Xue Tingting, Tao Yuchen, Wu Zong, Yuan Chenyue, Wang Yanlu, Xu Hao, Cai Shuyang, Wang Ziliang, Lu Jiahui
Shanghai Municipal Hospital of Traditional Chinese Medicine, Shanghai University of Traditional Chinese Medicine, Shanghai, China.
Shanghai Municipal Hospital of Traditional Chinese Medicine, Shanghai University of Traditional Chinese Medicine, Shanghai, China.
J Ethnopharmacol. 2025 Feb 11;341:119339. doi: 10.1016/j.jep.2025.119339. Epub 2025 Jan 7.
Myelodysplastic syndrome (MDS) is a hematologic malignancy that presents a unique opportunity for traditional Chinese medicine (TCM) to demonstrate its distinctive value in treatment. Realgar, a component of TCM, has shown notable potential in alleviating clinical symptoms and improving the prognosis of MDS patients. However, the precise mechanisms underlying the treatment of MDS with realgar, particularly its effects on apoptosis-related pathways, remain poorly understood.
This study aimed to investigate the pro-apoptotic effects of realgar on MDS cells and to elucidate the underlying molecular mechanisms.
We explored the targets and pathways of realgar's action on MDS using public databases, network pharmacology, and RNA sequencing. Various techniques were employed, including cell transfection, Cell Counting Kit-8 (CCK8) assay, Cellular Thermal Shift Assay (CETSA), Western blot (WB), quantitative real-time polymerase chain reaction (qRT-PCR), apoptosis and glycolysis assays, extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) measurements, dual-luciferase reporter assays, and immunofluorescence, to investigate the regulatory mechanisms involving STAT3, glycolysis, and apoptosis. Hematoxylin and eosin (HE) staining was utilized to assess realgar's toxicity. Apoptosis and hemogram changes were analyzed to evaluate the therapeutic effect of realgar on MDS transgenic mice.
Analysis of public data indicated that apoptosis-related genes are downregulated in MDS patients. Through network pharmacology, CETSA, qRT-PCR, WB, apoptosis assays, and STAT3 overexpression cell transfection, we discovered that realgar inhibits STAT3 expression. Further investigation using RNA sequencing suggested that glycolysis may be involved in this regulatory process. ECAR, OCR, glycolysis assays, WB, apoptosis assays, and glycolysis inhibitor experiments demonstrated that glycolytic function was inhibited. Additionally, GLUT1 expression was significantly decreased, and GLUT1 was found to directly bind to STAT3. In MDS mice, realgar treatment enhanced levels of white blood cells, red blood cells, hemoglobin, and platelets, and increased apoptosis levels.
Our findings reveal that realgar exerts a significant pro-apoptotic effect on MDS cells in both in vivo and in vitro models. Further analysis demonstrated that realgar regulates the STAT3 pathway, leading to GLUT1-mediated glycolysis alterations that ultimately induce apoptotic pathways, as represented by BCL2. These discoveries hold significant implications for the basic research and clinical diagnosis and treatment of MDS.
骨髓增生异常综合征（MDS）是一种血液系统恶性肿瘤，为传统中医（TCM）展现其独特治疗价值提供了一个独特机会。雄黄作为中药的一种成分，在缓解MDS患者临床症状和改善预后方面已显示出显著潜力。然而，雄黄治疗MDS的确切机制，尤其是其对凋亡相关途径的影响，仍知之甚少。
本研究旨在探讨雄黄对MDS细胞的促凋亡作用，并阐明其潜在的分子机制。
我们利用公共数据库、网络药理学和RNA测序技术，探索雄黄对MDS作用的靶点和途径。采用了多种技术，包括细胞转染、细胞计数试剂盒-8（CCK8）检测、细胞热位移分析（CETSA）、蛋白质免疫印迹法（WB）、定量实时聚合酶链反应（qRT-PCR）、凋亡和糖酵解检测、细胞外酸化率（ECAR）和耗氧率（OCR）测量、双荧光素酶报告基因检测以及免疫荧光，以研究涉及信号转导和转录激活因子3（STAT3）、糖酵解和凋亡的调控机制。采用苏木精-伊红（HE）染色评估雄黄的毒性。分析凋亡和血常规变化以评估雄黄对MDS转基因小鼠的治疗效果。
对公共数据的分析表明，凋亡相关基因在MDS患者中表达下调。通过网络药理学、CETSA、qRT-PCR、WB、凋亡检测以及STAT3过表达细胞转染，我们发现雄黄抑制STAT3表达。使用RNA测序进一步研究表明，糖酵解可能参与了这一调控过程。ECAR、OCR、糖酵解检测、WB、凋亡检测以及糖酵解抑制剂实验表明，糖酵解功能受到抑制。此外，葡萄糖转运蛋白1（GLUT1）表达显著降低，且发现GLUT1直接与STAT3结合。在MDS小鼠中，雄黄治疗提高了白细胞、红细胞、血红蛋白和血小板水平，并增加了凋亡水平。
我们的研究结果表明，雄黄在体内和体外模型中均对MDS细胞具有显著的促凋亡作用。进一步分析表明，雄黄调节STAT3途径，导致GLUT1介导的糖酵解改变，最终诱导以B细胞淋巴瘤-2（BCL2）为代表的凋亡途径。这些发现对MDS的基础研究以及临床诊断和治疗具有重要意义。
