The Effect Of Lead Exposure On The Expression Of SIRT1 And Resveratrol Intervention In Rat Hippocampus

Objective:Lead（Pb）has been widely used in some working condition and it is also ubiquitous in the living environment,can cause irreversible damage to the brain nervous system development if people has been long exposed,thus the nervous impairment induced by lead has been highly concerned.However,the mechanism of the nervous system damage by lead has not been fully elucidated.Recently,more and more studies have found that silent information regulator 2 homolog 1（SIRT1）played an important role in the process of the nervous system development and the protection of the nerve damage.Especially the critical role that SIRT1 regulated synaptic plasticity and maintained cognitive function by the means of regulating the expression of c AMP-response element binding protein（CREB）in post-transcriptional level had been revealed.These invoked reflections:whether the abnormal synaptic plasticity and cognitive dysfunction caused by Pb was due to the effect of SIRT1 on regulating its main downstream target gene CREB phosphorylation?Whether the SIRT1 is the target of lead neurotoxicity?Base on these questions,in our study,we established the lead exposed rat model with learning and memory impairment to explore the expression changes of SIRT1 and CREB phosphorylation in the rat hippocampus.Meanwhile,the SIRT1 activator Resveratrol（Res）was provided to survey its intervention effect for further clarity of lead neurotoxicity mechanism.Methods:（1）The effect of lead exposure on the expression of SIRT1 in the rat hippocampus:Sexually mature female SD rats were mated with the sexually mature males（2:1）.Then,the successful mating female rats were assigned to the different lead exposure groups randomly,which either 0,500 mg/L or 2000 mg/L lead acetate（containing 0,319mg/L,1275mg/L Pb2+）were contained in the distilled dranking water.This step was conducted during gestation period and lactation period till the pups being weaned.After being weaned,the male pups were divided into control（CON）,low lead exposed group（LLG）and high lead exposed group（HLG）,which were treated with 0,100mg/L and 500mg/L lead acetate（containing 0,64mg/L,319mg/L Pb2+）in the distilled drinking water corresponding to their mother.Detections were conducted at postnatal week 3（PNW3）and postnatal week 52（PNW52,1 year）.The learning and memory ability of rats were detected by Morris water maze system.Pb levels in blood and hippocampus were determined by inductively coupled plasma atomic emission spectroscopy（ICP-AES）.Reverse transcriptase polymerase chain reaction（RT-PCR）analysis was used to detect the mRNA level of SIRT1 and CREB in hippocampus.Western blotting（WB）analysis was applied to detect the protein expression of SIRT1 and CREB phosphorylation in the rat hippocampus.Immunofluorescent（IF）was used to detect the protein expression of SIRT1 in the different regions of rat hippocampus.（2）The intervention effect of Res on the SIRT1 expression change caused by lead exposure:Sexually mature female SD rats were mated with males（2:1）.And the successful mated females were randomly assigned to the different exposure groups,and drank distilled water containing either 0 or 500mg/L lead acetate(0,319mg/L Pb²⁺)during gestation and lactation until rat pups being weaned.Res intervention was implemented at weaning（PNW3）.Male weaned rats were dived into four groups:CON（solvent）,Res（Res,50 mg/kg·bw·d）,Pb（500 mg/L lead acetate）and Pb+Res（500 mg/L lead acetate+50 mg/kg·bw·d Res）group.The four groups were conducted by gavage per day and the intervention experiment lasted for 4 weeks.At PNW7,RT-PCR analysis was used to detect changes in expression level of SIRT1 mRNA and CREB mRNA in the rat hippocampus.And WB analysis was used to detect the protein expression of SIRT1 and CREB phosphorylation in the rat hippocampus.Results:（1）Lead exposure decreased the ability of learning and memory in rats.In the of Morris water maze test,we observed that there were significant difference in the escape latency after 4 days’place navigation experiments in the PNW3 and PNW52 groups,and the escape latency prolonged by the dose-dependent of Pb（P<0.05）.In the spatial probe test,the number of crossings of rats in LLG and HLG groups were significantly less than in the control group（P<0.05）,while there were no significant difference between the LLG and HLG groups（P>0.05）.（2）Lead exposure increased blood lead concentration and lead content in hippocampus.ICP-AES showed that the blood lead concentration and hippocampus lead content were increased by the lead concentration and the time of lead exposure,and there were dose-response relationship（P<0.05）.（3）Lead exposure influenced SIRT1 level in rat hippocampus.The RT-PCR analysis showed that the SIRT1 mRNA level decreased in the in PNW3 and PNW52rat hippocampus with lead exposure dose increased（P<0.05）.The WB analysis showed that the SIRT1 protein level decreased in the hippocampus of brain in PNW3and PNW52 rats with lead exposure dose increased（P<0.05）.The IF showed that the SIRT1 protein level decreased in the CA3 regions of brain hippocampus in PNW3rats with lead exposure dose increased（P<0.05）.And in PNW52 rats,the SIRT1protein level decreased in the CA1 and CA3 regions（P<0.05）.（4）Lead exposure influenced CREB mRNA expression and the protein phosphorylation in the rat hippocampus.The RT-PCR analysis showed that there were no significant difference in the CREB mRNA level（P>0.05）,while the WB analysis showed that lead exposure decreased the CREB phosphorylation in dose-depentdent relationship（P<0.05）.（5）Res intervention changed the expression of SIRT1 in rat hippocampus.The RT-PCR analysis showed that Res intervention increased the SIRT1 mRNA level（P<0.01）,what’s more,it suppressed the down regulation effect of lead（P<0.01）.The WB analysis showed that Res intervention increased the SIRT1 protein level（P<0.01）and suppressed the down regulation effect of lead（P<0.01）.（6）Res intervention changed the protein phosphorylational level of CREB in rat hippocampus.The RT-PCR analysis showed that there were no significant difference in the CREB mRNA level（P>0.05）,while the WB analysis showed that Res intervention increased the CREB phosphorylation and suppressed the down regulation effect of lead（P<0.01）.Conclusion:Lead exposure induced impairment of learning and memory may due to down regulating the expression of SIRT1 and the CREB phosphorylation in rat hippocampus,while Res can suppress this down regulation effect.Thus it may provide new clues to the mechanism of lead exposure induced nervous system damage,and provide theoretical basis for looking for new targets of drug treatment.