柚皮苷与替莫唑胺联合通过促进细胞凋亡抑制胶质母细胞瘤细胞的生长：网络药理学、体外实验和代谢组学研究

引言：胶质母细胞瘤是一种每年影响大量患者、诊断后平均存活期约14.6个月的最具致命性的原发性侵袭性脑肿瘤。目前，手术、放疗和替莫唑胺（TMZ）化疗是三大临床治疗手段。然而，治疗效果通常受限于TMZ耐药性。柚皮苷作为一种具有抗癌、抗氧化、金属螯合及降脂作用的生物类黄酮，已成为一种有潜力的治疗选择。方法：通过网络药理学分析、细胞ELISA、流式细胞术、免疫细胞化学、西方印迹和液相色谱高分辨质谱（LC-HRMS）代谢组学研究，探讨柚皮苷与TMZ在胶质母细胞瘤中的作用靶点和通路。结果：网络药理学分析提示，柚皮苷在增强胶质母细胞瘤化疗敏感性中的关键靶点包括Poly [ADP-ribose]聚合酶1（PARP-1）、O-6-甲基鸟嘌呤-DNA甲基转移酶（MGMT）和半胱天冬酶。功能富集分析显示这些靶点显著富集于p53信号通路、凋亡和DNA感应等重要通路。体外实验结果显示，在U87-MG和T98-G胶质母细胞瘤细胞中，联合使用TMZ和柚皮苷显著降低细胞存活率，抑制DNA修复酶PARP-1和MGMT以及PI3K/AKT信号通路，从而实现化疗敏感化并诱导凋亡，表现为p53和caspase-3表达升高，Bcl2表达降低。此外，利用液相色谱高分辨质谱技术对T98-G细胞进行代谢组分析，发现联合治疗组中C8-肉碱（-2.79）、L-六烷酰肉碱（-4.46）、DL-肉碱（-2.46）、乙酰-L-肉碱（-3.12）、腺嘌呤（-1.3）、胆碱（-2.07）、丙酰肉碱（-1.69）、肌酸（-1.33）、腺苷（-0.84）、精胺（-1.42）等代谢物降低，棕榈酸（+1.03）和神经酰胺（+0.89）则上调。讨论：总结认为，柚皮苷与TMZ联合使用能增强抗胶质母细胞瘤的化疗敏感性，并诱导肿瘤细胞凋亡。

Introduction: Glioblastoma, which affects a large number of patients every year and has an average overall lifespan of around 14.6 months following diagnosis stands out as the most lethal primary invasive brain tumor. Currently, surgery, radiation, and chemotherapy with temozolomide (TMZ) are the three major clinical treatment approaches. However, the ability to treat patients effectively is usually limited by TMZ resistance. Naringin, a bioflavonoid with anti-cancer, antioxidant, metal-chelating, and lipid-lowering effects, has emerged as a promising therapeutic option. Methods: To explore the targets and pathways of naringin and TMZ in glioblastoma network pharmacology, cell line-based ELISA, flow cytometry, immunocytochemistry, western blotting, and LC-HRMS based metabolomics study were used. Results: The findings through the network pharmacology suggested that the key targets of naringin in the chemosensitization of glioblastoma would be Poly [ADP-ribose] polymerase 1 (PARP-1), O-6-Methylguanine-DNA Methyltransferase (MGMT), and caspases. The functional enrichment analysis revealed that these targets were significantly enriched in important pathways such as p53 signaling, apoptosis, and DNA sensing. Further, the results of the in-vitro study in U87-MG and T98-G glioblastoma cells demonstrated that TMZ and naringin together significantly reduced the percentage of viability and inhibited the DNA repair enzymes PARP-1 and MGMT, and PI3K/AKT which led to chemosensitization and, in turn, induced apoptosis, which was indicated by increased p53, caspase-3 expression and decreased Bcl2 expression. Additionally, a metabolomics study in T98-G glioblastoma cells using liquid chromatography high-resolution mass spectrometry (LC-HRMS) revealed downregulation of C8-Carnitine (-2.79), L-Hexanoylcarnitine (-4.46), DL-Carnitine (-2.46), Acetyl-L-carnitine (-3.12), Adenine (-1.3), Choline (-2.07), Propionylcarnitine (-1.69), Creatine (-1.33), Adenosine (-0.84), Spermine (-1.42), and upregulation of Palmitic Acid (+1.03) and Sphingosine (+0.89) in the naringin and TMZ treatment groups. Discussion: In conclusion, it can be said that naringin in combination with TMZ chemosensitized TMZ antiglioma response and induced apoptosis in tumor cells.