【摘要】 目的 探讨镉（cadmium, Cd）、微塑料（microplastics, MPs）单独或联合暴露对认知功能损伤的发生机制。方法 按照体质量随机数字法将雌性小鼠分为空白对照组、Cd组（Cd:5 mg/kg·bw CdCl₂）、MPs组（MPs:10 mg/kg·bw）、Cd+MPs联合组（Cd+MPs:5 mg/kg·bw CdCl₂+10 mg/kg·bw MPs）,每组14只。每天分别经灌胃给予小鼠CdCl₂和MPs。12周末，采用神经行为学方法评价小鼠认知功能。实验结束后，每组随机取4只小鼠经体内灌注固定后，取大脑进行大脑神经元核抗原（neuronal nuclei antigen, NeuN）免疫组化检测；每组随机另取5只小鼠经尾静脉注射伊文思蓝检测血脑屏障（blood brain barrier, BBB）通透性；每组其余动物处死后取大脑组织采用Western blotting进行相关蛋白分子表达检测；采用石墨炉原子吸收分光光度计分析小鼠大脑Cd含量。结果 旷场试验结果显示，Cd组、MPs组和Cd+MPs联合组小鼠在周围区域停留时间均高于对照组（P<0.05）,在中央区活动时间均低于对照组（P<0.05）。新物体识别实验结果显示，与对照组相比，Cd组、MPs组、Cd+MPs联合组小鼠识别指数均表现为明显降低（P<0.05）。伊文思蓝检测结果显示，与对照组相比，Cd组、MPs组以及Cd+MPs联合组小鼠大脑匀浆OD值明显升高（P<0.05）,提示BBB通透性增加。大脑组织Cd含量分析结果显示，与对照组相比，Cd组、Cd+MPs联合组小鼠大脑Cd含量明显增加（P<0.05）,而且Cd+MPs联合组小鼠大脑Cd含量明显高于Cd组（P<0.05）。NeuN免疫组化染色后神经元计数结果显示，与对照组相比，Cd组、MPs组和Cd+MPs联合组大脑皮层神经元数目降低，差异均具有统计学意义（P<0.05）。Western blotting结果显示，Cd组、MPs组和Cd+MPs联合组小鼠大脑皮层突触素1、突触素、突触后密度蛋白95蛋白表达均降低（P<0.05）,其中Cd+MPs联合组降低幅度最大。与对照组相比，Cd组、MPs组和Cd+MPs联合组大脑皮层炎性关键调控蛋白NF-κB激活，表现为p-NF-κB/总NF-κB比值明显升高（P<0.05）。同时，焦亡轴下游消皮素D与NeuN免疫荧光双染显示，Cd组、MPs组以及Cd+MPs联合组小鼠大脑均观察到消皮素D与神经元共定位，以Cd+MPs联合组焦亡神经元最多。结论 Cd和MPs单独暴露均导致小鼠认知功能损伤，两者联合暴露大脑损伤加重，NF-κB激活，神经元炎性焦亡可能是Cd和MPs共同的作用机制之一。更多还原

【Abstract】 Objective To investigate the mechanism of cognitive impairment caused by cadmium（Cd） and microplastics（MPs） exposure alone or in combination. Methods Female mice were divided into control group, Cd group（Cd: 5 mg/kg·bw CdCl₂）, MPs group（MPs: 10 mg/kg·bw）, Cd+MPs combination group（Cd+MPs: 5 mg/kg·bw CdCl₂+10 mg/kg·bw MPs） by random number method according to body weight, with 14 mice in each group. The mice were given CdCl₂ and MPs once daily by gavage. At the end of 12 weeks, the neurobehavioral performances of the mice were evaluated. Subsequently, 4 mice from each group were randomly selected and transcardially perfused with 4% paraformaldehyde（PFA）, and the brains were dissected for immunohistochemical staining with NeuN. Another 5 mice from each group were treated with Evans blue by tail vein injection to determine the blood-brain barrier（BBB） permeability. The remaining mice of each group were sacrificed and the brains were subjected to Western blotting for detection of protein expression of interest. The Cd content in the brain of the mice was analysed by graphite furnace atomic absorption spectrophotometer. Results The results of the open field test showed that the dwell times in the peripheral area of mice from the Cd group, MPs group and Cd+MPs combination group were higher than those of the control group（P<0.05）, and the activity times in the central region were lower than those of the control group（P<0.05）. The results of the novel object recognition experiment showed that the recognition index of all mice in the Cd group, MPs group and Cd+MPs combination group was significantly decreased compared with the control mice（P<0.05）. Evans blue detection showed that the OD values of brain homogenate of mice from the Cd group, MPs group and Cd+MPs combination group significantly increased（P<0.05） compared with the control values, indicating the obvious increase of BBB permeability. The results of Cd analysis showed that the Cd content of mouse brain in Cd group and Cd+MPs combination group was significantly increased（P<0.05）, and the Cd level of mouse brain in Cd+MPs combination group was significantly higher than that of Cd group（P<0.05）. The neuron count results after NeuN immunohistochemical staining showed that compared with the control group, the decrease of neurons in the brain cortex of mice from Cd group, MPs group and Cd+MPs combination group was statistically significant（P<0.05）. Western blotting results showed that the protein expressions of synapsin1（Syn-1）, synaptonphysin（SYP） and postsynaptic density protein 95（PSD95） in the brain cortex of mice from the Cd group, MPs group and Cd+MPs combination group were decreased（P<0.05）, with the greatest decrease in the Cd+MPs combination group. Compared with the control group, the key inflammatory regulatory protein, NF-κB, was activated in the brain tissue of Cd, MPs and Cd+MPs combination groups, as evidenced by the p-NF-κB/total NF-κB ratio, which was significantly increased（P<0.05）. Immunofluorescence double staining of the downstream protein of the pyrotosis axis, Gasdermin D（GSDMD）, with NeuN showed that co-localization of GSDMD and NeuN-positive neurons was observed in the Cd group, the MPs group and the Cd+MPs combination group, and the most of the neurons with pyrotosis were observed in the Cd+MPs combination group. Conclusion Both Cd and MPs individual exposure could lead to cognitive impairment in mice, and their combined exposure aggravated the brain damage. The activation of NF-κB, inflammatory pyrotosis of neurons in the brain, may be one of the shared mechanisms of Cd and MPs.更多还原