Signaling Pathways Involved In Di-(2-Ethylhexyl) Phthalate Induced Anxiety-and Depression-like Behavior In Pre-pubertal Male Rats

Objective:To establish a whole animal model of pre-pubertal exposure of DEHP in immature rats,observe the changes of anxiety and depression-like behavior in young rats,and start from the pathological changes,oxidative stress response,GABA signaling pathway and ERK1/2-CREB signaling pathway to preliminarily explore the mechanism of neurotoxicity of DEHP pre-puberty exposure.Methods:SD male rats aged 3 weeks were divided into two groups according to body weight:Group A,80 male SD rats were randomly divided into 5 groups:solvent control group,1,10,50,200 mg/kg/d DEHP dose group;Group B,50 male SD rats were randomly divided into 5 groups:solvent control group,low dose control group(1mg/kg/d DEHP)),high-dose control group(200mg/kg/d DEHP),low-dose intervention group(1mg/kg/d DEHP+1 mg/kg/d Muscimol),and high-dose intervention group(200mg/kg/d)DEHP+1mg/kg/d Muscimol).After 3 weeks continuous gavage,forced swimming test,open field test and elevated plus maze test were performed to observe the anxiety-and depression-like behavior.After decapitation,the serum biochemical indexes were detected by automatic biochemical analyzer.MDA content in rat cerebral cortex was detected by TBA method.SOD activity of cerebral cortex was detected by WST-1 method.The nerve change of rat hippocampus was observed by Nissl staining.Expressions of GABA_ARα2,GAD65+67,ERK1/2,CREB,p-ERK1/2 and p-CREB in hippocampus were then detected by immunohistochemistry and Western blot.Result:1.Behavioral changes:(1)The rats in each group are generally in good condition,and there is no abnormality in eating or drinking.(2)Forced swimming test:Compared to control group,the time of swimming in 1 mg/kg/d group significantly increased,and the number of resting in 200 mg/kg/d group significantly decreased(P<0.05);(3)Open field experiment:Compared to solvent control group,central distance and exploration time of high-dose control group significantly reduced,and total distance of low-dose intervention group and high-dose intervention group significantly increased.The number of grooming significantly reduced(P<0.05);the total distance,central distance,and exploration time significantly increased in high-dose intervention group compared to the high-dose control group(P<0.05).(4)Elevated plus maze test:Compared to solvent control group,the number of open arm entrance and the open arm distance of low-dose control group and high-dose control group significantly decreased,and the ratio of time staying in open arm and total distance of high-dose control group significantly reduced,and the number of close arm entrance of high-dose intervention group significantly reduced(P<0.05).Compared to low-dose control group,the open arm distance significantly increased in low-dose intervention group(P<0.05);Compared to high-dose control group,the number of open arm entrance,open arm distance,and total distance significantly increased in high-dose intervention group(P<0.05).2.General toxicity:(1)Group A:The weight gain 10 mg/kg/d group in 21 days was significantly lower than that of the solvent group(P<0.05);the cardiac organ coefficient of 10,200mg/kg/d groups and the lung organ coefficient of the three high-dose groups significantly increased(P<0.05);The Glu level of 10 mg/kg/d group significantly decreased(P<0.05),and the UREA level of 50 mg/kg/d group significantly increased(P<0.05).(2)Group B:Compared to solvent control group,the lung organ coefficient of the high-dose control group significantly increased(P<0.05);The Glu level of high-dose control group and high-dose intervention group significantly increased compared to solvent control group(P<0.05).3.Pathological changes:(1)Group A:The number of Nissl bodies in each DEHP dose group significantly reduced compared to solvent control group,and the cells were obviously lightly stained and arranged loosely.(2)Group B:The number of Nissl bodies in DG area of each DEHP dose group significantly reduced compared to solvent control group,and the cells were obviously lightly stained and arranged loosely.4.Oxidative stress:(1)Group A:Compared to solvent control group,the SOD activity in the cerebral cortex of 200 mg/kg/d group significantly decreased(P<0.05);(2)Group B:Compared to solvent control group,the SOD activity in the cerebral cortex of high-dose control group significantly decreased(P<0.05);5.Mechanism exploration:(1)Group A:Western blot results showed that compared to solvent control group,the expression of CREB significantly decreased in200 mg/kg/d group,and the CREB phosphorylation level significantly increased in 1mg/kg/d group,the expression of ERK1/2 of 10,50,and 200 mg/kg/d groups,the expression of GAP65+67 of 50 and 200 mg/kg/d groups,and the expression GABA_ARα2 of 200 mg/kg/d dose group significantly decreased(P<0.05).(2)Group B:Western blot results showed that expression of GABA_ARα2 of each dose group and expression of GAD65+67 of high-dose control group in the hippocampus significantly decreased(P<0.05);Muscimol could reverse the decrease in GAD65+67 expression caused by DEHP exposure.The results of immunohistochemistry showed that expression level of GABA_ARα2 in CA2 region of high-dose control group,low-dose intervention group and high-dose intervention group and CA3 region of high-dose control group significantly decreased(P<0.05).Compared to high-dose control group,expression of GABA_ARα2 high-dose intervention group significantly increased(P<0.05).Compared to solvent group,expression of ERK1/2 CA3 region significantly decreased in high-dose intervention group(P<0.05).The expression level of ERK1/2 in CA3 region of high-dose intervention group was significantly lower than that of high-dose control group(P<0.05).Compared to solvent group,expression of CREB in CA1 region of high-dose intervention group significantly decreased,expression of CREB in CA1,CA2and CA3 regions of low-dose intervention group significantly decreased;Compared to low-dose control group,CREB expression in CA1 and CA2 regions of low-dose intervention group significantly decreased(P<0.05);Compared to high-dose control group,expression of CREB in CA1,CA2,and CA3 regions of high-dose intervention group significantly decreased(P<0.05).Conclusion:Under the experimental conditions,DEHP exposure may cause anxiety and depression-like behavior in male rats,and its mechanism may be related to oxidative stress,GABA signaling pathway and ERK1/2-CREB signaling pathway.The specific mechanism needs further study.