N-Acetyl-L-Cysteine Ameliorates Synaptic Vesicle Injury In Manganese Exposed Mice Hippocampal Neurons

Objective:Manganese is an essential trace element of human body.It is involved in the synthesis of a variety of enzymes and can activate enzymes.At the same time,it regulates a variety of physiological and biochemical processes in the body and maintains the balance of cells and even the internal and external environment of the body,to promote the health and activity of living organisms.After penetrating the blood-brain barrier,excessive manganese accumulates in multiple brain regions,which can cause degeneration of neurons in corresponding regions,resulting in"manganese poisoning".The cognitive dysfunction caused by manganese poisoning is mainly manifested in the defects of receiving new content and reviewing past events,which are closely related to hippocampal dysfunction.The mechanism of manganese-induced hippocampal participatory cognitive impairment is not fully understood.Oxidative stress is thought to be a potential mechanism for manganese-induced cognitive impairments.Mn causes a large amount of reactive oxygen species（ROS）to be generated,disrupts cell structure,and ultimately causes irreversible oxidative damage to tissues.Studies have shown that synapses are more sensitive to the toxic effects of manganese than neurons.Therefore,synaptic damage is important for manganese-induced neurodegeneration.Synaptic vesicle is an important organelle in the presynaptic structure.There is growing evidence that once synapses are designated to be destroyed,synaptic vesicles are also rapidly destroyed,and the damage of synaptic vesicles occurs even before synaptic degeneration,so research based on synaptic vesicle levels is important.In neurons,Rab26 protein has a specific connection with synaptic vesicle clusters and only localizes on the membrane of the functionally impaired synaptic vesicle,which provided a new pathway for us to screen and discover damaged synaptic vesicles.N-Acetyl-L-cysteine is a potent antioxidant that has been widely used in clinical treatment of neurodegenerative disorders and other neurological or psychiatric disorders,with positive effects in most diseases.In this study,we observed the neurobehavioral changes of mice and detected the synapse、synaptic vesicle damage and nerve cell oxidative injury in mice to evaluate the protective effect of NAC on manganese-induced synaptic vesicle damage in mouse hippocampal neurons.This provided a new basis for the study of the protective effect of NAC on manganese-induced neurotoxicity.Methods:In this study,40 10-week-old wild-type C57BL/6 mice were selected.The number of females and males was half.The feeding temperature was 24±2°C,and the humidity was 30-40%.Timely replacement of bedding and ventilation,supplement of rat food and drinking water,mice in the cage free movement and free feeding water.After 1week,mice were randomized into 4 groups according to body weight:control group:an intraperitoneal injection of normal saline solution,dorsal subcutaneous injection of normal saline;and manganese exposure group:intraperitoneal injection of a 100μmol/kg solution of Mn Cl_2,subcutaneous injection of normal saline onto the dorsum.Control group of NAC mice:intraperitoneal injection of normal saline,subcutaneous injection of 100 mg/kg NAC solution on the dorsum;NAC intervention group:subcutaneous injection of NAC solution100 mg/kg,1 h later,intraperitoneal injection of Mn Cl₂ solution 100μmol/kg.The injection volume was 5 ml/kg.Mn Cl₂ exposure and NAC pretreatment were performed every Monday to Friday for 6 weeks.The following tests were performed:the experiments to evaluate the neurobehavioral changes of mice included open field experiment,water maze experiment and jumping platform experiment.The manganese content in hippocampus of mice in each group was detected.The ratio of apoptotic cells to total cells and the ROS level of nerve cells were analyzed by flow cytometry.Nissl staining was used to evaluate the survival of neurons in specific sites.The number of spikes on neuronal dendrites was observed by Golgi staining.Ultrastructure of neuronal synapses observed under transmission electron microscope.The expressions of PSD-95,SYN 1,SYT 1,SYP and Rab26 were detected by western blotting.The co-localization levels of SYP protein and Rab26 protein were detected by immunofluorescence.The levels of GSH and MDA in nerve cells were detected.Results:Under the exposure of 100μmol/kg manganese chloride solution for 6 weeks,Mice showed abnormal neurobehavioral manifestations such as decreased autonomic activity and cognitive defects.The results of oxidative injury level showed that the levels of ROS and malonaldehyde increased significantly and the level of glutathione decreased significantly after manganese exposure.In the NAC control group,ROS and malonaldehyde levels decreased,while glutathione levels increased.Compared with the manganese exposure group,the levels of ROS and malonaldehyde in the NAC intervention group decreased,and the level of glutathione increased.This indicated that manganese induced oxidative stress and caused lipid oxidative injury,while NAC increased glutathione level and alleviated manganese-induced lipid oxidative injury.The manganese content and early apoptosis level of mice in the manganese exposure group were significantly increased.In the CA1 region,some nerve cells were folded,the volume was reduced,and the number of surviving neurons was reduced.In the manganese exposure group,the number of spines on dendrites was significantly reduced.The results of electron microscopy showed that the thickness of postsynaptic dense material decreased in manganese exposure group.At the same time,the number of presynaptic vesicles in manganese exposure group increased significantly.After pretreatment with 100 mg/kg NAC solution for 6 weeks,compared with the manganese exposure group,the proportion of apoptotic cells in the NAC intervention group decreased,the number of non-dead neurons increased,and the number of spines on dendrites increased.At the same time,the structure of postsynaptic dense substance was compensatory thickening,the abnormal aggregation of synaptic vesicles was alleviated less,and the number of synaptic vesicles decreased.The results of protein detection showed that the expression level of synapse-related proteins in the manganese exposure group decreased.On this basis,the NAC intervention group increased the expression level of synapse-related proteins.The expression levels of SYT 1,SYP and Rab26 in the manganese exposure group were increased,and the expression levels of SYT 1,SYP and Rab26 in the NAC intervention group were lower than those in the manganese exposure group.Fluorescence staining results showed that the co-localization area of SYP protein and Rab26 protein in the manganese exposure group increased,and the co-localization area of SYP protein and Rab26 protein in the NAC intervention group decreased.These results indicated that manganese exposure led to neuronal synaptic damage,resulting in the accumulation of damaged synaptic vesicles and the aggravation of synaptic vesicle damage.NAC alleviated manganese-induced neuronal,neuronal synapse and synaptic vesicle damage.Conclusion:Manganese-induced excessive ROS production causes oxidative damage,and leads to the accumulation of damaged synaptic vesicles in hippocampal neurons,resulting in synaptic vesicle damage and synaptic damage.NAC may alleviate manganese-induced synaptic vesicle damage and reduce manganese neurotoxicity through antioxidant effect.