The Effects Of Mixed Exposure Of Low-dose Heavy Metals On Rat Synapses And Their Regulatory Proteins

Objectives:Heavy metal pollution has become the most important type of environmental pollution today.Due to its wide distribution in the environment,it is not degradable by microorganisms,and it is a major hazard to human health.Environmental heavy metal pollution has always been a public health problem that affects the health of people.Exposure to these metals at the same time or in succession through a variety of ways,low-dose exposure of the three heavy metals lead,cadmium and mercury may not cause damage to the body,but when they are exposed together,they may cause public health problems.Therefore,this study aims In the establishment of animal models of three heavy metal mixed exposures of lead,cadmium and mercury at different ages,the effects of low-dose mixed exposure on the motor and learning and memory abilities of rats and the related synaptic regulatory mechanisms that may be involved were explored to explain the effects of mixed heavy metal exposure on rats Changes in learning and memory abilities,dendritic spine density,and related regulatory proteins provide a basis for dynamic changes with development.Methods:1.Establishment of animal models8-week-old sexually mature SPF female and male SD rats,after one week of adaptive feeding,taking into account the weight factor,the rats were caged according to the ratio of male to female 2: 1,and the pregnant rats were detected by vaginal smear the next day.The obtained pregnant rats were divided into a blank control group(Control,drinking distilled water),a 1 × MM group(drinking 20 mg / L lead acetate trihydrate,7 mg / L cadmium chloride and 0.1 mg / L of mercury chloride mixed poisoning solution),5 × MM group(drinking 5 times 1 × MM mixed poisoning solution),male offspring rats are used as experimental objects,and during pregnancy and lactation,the offspring drink through mother rats The poisoned solution was exposed until weaning.After weaning,the blank control group continued to drink distilled water.1 × MM rats drink 6.67 mg / L lead acetate trihydrate,1mg / L cadmium chloride,0.03 mg / L chlorine Mercury mixed poisoning solution,the 5 × MM group drank the mixed poisoning solution containing 5 times the concentration of 1 × MM,reared until 54 days after birth,and the body weight and growth status of the rats were detected,respectively.),Weaning(PND24),puberty(PND54),the rats were dissected and sampled for testing.2.Detection of heavy metal content in rat whole blood and brain tissueThe Nex ION 300 X inductively coupled plasma mass spectrometer was used to determine the content of lead and cadmium,and the AFS-8330 atomic fluorescence photometer was used to determine the content of mercury.3.Detection of learning and memory abilityThe dark avoidance test,the open field test,the new object recognition test and the Morris water maze test were used to test the learning and memory abilities of weaning and adolescent rats.4.Growth and development and early neurobehavioral testingAfter birth,the offspring rats were monitored for changes in body weight and early neurobehavioral development indicators were detected at specific times.Seven-dayold rats underwent plane reflex,negative geotaxis and cliff avoidance The behavioral test of the 10-day-old answer book was conducted for the forelimb suspension and tailflick test,and the 12-day-old rat was tested for the hind limb support test.After birth,the offspring weight change is monitored,and the offspring ’s early neurobehavioral development indicators are detected at a specific time.The seven-dayold baby rat conducts plane reflex,negative ground motion,cliff avoidance For academic testing,ten-day-old pups were tested for behaviors of forelimb suspension and tail-flick test,and twelve-day-old pups were tested for hind limb propulsion test.5.Detection of changes in dendritic spine structureGolgi-Cox method was used to detect the density of dendritic spines in various regions of rat brain.6.Detection of the expression levels of dendritic spines related regulatory proteins Arp3,N-WASp,Cofilin and Cofilin related regulatory proteins LIMK1 and SSH1LWestern Blot method was used to detect the protein expression levels of Arp3,NWASp,Cofilin,Cofilin,LIMK1,p LIMK1,SSH1 and p-SSH1 at different ages.Results:1.The effect of mixed exposure of heavy metals on the content of three heavy metals in lead blood,cadmium and mercury in the blood and brain tissues of offspring ratsThe three heavy metals of lead,cadmium and mercury in the blood and brain of rats increased significantly with the increase of the exposure dose(P <0.05),and the heavy metal contents of the 1 × MM group in the weaning blood were(lead 26.72 ±7.56),(Cadmium 0.50 ± 0.30),(mercury 1.41 ± 0.23),and the heavy metal contents of the 1 × MM group in the blood of adolescent rats were(lead 30.05 ± 6.58),(cadmium0.76 ± 0.17),(mercury 1.34 ± 0.49),all It is consistent with the levels of lead,cadmium,mercury and heavy metals in the blood of the general population in China.2.Effects of mixed exposure of heavy metals on growth and development and early neurobehavior of young ratsThe offspring rats grow well after birth,and the weight of the rats in each group shows a gradual increase.The weight of each group at birth is not statistically significant.From the ninth day after birth to the 49 th day after birth,each Compared with the control group,the weight of the poison group was significantly lower than that of the control group.The plane correction score and adjusted hindlimb support coefficient decreased with the increase of the dose group,and the difference was statistically significant in the 5 × MM group(P <0.05);the plane correction time,cliff avoidance score,and cliff avoidance time were 1 There was a difference in the × MM group(P <0.05);although the negative geotaxis score,tail flick test and forelimb suspension time had a certain dose-response relationship,no significant difference was observed(P> 0.05)3.The effect of mixed exposure of heavy metals on learning and memory in ratsThe field test results of weaning period and puberty show that compared with the control group,the stay time of rats in the central area of each exposure group was not significantly prolonged,but compared with that of the weaning rats,For a long time,there is a tendency to produce anxiety.Compared with the control group,the results of the dark avoidance experiment in weaning rats showed that the memory latency of the rats in the exposure group was significantly prolonged and there was a statistical difference from the 1 × MM group,and the number of memory errors in the 1 × MM group was also significant The number of memory errors in the 5 × MM group also increased but the difference was not significant.The results of the new object recognition test showed that there was no difference in the time of sniffing new objects among the groups of weaning rats.The identification index DI of the heavy metal mixed exposure group had a downward trend,but there was no statistical significance(P> 0.05).The sniffer time of new objects and the identification index DI decreased significantly with the increase of the exposure dose,and the difference was statistically significant in the 5 × MM group(P <0.05).Morris water maze test results showed that with the increase of training days in weaning and puberty rats,the escape latency of the heavy metal mixed exposure group was longer than that of the control group,and both were 5 × MM on the fourth day of training.The escape latency of the group was significantly prolonged(P <0.05).After standardization,it was found that the escape latency of rats in the pubertal exposure group was longer than that of weaning rats.4.The effect of mixed exposure of heavy metals on dendritic spine density at different agesMixed exposure of heavy metals will lead to a decrease in dendritic spine density in various regions of the brain tissue of the weaning pups.The density in the CA1 and DG area exposure groups is significantly lower than that in the control group.The density of dendritic spines in the brain tissue of the pubertal young rats mixed with heavy metal exposure group showed a decreasing trend.The overall density of adolescent dendritic spines in the 1×MM group was significantly lower than that during weaning,suggesting that mixed exposure of heavy metals interfered with dendritic spinal density changes during normal development.5.Effects of mixed exposure of heavy metals on the expression of dendritic spinerelated regulatory proteins NWASp-Arp3,SSH1/ LIMK-CofilinThe expression of Arp3 and N-WASp protein in the low-dose mixed exposure group of neonatal pups had a downward trend,and the difference between the control group N-WASp and 1×MM group was statistically significant(P<0.05).Cofilin activity of neonatal mixed exposure group decreased,LIMK1 and SSH1 activity decreased,hippocampal and cortical Arp3 and N-WASp expression trends during weaning and puberty were consistent with neonatal period,and Cofilin,LIMK1 and SSH1 expression trends changed It is also consistent with the freshman period.The test results of different age groups in the same dose group found that first,mixed exposure of heavy metals would damage the expression of dendritic spine-related regulatory protein SSH1/LIMK1-Cofilin during normal development..Conclusions:1.The mixed exposure of three heavy metals,lead,cadmium and mercury,will cause damage to the learning and memory ability of rats,and the damage is dosedependent and time-accumulative.2.The mixed exposure of three heavy metals,lead,cadmium and mercury,can damage the plasticity of the synaptic structure in the hippocampus and cortex of the rat brain,and the damage has cumulative effects in different developmental processes.3.The damage of learning and memory ability and the changes of synapse structure caused by the mixed exposure of heavy metals may be related to the abnormal expression of Arp3,N-WASp,Cofilin and p-Cofilin.