Effects Of Ethanol Combined With 1,2-Dichloroethane On Brain Tissue Damage And Neuroinflammatory Response In Mice

Objective:1,2-Dichloroethana(1,2-DCE)is a halogenated hydrocarbon compound that has been used as a binder in industrial production such as the plastics and footwear industries.Subacute 1,2-DCE occupational poisoning is a unique occupational disease in China,which mainly damages brain tissue and causes toxic encephalopathy.In vivo,1,2-DCE is mainly metabolized by cytochrome P4502E1(CYP2E1),producing active intermediate metabolites such as chloroacetaldehyde and reactive oxygen species(ROS),which in turn damage brain tissue.Research suggests that chronic or heavy alcohol consumption in humans may induce CYP2E1 expression in brain tissue and participate in ethanol metabolism in brain tissue.Since both ethanol and 1,2-DCE exposure can induce CYP2E1 expression,the increase in CYP2E1 expression level can enhance the metabolic process of ethanol and 1,2-DCE in brain tissue,producing a large number of active intermediate metabolites such as acetaldehyde,chloroacetaldehyde and ROS.Therefore,combined exposure of ethanol to 1,2-DCE can aggravate brain tissue damage.In this study,we intend to simulate occupational exposure of drinking workers with 1,2-DCE by establishing a mouse experimental model of combined exposure of drinking alcohol and1,2-DCE,explore the effect of ethanol intake on 1,2-DCE-induced neuroinflammation and brain injury,broaden the influencing factors of occupational 1,2-DCE exposure on the health hazards of workers,provide reference data for further revealing the mechanism of1,2-DCE toxic cerebral edema,and provide scientific basis for the prevention and control of occupational 1,2-DCE exposure workers.Methods:1.Animal grouping and treatment:60 healthy and clean Kunming mice with a weight of 20-22 grams were selected,divided into 6 groups,namely the control group,the 1,2-DCE simple infection group(hereinafter referred to as the simple infection group),the ethanol exposure group and the combined exposure group(hereinafter referred to as the"low-dose combination group","medium-dose combination group"and"high-dose combination group"),with 10 mice in each group.1.1 Pretreatment before infection:0.2 ml normal saline gavage was given to mice in the control group and simple infection group,0.2 ml ethanol aqueous solution was given to mice in the ethanol exposure group and the combined exposure group,and the ethanol intake dose of ethanol intake in the ethanol exposure group and the low,medium and high dose combined exposure group was 3.0,0.75,1.5 or 3.0 g/kg,respectively,once a day for3 consecutive days.1.2 Poison treatment:After 3 days of continuous gavage,each group of mice was placed in a 100 L infection cabinet for 3.5h(5 mice per cabinet)after daily pretreatment,1,2-DCE was added to the infection cabinet of mice in the control group and ethanol exposure group,and 1.0 g/m~3 1,2-DCE was added to the infection cabinet of mice in the simple infection group and the combined exposure group.The behavioral changes and poisoning manifestations of mice were observed daily,and changes in mouse weight were recorded.After 3 days of intravenous inhalation of 1,2-DCE in each group,mice were killed the day after the last infection,and brain tissues were taken for follow-up experiments.2.Basic indications of ethanol-induced 1,2-DCE animal model and expression of CYP2E1 in the brain:animal weight was monitored daily during model establishment and statistically analyzed.Western blotting was used to determine CYP2E1 protein expression levels in the brain of each group of mice.RT-qPCR was used to determine the expression level of CYP2E1 mRNA in the brain of each group of mice.3.Effect of 1,2-DCE on microglia and astrocytes activation in the brain of mice under ethanol induction:Western blotting was used to test the protein expression in the brains.4.Effects of 1,2-DCE on brain tissue inflammation and NF-κB signaling pathway under ethanol induction:Western blotting was used to detect the protein expression levels of ICAM-1,VCAM-1,AQP4,MMP-9,TNF-α,IL-1β,p65 and p-p65 in the brains of mice in each group.5.Effect of 1,2-DCE on brain injury in mice under ethanol induction:HE staining was observed in the tissue of each group of mice.The Western blotting was used to detect the expression levels of protein,such as caspase-3,cleaved caspase-3,Bax and Bcl-2.Apoptosis was monitored in the brain tissue of each group of mice using the TUNEL method.Result:The weight of mice in each group decreased during the poisoning period,and the detection indexes in the brain tissues of mice in the simple infection group and the ethanol exposure group were not statistically significant compared with the control group.Compared to the single infection group,the levels of ICAM-1,VCAM-1 and Bax/Bcl-2 protein expression were significantly increased in the brain of mice in the low-dose combination treatment group(P<0.05),and the expression levels of CYP2E1,ICAM-1,VCAM-1,AQP4,MMP-9,IL-1β,p-p65,caspase-3,Bax/Bcl-2 protein and CYP2E1mRNA were significantly increased in females of the low combined exposure group(P<0.05),and the expression levels of all proteins and CYP2E1 were significantly increased in females of the high combined exposure group.MMP-9 protein expression levels in mouse brain were significantly increased in the medium combined dose group(P<0.05)and Iba-1,GFAP,AQP4,MMP-9,IL-1β,TNF-α,p-p65,cleaved caspase-3 protein and CYP2E1 mRNA expression levels were significantly increased in the high combined dose group(P<0.05).TNF-α,cleaved caspase-3 protein and CYP2E1 mRNA expression levels in the brain of mice in the high dose combined exposure group were significantly increased(P<0.05).Factor analysis results using a control group,,simple infection group,ethanol exposure group and high-dose combined exposure group showed that ethanol and 1,2-DCE combined exposure could synergistically upregulate the expression of all proteins except ICAM-1 in mouse brain tissues(P<0.05).Observations of pathological tissues showed that there were no obvious abnormal changes in the brain tissue structure of mice in the control and ethanol-exposed groups.In the brain tissues of mice in the simple infection group and the low,medium and high dose combined exposure group,interstitial hyperstitis was loosened,the space around the nucleus was widened,the cytoplasm was lightened,the swelling and degeneration of the cell body and the edges were blurred.Some cells are round or oval masses,and the cytoplasm is dense purple,and the abnormal cytopathological changes of the high-dose combined exposure group are more obvious.The TUNEL results showed that there was no apparent apoptosis in the brains of mice from the control and ethanol-exposed groups,and increasing ethanol concentration and dose resulted in a progressive increase in apoptosis in brain cells of mice in the singly infected group and in the low,medium and high combined dose groups.Conclusion:1.The combined exposure of ethanol and 1,2-DCE can synergistically upregulate the expression level of CYP2E1 in the brain of mice,causing apoptosis and brain tissue damage in brain tissue cells.2.Combined exposure of ethanol and 1,2-DCE can synergistically induce and aggravate the inflammatory response in the brain of mice.3.Ethanol and 1,2-DCE combined exposure can synergize to activate the NF-κB signaling pathway.