Effects Of Subchronic 1,2-DCE Exposure On CAMP-PKA-CREB Signaling Pathway And Synapse-associated Protein Expression In Mouse Hippocampal Tissue

Objective:The halogenated hydrocarbon 1,2-dichloroethane（1,2-DCE）is not only a common organic solvent in factories,but also a widespread environmental pollutant.1,2-DCE is highly toxic in the central nervous system toxicant class,and its subchronic exposure has been shown to cause learning and memory dysfunction,but the exact mechanism is unclear.Long-term potentiation（LTP）in the hippocampus is not only an ideal model for synapses,but also an important neurobiological basis for learning memory.Many studies have shown that the induction and maintenance of LTP depends on the opening of calcium(Ca²⁺)channels in the N-methyl-D-aspartate receptor（NMDAR）,which allows Ca²⁺to flow into the postsynaptic membrane and bind to substrate proteins to exert a series of biological effects.Numerous studies have shown that the cyclic adenosine monophosphate-protein kinase A-c AMP response element binding protein（c AMP-PKA-CREB）signaling pathway,regulated by Ca²⁺binding to calmodulin（Ca M）,is essential for the maintenance of LTP.In addition,synaptophysin（SYP）,a calcium-binding protein,is specifically present in the presynaptic vesicle membrane and mainly regulates the release of neurotransmitter stores in vesicles and vesicle transport.Postsynaptic density protein 95（PSD 95）is located in the dense region of the postsynaptic membrane and is not only closely related to LTP induction and maintenance,but also an important indicator of synaptic development.The aim of this study was to investigate the changes of c AMP-PKA-CREB signaling pathway and synaptic-related protein expression in hippocampal tissue by subchronic 1,2-DCE exposure,and to provide an experimental reference for revealing the mechanism of learning memory dysfunction.Methods:Healthy female albino Kunming mice were randomly assigned to the control and 0.225 g/m³,0.45 g/m³ and 0.9 g/m³ 1,2-DCE exposure groups after one week of acclimatization feeding.The mice were exposed by static inhalation for 3.5 h per day for8 weeks to establish a subchronic 1,2-DCE-exposed animal model.Firstly,we investigated the effects of subchronic 1,2-DCE exposure on the learning and memory functions of mice through novelty recognition and water maze experiments;at the end,we anesthetized the mice and observed the morphological pathological changes of cone cells in CA1 area of hippocampus by HE staining.The pathological changes of subchronic 1,2-DCE exposure on the synaptic ultrastructure of neurons in CA1 area of mouse hippocampus were observed by transmission electron microscopy.In addition,the expression of AC and c AMP in mouse hippocampus was detected by ELISA;the protein and m RNA of pathway-related proteins Ca M,PKA,PKA-C,CREB,p-CREB,BDNF,c-fos,c-Jun and synapse-related proteins SYP and PSD 95 were detected by Western blotting and RT-PCR.expression levels.Finally,the expression of PKA,SYP and PSD 95 in various hippocampal regions was observed by immunofluorescence.Result:Subchronic 1,2-DCE exposure impaired learning memory behavior in mice.In novelty recognition,the recognition index of old and new objects in the training period and the recognition index and discrimination index of old and new objects in the test period decreased with increasing dose.In addition,the localization cruising phase of the water maze experiment showed a gradual increase in escape latency in the exposed group compared to the control group after the fourth day of training.Not only that,the percentage of spent time in the target quadrant showed a dose-dependent decrease in the spatial exploration experiments of the exposed group of mice during the test period,but also the heat map and trajectory plot showed purposelessness and dispersion.The pathological changes such as reduced number and loose distribution of cone cells in the CA1 area of the exposed mice were observed.In addition,the expression of AC and c AMP in the hippocampus of the exposed mice decreased,and the protein and m RNA expression levels of pathway-related proteins Ca M,PKA,PKA-C,CREB,p-CREB,BDNF,c-fos,and c-Jun decreased in a dose-dependent manner.Not only that,the fluorescence expression of PKA in DG,CA3 and CA1 regions of hippocampus was consistent with the trend of protein resultant expression.The number of synapses,PSD thickness,and AZ active band length in the CA1 region of hippocampus of exposed mice decreased with increasing dose of dosing and the synaptic gap widened with increasing dose of dosing compared with the control group,as observed by transmission electron microscopy.The protein and m RNA expressions of synapse-related proteins SYP and PSD 95 and their fluorescence-positive expressions decreased with the increase of the dose.Conclusion:Subchronic 1,2-DCE exposure leads to learning memory dysfunction in mice,and the mechanism of the impairment may be related to the inhibition of the c AMP-PKA-CREB signaling pathway in the hippocampus,damage to synaptic structures and down-regulation of synaptic-related protein expression,thereby affecting the efficacy of synaptic transmission.