镉致肾细胞损伤中氧化应激及N^6-甲基腺苷修饰酶表达变化  

Changes of oxidative stress and expression of N^6 methyladenosine modification enzymes during cell damage induced by cadmium in renal cells

作　　者：何作顺 李萌竹 谷仕艳 HE Zuo-shun;LI Meng-zhu;GU Shi-yan(Institute of Preventive Medicine,School of Public Health,Dali University,Dali,Yunnan 671000,China)

机构地区：[1]大理大学公共卫生学院,大理大学预防医学研究所,云南大理671000

出　　处：《环境与职业医学》2020年第9期897-902,共6页Journal of Environmental and Occupational Medicine

基　　金：2019年云南省教育厅科学研究基金项目(2019Y0255);大理大学博士科研启动项目(KYBS2018006)。

摘　　要：[背景]N^6-甲基腺苷(m^6A)修饰酶可参与细胞损伤过程,但m6A修饰酶在镉引起肾细胞损伤中有何变化尚不清楚。[目的]观察CdSO4致肾小管上皮细胞(HK-2细胞)损伤过程中m6A修饰酶的变化水平。[方法]以不同浓度CdSO4(0、4、8、16μmol·L^-1)处理HK-2细胞24 h后对相应指标进行检测。用Hoechst染色法观察细胞凋亡水平,镜下随机选取视野计数正常细胞和凋亡细胞,计算细胞凋亡率,利用相应试剂盒检测细胞中超氧化物歧化酶(SOD)活性、谷胱甘肽过氧化物酶(GSH-PX)活性、丙二醛(MDA)含量,以蛋白免疫印迹法检测m^6A甲基转移酶中的甲基转移酶样3(METTL3)和脂肪量与肥胖相关蛋白(FTO)的蛋白表达水平,荧光定量PCR法检测YTH结构域家族蛋白1(YTHDF1)和YTH结构域家族蛋白3(YTHDF3)的mRNA水平。[结果]经0、4、8、16μmol·L^-1 CdSO4处理后,细胞凋亡率依次为6.83%、5.17%、21.17%、35.80%;4、8、16μmol·L^-1组SOD活性依次是(15.41±0.96)、(13.57±1.42)、(10.96±0.61)×10^3U·g^-1,与"0"组相比,各组均有降低(P<0.05)。GSH-PX活性在CdSO4浓度为8μmol·L^-1时降低至(8.74±3.91)U·g^-1,而在8、16μmol·L^-1 CdSO4作用时,MDA含量分别升高至(87.69±3.05)、(93.42±4.71)μmol·g^-1(P<0.05)。蛋白免疫印记结果显示,METTL3蛋白相对表达水平在4、8、16μmol·L^-1时分别升高至"0"组的1.09、1.25、1.33倍,FTO蛋白相对表达水平在8、16μmol·L^-1时分别为0.81和0.74(P<0.05)。在CdSO4浓度为4μmol·L^-1时,YTHDF1的mRNA水平是"0"组的7.62倍,YTHDF3的mRNA水平在4、16μmol·L^-1 CdSO4作用时是"0"组的2.65、2.26倍(P<0.05)。[结论]在CdSO4致HK-2细胞发生损伤过程中,m^6A修饰酶METTL3和FTO的蛋白水平以及YTHDF1和YTHDF3的mRNA水平发生不同程度的变化。[Background]N^6 methyladenosine(m^6A)modification enzymes participate in the process of cell damage.However,the changes of m^6A modification enzymes in renal cell damage induced by cadmium remain unclear.[Objective]This experiment investigates changes of m6A modification enzymes during the process of CdSO4-induced cell damage in human renal tubules cells(HK-2 cells).[Methods]HK-2 cells were treated with different concentrations of CdSO4(0,4,8,and 16μmol·L^-1)for 24 h and corresponding indicators were tested.The apoptotic level was observed after Hoechst staining by counting normal cells and apoptotic cells in a random field under microscope and the apoptotic rate was calculated;the activity of superoxide dismutase(SOD),the activity of glutathione peroxidase(GSH-PX),and the content of malondialdehyde(MDA)were detected using corresponding kits;the protein expression levels of methyltransferase like 3(METTL3)and fat mass and obesity associated protein(FTO)were detected by Western blotting,and the mRNA expression levels of YTH domain family protein 1(YTHDF1)and YTH domain family protein 3(YTHDF3)were detected by real-time PCR.[Results]After administration with CdSO4 at 0,4,8,and 16μmol·L^-1,the cell apoptotic rates were 6.83%,5.17%,21.17%,and 35.80%,respectively.Compared with the 0μmol·L^-1 CdSO4 group,the SOD activities of the 4,8,and 16μmol·L^-1 CdSO4 groups were decreased[(15.41±0.96),(13.57±1.42),and(10.96±0.61)×10^3 U·g^-1,respectively](P<0.05),the GSH-PX activity of the 8μmol·L^-1 CdS O4 group was decreased to(8.74±3.91)U·g^-1,and the MDA content of the 8 and 16μmol·L^-1 CdS O4 groups were increased to(87.69±3.05)and(93.42±4.71)μmol·g^-1(P<0.05).The results of Western blotting showed that the protein expression levels of METTL3 of the 4,8,and 16μmol·L^-1 CdSO4 groups increased to 1.09,1.25,and 1.33 times of the 0μmol·L^-1 CdSO4 group respectively,and the protein expression levels of FTO of the 8 and 16μmol·L^-1 CdSO4 groups decreased to 0.81 and 0.74 times of the 0μmol·L^-1 CdSO4

关 键 词：HK-2细胞 氧化应激 甲基转移酶 去甲基化酶 甲基结合蛋白 

分 类 号：R114[医药卫生—卫生毒理学]