| Background:China has become the world’s largest producer of copper processing materials,and human activities such as a large number of mining,industrial wastewater discharge,sewage irrigation and others have aggravated the problem of copper pollution.The content of copper in some areas obviously exceeds the environmental capacity.The increase of copper content in environmental media has greatly increased the chance of human contact,and the risk of environmental exposure is increasing day by day.Many studies have shown that copper metabolism disorders are closely related to the occurrence and development of neurodegenerative diseases.However,at present,there are relatively few studies on copper neurotoxicity,and its specific mechanism is still not clear.Therefore,it is of great significance to explore the neurotoxic mechanism of copper.Objective:In this study,C.elegans was used as the research object to explore the role of oxidative stress in copper-induced nerve damage and its molecular mechanism,so as to provide a scientific basis for assessing the health risks of copper exposure and carrying out related research.Methods:In this study,the corresponding C.elegans strains were selected for related experiments according to different experimental purposes.The copper exposed doses of the C.elegans strains used were 0.01,0.1,1 and 10mg/L,respectively and exposed time ranged from L4 larval to adult stage.1.Effect of copper exposure on nervous system of C.elegansIn this study,the head swinging frequency,body bending frequency,pharyngeal pump frequency,defecation rate,chemical tendency test and other indexes of N2 nematode were observed by stereomicroscope to reflect the neurotoxic effects caused by copper exposure from the perspective of neurobehavior.The neuronal structures of BZ555,EG1285,LX929 and DA1240 strains were observed under a fluorescence microscope and the fluorescence intensity of the neurons was quantified to evaluate the effect of copper on the neurons of reporter gene strains.RT-q PCR was used to determine whether copper could affect the gene transcription of glutamate,serotonin and dopamine in N2nematodes at the gene level.2.Effects of copper on oxidative stress of beautiful nematode.The levels of ROS,mitochondrial membrane potential and the expression of SOD and GSH-Px genes in N2 nematodes were detected by H2DCFDA probe,JC-1 probe and RT-q PCR.Fluorescence microscope was used to observe the reporter gene strain CF1553 to reflect the expression level of SOD-3 protein,so as to explore the role of oxidative stress in nerve injury.3.The p38 MAPK signaling pathway targeting SKN-1/Nrf2 regulates nerve damage induced by copper in C.elegansThe head swing frequency,pharyngeal pump frequency,defecation rate and other indicators of KU25（pmk-1 gene mutant encoding MAPK）were observed under a volumetric microscope,and the SKN-1 nuclear transfer of LD1 was observed under a fluorescence microscope to analyze whether copper regulates SKN-1/Nrf2-induced oxidative damage through p38 MAPK signaling pathway.This can lead to neurological dysfunction.Results:1.Effects of copper on nervous system of C.elegans1.1 Damage of copper to neural behavior of C.elegans（1）The body bending and head swinging frequencies of N2 nematodes in the infected groups were significantly different from those in the control group,and decreased with the increase of copper concentration（P<0.01）.（2）The pharyngeal pump frequency of N2 nematodes in 0.1mg/L,1mg/L and 10mg/L groups decreased gradually with the increase of copper exposure,and compared with the control group,there were statistically differences among them.（P<0.01）.（3）With the increase of copper concentration,the defecation time interval of N2nematodes gradually prolonged and the defecation rate decreased.There were significant differences between each treatment group and the control group（P<0.01）.（4）The chemical trend index of N2 nematodes decreased with the increase of copper concentration.The trend index of 1mg/L group and 10 mg/L group was negative,and the differences were statistically significant compared with the control group（P<0.01）.1.2 Damage of neurons and synapses in C.elegans by copper（1）Compared with the unexposed group,the fluorescence area and intensity of Glutamate,Glu and DA1240 neurons in the head of nematode in 0.01mg/L,0.1 mg/L and 1mg/L groups were not changed,and the differences were not statistically significant（P>0.05）.However,the fluorescence area and intensity of the neural ring in the head of nematodes in the 10 mg/L group were significantly decreased,with statistical significance（P<0.01）.（2）γ-aminobutyric acid（GABA,EG1285）neurons were broken and lost to different degrees in all the infected groups,and the degree of broken and lost neurons in the high concentration group was higher than that in the low concentration group.Compared with the control group,the fluorescence intensity of GABAgic neurons after copper exposure gradually decreased with the increase of exposure concentration,with statistical significance（P<0.01）（3）Acetylcholine（Ach,LX929）neurons of the tested strains in all the infected groups were broken and lost to varying degrees,among which the broken and lost neurons in the high concentration group were more serious.Compared with the control group,the fluorescence intensity of cholinergic neurons in all exposed groups was decreased,and the differences were statistically significant（P<0.01）.（4）Compared with the non-exposed group,the fluorescence area of dopaminergic neurons（DA,BZ555）in the copper-exposed group was decreased,and the fluorescence area of dopaminergic neurons（DA,BZ555）in the copper-exposed group was significantly lower than that in the control group.Compared with the control group,the fluorescence intensity of dopaminergic neurons in all the exposed groups was decreased,with statistical significance（P<0.01）.（5）After copper exposure,the expression levels of glutamate transporter and glutamate receptor-related genes were decreased in different degrees compared with the control group,with statistical significance（P<0.01）.The expressions of dopamine transporter and dopamine receptor-related genes were down-regulated to varying degrees,with statistical significance（P<0.01）.Compared with the control group,the expression levels of serotonin transporter and serotonin receptor-related genes were decreased to different degrees,with statistical significance（P<0.01）.2.Effects of copper on oxidative stress of C.elegans2.1 Effects of copper on oxidative damage of ROS and mitochondria of C.elegans（1）With the increase of copper exposure,the ROS level of N2 nematodes increased gradually.The differences between the treatment groups and the control group were statistically significant（P<0.01）.（2）After copper exposure,mitochondria of N2 nematodes were damaged and mitochondrial membrane potential decreased.The differences between the treatment groups and the control group were statistically significant（P<0.01）.2.2 Effect of copper on the expression level of antioxidant enzymes in C.elegans（1）After copper exposure,GSH-Px-related genes of N2 nematodes were down-regulated to varying degrees compared with the control group,and the difference was statistically significant（P<0.01）.（2）Compared with the control group,SOD-related genes of N2 nematodes were down-regulated to varying degrees after copper exposure,with statistical significance（P<0.01）.（3）The green fluorescence intensity of CF1553 represents the expression level of SOD-3 protein in nematodes.After copper exposure,the green fluorescence area of C.elegans decreased gradually,and the mean fluorescence intensity of each dose group decreased compared with the control group,with statistical significance（P<0.01）.In other words,the expression level of SOD-3 protein in nematode decreased gradually after exposure.3.The p38 MAPK signaling pathway targeting SKN-1/Nrf2 regulates nerve damage induced by copper in C.elegans3.1 Role of p38 MAPK pathway in copper-induced nerve injury（1）Under natural conditions,the head swing frequency of KU25 was lower than that of N2,and the difference was statistically significant（P<0.01）.After 10 mg/L copper exposure,the head swing frequency of N2 nematodes decreased significantly compared with the control group,and the difference between the two groups was statistically significant（P<0.01）.There was no significant change in the head swing frequency of KU25 compared with the control group（P>0.05）.（2）Under natural conditions,the pharyngeal pump frequency of KU25 was lower than that of N2,and the difference was statistically significant（P<0.01）.After 10 mg/L copper exposure,the frequency of N2 pharyngeal pump was significantly slower than that of the control group（P<0.01）.On the contrary,the pharyngeal pump frequency did not significantly slow down after KU25 exposure,and the difference was not statistically significant compared with the control group（P>0.05）.（3）Under natural conditions,the defecation rate of KU25 was lower than that of N2,and the difference was statistically significant（P<0.05）.After 10 mg/L copper exposure,the defecation rate of N2 nematodes decreased compared with the control group,and the difference was statistically significant（P<0.01）.Compared with the control group,the defecation rate of KU25 was similar,and the difference was not statistically significant（P>0.05）.3.2 Copper promotes SKN-1 nuclear transfer through the p38 MAPK signaling pathway（1）With the increase of copper concentration,the expression of SKN-1 gene in N2 nematode was gradually increased,and the difference between the infected groups and the control group was statistically significant（P<0.01）.（2）After copper exposure,SKN-1 protein was significantly accumulated in the intestinal nucleus of C.elegans,and the difference was statistically significant compared with the control group（P<0.01）.（3）After 10 mg/L copper exposure,ROS levels of N2 nematodes were increased,there was statistical significance compared to the non-exposed group,（P<0.01）.There was no significant change in ROS level between KU25 and the control group with no statistically significance（P>0.05）.（4）After copper exposure,the expression of SKN-1 gene in N2 nematodes was up-regulated,and the difference was statistically significant compared with the control group（P<0.01）.On the contrary,there was no significant change in SKN-1 gene expression after KU25 exposure,and the difference was not statistically significant compared with the control group（P>0.05）.Conclusion:1.Copper exposure leads to neurobehavioral defects,neuronal damage and affects the normal conduction and reception of neurotransmitters.Copper has a neurotoxic effect.2.Copper exposure can increase ROS,damage mitochondria,and inhibit the expression of antioxidant enzymes in nematodes,thus increasing the level of oxidative stress.3.Copper may regulate SKN-1/Nrf2-induced oxidative damage through p38 MAPK signal pathway,resulting in neurological dysfunction. |
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