硫芥对人神经母细胞瘤SH-SY5Y细胞的毒效应及机制  

Toxic effects and mechanism of sulfur mustard on human neuroblastoma SH-SY5Y cells

作　　者：张赔 徐斌 蒋宁 周文霞 ZHANG Pei;XU Bin;JIANG Ning;ZHOU Wen-xia(School of Pharmacy,Yantai University,Yantai 264005,China;State Key Laboratory of Toxicology and Medical Countermeasures,Institute of Pharmacology and Toxicology,Academy of Military Medical Sciences,Beijing 100850,China)

机构地区：[1]烟台大学药学院,山东烟台264005 [2]军事科学院军事医学研究院毒物药物研究所,抗毒药物与毒理学国家重点实验室,北京100850

出　　处：《中国药理学与毒理学杂志》2023年第4期263-273,共11页Chinese Journal of Pharmacology and Toxicology

摘　　要：目的建立硫芥(SM)染毒的神经细胞损伤模型,研究SM的神经细胞毒性效应及潜在机制。方法采用SM 0(溶剂,0.2%乙醇),0.1,1.0,10,50,100,200,400和800μmol·L^(-1)孵育人神经母细胞瘤SH-SY5Y细胞30 min,而后去除药物,换用正常培养基孵育24 h。应用IncuCyte ZOOM长时间动态活细胞成像及数据分析系统观察细胞融合率和细胞形态;CCK-8法检测细胞活力;SM 0,1.0,10,25,50,100和400μmol·L^(-1)以同样方式处理细胞,calcein-AM/PI荧光染色检测活细胞比例和死细胞比例;试剂盒检测细胞乳酸脱氢酶(LDH)释放率;5-乙炔基-2′-脱氧尿苷(EdU)-488细胞增殖试剂盒检测细胞增殖;免疫荧光法测定细胞DNA双链断裂标志物γ-H2AX。SM 0,1.0,10和100μmol·L^(-1)以同样方式处理细胞后,流式细胞仪检测细胞内活性氧(ROS)相对含量;试剂盒检测细胞内NAD+/NADH和ATP含量;SM 10μmol·L^(-1)处理细胞后,超高效液相色谱-串联质谱法检测细胞内SM-DNA加合物种类。结果与溶剂组相比,SM染毒后2 h时,SM 800μmol·L^(-1)组细胞融合率降至<5%且不再增加;SM染毒6~8 h后,SM 10~400μmol·L^(-1)组细胞融合率曲线逐渐降低。SM染毒后24 h时,细胞活力呈浓度依赖性降低(P<0.05,P<0.01,r=-0.6681),半数抑制浓度(IC50)为15.92μmol·L^(-1)。SM 25~400μmol·L^(-1)显著增加死细胞比例(P<0.01),浓度依赖性显著增加LDH释放率(P<0.01,r=0.9401);SM 400μmol·L^(-1)显著降低EdU阳性细胞比例(P<0.01)。SM 1.0~100μmol·L^(-1)均可显著增加细胞内ROS含量(P<0.01),且显著降低细胞内NAD+/NADH和ATP含量(P<0.01),均呈浓度依赖性(r分别为0.8074,-0.8885和-0.9514);SM 25~400μmol·L^(-1)可浓度依赖性增加γ-H2AX阳性细胞比例(P<0.01,r=0.8550)。在SM 10μmol·L^(-1)染毒细胞中检测到2种SM与DNA加合物——单加合物N7-HETEG和双加合物Bis-G。结论SM对SH-SY5Y细胞具有明显的细胞毒性作用,可破坏细胞膜结构,阻碍细胞增殖,降低细胞存活率,其机制可能与引起神�OBJECTIVE To establish a model of nerve cell injury induced by sulfur mustard(SM)and investigate the toxic effects and potential toxic mechanism of SM.METHODS Human neuroblastoma cells(SH-SY5Y)were treated with SM of 0(vehicle,0.2%ethanol),0.1,1.0,10,50,100,200,400 and 800μmol·L^(-1) for 30 min before the drug was replaced with normal medium for 24 h of incubation.The long-term dynamic living cell imaging and data analysis system of IncuCyte ZOOM was used to observe cell confluence and morphology.Cells were treated with SM of 0.1,1.0,10,50,100,200,400 and 800μmol·L^(-1) as mentioned above,and the cell viability was dected by CCK-8 assay.Cells were treated with SM of 0,1.0,10,25,50,100 and 400μmol·L^(-1) in the same way as above.The percentage of living cells and dead cells was detected by calcein AM/PI immunofluorescence staining,the cytotoxic lactate dehydro⁃genase(LDH)detection kit was used to detect the release rate of LDH,the EdU-488 cell proliferation kit was used to detect cell proliferation,the DNA double-strand break markerγ-H2AX was determinedby immunofluorescence assay.Cells were treated with SM of 0,1.0,10 and 100μmol·L^(-1),the relative content of intracellular reactive oxygen species(ROS)was detected by flow cytometry,and the ATP and NAD+/NADH detection kits were used to detect intracellular ATP and NAD+/NADH levels.Cells were treated with SM 10μmol·L^(-1),and the types of intracellular SM-DNA adducts were detected by ultraperformance liquid chromatography-tandem mass spectrometry(UPLC-MS/MS).RESULTS Compared with the vehicle group,the cell confluence of the SM 800μmol·L^(-1) group decreased to below 5%and ceased to increase after 2 h of SM exposure.The cell confluence in the concentration range of SM 10-400μmol·L^(-1) began to gradually decrease after 6 to 8 h of exposure.SM reduced cell viability in a concentra⁃tion-dependent manner 24 h after exposure(P<0.05,P<0.01,r=-0.6681),and the median inhibitory concentration of SM on SH-SY5Y cells was 15.92μmol·L^(-1).The percentage of

关 键 词：硫芥 人神经母细胞瘤细胞 DNA损伤 氧化应激 能量代谢 

分 类 号：R99[医药卫生—毒理学]