NR4A1通过上调NRF2表达抑制顺铂诱导的近端肾小管上皮细胞铁死亡  

NR4A1 suppresses cisplatin-induced ferroptosis in renal proximal tubular epithelial cells by up-regulating the expression of NRF2

作　　者：薛嵘[1] 马金刚 黄军悦[1] 李莹屏[1] 高佩娟 黄文辉[1] 杨晓军[3] 钱睿 赵娟[1] Xue Rong;Ma Jingang;Huang Junyue;Li Yingping;Gao Peijuan;Huang Wenhui;Yang Xiaojun;Qian Rui;Zhao Juan(Nephrology Department,Gansu Provincial Hospital,Lanzhou 730000,China;Institute for Drug and Instrument Control of Xining Joint Logistics Support Center,Lanzhou 730050,China;Department of General Surgery,Gansu Provincial Hospital,Lanzhou 730000,China)

机构地区：[1]甘肃省人民医院肾内科,兰州730000 [2]西宁联勤保障中心药品仪器监督检验站,兰州730050 [3]甘肃省人民医院普外科,兰州730000

出　　处：《中华肾脏病杂志》2023年第8期600-609,共10页Chinese Journal of Nephrology

基　　金：国家自然科学基金(81660398);甘肃省自然科学基金项目(21JR7RA625,22JR11RA256);兰州市科技计划项目(2020-ZD-23)。

摘　　要：目的探究核受体亚家族4 A组成员1(nuclear receptor subfamily 4 group A member 1,NR4A1)在缓解顺铂对近端肾小管上皮细胞毒性的作用及其分子机制。方法通过"Tabula-muris"单细胞转录组测序数据库分析肾脏组织各细胞亚群NR4A1基因的表达。在近端肾小管上皮细胞HK-2细胞系及原代细胞中,通过慢病毒感染以过表达NR4A1基因。采用细胞增殖与毒性检测试剂盒(cell counting kit-8,CCK-8)检测顺铂的细胞毒性。细胞用碘化丙啶(propidium iodide,PI)单染后通过流式细胞仪检测细胞的死亡比例。通过实时荧光定量PCR和Western印迹法检测细胞中NR4A1和核因子E2相关因子2(nuclear factor erythroid 2-related factor 2,NRF2)基因的表达。通过检测丙二醛(malondialdehyde,MDA)和氧化型谷胱甘肽(oxidized glutathione,GSSG)以及脂质活性氧(reactive oxygen species,ROS)的含量分析细胞铁死亡程度。结果单细胞转录组数据分析的结果表明NR4A1基因表达在肾脏组织的近端肾小管上皮细胞亚群中最低。50μmol/L和100μmol/L顺铂能够显著诱导近端肾小管上皮细胞MDA、GSSG和脂质ROS含量的上升(均P<0.01),且顺铂浓度越高诱导MDA、GSSG和脂质ROS增加越多。相比对照HK-2细胞,过表达NR4A1的HK-2细胞脂质ROS含量及铁离子含量显著较低(均P<0.01),过表达NR4A1抑制了顺铂对近端肾小管上皮细胞的毒性及其诱导的铁死亡。分子机制研究发现,在近端肾小管上皮细胞中,过表达NR4A1上调了抗铁死亡基因NRF2的表达(P<0.01)。进一步单细胞转录组数据分析的结果表明,与NR4A1在肾脏组织细胞亚群的表达状态相似,NRF2表达在近端肾小管上皮细胞中也最低。结论顺铂能够诱导近端肾小管上皮细胞发生铁死亡,且浓度越高越明显。NR4A1通过上调近端肾小管上皮细胞NRF2的表达抑制顺铂诱导的细胞铁死亡,从而缓解顺铂对细胞的毒性。Objective To explore the role and mechanism of nuclear receptor subfamily 4 group A member 1(NR4A1)in suppressing cisplatin nephrotoxicity.Methods The expression of NR4A1 gene in renal cell subpopulations was analyzed using the"Tabula-muris"single cell transcriptome sequencing database.NR4A1 gene was over-expressed by lentivirus infection in HK-2 cell line and primary renal proximal tubular epithelial cells.Cell counting kit-8 was used to detect the cytotoxicity of cisplatin.The cell death ratio was analyzed using propidium iodide(PI)staining by flow cytometry.The expression of NR4A1 and nuclear factor erythroid 2-related factor 2(NRF2)was detected by real-time fluorescent quantitative PCR and Western blotting.Ferroptosis was analyzed by detecting the contents of malondialdehyde(MDA),oxidized glutathione(GSSG)and lipid reactive oxygen species(ROS).Results The single cell transcriptome sequencing database showed that NR4A1 gene was the lowest expression in renal proximal tubular epithelial cell subsets.Cisplatin(50μmol/L or 100μmol/L)could significantly induce MDA,GSSG and lipid ROS production in renal proximal tubular epithelial cells(all P<0.01),and higher cisplatin concentration accompanied with a more increase of MDA,GSSG and lipid ROS.Compared with the control HK-2 cells,the lipid ROS content and iron ion content of HK-2 cells over-expressing NR4A1 were significantly lower(all P<0.01),and the over-expression of NR4A1 inhibited cisplatin-induced cytotoxicity and ferroptosis in renal proximal tubular epithelial cells.Mechanistically,NR4A1 up-regulated the expression of anti-ferroptosis gene NRF2 in proximal renal tubular epithelial cells(P<0.01).Furthermore,single cell data analysis showed that,similar to the expression of NR4A1 in renal tissue subsets,NRF2 was also the lowest in renal proximal tubular epithelial cells.Conclusions Cisplatin can induce ferroptosis of renal proximal tubular epithelial cells in a dose-dependent manner.NR4A1 can inhibit cisplatin-induced ferroptosis by up-regulating NRF2 in renal

关 键 词：铁死亡 顺铂 上皮细胞 肾小管 近端 核受体亚家族4 A组 成员1 NRF2 

分 类 号：R692.6[医药卫生—泌尿科学]