Zhang Yong-Gen, Yan Xiao-Fei, Liu Feng, Hao Wen-Zhe, Cai Yue, Liu Ying, Liu Lan-Lin, Li Xue-Jun
Anhui University of Chinese Medicine Hefei 230012, China.
the Second Affiliated Hospital of Anhui University of Chinese Medicine Hefei 230061, China.
Zhongguo Zhong Yao Za Zhi. 2024 Dec;49(23):6459-6467. doi: 10.19540/j.cnki.cjcmm.20240919.501.
This study primarily investigated the mechanism of Astragalus polysaccharides(APS), a Chinese medicinal material, in regulating the Nrf2/SLC7A11/GPX4 signaling pathway to induce ferroptosis in ovarian cancer cells(Caov-3 and SKOV3 cells). Caov-3 and SKOV3 cells were divided into control(Vehicle) group, APS group, glutathione peroxidase 4 inhibitor(RSL3) group, and APS+RSL3 group. After 48 h of intervention, the activity and morphology of the cells in each group were observed. The cell counting kit-8(CCK-8) method was used to determine the half-maximal inhibitory concentration(IC_(50)), while colony formation and EdU assays were conducted to assess cell proliferation. Biochemical reagents were used to detect lipid reactive oxygen species(L-ROS), malondialdehyde(MDA), divalent iron ions(Fe(2+)), and glutathione(GSH) in Caov-3 cells. Transmission electron microscopy was employed to observe the morphological changes of mitochondria in Caov-3 cells. Bioinformatics analysis were used to screen potential target genes of APS in ovarian cancer cells. Western blot and RT-PCR were applied to measure the protein and mRNA expression of Nrf2, SLC7A11, and GPX4. The results revealed that APS effectively inhibited the activity and proliferation of ovarian cancer cells, significantly increased the expression levels of L-ROS, MDA, and Fe(2+)(P<0.001), and significantly reduced the expression level of GSH(P<0.001). Under electron microscopy, the mitochondria of Caov-3 cells appeared significantly smaller, with a marked increase in the density of the bilayer membrane, disappearance of mitochondrial cristae, and rupture of the outer mitochondrial membrane. These effects were more pronounced when APS was combined with RSL3. Bioinformatics screening identified Nrf2, SLC7A11, and GPX4 as potential target genes for APS in ovarian cancer cells. APS was shown to reduce the protein and mRNA expression of Nrf2, SLC7A11, and GPX4(P<0.01), with the APS+RSL3 showing even more significant effects(P<0.001). In conclusion, APS can induce ferroptosis in ovarian cancer cells, and its mechanism may be related to the regulation of the Nrf2/SLC7A11/GPX4 signaling pathway, providing an experimental basis for the use of APS injections in the treatment of ovarian cancer.
本研究主要探讨中药黄芪多糖（APS）调控Nrf2/SLC7A11/GPX4信号通路诱导卵巢癌细胞（Caov-3和SKOV3细胞）铁死亡的机制。将Caov-3和SKOV3细胞分为对照组（溶媒）、APS组、谷胱甘肽过氧化物酶4抑制剂（RSL3）组和APS+RSL3组。干预48小时后，观察各组细胞的活性和形态。采用细胞计数试剂盒-8（CCK-8）法测定半数抑制浓度（IC50），同时进行集落形成和EdU检测以评估细胞增殖。使用生化试剂检测Caov-3细胞中的脂质活性氧（L-ROS）、丙二醛（MDA）、二价铁离子（Fe2+）和谷胱甘肽（GSH）。采用透射电子显微镜观察Caov-3细胞线粒体的形态变化。利用生物信息学分析筛选APS在卵巢癌细胞中的潜在靶基因。应用蛋白质免疫印迹法和逆转录聚合酶链反应检测Nrf2、SLC7A11和GPX4的蛋白和mRNA表达。结果显示，APS能有效抑制卵巢癌细胞的活性和增殖，显著提高L-ROS、MDA和Fe2+的表达水平（P<0.001），并显著降低GSH的表达水平（P<0.001）。电镜下，Caov-3细胞的线粒体明显变小，双层膜密度显著增加，线粒体嵴消失，线粒体外膜破裂。当APS与RSL3联合使用时，这些效应更为明显。生物信息学筛选确定Nrf2、SLC7A11和GPX4为APS在卵巢癌细胞中的潜在靶基因。APS可降低Nrf2、SLC7A11和GPX4的蛋白和mRNA表达（P<0.01），APS+RSL3组的作用更显著（P<0.001）。综上所述，APS可诱导卵巢癌细胞铁死亡，其机制可能与调控Nrf2/SLC7A11/GPX4信号通路有关，为APS注射液用于治疗卵巢癌提供了实验依据。
