邻苯二甲酸二乙基己酯与双酚A联合染毒对小鼠学习记忆障碍及淀粉样前体蛋白酶解通路的影响

Effects of Combined Exposure of Diethylhexyl Phthalate and Bisphenol A on Learning and Memory Impairment and Amyloid Precursor Protein Processing Pathway in Mice

研究邻苯二甲酸二乙基己酯(diethylhexyl phthalate,DEHP)与双酚A(bisphenol A,BPA)联合染毒对小鼠学习记忆障碍及淀粉样前体蛋白(APP)酶解通路的影响。选用昆明种小鼠80只,随机分为空白对照(CT)组、BPA染毒组(1 mg·L^(-1))、不同浓度的DEHP染毒组(100、250和500 mg·kg^(-1))与联合染毒组。经口灌胃染毒,以0.01 mL·g^(-1)的剂量灌胃,7次/周,持续6周。使用Morris水迷宫、避暗、跳台的行为学实验方法检测2种污染物单独及联合染毒对小鼠学习记忆的影响;检测小鼠大脑组织中丙二醛(MDA)的含量和单胺氧化酶(MAO)、过氧化氢酶(CAT)、乙酰胆碱酯酶(AChE)的活性;采用酶联免疫分析(ELISA)法检测脑组织中的β、γ-分泌酶活性和Aβ_(1-42)的含量;Western Blot法检测APP、PS1、BACE1和Tau的蛋白表达水平;免疫组化法观察小鼠脑组织海马区HE染色情况。行为学测试结果表明,与CT组比较,单独染毒处理组小鼠学习记忆能力出现障碍,空间探索能力较弱;联合染毒组与单独染毒组相比,小鼠于目标象限停留时间短,穿台次数少,差异具有统计学意义(P<0.05);生化学指标结果显示,随着毒物浓度增加,脑组织中MAD含量、MAO和AChE活性逐渐增加,CAT活性逐渐降低,联合染毒组的变化更为明显,染毒处理组β、γ-分泌酶活性和Aβ_(1-42)含量增加,联合染毒组比单独染毒组酶活性增加更明显;各组差异均具有统计学意义(P<0.05)。DEHP与BPA单独染毒,对小鼠脑组织均显示有神经毒性作用,会造成小鼠学习记忆功能障碍;随着联合染毒组DEHP浓度的增加,联合染毒组与对应剂量的单独染毒组相比,小鼠行为学测试结果与生化指标变化更加明显,采用效应相加模型对其联合毒性作用进行评价,表现为协同作用;染毒组小鼠脑组织神经递质明显减少,具体机制可能与氧化应激及APP酶解通路的作用有关。

To study the effects of combined exposure of diethylhexyl phthalate(DEHP)and bisphenol A(BPA)on learning and memory impairment and amyloid precursor protein(APP)processing pathway in mice.80 Kunming mice were selected and randomly divided into a blank control(CT)group,a BPA exposure group(1 mg·L^(-1)),DEHP exposure groups of different concentrations(100,250 and 500 mg·kg^(-1))and combined exposure groups.The mice were exposed to the toxin by oral gavage at 0.01 mL·g^(-1),7 times a week for 6 weeks.Morris water maze,step-through training and jumping platform experiment methods were used to detect the effects of two chemicals and their combined exposure on learning and memory of mice.The content of malondialdehyde(MDA)and monoamine oxidase(MAO),catalase(CAT)and activities of acetylcholinesterase(AchE)were detected from brain tissue;enzyme-linked immunoassay(ELISA)method were used to detect the activity ofβ-secretase andγ-secretase and the content of Aβ_(1-42) in brain tissue;Western Blot method was used to detect the protein expression levels of APP,PS1,BACE1 and Tau;immunohistochemistry was used to observe HE staining in the hippocampus of mouse brain tissue.Compared with the CT group,the behavioral test results showed that mice in the single chemical exposure group had dysfunction in the learning and memory ability,and the spatial exploration capability was weaker.In the combined exposure group the target quadrant stay time was shorter and the number of crossings was less than the single-exposure group,the difference was statistically significant(P<0.05).Biochemical indicators show that as the concentration of the chemical increases,the content of MAD,the activities of MAO and AChE were gradually increased,and CAT activity was gradually decreased in the brain tissue.This changes in the combined exposure group showed more obviously.In the exposure groups,the activity ofβ-secretase andγ-secretase and the content of Aβ_(1-42) were increased,and the enzyme activity of the combined exposure group were increased more significantly than single exposure group;the differences in each group were statistically significant(P<0.05).DEHP and BPA exposure have separate neurotoxic effects on mouse brain tissues,and both cause learning and memory dysfunction in mice;with the increase of DEHP concentration in the combined exposure group,the behavioral test results and biochemical indicators were changed more obviously compared with the single exposure group with the corresponding dose.The combined effect mode was evaluated by the effect additive model,which showed a synergistic effect;the neurotransmitters of brain tissue were significantly reduced in the exposure group,and the specific mechanism may be related to the oxidative stress and APP processing pathway.