无机砷介导As3MT上调对16HBE细胞增殖、凋亡及AP-1表达的影响  

Effects of inorganic arsenic-mediated up-regulation of As3MT on proliferation,apoptosis and AP-1 expression of 16HBE cells

作　　者：古云 成会荣[2] 陈倩 于甜乐 张启航 文卫华[3] GU Yun;CHENG Huirong;CHEN Qian;YU Tianle;ZHANG Qihang;WEN Weihua(School of Public Health,Dali University,Dali Yunnan,671000,China;Institute for Prevention and Treatment of Chronic Noncommunicable Diseases,Yunnan Center for Disease Control and Prevention,Kunming Yunnan,650022,China;Institute of Occupational Health and Radiological Hygiene,Yunnan Center for Disease Control and Prevention,Kunming Yunnan,650022,China;Weihai Central Hospital,Weihai Shandong,264400,China;Graduate School,Kunming Medical University,Kunming Yunnan,650500,China)

机构地区：[1]大理大学公共卫生学院,云南大理671000 [2]云南省疾病预防控制中心慢性非传染性疾病防制所,云南昆明650022 [3]云南省疾病预防控制中心职业健康与放射卫生所,云南昆明650022 [4]威海市中心医院,山东威海264400 [5]昆明医科大学研究生院,云南昆明650500

出　　处：《职业与健康》2024年第5期613-619,624,共8页Occupation and Health

基　　金：国家自然科学基金项目(82060591);云南省兴滇英才计划项目(YNWR-MY-2018-012);云南省卫生健康委员会医学领军人才培养项目(L-2018016)。

摘　　要：目的了解无机砷(inorganic arsenic,i As)介导砷(+3氧化态)甲基转移酶[arsenic(+3 oxidation state)methyltransferase,As3MT]上调对16HBE细胞增殖、凋亡及激活蛋白-1(Activation of protein-1,AP-1)表达的影响,从而深入了解无机砷在细胞生物学中的作用机制。方法将16HBE细胞经体外培养后随机分成两大组:以不同浓度(0、2、4、6μmol/L)亚砷酸钠(sodium arsenite,NaAsO_(2))为一组和相同浓度(0、6、6、6μmol/L)一甲基砷酸(monomethylarsonic acid,MMA)、二甲基砷酸(dimethylarsinic acid,DMA)、NaAsO_(2)为一组染毒人支气管上皮样细胞(16HBE)48 h。Western Blot检测As3MT蛋白在细胞中的表达。通过小干扰RNA(small disturbance RNA,siRNAs)转染沉默As3MT可对染毒后细胞产生一系列生物学效应。CCK-8试剂盒来检测细胞活力,JC-1试剂盒检测早期细胞凋亡和HO/PI双染试剂盒检测晚期细胞凋亡和坏死。采取双萤光素酶报告基因检测转录因子AP-1的转录活性。Western Blot检测c-Jun(p-c-Jun)和c-Fos(p-c-Fos)的蛋白表达水平。结果实验发现,无机砷处理能够显著上调16HBE细胞中As3MT的表达水平,并对细胞的增殖和凋亡产生影响。与对照组相比,染毒后的16HBE细胞中As3MT表达随着NaAsO_(2)浓度的增加而增加,呈剂量-效应关系。相同浓度下,诱导As3MT表达的能力NaAsO_(2)>DMA>MMA。细胞转染技术提示,成功沉默As3MT。沉默As3MT后,CCK-8法显示细胞活力下降(NCmean=1.00,S1mean=0.94,S2mean=0.71),siAs3MTb1组细胞活力明显低于siCtrl组(P<0.05);siAs3MTb2组细胞活力明显低于siCtrl组(P<0.05)。JC-1检测提示细胞凋亡增加(NCmean=1.80,S1mean=1.40,S2mean=1.18),siAs3MTb 1组550 nm/485 nm比值明显低于siCtrl组(P<0.05);siAs3MTb2组550 nm/485 nm比值明显低于siCtrl组(P<0.05)。HO/PI双染提示细胞凋亡和坏死增加,siAs3MT组较siCtrl组细胞染色更为明显。双萤光素酶报告基因分析的结果AP-1活性上升(NCmean=48.83,S1mean=132.67,S2mean=163.83),siAs3MTb 1组AP-Objective To explore the effect of inorganic arsenic(iAs)mediated arsenic(+3 oxidation state)methyltransferase[arsenic(+3 oxidation state)methyltransferase,As3MT]on the proliferation,apoptosis of 16HBE cells,activation of protein-1(AP-1)expression,so as to gain insight into the mechanism of action of inorganic arsenic in cell biology.Methods 16HBE cells were cultured in vitro and randomly divided into 2 groups:sodium arsenite(NaAsO_(2))with different concentrations(0,2,4,6μmol/L)and the same concentration(O,6,6,6μmol/L)monomethylarsenic acid(MMA),dimethylarsenic acid(DMA)and NaAsO_(2)were used as a group to infect 16HBE cells for 48 hours.The expression of As3MT protein in cells was detected by Western Blot.Silencing As3MT by small interfering RNA(siRNAs)transfection can produce a series of biological effects on the infected cells.CCK-8 kit was used to detect cell viability,JC-1 kit was used to detect early cell apoptosis and HO/PI double staining kit was used to detect late cell apoptosis and necrosis.The transcriptional activity of transcription factor AP-1 was detected by dual luciferase reporter gene.The protein expression levels of c-Jun(p-c-Jun)and c-Fos(p-c-Fos)were detected by Western Blot.Results It was found that the treatment of inorganic arsenic could significantly up-regulate the expression level of As3MT in 16HBE cells,and affect the proliferation and apoptosis of cells.Specifically,compared with the control group,the expression of As3MT in 16HBE cells after exposure increased with the increase of NaAsO_(2)concentration,showing a dose-effect relationship.At the same concentration,the ability to induce the expression of As3MT was NaAsO_(2)>DMA>MMA.Cell transfection technology suggested that As3MT was successfully silenced.After silencing As3MT,the CCK-8 method showed that the cell viability decreased(NCmean=1.00,S1mxan=0.94,S2mm=0.71),and the cell viability of the siAs3MTbl group was significantly lower than that of the siCtrl group(P<0.05),the cell viability of the siAs3MTb2 group was significantl

关 键 词：砷 砷(+3氧化态)甲基转移酶 激活蛋白-1 人支气管上皮样细胞 细胞凋亡 细胞增殖 

分 类 号：R114[医药卫生—卫生毒理学]