| 摘要: |
| 目的： 探讨青蒿琥酯对膀胱癌T24细胞的抑制作用及诱导铁死亡的作用机制。方法： 以不同浓度的青蒿琥酯处理膀胱癌T24细胞,CCK-8法检测细胞增殖抑制率,计算IC50,并筛选出合适浓度范围进行后续实验；流式细胞术检测细胞周期变化；细胞划痕法检测细胞迁移活性；Transwell侵袭法检测细胞侵袭能力。CCK-8法检测青蒿琥酯单用以及分别与铁死亡抑制剂(DFOM)、凋亡抑制剂(Z-VAD-FMK)、坏死抑制剂(Necrostatin-1)、自噬抑制剂(CQ)联合处理后细胞的活性变化；透射电镜观察青蒿琥酯处理细胞超微结构变化；荧光探针检测青蒿琥酯单用及联合DFOM后细胞ROS水平；比色法试剂盒检测青蒿琥酯单用及联合DFOM后细胞Fe2+、GSH、MDA水平；Western blotting检测青蒿琥酯单用及联合DFOM后细胞内FTH1、GPX4及Nrf2蛋白水平。结果： 与空白对照组相比,青蒿琥酯可显著抑制T24细胞增殖(P＜0.01)；诱导T24细胞发生G1、G2期阻滞(P＜0.05),抑制细胞迁移能力(P＜0.01)和侵袭能力(P＜0.01)。与青蒿琥酯单用相比,青蒿琥酯分别与DFOM、Z-VAD-FMK、CQ、的Nec-1联用后,青蒿琥酯引起的细胞活性降低均可发生不同程度逆转(P＜0.05)。青蒿琥酯处理后的细胞线粒体体积减小、膜增厚、嵴断裂或减少；与空白对照组相比,青蒿琥酯组的ROS、Fe2+、MDA水平显著升高(P＜0.05),GSH水平降低(P＜0.05),FTH1、GPX4的蛋白表达水平降低(P＜0.05),Nrf2水平升高(P＜0.01)；与青蒿琥酯组相比,青蒿琥酯+DFOM组的ROS、Fe2+、MDA水平较青蒿琥酯组降低(P＜0.05),GSH水平升高(P＜0.01)；FTH1、GPX4蛋白表达水平升高(P＜0.05),Nrf2水平降低(P＜0.05)。结论：青蒿琥酯可以抑制膀胱癌T24细胞增殖、迁移和侵袭,阻滞细胞周期,并通过调控GSH/GPX4诱导其铁死亡,同时可能激活了其铁死亡抗性。 |
| 关键词: 青蒿琥酯 膀胱癌 铁死亡 增殖 |
| DOI： |
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| 基金项目:国家自然科学基金（81402344）；陕西省自然科学基础研究计划项目（2023-JC-YB-745、2020JM-595） |
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| To investigate the effect of artesunate on ferroptosis in bladder cancer T24 cells based on GSH/GPX4 |
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wangsiyu, wang yu
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Shaanxi University of Chinese Medicine
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| Abstract: |
| ABSTRACT: OBJECTIVE To investigate the inhibitory effect of artesunate on bladder cancer cells T24 and the mechanism of inducing ferroptosis. METHODS Different concentrations of artesunate to treat T24 cells of bladder cancer, CCK-8 method was used to detect the cell proliferation inhibition rate, calculate IC50, and select the appropriate concentration range for subsequent experiments; Cell cycle changes were detected by flow cytometry; Cell migration activity was detected by cell scratch method; Transwell invasion method was used to detect the invasion ability of cells.CCK-8 method was used to detect the changes of cell activity after artesunate was used alone or combined with ferroptosis inhibitor, desferriamine mesylate (DFOM), apoptosis inhibitor (Z-VAD-FMK), necrosis inhibitor (Necrosin-1) and autophagy inhibitor (CQ); The ultrastructural changes of artesunate treated cells were observed by transmission electron microscopy; The ROS level of cells after artesunate alone and combined with DFOM was detected by fluorescence probe; The levels of Fe2+, GSH and MDA in cells after artesunate alone and in combination with DFOM were detected by colorimetric kit; Western blotting was used to detect the levels of FTH1, GPX4 and Nrf2 protein in the cells after artesunate alone and in combination with DFOM. RESULTS Compared with control group, artesunate significantly inhibited the proliferation of T24 cells (P < 0.01). It induced G1 and G2 phase arrest of T24 cells (P < 0.05), and inhibited cell migration ability (P < 0.01) and invasion ability (P < 0.01). Compared with artesunate alone, artesunate combined with DFOM, Z-VAD-FMK, CQ and Nec-1, respectively, reversed the decrease in cell activity induced by artesunate to varying degrees (P < 0.05). After artesunate treatment, mitochondrial volume decreased, membrane thickened, ridge fracture or reduction. Compared with blank control group, ROS, Fe2+ and MDA levels in artesunate group were significantly increased (P < 0.05), GSH level was decreased (P < 0.05), protein expression levels of FTH1 and GPX4 were decreased (P < 0.05), and Nrf2 level was increased (P < 0.01). Compared with artesunate group, ROS, Fe2+ and MDA levels in artesunate +DFOM group were decreased compared with artesunate group (P < 0.05), while GSH level was increased (P < 0.01). The protein expression levels of FTH1 and GPX4 were increased (P < 0.05), while the level of Nrf2 was decreased (P < 0.05). CONCLUSION Artesunate can inhibit proliferation, migration and invasion of bladder cancer T24 cells, arrest cell cycle, induce ferroptosis by regulating GSH/GPX4, and may activate ferroptosis resistance. |
| Key words: artesunate bladder cancer ferroptosis proliferation |
