To Investigate The Effects Of Pulmonary Exposure To Copper Oxide Nanoparticles On Cerebral Cortex And Its Molecular Mechanism

Objectives: Copper oxide nanoparticles(CuONPs)are one of the nanoscale transition metal oxides.They are widely used in industry,medicine,food,semiconductor and other fields due to their good antibacterial and antiviral properties.However,they have been proven to enter the human body through the respiratory tract,digestive tract,skin,etc.,and react with cells,tissues,and organs in the body,resulting in lung,liver,and kidney damage because of their small size and high surface activity.The respiratory tract has been shown to be the main route of exposure to CuONPs in occupational populations.However,studies on the toxic effects of inhaled CuONPs on organisms have mainly focused on the lung tissue.Whether inhalation of CuONPs affects the brain and its underlying mechanisms have not been fully elucidated.Therefore,in this study,a model of acute pulmonary exposure to CuONPs was established by a single intratracheal instillation of CuONPs to investigate the effects of pulmonary exposure to CuONPs on cerebral cortex and its potential toxic mechanism,so as to provide experimental basis for the safety evaluation of CuONPs.Methods: In this study,healthy C57BL/6J male mice aged 8-12 weeks and weighing 18-22 g were randomly divided into control group(0μg/animal),low dose of CuONPs(30 μg/animal),and medium dose of CuONPs(50 μg/animal)and high doses of CuONPs(100 μg/animal),10 mice per group.The control group was given a single intratracheal instillation of normal saline and the treatment group was given a single intratracheal instillation of CuONPs with different doses.Three days later,the mice were anesthetized and sacrificed,and the mouse cerebral cortex was isolated.The pathological changes of the mouse cortex were detected by H&E staining and Nissl staining.The m RNA expression levels of the proinflammatory factors Tnf,Il-6 and IL-1β,and the oxidative stress-related genes Hmox-1,Txn1,Txn2,Nqo1,p62,and Gclm were detected by q PCR assay.Western blot assay was performed to detect the expression levels of oxidative stress-related proteins HO-1,TXN,NQO1,p62 and GCLM,and mitochondrial function-related proteins Tim23,TFAM and MFN2.Immunohistochemistry was used to detect the protein expression level of macrophage infiltration marker CD68 in mouse cortex.Results: Our results showed that compared with the control group:(1)The number of neurons was significantly reduced,the nuclei were shrunken and deformed,and the chromatin of neurons was condensed with the increase of CuONPs concentration.The number of Nissl bodies was reduced,the vacuoles were degenerated,and the staining became lighter with the increase of CuONPs concentration.These results indicated that a dose-dependent damage on the cerebral cortex was caused by the treatment with CuONPs in mice.(2)The m RNA expression levels of pro-inflammatory factors Tnf and Il-6 were increased after treatment with CuONPs.(3)The m RNA levels of oxidative stress-related genes Hmox-1,Txn1 and Txn2 were increased in the high-dose CuONPs group,and the m RNA level of Gclm was significantly decreased.In addition,the expression levels of oxidative stress-related proteins HO-1 and TXN in the high-dose CuONPs group were increased,and the expression level of p62 protein in the CuONPs-treated group was significantly decreased.(4)The expression levels of mitochondria-related proteins Tim23,TFAM and MFN2 in the CuONPs treatment group showed a downward trend.Conclusion: These results indicate that pulmonary exposure to CuONPs induces pathological damage,inflammation,oxidative stress,and mitochondrial dysfunction in the cerebral cortex.