水凝胶负载Erastin诱导子宫内膜癌细胞铁死亡的作用研究

Ferroptosis in Endometrial Cancer cells Induced by Erastin-Loaded Hydrogels

目的:利用温敏性水凝胶泊洛沙姆407(P407)负载Erastin缓释系统诱导人子宫内膜癌细胞(Ishikawa)铁死亡。方法:构建并评估负载Erastin水凝胶复合物(P407^(Era))的物理表征、相变时间和缓释性能。实验分为对照(control,Ctrl)组、水凝胶(P407)组、Erastin处理(Erastin)组和水凝胶负载Erastin(P407^(Era))组。CCK-8检测细胞增殖情况,ROS试剂盒检测细胞内活性氧含量,TUNEL原位细胞凋亡试剂盒检测细胞凋亡,Mito-Tracker Red CMXRos检测细胞线粒体膜电位,Mito-FerroGreen检测线粒体内Fe2+含量,免疫荧光和RT-qPCR检测铁死亡相关分子的表达水平。结果:P407水凝胶的相变时间与浓度呈负相关,18%浓度的P407水凝胶复合物药物缓释性能较优。与对照组相比,P407组、Erastin组和P407^(Era)组的Ishikawa细胞增殖、迁移和侵袭均受明显抑制,且P407^(Era)组比Erastin组抑制增殖、迁移和侵袭作用更加明显;Erastin组及P407^(Era)组细胞活性氧水平、细胞凋亡相对数量显著升高;细胞线粒体膜电位和线粒体Fe^(2+)结果显示,Erastin组和P407^(Era)组细胞线粒体膜电位显著下降,且线粒体Fe2+含量显著升高;免疫荧光结果显示Erastin组及P407^(Era)组GPX4含量显著降低,NRF2含量无明显变化;Erastin组及P407^(Era)组TFR1、NCOA4和P53的mRNA表达水平显著升高。结论:温敏性水凝胶泊洛沙姆407负载Erastin缓释系统诱导人子宫内膜癌细胞发生铁死亡。

Objective To induce ferroptosis in human endometrial cancer cells(Ishikawa)by a sustained-release thermo⁃sensitive hydrogel based on poloxamer 407(P407)containing Erastin.Methods The physical characterization,phase tran⁃sition time,and sustained release properties of Erastin-loaded hydrogel complex(P407^(Era))were constructed and evaluated.The experiment was divided into a control control(Ctrl)group,hydrogel(P407)group,erastin-treated(Erastin)group,and hydrogel-loaded Erastin(P407^(Era))group.CCK-8 assay,ROS kit,and TUNEL assay kit were used to detect cell pro⁃liferation,intracellular ROS,and cell apoptosis,respectively.Cell mitochondrial membrane potential and Fe^(2+)in mitochon⁃dria were observed using Mito-Tracker Red CMXRos and Mito-FerroGreen,respectively.Immunofluorescence and RTqPCR were employed to determine the expressions of ferroptosis-related molecules.Results The phase change time of P407 was negatively correlated with concentration,and the P407 with a concentration of 18%had better drug sustained release performance.Compared with the conditions in the Ctrl group,the Ishikawa cell proliferation,migration and invasion were significantly inhibited in the P407,Erastin,and P407^(Era)groups,and the P407^(Era)group had more inhibition than the Erastin group.Compared with the Ctrl group,the Erastin and P407^(Era)groups had significantly increased ROS and relative amount of cell apoptosis,decreased mitochondrial membrane potential of cells,elevated Fe2+in mitochondria,and reduced GPX4,with no marked change in the NRF2.Additionally,the mRNA expressions of TFR1,NCOA4,and P53 in the Erastin group and the P407^(Era)group were significantly increased.Conclusion Erastin-c2ʍloaded P407 induces ferroptosis in human endome⁃trial cancer cells.