Studies On The Effects Of Lead Exposure On PKC And CaM Expression In Signaling Pathway In Nerve Cells

Objective To observe the effect of chronic lead contaminant on mRNA and protein expression of PKC and CaM in hippocampus of baby-rats and U251 cell line.Methods1 The effect of lead on learning and memory ability and relevant index in baby-rats:The pregnant Wistar rats were randomly divided into 3 groups fed with deionized water and lead contained water(0.2%and 1%lead acetate) respectively.The lead-exposure period was from the 0 day of pregnancy to the offspring weaned.Then the baby-rats were fed with lead water the same as their mothers.Carry out cliff avoidance reflex within PN 8 and step down test at PN 50. Then pups were killed at PN 8 and PN 50 respectively.Atomic absorption spectrometry was used to determine lead content of rats' brain.Kits were used to test acetylcholinesterase activity.RT-PCR and western blot were used to observe mRNA and protein expression of PKC and CaM in hippocampus of baby-rats.2 The effect of lead on PKC and CaM expression in U251 cell line:U251 cell were divided into four groups:5×10^-8,5×10^-6,5×10^-4mol/L lead acetate and normal saline.Inoculation density was 5.5×10⁵cell/ml,5ml for every culture bottle,exposure to lead for 24 hours after adherent.MTT assay was used to observe the effect of lead on U251 cell viability,immunocytochemistry to observe expression of PKC and CaM in cells,RT-PCR and western blot to observe mRNA and protein expression of PKC and CaM in cells.Results1 The effect of lead on learning and memory ability and relevant index in baby-rats:(1)The body weight of test groups were much lighter than that of the control group.The body length of test groups were much shorter than that of the control group.(2)The brain lead contents of test groups were much higher than that of the control group.The brain lead contents of PN 50d were higher than that of PN 8d at the same dose.(3)The completion rate of surface righting reflex of the test groups were lower than control group(P＜0.01).The completion rate of surface righting reflex of 1%lead acetate group was lower than 0.2%lead acetate group(P＜0.01).(4)The completion rate of cliff avoidance reflex of the test groups were lower than control group(P＜0.01).The completion rate of cliff avoidance reflex of 1%lead acetate group was lower than 0.2%lead acetate group (P＜0.01).(5)Lead can damage learning and memory ability of baby rats.The reaction time of test groups was longer,incubation period shorter,error numbers more than that of control group(P＜0.05,P＜0.01).(6)Cholinesterase activity of 1%lead acetate group of PN 8d was lower than that of control group(P＜0.01). Cholinesterase activity of 0.2%,1%lead acetate group of PN 50d were both lower than that of control group(P＜0.05,P＜0.01).(7)PKC mRNA expression of 1%lead acetate group of PN 8d and PN 50d were both downtrend(P＜0.05, P＜0.01).PKC mRNA expression of 0.2%lead acetate groups were not downtrend (P＞0.05).PKC mRNA expression of 1%lead acetate group of PN 50d was lower than that of PN 8d;but there is no significance.(8)Both at PN 8d and PN 50d, PKC protein expression of 0.2%and 1%lead acetate group were downtrend (P＜0.01).PKC protein expression of 1%lead acetate group of PN 50d was lower than that of PN 8d;but there is no significance.(9)CaM mRNA expression of 1% lead acetate group of PN 8d was downtrend(P＜0.05).But that of 0.2%lead acetate group of PN 8d were not downtrend(P＞0.05).CaM mRNA expression of 0.2%and 1%lead acetate group of PN50d were both downtrend(P＜0.05, P＜0.01).CaM mRNA expression of 1%lead acetate group of PN 50d was lower than that of PN 8d;but there is no significance.(10)CaM protein expression of 1%lead acetate group of PN 8d was downtrend(P＜0.01).But that of 0.2%lead acetate group of PN 8d were not downtrend(P＞0.05).CaM protein expression of 0.2%and 1%lead acetate group of PN50d were both downtrend (P＜0.05,P＜0.01).CaM protein expression of 0.2%and 1%lead acetate group of PN 50d was lower than that of PN 8d;but there is no significance.2 The effect of lead on PKC and CaM expression in U251 cell line:(1)The cell viability of 5×10^-6,5×10^-5,5×10^-4mol/L lead acetate groups are lower than that of control group(P＜0.01).The cell viability decreased when the dose increased.(2)Through immunocytochemistry we can see that PKC and CaM exist in the cytoplasm.The number of positive cells was decreased when the dose increased.(3)PKC mRNA expression of 5×10^-4mol/L lead acetate group were downtrend(P＜0.05).CaM mRNA expression of 5×10^-8,5×10^-6,5×10^-4mol/L lead acetate group were all downtrend(P＜0.01)and had a dose-response relationship. (4)Compared with control group,PKC and CaM protein expression of lead exposure group were all downtrend(P＜0.01)and had a dose-response relationship.Conclusion1 By maternal exposure to lead through pregnancy and breast-feeding period, lead can accumulate in the brain of baby-rats,decline learning and memory abilities of baby-rats.Cholinesterase activity was decreased.PKC and CaM mRNA and protein expression of the different stages of the rat hippocampus were all decreased,which may be one of the mechanisms of lead neurotoxicity.2 Lead can decline the cell viability of U251 cell.PKC and CaM mRNA and protein expression of U251 cell were decreased,which may be one of the mechanisms of lead neurotoxicity to human being.In a word,the results of animal experiments are consistent with that of cell experiments.The effect of lead on PKC and CaM expression in signal transduction pathway may be one of the mechanisms of its neurotoxicity.