锰对下丘脑GT1-7神经细胞氧化应激与凋亡的影响  

Effects of manganese on oxidative stress and apoptosis in hypothalamus GT1-7 neurons

作　　者：杨纯 邓月 易湘 彭东杰[1] 姜岳明[1] 李少军[1] 卢国栋[1] YANG Chun;DENG Yue;YI Xiang;PENG Dong-jie;JIANG Yue-ming;LI Shao-jun;LU Guo-dong(Department of Health Toxicology,School of Public Health,Key Laboratory of Prevention and Control of High Incidence Diseases,Guangxi Medical University,Nanning Guangxi 530021,China)

机构地区：[1]广西医科大学公共卫生学院卫生毒理教研室高校高发疾病预防与控制研究重点实验室,广西南宁530021

出　　处：《毒理学杂志》2022年第2期163-169,共7页Journal of Toxicology

基　　金：国家自然科学基金(81803281,81773476);广西自然科学基金(2018GXNSFBA050060)。

摘　　要：目的建立锰(Mn)致下丘脑GT1-7神经细胞毒作用研究的体外模型,探讨Mn对下丘脑神经细胞氧化应激与凋亡的影响。方法取对数生长期GT1-7细胞分别经:(1)0、50、100、300和500μmol/L MnCl_(2)作用3、6、12、24和48 h;(2)经100μmol/L抗氧化剂(NAC)或等体积的1%DMSO预处理1 h后,再经0、50和300μmol/L MnCl_(2)处理12 h。然后,收集细胞和培养基,分别采用噻唑蓝(MTT)法和乳酸脱氢酶(LDH)试剂盒法测定细胞的存活率和LDH释放量;(3)经0、50、100、300和500μmol/L MnCl_(2)作用1、3、6和12 h;(4)经100μmol/L NAC或等体积的1%DMSO预处理1 h后,再经0、50和300μmol/L MnCl_(2)处理1、3、6和12 h。处理结束后,测定细胞内ROS水平。结果经染Mn 3 h后,100、300和500μmol/L MnCl_(2)处理组GT1-7细胞存活率比对照组低;经染Mn 6、12、24和48 h后,Mn浓度达到50μmol/L即可使GT1-7细胞存活率降低(P<0.05)。染Mn 12和24 h时,与对照组比较,100、300和500μmol/L MnCl_(2)处理组GT1-7细胞培养液LDH活性升高(P<0.05)。染MnCl_(2)48 h时,与对照组比较,染Mn浓度达到50μmol/L可使GT1-7细胞LDH释放量升高(P<0.05)。染MnCl_(2)3 h及以上时,50、300和500μmol/L MnCl_(2)处理组GT1-7细胞产生活性氧(ROS)比对照组高(P<0.05);NAC预处理对Mn致GT1-7细胞存活率下降、LDH释放和ROS含量升高有拮抗作用。结论过量Mn暴露可使下丘脑GT1-7神经细胞ROS升高,并使LDH释放量升高和存活率降低,NAC对上述损伤有一定的拮抗效应,提示氧化应激可能是Mn致下丘脑神经细胞损伤的毒作用机制之一。Objective To explore the mechanism of manganese(Mn)-induced oxidative stress and apoptosis in hypothalamic nerve cells via establishing an in vitro model of Mn-induced neurocytotoxic effects of GT1-7 in hypothalamus.Methods The GT1-7 neurons at logarithmic growth phase were seeded into plates and separated by:(1)0,50,100,300,500μmol/L MnCl_(2)for 3,6,12,24 and 48 h.(2)After pretreatment with 100μmol/L NAC or equal volume 1%DMSO for 1 h,cells were treated with 0,50,300μmol/L MnCl_(2)for 12 h.Then,cells and culture medium were collected to determine cell viability and LDH release by Methyl thiazolyl diphenyl-tetrazolium bromide(MTT)and LDH kits.(3)0,50,100,300,500μmol/L MnCl_(2)for 1 h,3 h,6 h,12 h;(4)After being pretreated with 100μmol/L NAC or equal volume 1%DMSO for 1 h,cells were treated with 0,50 and 300μmol/L MnCl_(2)for 1 h,3 h,6 h and 12 h.After treatment,cells were collected to determine intracellular ROS levels.Results The survival rates of GT1-7 cells in the 100,300 and 500μmol/L MnCl_(2)treatment group were lower than those in the control after treated for 3 h.After treatment with Mn for 6,12,24 and 48 h,the survival rate of GT1-7 cells was decreased when the concentration of MnCl_(2)approached 50μmol/L(P<0.05).Moreover,compared with the control group,the releases of LDH in the culture medium of 100,300 and 500μmol/L MnCl_(2)treatment groups were increased(P<0.05).When the concentration of Mn was up to 50μmol/L,the LDH release of GT1-7 cells was higher than those in the Control(P<0.05).ROS levels in GT1-7 cells exposed to 50,300 and 500μmol/L MnCl_(2)for 3 h and above was higher than those in the Control(P<0.05).NAC had antagonistic effect on the above-mentioned changes in GT1-7 cells induced by Mn.Conclusion Excessive Mn exposure may reduce the survival rate of GT1-7 cells,increase ROS and LDH release.NAC has antagonistic effect on the above-mentioned changes,suggesting that oxidative stress may be one of the mechanisms of Mn induced hypothalamic nerve cell injury.

关 键 词：锰 下丘脑 GT1-7神经细胞 氧化应激 凋亡 

分 类 号：R114[医药卫生—卫生毒理学] R99[医药卫生—公共卫生与预防医学]