| Background:Cadmium,a widespread toxic heavy metal can accumulate in multiple organs by diet and breath.Direct exposure to Cd can cause toxicological damage to organs such as liver,kidney,testis,lung,and brain,and many studies focused on this field have been performed.However,the underlying mechanism of Cd exposure in the offspring to the central nervous system has not been fully discovered.In our previous study,we have found that the phospholipase PLC?4 in the hippocampus of male offspring is showed abnormally high expression after maternal Cd exposure during pregnancy and lactation.As an essential neurotrophic factor,exogenous BDNF could reverse the up-regulation of PLC?4,suggesting that there may be a relationship between PLC?4and BDNF.PLC?1,an isoenzyme of PLC?4,is also widely involved in the regulation of central nervous growth and development.The studies on the damage of brain development caused by Cd exposure in fetus and children have been focused on epidemiology,and the target proteins and molecular mechanism involved are rarely reported.It is of great significance to elucidate the molecular mechanism of the damage to the development of the central nervous system of the offspring caused by the exposure of maternal CD.These results help to provide the laboratory data and theoretical basis for the therapy of children's learning and memory impairment.Objective:It aims to disclose the molecular mechanism of phospholipase PLC?4/1 involved in the damage of learning and memory ability of male offspring by exposure to maternal Cd.Methods:Experiments in vivo:Adult female and male SD rats were mated at a ratio of 1: 1 or 1: 2 for 3-4 days.After confirming the successful conception,female rats were randomly divided into blank groups(Ctrl: 0.9% Normal saline),the low-dose group(Cd1: 1 mg / kg)and the high-dose group(Cd5: 5 mg / kg Animals received cadmium chloride(Cd Cl2,Sigma,St.Louis,MO,USA;1?mg/kg b.w.defined as Cd1,5?mg/kg b.w.defined as Cd5)solution or water by gavage during pregnancy and lactation.F1 female offspring at postnatal day(PND)21,35 and 56 were sacrificed and tissue samples were collected for examination.Hippocampus was fixed with paraformaldehyde to prepare frozen sections.The liquid chromatograph-mass spectrometer(LC-MS)was used to determine the endogenous cannabinoid 2-AG content in hippocampus.WB was used to determine the protein levels of signal pathway mGlu R5/PLC?1/DGL? and PGRMC1/BDNF pathway mediated by PLC?4.The offspring rats were subjected to Y maze behavioral experiments at PND 35 and PND 56,and old male offspring were subjected to water maze behavioral experiments to evaluate the impairment of the learning and memory abilities.Magnetic Resonance imaging Diffusion Kurtosis Imaging(MRI-DKI)was performed to evaluate tissue complexity and neuron loss in the CA1 area.Experiments in vitro:Primary hippocampal neurons were separated from brains of newborn Sprague-Dawley rats.Cells were exposed to Cd in the concentration 2 ?M,4 ?M,and6 ?M,respectively.After 24 hours,the expression levels of mGlu R5,PLC?1,and DGL? protein molecules in neurons were determined by WB.The expression of PLC?4 in PC12 cells and hippocampal neurons were knocked down using si RNA interference technology,and then the expression levels of PGRMC1,BDNF and PLC?4 were detected.Exogenous progesterone(P4)was added into the culturemedium of cells exposed to Cd(2 ?M),and then the protein expression levels of PGRMC1,BDNF and PLC?4 in the cells were determined.Results:1.The spontaneous alternation rate in Y maze experiment was significantly decreased in adolescent rats of group Cd1 and Cd5(P<0.01).When the rats grew to adulthood(PND 56),the spontaneous rate still kept decreased significantly(P<0.001).2.In the Morris water maze experiment,the escape latency in aged rats of Cd5 group was significantly prolonged(P<0.05),and the number of crossing platforms was also significantly reduced(P <0.05).3.In the MRI-DKI images of the aged male offspring,the average water molecule diffusion rate(Diffusion Kurtosis Imaging-Mean diffusivity,DKI-MD)value increased significantly in the rats of group Cd5;the escape latency and DKI-MD were highly positively correlated(r = 0.9783,P<0.0001)in the Pearson's correlation test);while the average diffusion peak value of water molecules(Diffusion Kurtosis Imaging-Mean kurtosis,DKI-MK)was significantly reduced,and it showed a highly negative correlation with escape latency in water maze(r =-0.9708,P<0.0003).4.Compared with Ctrl group,the male offspring at PND 21 had significantly higher protein level of PLC?4 in hippocamous of Cd5 group(P<0.05),and significantly lower levels of MMP9,PGRMC1,and BDNF(P <0.05).Compared with Ctrl group,the male offspring at PND 35 had significantly higher level of PLC?4 in Cd1 and Cd5 groups(P<0.01),and significantly lower levels of MMP9,PGRMC1,and BDNF in Cd5 group(P <0.05);the expression level of BDNF in Cd1 group was significantly down-regulated(P<0.01).Compared with Ctrl group,the male offspring at PND 56 had significantly higher level of PLC?4 in Cd5 group(P<0.05),andsignificantly lower levels of MMP9,PGRMC1,and BDNF in the Cd1 group(P<0.05);the expression levels of MMP9,PGRMC1 and BDNF were significantly down-regulated in Cd5 group(P<0.05).Compared with the Ctrl group,the aged male offspring had significantly higher level of PLC?4 in Cd5 group(P<0.05),and significantly lower levels of MMP9,PGRMC1,BDNF,p-Trk?,and Trk?(P<0.05);the phosphorylation of Trk? in Cd1 group was significantly down-regulated(P<0.001).5.In the si RNA interference experiment of PC12 cell line and hippocampal neurons,PC12 cells were treated with Cd(Cd: 4 ?M)after gene silencing.The expression level of PLC?4 in the cells of 4 ?M group was significantly increased(P<0.01),while BDNF protein level was significantly down-regulated(P<0.05).Compared with the 4 ?M group,the expression of PLC?4 in the cells of 4 ?M + si group and the si group was significantly down-regulated(P<0.001),while the expression level of BDNF was significantly increased(P<0.001).After silencing PLC?4 in hippocampal neurons after Cd treatment(Cd: 2 ?M),compared with 2 ?M group,the expression level of PLC?4 in cells of 2 ?M + si group and si group was significantly down-regulated(P<0.001),while PGRMC1 protein level was significantly increased(P<0.05,P<0.001),and the expression level of BDNF was also significantly increased(P<0.01,P<0.001).6.In the si RNA interference experiment of male hippocampal neurons,after PGRMC1 silencing,compared with the negative control(negative control,NC),the protein expression levels of PGRMC1,MMP9 and BDNF in the si-634 group were significantly down-regulated(P<0.01,P<0.01,P<0.05),and PLC?4 protein expression level was significantly up-regulated(P<0.05).7.The hippocampal neurons were treated with progesterone(P4)while receiving Cd exposure.Compared with the Cd exposure group(Cd: 2 ?M),the protein expression of PLC?4 in the cells of 2 ?M + P1 group(P1: progesterone 1 n M),2 ?M+P3 group(P3: progesterone 3 n M)and 2 ?M + P9 group(P9: progesterone 9 n M)wassignificantly down-regulated(P<0.01,P<0.001,P<0.001),while the protein expression level of PGRMC1 was significantly increased(P<0.05,P<0.01,P<0.01),and the protein expression level of BDNF was also significantly increased(P<0.01,P<0.05,P<0.001).8.The content of 2-AG in hippocampus was determined by high performance liquid mass spectrometry.The content of endogenous cannabinoid 2-AG had no significant change in the hippocampus of male offspring with different treatments during childhood(PND 21).In adolescence(PND 35),the endogenous cannabinoid2-AG content decreased significantly in the hippocampus of rats in the Cd5 group(P<0.001).In adulthood(PND 56),the endogenous cannabinoid 2-AG content decreased significantly in the hippocampus of rats in the Cd1 group and the Cd5group(P <0.001,P <0.05).9.Compared with the control group of corresponding age,the protein expression level of mGlu R5,PLC? 1 and DGL? were significantly reduced in hippocampus of rats in group Cd5 at PND 21(P <0.05),in group Cd1 and Cd5 at PND 35 and 56(P<0.05).When male offspring were aged,the expression levels of mGlu R5,PLC?1 and DGL? in in hippocampus of rats in group Cd5 were also significantly reduced(P<0.05).10.When the hippocampal neurons were exposed to Cd,compared with the control group,mGlu R5,PLC?1 and DGL? of the endogenous cannabinoid 2-AG generation pathway in the 4 ?M group and the 6 ?M group showed a significant downward trend(P<0.01).Conclusion:(1)Cd exposure during pregnancy and lactation can damage the learning and memory abilities of male offspring for a long time and irreversibly until the old age.(2)Cd negatively regulates progesterone mediated BDNF production pathway PGRMC1/BDNF by targeting on PLC?4,which ultimately reduces BDNF production in hippocampus;meanwhile,BDNF can negatively feedback and regulate the expression level of PLC?4,and there is a regulatory relationship between PLC?4 and BDNF.(3)After exposure to Cd during maternal and lactation,in the male offspring,Cd negatively regulates the endocannabinoid 2-AG generation signal pathway mGlu R5/PLC?1/DGL?,thereby reducing the generation of 2-AG in the hippocampus and ultimately impairing the learning and memory abilities of male. |
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