G6PD介导的还原应激在砷致细胞恶性转化中的作用  

The role of glucose-6-phosphate dehydrogenase-mediated reduction stress in arsenic-induced cell malignant transformation

作　　者：兰斓 胡淮 吴昊[1] 沈彬晴 陈慧婷 杨乾磊 安艳[1] Lan Lan;Hu Huai;Wu Hao;Shen Binqing;Chen Huiting;Yang Qianlei;An Yan(Department of Toxicology,School of Public Health,Suzhou Medical College,Soochow University,Suzhou 215021,China;China-Japan Joint Laboratory on Environmental Health,School of Public Health,Suzhou Medical College,Soochow University,Suzhou 215021,China)

机构地区：[1]苏州大学苏州医学院公共卫生学院卫生毒理学系,苏州215021 [2]苏州大学苏州医学院公共卫生学院中日环境与健康联合实验室,苏州215021

出　　处：《中华地方病学杂志》2024年第6期431-439,共9页Chinese Journal of Endemiology

基　　金：国家自然科学基金(81872646、81811540034、81573173、82381240027);国家级和江苏省大学生创新创业训练计划项目(202210285067Z、202210285069Z、202210285084P、202210285197K)

摘　　要：目的探讨糖代谢关键酶葡萄糖-6-磷酸脱氢酶(G6PD)介导的还原应激在砷致细胞恶性转化中的作用以及具体机制。方法以0.0(对照组)、1.0μmol/L(染砷组)亚砷酸钠(NaAsO_(2))处理永生化人皮肤角质形成细胞(HaCaT细胞),利用细胞生长动力学、细胞划痕和软琼脂集落形成实验检测细胞恶性转化指标。在砷处理不同时期(0、1、7、14、21、28、35代细胞),通过相应试剂盒和免疫印迹(Western blot)检测NaAsO_(2)对HaCaT细胞糖代谢[葡萄糖-6-磷酸(G6P)、乳酸、乙酰辅酶A、G6PD水平及己糖激酶2(HK-2)、6-磷酸果糖-2-激酶/果糖-2,6-二磷酸酶3(PFKFB3)、丙酮酸脱氢酶激酶1(PDK1)、6-磷酸葡萄糖脱氢酶(PGD)、G6PD蛋白表达水平]的影响;提取线粒体,通过相应试剂盒检测NaAsO_(2)对HaCaT细胞及线粒体氧化还原[还原型烟酰胺腺嘌呤二核苷酸磷酸(NADPH)/烟酰胺腺嘌呤二核苷酸磷酸(NADP+)比值、还原型谷胱甘肽(GSH)/氧化型谷胱甘肽(GSSG)比值]的影响;使用小干扰RNA(siRNA)干预方法,检测G6PD对还原应激及NaAsO_(2)诱导的HaCaT细胞恶性转化的影响。结果1.0μmol/L NaAsO_(2)培养HaCaT细胞至35代(T-HaCaT细胞),与传代匹配的0.0μmol/L NaAsO_(2)培养HaCaT细胞相比[(33.797±0.280)h、0.177±0.015、(13.667±2.625)个],倍增时间[(24.042±0.479)h]较短(t=30.45,P<0.001),细胞迁移率(0.396±0.039)较高(t=9.08,P<0.001),软琼脂集落数量[(73.667±4.450)个]较多(t=20.11,P<0.001)。与同组0代和同代对照组细胞相比,染砷组细胞糖代谢指标G6P水平在1、7、14、21、28、35代均较高(均P<0.05),乳酸和G6PD水平在14、21、28、35代均较高(均P<0.05),乙酰辅酶A水平在21、28、35代均较低(均P<0.05),HK-2、PFKFB3、PDK1、PGD、G6PD蛋白表达水平在7、14、21、28、35代均较高(均P<0.05);细胞NADPH/NADP+比值在1、14、21、28、35代均较高(均P<0.05),GSH/GSSG比值在21、28、35代均较高(均P<0.05);线粒体NADPH/NADP+比值在1、7、21、28、35代Objective To study the role and specific mechanisms of glucose-6-phosphate dehydrogenase(G6PD),a key enzyme for glycometabolism,mediated reduction stress in arsenic-induced malignant transformation of cells.Methods Immortalized human skin keratinocyte-forming cells(HaCaT cells)were treated with sodium arsenite(NaAsO_(2))at a concentration of 0.0(control group)and 1.0μmol/L(arsenic group),and malignant transformation indicators were tested using cell growth kinetics assay,cell scratch assay,and soft agar colony formation assay.At different stages of arsenic treatment(0,1,7,14,21,28,35 passages of cells),the effects of NaAsO_(2)on glycometabolism in HaCaT cells were determined using corresponding reagent kits and Western blot,including glucose-6-phosphate(G6P),lactate,acetyl CoA,G6PD levels,as well as protein expression levels of hexokinase 2(HK-2),6-phosphofructose-2-kinase/fructose-2,6-diphosphatase 3(PFKFB3),pyruvate dehydrogenase kinase 1(PDK1),6-phosphoglucose dehydrogenase(PGD),and G6PD.Mitochondria were extracted,and the effects of NaAsO_(2)on HaCaT cells and mitochondrial redox[reduced nicotinamide adenine dinucleotide phosphate(NADPH)/nicotinamide adenine dinucleotide phosphate(NADP+)ratio,reduced glutathione(GSH)/oxidized glutathione(GSSG)ratio]were determined using corresponding reagent kits.The effect of G6PD on reduction stress and NaAsO_(2)-induced malignant transformation of HaCaT cells was determined using small interfering RNA(siRNA)intervention method.Results Compared 1.0μmol/L NaAsO_(2)-cultured HaCaT cells up to 35 generations(T-HaCaT cells)with matching passage 0.0μmol/L NaAsO_(2)-cultured HaCaT cells[(33.797±0.280)h,0.177±0.015,13.667±2.625],the multiplication time[(24.042±0.479)h]was shorter(t=30.45,P<0.001),the cell migration rate(0.396±0.039)was higher(t=9.08,P<0.001),and the number of colonies formed in soft agar(73.667±4.450)was higher(t=20.11,P<0.001).Compared with matching passage control group cells and 0 generation of the same group,G6P level in the arsenic group was higher a

关 键 词：亚砷酸盐类 恶性转化 糖代谢 葡萄糖-6-磷酸脱氢酶 还原应激 

分 类 号：R114[医药卫生—卫生毒理学]