Zhang Ting, Wang Cui-Cui, Zhu Cong, Zhou Xin-Yu, Jia Xiu-Hong
Department of Pediatrics， Binzhou Medical University Affiliated Hospital， Binzhou 256603， Shandong Province， China.
Zhongguo Shi Yan Xue Ye Xue Za Zhi. 2025 Aug;33(4):1007-1015. doi: 10.19746/j.cnki.issn.1009-2137.2025.04.011.
To investigate the effect of piperlongumine(PL) on the proliferation and ferroptosis of human adriamycin-resistant chronic myeloid leukemia K562/ADR cells, and to explore its possible molecular mechanism.
CCK-8 assay was used to detect the effect of PL on the survival rate of K562/ADR cells and to screen the appropriate drug concentration. After K562/ADR cells were treated with low, medium and high concentrations of PL(2, 4, and 6 μmol/L), EdU proliferation assay and plate colony formation assay were used to detect cell proliferation and colony formation ability. CCK-8 assay was used to detect the effects of different inhibitors (Fer-1, Z-VAD, Nec-1) combined with PL on cell proliferation. The intracellular Fe, ROS, malondialdehyde(MDA) and glutathine(GSH) contents were respectively detected by iron ion colorimetry, DCFH-DA fluorescent probe, MDA and GSH kits. RT-qPCR and Western blot were respectively used to detect the expression level of mRNA and protein in cells. Bioinformatics websites predicted miRNA that could target and regulate . RT-qPCR was used to detect the effects of different concentrations of PL on the expression levels of the predicted miRNA. Dual luciferase gene reporter assay was used to verify the targeting relationship between and . After treating cells with PL or PL+ inhibitor, the Fe, ROS, MDA, GSH centents and GPX4 protein expression levels in cells were detected.
PL inhibited K562/ADR cell proliferation in a concentration-dependent manner（ =0.979）. Compared with the blank control group, the survival rate, EdU positive cells rate in low, medium and high concentration PL groups were significantly decreased ( < 0.01). Compared with the PL group alone, the survival rate of cells in the Z-VAD+PL group was increased slightly ( < 0.05). The cell survival rate was significantly increased in medium or high concentration PL+Fer-1 group ( < 0.01). Compared with blank control group, ROS expression level in low concentration PL group was slightly increased ( < 0.05), and GSH content was slightly decreased ( < 0.05). In medium and high concentration PL groups, the contents of Fe, ROS and MDA were significantly increased ( < 0.01), while the contents of GSH, expression of mRNA and protein were significantly decreased( < 0.01). Bioinformatics prediction and double luciferase reporter gene experiment confirmed the targeting relationship between and . Compared with the blank control group, the expression level of in cells of medium and high concentration PL groups was significantly increased ( < 0.01). Compared with PL group alone, the intracellular Fe, ROS and MDA contents in PL+ inhibitor group were all decreased ( < 0.01), while GSH content and GPX4 protein expression levels were significantly increased ( < 0.01).
Medium and high concentrations of PL can inhibit the proliferation of K562/ADR cells by inducing ferroptosis, which is related to the regulation of pathway.
探讨胡椒碱（PL）对人耐阿霉素慢性髓系白血病K562/ADR细胞增殖和铁死亡的影响，并探究其可能的分子机制。
采用CCK-8法检测PL对K562/ADR细胞存活率的影响，筛选合适的药物浓度。将K562/ADR细胞分别用低、中、高浓度的PL（2、4和6 μmol/L）处理后，采用EdU增殖实验和平板克隆形成实验检测细胞增殖及克隆形成能力。采用CCK-8法检测不同抑制剂（Fer-1、Z-VAD、Nec-1）与PL联合应用对细胞增殖的影响。分别采用铁离子比色法、DCFH-DA荧光探针、丙二醛（MDA）和谷胱甘肽（GSH）试剂盒检测细胞内铁、活性氧（ROS）、MDA和GSH含量。分别采用RT-qPCR和蛋白质免疫印迹法检测细胞中mRNA和蛋白质的表达水平。利用生物信息学网站预测可靶向调控 的微小RNA（miRNA）。采用RT-qPCR检测不同浓度PL对预测的miRNA表达水平的影响。采用双荧光素酶基因报告实验验证 与 之间的靶向关系。用PL或PL+抑制剂处理细胞后，检测细胞内铁、ROS、MDA、GSH含量及谷胱甘肽过氧化物酶4（GPX4）蛋白表达水平。
PL以浓度依赖性方式抑制K562/ADR细胞增殖（ =0.979）。与空白对照组相比，低、中、高浓度PL组的细胞存活率、EdU阳性细胞率均显著降低（ < 0.01）。与单独PL组相比，Z-VAD+PL组细胞存活率略有升高（ < 0.05）。中或高浓度PL+Fer-1组细胞存活率显著升高（ < 0.01）。与空白对照组相比，低浓度PL组ROS表达水平略有升高（ < 0.05），GSH含量略有降低（ < 0.05）。中、高浓度PL组铁、ROS和MDA含量显著升高（ < 0.01），而GSH含量、 mRNA和蛋白表达显著降低（ < 0.01）。生物信息学预测和双荧光素酶报告基因实验证实了 与 之间的靶向关系。与空白对照组相比，中、高浓度PL组细胞中 的表达水平显著升高（ < 0.01）。与单独PL组相比，PL+抑制剂组细胞内铁、ROS和MDA含量均降低（ < 0.01），而GSH含量和GPX4蛋白表达水平显著升高（ < 0.01）。
中、高浓度PL可通过诱导铁死亡抑制K562/ADR细胞增殖，这与 通路的调控有关。
