南方医科大学学报

南方医科大学学报 ›› 2023, Vol. 43 ›› Issue (5): 764-771.doi: 10.12122/j.issn.1673-4254.2023.05.12

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吡罗克酮乙醇胺盐通过PI3K/AKT通路破坏胶质瘤细胞的线粒体动力学

徐文琴，叶静静，王 飞，陈天兵   

  1. 皖南医学院重大疾病非编码RNA转化研究安徽普通高校重点实验室，安徽 芜湖 241002；弋矶山医院中心实验室，安徽 芜湖 241001；芜湖市中医医院，安徽 芜湖 241001
  • 出版日期:2023-05-20 发布日期:2023-06-12

Piroctone olamine disrupts mitochondrial dynamics in glioma cells through the PI3K/AKT pathway

XU Wenqin, YE Jingjing, WANG Fei, CHEN Tianbing   

  1. Key Laboratory of Noncoding RNA Transformation Research of Anhui Higher Education Institution, Wannan Medical College, Wuhu 241002, China; Central Laboratory of Yijishan Hospital, Wuhu 241001, China; Wuhu Hospital of Traditional Chinese Medicine, Wuhu 241001, China
  • Online:2023-05-20 Published:2023-06-12

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摘要： 目的 探究吡罗克酮乙醇胺盐（PO）对胶质瘤细胞U251和U373增殖抑制和促凋亡的作用及其机制。方法 培养细胞，经不同浓度的PO处理，CCK-8法和EdU实验检测细胞增殖情况；克隆形成实验检测细胞克隆形成；流式细胞术检测细胞凋亡；JC-1检测线粒体膜电位水平；荧光探针检测线粒体形态学改变，Western blot检测线粒体分裂蛋白DRP1和融合蛋白OPA1；转录组测序和差异基因富集分析后进行Western blot检测验证PI3K，AKT和p-AKT蛋白表达水平。结果 CCK-8结果显示，PO可以抑制U251和U373细胞增殖，并呈时间和剂量依赖性（P<0.001）；EdU实验显示，PO处理组的细胞增殖水平明显下降，克隆形成实验的细胞克隆数也明显减少（P<0.01）；流式细胞术显示，PO处理组细胞凋亡率增加（P<0.05）；荧光探针检测显示，线粒体膜电位水平下降并可致线粒体形态学改变；富集分析显示，下调的基因显著富集于PI3K/AKT通路；Western blot结果显示，PO下调PI3K，AKT和p-AKT蛋白表达水平（P<0.05）。结论 PO通过PI3K/AKT通路干扰线粒体融合裂变功能，进而抑制胶质瘤细胞的增殖和增加凋亡。

关键词: 吡罗克酮乙醇胺盐；线粒体裂变；胶质瘤；PI3K/AKT通路

Abstract: Objective To investigate the growth-inhibitory and pro-apoptotic effects of piroctone olamine (PO) on glioma cells and explore the underlying mechanism. Methods Human glioma cell lines U251 and U373 were treated with PO and the changes in cell proliferation were detected using CCK-8 assay and EdU assay. Clone formation assay and flow cytometry were used to examine the changes in clone formation ability and apoptosis of the treated cells. Mitochondrial membrane potential of the cells and morphological changes of the mitochondria were detected using JC-1 staining and a fluorescence probe, respectively. The expressions of mitochondrial fission protein DRP1 and the fusion protein OPA1 were determined with Western blotting. Transcriptome sequencing and differential gene enrichment analysis was performed, and the expression levels of PI3K, AKT and p-AKT in the treated cells were verified using Western blotting. Results CCK-8 assay showed that PO significantly inhibited the proliferation of U251 and U373 cells in a time- and dose-dependent manner (P<0.001). EdU test showed that the proliferative activity of PO-treated cells was significantly decreased, and the number of cell colonies also decreased significantly (P<0.01). PO treatment significantly increased apoptotic rates (P<0.01), decreased mitochondrial membrane potential and caused obvious changes in mitochondrial morphology of the cells. Pathway enrichment analysis showed that the down-regulated genes were significantly enriched in the PI3K/AKT pathway, which was verified by Western blotting showing significantly down-regulated expression levels of PI3K, AKT and p-AKT in PO-treated cells (P<0.05). Conclusion PO interferes with mitochondrial fusion and fission function through the PI3K/AKT pathway, thereby inhibiting the proliferation and increasing apoptosis of glioma cells.

Key words: piroctone olamine; mitochondrial fission; glioma; PI3K/AKT pathway