基于PERK/Nrf2信号通路研究山豆根提取物诱导鼻咽癌细胞铁死亡作用机制  

Mechanism of Sophorae Tonkinensis Radix et Rhizoma extract on iron death of nasopharyngeal carcinoma cells based on PERK/Nrf2 signaling pathway

作　　者：丁虹[1] 吴紫陆 蔡纪堂[2] DING Hong;WU Zi-lu;CAI Ji-tang(Second Clinical Medical College,Henan University of Chinese Medicine,Zhengzhou,450002,China;Henan Province Hospital of Traditional Chinese Medicine,Zhengzhou,450002,China)

机构地区：[1]河南中医药大学第二临床医学院,河南郑州450002 [2]河南省中医院,河南郑州450002

出　　处：《中草药》2023年第8期2471-2479,共9页Chinese Traditional and Herbal Drugs

基　　金：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)。

摘　　要：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 member11,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),G_(0)/G_(1)期和S期细胞比例显著降低(P<0.05、0.01),G_(2)/M期细胞比例显著增加(P<0.05、0.01),Fe^(2+)、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Fe^(2+)、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Fe^(2+)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/Nrf2Objective 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 G_(0)/G_(1) 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),Fe^(2+),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 Fe^(2+),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

关 键 词：山豆根提取物 鼻咽癌细胞 铁死亡 蛋白激酶R样内质网激酶 核因子E2相关因子2 苦参碱 氧化苦参碱 

分 类 号：R285.5[医药卫生—中药学]