling pathway, cancer-related signaling pathways, and the IL-17 signaling pathway. In vitro experiments confirmed that Scutellaria barbata D. Don inhibited Huh7 cell proliferation in a concentration-dependent manner. Compared with the control group, the Scutellaria barbata D. Don group showed increased intracellular ROS and Fe ²⁺ levels and decreased mitochondrial membrane potential. Among ferroptosis-related indicators, the mRNA and protein expression levels of GPX4 and SLC7A11 were downregulated, while those of IREB2 and ACSL4 were upregulated. The mRNA and protein expression levels of the network pharmacology-screened targets MAPK1, TP53, MAPK14, and HIF-1α were increased, whereas RELA expression was decreased. In vivo experiments showed that tumor volume and weight in mice in the Scutellaria barbata D. Don group were lower than those in the control group. Tumor tissue cells were arranged irregularly, with obvious necrotic foci observed. Conclusion Scutellaria barbata D. Don can effectively inhibit the malignant progression of HCC both in vivo and in vitro, and its mechanism may be associated with the induction of ferroptosis in HCC cells.

关键词

肝癌 / 铁死亡 / 半枝莲 / 网络药理学

Key words

Hepatocellular carcinoma / Ferroptosis / Scutellaria barbata D. Don / Network pharmacology

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基金资助

广东省中医药局面上基金项目(20241047)

广东省中医药局面上基金项目(20241055)

中西医协同“旗舰”科室建设项目(国中医药综结合函〔2024〕221号)