TBBPA-DHEE暴露对大鼠肾上腺髓质嗜铬细胞瘤PC12细胞的影响及机制  

Effect of TBBPA-DHEE exposure on rat adrenal pheochromocytoma PC12 cells and its mechanism

作　　者：曾政嘉 冯伟伟 丁阳阳 钱显 罗梦娜 茆广华 仰榴青[3] 吴向阳 ZENG Zhengjia;FENG Weiwei;DING Yangyang;QIAN Xian;LUO Mengna;MAO Guanghua;YANG Liuqing;WU Xiangyang(School of the Environment and Safety Engineering,Jiangsu University,Zhenjiang Jiangsu 212013,China;Laboratory Animal Research Center,Jiangsu University,Zhenjiang Jiangsu 212013,China;School of Chemistry and Chemical Engineering,Jiangsu University,Zhenjiang Jiangsu 212013,China)

机构地区：[1]江苏大学环境与安全工程学院,江苏镇江212013 [2]江苏大学实验动物中心,江苏镇江212013 [3]江苏大学化学化工学院,江苏镇江212013

出　　处：《江苏大学学报（医学版）》2023年第3期225-230,244,共7页Journal of Jiangsu University：Medicine Edition

基　　金：国家自然科学基金面上项目(21976072)。

摘　　要：目的:研究四溴双酚A双(2-羟基乙基)醚[tetrabromobisphenol A bis(2-hydroxyetyl)ether,TBBPA-DHEE]对大鼠肾上腺髓质嗜铬细胞瘤(PC12)细胞的影响及潜在的作用机制。方法:将PC12细胞随机分为5组,分别为空白对照组、溶剂对照组(0.05%二甲基亚砜)、TBBPA-DHEE低剂量组(5μg/mL)、中剂量组(20μg/mL)和高剂量组(35μg/mL),按分组对应处理48 h;通过MTT实验分析TBBPA-DHEE对PC12细胞的半数抑制浓度(IC_(50));采用试剂盒检测PC12细胞中超氧化物歧化酶(SOD)活力、丙二醛和活性氧含量,以及细胞培养液中NO和Ca^(2+)含量;采用ELISA法检测PC12胞内电压门控钙离子通道(voltage-gated calcium channel,VGCC)含量;采用蛋白质免疫印迹法测定钙离子信号通路相关蛋白表达。结果:TBBPA-DHEE(≥20μg/mL)暴露显著抑制PC12细胞活力,半数抑制浓度为33.4μg/mL;与对照组相比,5、20和35μg/mL TBBPA-DHEE组PC12细胞SOD活力、丙二醛及活性氧含量显著升高(P<0.05),细胞培养液中NO和Ca^(2+)含量显著增加(P<0.01或P<0.05),VGCC含量显著降低(P<0.01);5μg/mL TBBPA-DHEE组钙离子信号通路中p-CaMKⅡ、p-ERK1/2和p-CREB蛋白相对表达水平显著降低(P<0.01),20和35μg/mL TBBPA-DHEE组CaM、MKP-1、p-CaMKⅡ、p-ERK1/2和p-CREB蛋白相对表达水平显著降低(P<0.01)。结论:TBBPA-DHEE暴露可显著抑制PC12细胞活力并导致细胞氧化损伤,其可能通过下调VGCC表达,影响细胞钙离子稳态,进而影响钙离子信号通路相关蛋白的表达,从而产生神经毒性。Objective:To study the effect of tetrabromobisphenol A bis(2-hydroxyetyl)ether(TBBPA-DHEE)on rat adrenal pheochromocytoma PC12 cells and mechanisms.Methods:PC12 cells were randomly divided into 5 groups:control group,solvent control group(0.05%dimethyl sulfoxide),TBBPA-DHEE low-dose group(5μg/mL),medium-dose group(20μg/mL)and high-dose group(35μg/mL).Experimental exposure lasted for 48 hours according to corresponding groups.MTT assay was used to analyze the half maximal inhibitory concentration(IC_(50))of TBBPA-DHEE on PC12 cells.The activity of superoxide dismutase(SOD),and contents of malondialdehyde(MDA)and reactive oxygen species(ROS)in PC12 cells were detected by corresponding assay kits.In addition,the contents of nitric oxide(NO)and Ca^(2+)in cell culture medium were also detected.The content of PC12 cells voltage-gated calcium channel(VGCC)was determined by ELISA method.The expression of proteins in calcium signaling pathway was determined by Western blotting.Results:TBBPA-DHEE(≥20μg/mL)exposure significantly inhibited the viability of PC12 cells,with IC_(50)of 33.4μg/mL.Compared with the control group,5,20 and 35μg/mL TBBPA-DHEE groups PC12 cells SOD activity,MDA and ROS contents were significantly increased(P<0.05),the contents of NO and Ca^(2+)in the cell culture medium were significantly increased(P<0.01 or P<0.05),and the content of VGCC was markedly decreased(P<0.01);the relative expression levels of p-CaMKⅡ,p-ERk1/2 and p-CREB proteins in calcium ion signaling pathway of 5μg/mL TBBPA-DHEE group were remarkably decreased(P<0.01);the relative expression levels of CaM,MKP-1,p-CaMKⅡ,p-ERk1/2 and p-CREB proteins in 20 and 35μg/mL TBBPA-DHEE groups were significantly decreased(P<0.01).Conclusion:Exposure to TBBPA-DHEE could significantly inhibit the viability of PC12 cells and lead to cellular oxidative damage.TBBPA-DHEE may cause calcium dyshomeostasis by down-regulating the expression of VGCC,and then disrupt the expression of calcium signaling pathway related proteins,thus producing neu

关 键 词：四溴双酚A双(2-羟基乙基)醚 大鼠肾上腺髓质嗜铬细胞瘤细胞 电压门控钙离子通道 氧化应激 神经毒性 

分 类 号：R994[医药卫生—毒理学]