背景：

胶质母细胞瘤（glioblastoma, GBM）是中枢神经系统最具侵袭性的恶性肿瘤之一，治疗手段十分有限。本研究旨在探讨铷离子（rubidium ions, Rb⁺）对抗GBM的效应及其潜在分子机制，为Rb⁺在抗肿瘤治疗中的应用提供理论依据和实验支持。

方法：

采用CCK-8法评估Rb⁺在不同浓度下对多种常见肿瘤细胞增殖的影响。并以U87和U251细胞为代表，通过细胞毒性检测、克隆形成实验、透射电子显微镜（transmission electron microscopy, TEM）观察、能谱分析（energy dispersive spectroscopy, EDS）、细胞凋亡检测、细胞周期评估、侵袭及迁移能力测试、凋亡蛋白分析等方法系统评估Rb⁺对GBM细胞表型的调控作用。

2. 通过转录组测序（RNA sequencing, RNA-seq）筛选差异表达基因，结合京都基因与基因组百科全书（Kyoto Encyclopedia of Genes and Genomes, KEGG）分析识别潜在关键信号通路，相关通路蛋白的表达变化通过western blot来验证。进一步使用通路激活剂MHY1485验证该信号通路在Rb⁺抗瘤机制中的核心作用。

3. 结合非靶向代谢组学分析Rb⁺对GBM细胞代谢的影响，检测细胞内乳酸脱氢酶（lactate dehydrogenase, LDH）、活性氧（reactive oxygen species, ROS）、谷胱甘肽（glutathione, GSH）、丙二醛（malondialdehyde, MDA）等相关代谢指标的改变，探究Rb⁺诱导GBM细胞发生代谢相关死亡的分子机制。

4. 基于BALB/c裸鼠皮下成瘤模型，评估Rb⁺的体内抑瘤效果，并通过差异剂量干预探究其有效抑瘤剂量范围。通过血清生化分析、主要脏器检测和行为学实验评价有效剂量下Rb⁺的药物使用安全性。

结果：

Rb⁺对多种肿瘤细胞系表现出抑制作用，并呈现明显的剂量依赖性和时间依赖性。Rb⁺显著诱导U87和U251发生细胞凋亡并导致细胞周期阻滞于G2/M期，抑制细胞迁移和侵袭能力，呈现浓度和时间依赖性。

Rb⁺通过PI3K/AKT/mTOR信号通路影响凋亡发生。功能拯救实验显示通路激活剂MHY1485可部分逆转Rb⁺的抑制作用，提示该通路在Rb+抗瘤效应中的重要作用。

Rb⁺显著增加细胞内ROS和亚铁离子含量，降低GSH水平并升高MDA。非靶向代谢组学分析显示Rb⁺可以影响脂质代谢、铁稳态和抗氧化系统，诱导铁死亡，促进肿瘤细胞死亡。

动物实验显示，Rb⁺（900 mg/kg）显著抑制皮下肿瘤生长。血清生化检测显示肝、肾、心功能无异常，主要脏器未见明显损伤，行为学实验无不良表现。证明Rb⁺具有良好的安全性。

结论：

本研究系统揭示了Rb⁺通过两种途径发挥抗GBM作用的分子机制：一是通过抑制PI3K/AKT/mTOR通路促进细胞凋亡，二是通过ROS水平升高及GSH耗竭激活铁死亡过程。体内外实验结果验证了Rb⁺的抗瘤效果及其良好的用药安全性。该研究为金属离子参与胶质瘤治疗提供了新的研究方向与理论支持，具备创新性科学意义和巨大的临床转化潜力。

Background:

Glioblastoma (GBM) represents one of the most aggressive malignant tumors in the central nervous system, with few available treatments. This study aims to investigate the antitumor effects of rubidium ions (Rb⁺) against GBM and to elucidate the underlying molecular mechanisms, thereby providing both theoretical rationale and experimental evidence for the potential application of Rb⁺ in GBM therapy.

Methods: 

CCK-8 method was used to evaluate the effect of Rb⁺ on the proliferation of various common tumor cells at different concentrations. U87 and U251 GBM cells were selected as representative models. The regulatory effects of Rb+ on GBM cell phenotypes were systematically evaluated through cytotoxicity assays, colony formation assays, transmission electron microscopy (TEM), energy dispersive spectroscopy (EDS), apoptosis assays, cell cycle analysis, invasion and migration assays, and apoptosis-related protein detection.

2. The differential expression genes were screened by RNA sequencing (RNA-seq), then analyzed in combination with Kyoto Encyclopedia of Genes and Genomes (KEGG) to identify potential key signaling pathways. Changes in the expression of related pathway proteins were verified by western blot. Furthermore, the pathway activator MHY1485 was used to validate the central role of this pathway in the antitumor mechanism of Rb⁺.

3. Untargeted metabolomics analysis was performed to assess the metabolic impact of Rb⁺ on GBM cells. Intracellular lactate dehydrogenase (LDH), reactive oxygen species (ROS), glutathione (GSH), and malondialdehyde (MDA) levels were measured to systematically explore the molecular mechanisms of Rb⁺-induced metabolism-related cell death in GBM cells.

4. A subcutaneous xenograft model was established in BALB/c nude mice to evaluate the antitumor efficacy of Rb⁺ in vivo. Different doses were administered to explore the effective dose range for tumor suppression. The safety of Rb⁺ was systematically evaluated by serum biochemical analysis, major organ detection, and behavioral tests.

Results: 

Rb⁺ exhibited significant inhibitory effects on multiple tumor cell lines in a dose- and time-dependent manner. Treatment with Rb⁺ in U87 and U251 cells significantly induced apoptosis and arrested the cell cycle at the G2/M phase, while suppressing cell migration and invasion in a manner dependent on time and concentration.

2. By downregulating the PI3K/AKT/mTOR pathway, Rb⁺ facilitated the induction of apoptosis in cancer cells.. Functional rescue experiments demonstrated that the mTOR activator MHY1485 partially reversed the inhibitory effects of Rb⁺, indicating the critical role of this pathway in the antitumor activity of Rb⁺.

3. Rb⁺ caused a significant rise in intracellular ROS and Fe²⁺ levels, a decrease in GSH, and an increase in MDA levels.. Through untargeted metabolomics, it was found that Rb⁺ disturbed lipid metabolism, iron regulation, and the antioxidant system, causing ferroptosis and encouraging the death of tumor cells.

4. Rb⁺ at 900 mg/kg was found to significantly hinder the growth of subcutaneous tumors in nude mice, according to in vivo experiments. Serum biochemical evaluations revealed no abnormalities in liver, kidney, or cardiac function, and examinations of major organs and behavior tests showed no significant adverse effects, supporting the safety of Rb⁺.

Conclusions:

This research comprehensively uncovered the molecular mechanism of Rb⁺ anti-GBM via two routes: one involves promoting apoptosis by suppressing the PI3K/AKT/mTOR pathway, and the other involves triggering the ferroptosis by elevating ROS levels and depleting GSH. Furthermore, the antitumor effectiveness and safety of Rb⁺ were confirmed both in vitro and in vivo. This investigation presents a new direction in research and theoretical support for the use of metal ions in treating GBM, showcasing its innovative scientific value and considerable potential for clinical transformation.