目的 基于蛋白激酶R样内质网激酶（protein kinase R-like endoplasmic reticulum kinase，PERK）/核因子E2相关因子2（nuclear factor erythroid-2-related factor 2，Nrf2）通路探究山豆根提取物（Sophorae Tonkinensis Radix et Rhizoma extract，TSRE）诱导鼻咽癌细胞铁死亡作用机制。方法 为研究TSRE对鼻咽癌CNE1细胞的抑制作用，设置对照组和TSRE低、中、高剂量（100、200、400 mg/L）组；为研究铁死亡在TSRE致CNE1细胞增殖抑制中的作用以及PERK/Nrf2信号通路对铁死亡的影响，TSRE处理过程中分别用铁死亡抑制剂Fer-1、PERK抑制剂GSK2606414、Nrf2 siRNA进行干预。MTT法检测细胞活性；流式细胞术检测细胞周期；荧光探针检测活性氧（reactive oxygen species，ROS）和脂质过氧化水平；比色法检测铁离子和谷胱甘肽（glutathione，GSH）水平；Western blotting检测溶质载体家族7成员11（solute carrier family 7 member 11，SLC7A11）、谷胱甘肽过氧化物酶4（glutathione peroxidase 4，GPX4）、p-PERK、Nrf2和血红素氧合酶-1（heme oxygenase-1，HO-1）蛋白表达；免疫荧光检测p-PERK表达和Nrf2入核情况。结果TSRE组细胞存活率以及SLC7A11和GPX4蛋白表达水平均显著降低（P＜0.05、0.01），G₀/G₁期和S期细胞比例显著降低（P＜0.05、0.01），G₂/M期细胞比例显著增加（P＜0.05、0.01），Fe²⁺、ROS和脂质过氧化水平升高（P＜0.01），GSH水平显著降低（P＜0.01）；与TSRE组比较，Fer-1干预缓解了TSRE诱导的细胞存活率降低（P＜0.01），降低CNE1细胞中Fe²⁺、ROS及脂质过氧化水平（P＜0.01），提高细胞中GSH水平（P＜0.01）。机制研究表明，TSRE诱导Nrf2表达水平的升高并促进Nrf2入核（P＜0.01）；与NC siRNA组比较，Nrf2 siRNA能抑制TSRE引起的细胞脂质过氧化和Fe²⁺水平升高和GSH水平降低（P＜0.01）。进一步研究发现，TSRE组细胞中的p-PERK蛋白表达水平较对照组明显升高（P＜0.01）；PERK抑制剂GSK2606414减弱了TSRE诱导的Nrf2信号活化（P＜0.01）。结论TSRE诱导的鼻咽癌细胞铁死亡可能与其激活PERK/Nrf2信号通路有关。

Objective To explore the effect of Shandougen (Sophorae Tonkinensis Radix et Rhizoma) extract (TSRE) on iron death of nasopharyngeal carcinoma cells based on protein kinase R-like endoplasmic reticulum kinase (PERK)/nuclear factor E2-related factor 2 (Nrf2) pathway. Methods In order to study the inhibitory effect of TSRE on nasopharyngeal carcinoma CNE1 cells, control group, low-, medium- and high-dose (100, 200, 400 mg/L) TSRE groups were set up. In order to study the role of iron death in proliferation inhibition of CNE1 cells induced by TSRE and influence of PERK/Nrf2 signaling pathway on iron death, iron death inhibitor Fer-1, PERK inhibitor GSK2606414 and Nrf2 siRNA were used to intervene in TSRE treatment. Cell activity was detected by MTT assay; Cell cycle was detected by flow cytometry. The levels of reactive oxygen species (ROS) and lipid peroxidation were detected by fluorescence probe. The levels of iron ion and glutathione (GSH) were detected by colorimetry. Western blotting was used to detect the protein expressions of solute carrier family 7 member 11 (SLC7A11), glutathione peroxidase 4 (GPX4), p-PERK, Nrf2 and heme oxygenase-1 (HO-1). Immunofluorescence was used to detect the expression of p-PERK and nucleation of Nrf2. Results In TSRE group, cell survival rate and expression levels of SLC7A11 and GPX4 protein were significantly decreased (P < 0.05, 0.01), proportion of G₀/G₁ and S phase cells were significantly decreased (P < 0.05, 0.01), proportion of G₂/M phase cells was significantly increased (P < 0.05, 0.01), Fe²⁺, ROS and lipid peroxidation levels were increased (P < 0.01), while GSH level was decreased (P < 0.01). Compared with TSRE group, the intervention of Fer-1 alleviated the decrease of cell survival rate induced by TSRE (P < 0.01), decreased the levels of Fe²⁺, ROS and lipid peroxidation in CNE1 cells (P < 0.01) and increased the level of GSH in cells (P < 0.01). The mechanism study showed that TSRE induced the increase of Nrf2 expression level and promoted Nrf2 nucleation (P < 0.01); Compared with NC siRNA group, Nrf2 siRNA could inhibit the increase of lipid peroxidation, Fe²⁺ levels and the decrease of GSH level induced by TSRE (P < 0.01). Further study showed that the expression level of p-PERK protein in TSRE group was significantly higher than that in control group (P < 0.01), PERK inhibitor GSK2606414 weakened the activation of Nrf2 signal induced by TSRE (P < 0.01). Conclusion TSRE-induced iron death in nasopharyngeal carcinoma cells may be related to its activation of PERK/Nrf2 signaling pathway.

河南中医药大学2021年度研究生科研能力创新提升计划（2021KYCX025）；河南省中医药科学研究专项课题（2022ZY1057）