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.

Key words: Cadmium, Microsplastics, Cognitive function, Neuron, Pyrotosis