纳米氧化铝暴露对小鼠神经行为学的持续影响及其机制探讨

The effects of aluminum oxide nanoparticles exposure on the neurobehavioral of mice and its mechanism

目的探讨纳米氧化铝吸入暴露染毒对小鼠神经行为学的持续影响及其可能存在的机制。方法将40只健康ICR小鼠随机分成4组,每组10只,1组为对照组,3组为纳米氧化铝暴露染毒。应用动式吸入染毒柜,同时给予各暴露染毒组小鼠纳米氧化铝动态吸入染毒,染毒浓度1 mg/m3,每天暴露8 h,连续染毒14 d,于染毒开始前1 d(对照组),染毒结束后1 d(暴露后1 d组)、60 d(暴露后60 d组)、90 d(暴露后90 d组),采用旷场试验和Morris水迷宫试验检测小鼠的空间学习和记忆能力,取大脑皮层组织作苏木素-伊红(HE)染色和淀粉样蛋白前体(amyloid precursor protein,APP)免疫组化染色,qRT-PCR检测APP mRNA的表达水平,并用试剂盒法检测脑组织中腺嘌呤核苷三磷酸(ATP)、丙二醛(MDA)、活性氧(ROS)含量。结果旷场试验中,与对照组相比,除暴露后1 d组小鼠在进入中心区频率差异无统计学意义,其余各组小鼠各项指标均降低且差异有统计学意义(P<0.05);水迷宫试验中,各暴露染毒组小鼠平台象限停留时间均降低,差异均有统计学意义(P<0.05);穿越平台区频率均降低,仅暴露后90 d组小鼠差异有统计学意义(P<0.05)。大脑皮层的HE染色均未见明显的病理改变,APP免疫组化染色显示暴露后90 d组APP阳性细胞明显增加;qRT-PCR检测显示,APP mRNA的表达水平随暴露后天数增加而升高,且差异均有统计学意义(P<0.05);脑组织中ATP水平与对照组相比均降低,暴露后60和90 d组小鼠ATP水平差异有统计学意义(P<0.05),MDA含量和ROS含量均升高且差异均有统计学意义(P<0.05)。结论经纳米氧化铝暴露染毒,脱离暴露环境后,仍然会提高小鼠脑组织氧化应激水平并改变其神经行为学。APP的过度表达以及脑组织氧化应激反应可能是纳米氧化铝导致小鼠神经行为学改变的重要机制之一。

Objective To study continuous effects and potential mechanisms of neurobehavioral changes upon aluminum oxide nanoparticle inhalation exposure. Methods A total of 40 ICR mice were randomly divided into control group and 3 test groups which were subjected to 8 h dynamic inhalation exposure of 1 mg / m3 aluminum oxide nanoparticle for 14 consecutive days. Mice in control group were sacrificed 1 d before exposure; mice in exposure groups were sacrificed 1 d after exposure（ 1 d group）,60 d after exposure（ 60 d group） and90 d after exposure（ 90 d group）,respectively. Open field test and Morris water maze test were employed to detect spatial learning and memory capacity; cerebral cortex tissue HE staining and amyloid precursor protein（ amyloid precursor protein,APP） immunohistochemical staining were performed; APP mRNA expression was detected by qRT-PCR. The concentrations of adenine nucleoside three phosphoric acid（ ATP）,malondialdehyde（ MDA） and reactive oxygen species（ ROS） in brain tissue were detected by corresponding analysis kits. Results Compared with control group,mice except for those in 1 d group had comparable frequency to enter central zone,all indices of other test groups were lower,all differences were significantly statistical（ P〈0. 05）. In water maze test,all mice in test groups had shorter platform quadrant residence time than control group,all with statistical difference（ P〈0. 05）; the cross platform area frequency decreased for all test groups,only 90 d group had statistical significance（ P〈0. 05）. No obvious pathological changes were observed by HE staining in cerebral cortex tissue. Immunohistochemistry staining showed APP positive cells in 90 d group increased significantly; qRT-PCR showed APP mRNA expression increased upon incensement of days after exposure,with significant statistically difference（ P〈0. 05）. ATP levels in brain tissues of test groups were lower than control group; statistical significant difference was observed between 60 d and 90 d groups. MDA and ROS concentration both increased significantly（ P〈0. 05）. Conclusion The aluminum oxide nanoparticle exposure had continuous effect on improving oxidative stress and inducing neurobehavioral changes of mice brain after the exposure. APP over expression and oxidative stress response in brain tissues may serve as an important mechanism for mice neurobehavioral changes upon aluminum oxide nanoparticle exposure.