A Comparative Study On The Effects Of Maternal Cd Exposure On Learning Cognition Of Male And Female Offspring And Its Mechanism

Background:Cadmium(Cd)is a kind of heavy metals with long half-life and accumulative properties.Although many studies have preliminarily confirmed the toxicological effects of Cd on liver,kidney,testis,lung,brain and other organs,the detailed mechanism of Cd on central nervous system injury is still unclear,and there is no report on the molecular mechanism of maternal cadmium exposure during gestation and lactation for the development of offspring’s nervous system.At present,the study of Cd neurotoxicity on the brain development of fetuses and young children is only focused on epidemiology,lacking of support and demonstration of laboratory data.Objective:By comparing the effects of maternal Cd exposure on learning and cognitive development of female and male offspring,we investigated the relationship between brain derived neurotrophic factor(BDNF)related signaling pathways regulating synaptic plasticity and Cd-injured neurons,and screened differentially expressed proteins in female and male offspring’s hippocampus by iTRAQ proteomics technology in order to elucidate the toxicological molecular mechanism of maternal Cd exposure impairing the ability of learning and cognitive in offspring.This study will provide laboratory evidence for the pathogenesis and treatment of impaired learning and memory ability in children caused by Cd pollution.Methods:SD pregnant rats were randomly divided into control group(Ctrl),low dose group(1 mg/kg,Cd1)and high dose group(5 mg/kg,Cd5).From the day of pregnancy to 21 days after the birth(PND)of offspring,Cd was exposed to the pregnant rats by gavage once a day according to their weight.After the offspring were born for 35 and 56 days,the male and female offspring were tested for learning and cognitive ability by water maze behavior test;at PND 21,35 and 56,the offspring rats were anesthetized and killed,and the blood,cerebral cortex and hippocampal tissue were collected for testing;the content of Cd in cerebral cortex and hippocampus was detected by atomic absorption spectrometry(AAS);the morphological changes of synaptic structure in hippocampal CA1 region of male and female offspring were observed by transmission electron microscopy(TEM);the expression of progesterone receptor membrane component 1(PGRMC1)and BDNF in hippocampus was detected by immunofluorescence(IF)and immunohistochemistry(IHC);and the expression of PGRMC1,MMP-9(matrix metallopeptidase 9),BDNF,tyrosine kinase B(Trk B)and p-Trk B in hippocampus were detected by WB technology;the express levels of BDNF in hippocampus tissue and serum were detected by ELISA;astrocytes were separated from neonatal rats exposed to Cd during gestation,and co-cultured with hippocampal neurons of normal neonatal rats,and the expression of PGRMC1-MMP 9-BDNF and BDNF-Trk B/p-Trk B were detected by WB.The hippocampal tissues of neonatal rat exposed to Cd during gestation were separated and identified by iTRAQ proteomics;the differentially expressed proteins of male and female offspring in Cd1 group compared with control group were obtained by protein enrichment and pathway analysis;the expression levels of differentially expressed proteins of female and male offspring in different periods were verified by IF and WB techniques;After the hippocampal neurons of neonatal female and male rats were separated to construct cell model of Cd exposed and BDNF was added simultaneously,the express levels of differentially expressed proteins were detected and the upstream and downstream regulation relationship between differentially expressed proteins and BDNF were investigated.Results:1.Cd was detected in the cerebral cortex of both male and female offspring after gestation and lactation exposure to Cd(Cd5: p<0.05),but not in hippocampus(< 0.001 mg/kg).2.The results of Morris water maze showed that at PND 35 the escape latency of male Cd5 group in positioning navigation experiment was significantly higher than that in Ctrl group at the first three days(p<0.05),and the number of quadrants crossing the original platform in space exploration experiment was significantly lower compared with Ctrl group(p<0.05),while the escape latency of female Cd1 group was significantly lower than that in Ctrl group(p<0.01),and in space exploration experiment,the number of quadrants in female Cd1 group was significantly higher compared with Ctrl group(p<0.05).At PND56,the escape latency of male Cd5 group was still significantly higher than that in Ctrl group(p<0.01),and the number of quadrants crossing the original platform in space exploration experiment was significantly lower compared with Ctrl group(Cd5: p<0.05),while the escape latency of female model group was significantly lower compared with Ctrl group(Cd1: p<0.01,Cd5: p<0.01),but there was no significant difference in the number of quadrants crossing the original platform between the female model group and the Ctrl group.3.The results of synaptic morphology showed that at PND 21,compared with Ctrl group,the length of active zone in hippocampus CA1 region of male offspring decreased significantly(Cd1: p<0.05,Cd5: p<0.01),the mean PSD thickness became thinner(Cd1: p<0.05,Cd5: p<0.01),and the width of synapses cleft increased significantly(Cd1: p<0.01,Cd5: p<0.01),and these trends lasted to PND35 and PND56 in male offspring.In the female offspring,compared with Ctrl group,the mean PSD thickness decreased(Cd1: p<0.05,Cd5: p<0.01),the width of synapses cleft increased(p<0.01);at PND 35,the mean PSD thickness increased significantly(p<0.05),while the width of synapses cleft increased inCd5 group(p<0.05);at PND 56,the mean PSD thickness in Cd5 group increased significantly(p<0.05),and the width of synapses cleft did not change significantly in both Cd1 and Cd5 group;specially,the length of active zone in female offspring did not change significantly in these three periods.4.The results of WB showed that the express levels of PGRMC1,MMP-9,BDNF,TrkB and p-TrkB were all down-regulated in Cd1 and Cd5 group in male offspring during thees three periods(Cd1: p<0.05,Cd5: p<0.01).In female offspring,at PND 21,the express level of PGRMC1,MMP-9,BDNF,TrkB and p-TrkB were downregulated in Cd1 group and Cd5 group,compared with Ctrl group.(Cd1: p< 0.05,Cd5: p<0.01).At PND 35,the express levels of PGRMC1,MMP-9,BDNF,TrkB and pTrkB in Cd1 group were up-regulated(p<0.01),but there was no significant change in these five proteins in Cd5 group.At PND 56,PGRMC1,MMP-9,BDNF,TrkB and p-TrkB in Cd1 group and Cd5 group were all up-regulated(Cd1: p<0.05,Cd5: p<0.05).5.The results of ELISA,IF,IHC in hippocampus were consistent with the results in WB.By detecting the content of BDNF in the serum of offspring,the results show that the content of serum BDNF in male model group increased in these three periods(Cd1: p<0.05,Cd5: p<0.01),but decreased in hippocampus,correspondingly.(Cd1: p<0.05,Cd5: p<0.01).In female Cd5 group,the serum BDNF content increased at PND 21(p<0.01),but decreased in hippocampus(p<0.01);at PND35,the serum BDNF content in the Cd1 group decreased(p<0.01)but increased in hippocampus(p<0.01);at PND 56,the serum BDNF content decreased in model group(Cd1: p<0.05,Cd5: p<0.01),but increased in hippocampus Cd1: p<0.05,Cd5: p<0.01).6.Astrocytes and hippocampal neurons were successfully separated from neonatal rats,and astrocytes were purified and identified.WB technique was used to detect the expression of PGRMC1,MMP-9,BDNF,TrkB and p-TrkB in co-culture system.The results showed that the expression of PGRMC1、MMP-9、BDNF、TrkB、p-TrkB in male offspring down-regulated compared with Ctrl group(Cd1:p<0.05,Cd5: p<0.05),while that in female offspring was no significance difference.7.The hippocampal tissues of newborn offsprings in Cd1 and Ctrl group were separated and identified by proteomics.The abundance of differentially expressed proteins were obtained by protein enrichment and pathway analysis.The significant differentially expressed protein phospholipase C β4(PLC β4)was screened out.Compared with Ctrl group,the expression of PLC β4 was up-regulated by 6.8 times in male Cd1 group and was down-regulated by 0.86 time in female Cd1 group.8.The results of WB and IF showed that the express level of PLC β4 in male model group were up-regulated(Cd1: p<0.05,Cd5: p<0.01),while the express levels of postsynaptic density 95(PSD95)were down-regulated in these three periods(Cd1: p<0.05,Cd5: p<0.01).In female offspring,at PND21,the express level of PLC β4 in model group was similar to that of male offspring(Cd5: p< 0.01);At PND35,the express level of PLC β4 in Cd1 group was down-regulated(Cd1: p<0.05),while PSD95 was up-regulated(p<0.05);at PND 56,the expression trend of PLC β4 in Cd5 group was down-regulated(p<0.01),and PSD95 was up-regulated(Cd5: p<0.05).The results of IF were consistent with those in WB.It was suggested that the expression of PLC β4 was significantly different in the dentate gyrus(DG)of hippocampus.9.The hippocampal neurons of Cd-exposure model and Ctrl group was successfully constructed.The relationship between PLC β4 and BDNF was investigated by adding BNDF.WB results showed that in male hippocampal neurons,compared with Ctrl group,PLC β4 in Cd2 group of male hippocampal neurons was significantly up-regulated(Cd2: p<0.01),and BDNF was significantly downregulated(Cd2: p<0.01).After treated with exogenous BDNF,compared with the corresponding groups before treatment,PLC β4 was both in Ctrl+BDNF group(p<0.01 vs Ctrl)and Cd2+BDNF group(p<0.01 vs Cd2).The expressions of BDNF in model group were down-regulated because of the exogenous BDNF(p<0.05,p<0.01).After Cd exposure in female hippocampal neurons,there was no difference in the expression of PLC β4 and BDNF.Similarly,after adding exogenous BDNF,there was no change in the expression of PLC β4 and BDNF Conclusion:(1)After maternal exposure to Cd during gestation and lactation,higher doses of Cd can accumulate in the cerebral cortex of offspring,leading to the plasticity damage of hippocampal synaptic structure,which cannot be reversed in male offspring lasting to adulthood,leading to learning and cognitive impairment in both young and adult males;compared with male offspring,there is less damage to synaptic structure in young female offspring,and the damage is gradually repaired with the development of female offspring and reversed in adulthood.Therefore,female rats in Cd5 model group have learning and cognitive impairment at PND 35,but at PND 56,the damage was reversed and learning and cognitive impairment disappeared.(2)Cd affects synaptic plasticity by affecting the synthesis of BDNF and BDNFTrk B signaling pathway in male and female offspring,thus affecting the biological function of synapses.(3)Based on proteomic data analysis of the hippocampus of the newborn offspring,it was found that PLC β4 increased 6.8 times in the male Cd1 group and participated in regulating several signal pathways related to synaptic structure and function.The expression trend of PLC β4 was verified in animal and cell Cd exposure models respectively.The expression of PLC β4 in model group was higher than that in male Ctrl group throughout the whole developmental period,while the expression of PLC β4 in female model group was gradually decreases accompanying growth.Through addition of exogenous BDNF to hippocampal neurons exposed to Cd it indicated that BDNF and PLC β4 were mutually regulated.