Study On Learning And Memory Dysfunction Induced By Lead Exposure Through Apoptosis Induced By HDAC5/NPAS4

Objective:Lead(Plumbum,Pb)is a toxic heavy metal widely found in the environment.Long-term exposure to a low-dose lead environment can cause harm to the central nervous system,leading to learning and memory dysfunction.However,the specific damage mechanism of Pb-induced neurotoxicity is not clear.Neuronal PAS domain protein 4(NPAS4)is a confirmed nerve damage protection factor that can regulate apoptosis factors.Histone deacetylase 5(HDAC5)can specifically bind to the enhancer of NPAS4,but it is not clear whether they are involved in learning and memory disorders caused by Pb exposure.Therefore,this study constructed a Pb exposure SD rat model and an HT22 cell model to detect the effects of different concentrations of Pb on apoptosis,and observed the effect of Pb exposure on the expression of HDAC5 and NPAS4.In addition,we also silent HDAC5 in the cell model to clarify the relationship between HDAC5 and NPAS4.NPAS4 was expressed in the Pb-exposed HT22 cell model to explore the effect of NPAS4 on apoptosis.We established a model of Pb exposure in vivo and vitro,explored the mechanism of Pb damage to learning and memory impairment,and provides a new theoretical basis for the neurotoxicity treatment of Pb exposure.Methods:1.Effects of Pb exposure on learning and memory and the apoptosis of hippocampus and HT22 cells..Establish a Pb-exposed SD rat model.Select sexually mature rats for 12 weeks:female and male rats are caged by a 2:1 ratio.After the cage,the rats were randomLy divided into three groups,drinking 0g/L,0.5g/L and 2.0g/L lead acetate aqueous solutions.The 21-day-old rats were used for the following experiments.Determine the lead concentration in the wholeblood and hippocampus of rats in each group.The Morris Water Maze tested the learning and memory ability of each group of rats.A lead-exposed HT22 cell model was established,and cells were cultured by different culture medium containing 0μM,1μM and 100μM lead acetate.CCK-8 detected cell vitality,and flow cytometer detected apoptosis.Reverse transcriptase polymerase chain reaction(RT-PCR)and Western blotting(WB)detected expression of apoptosis marker genes Bcl-2 and Caspas-3 in rat hippocampus and HT22 cells.2.The effect of Pb exposure on the expression of NPAS4 and the effect of NPAS4 overexpression on apoptosis.RT-PCR,WB and lmmunofluorescence technique(IHC)were used to detect the mRNA and protein levels of NPAS4 in rat hippocampus and HT22 cells.HT22 cells were divided into 100μM lead acetate+empty plasmid(negative control)group and 100μM lead acetate+NPAS4 overexpression plasmid group.RT-PCR and WB technology detected the expression levels of NPAS4,Bcl-2 and Caspas-3.CCK-8 detected cell vitality and flow cytometer detected apoptosis levels.3.The effect of Pb exposure on the expression of HDAC5 and the effect of silent HDAC5 on NPAS4.RT-PCR,WB and IHC were used to detect the mRNA and protein levels of HDAC5 in rat hippocampus and HT22 cells.HT22 cells were divided into 100μM lead acetate+NC siRNA(negative control group)and 100μM lead acetate+HDAC5 siRNA group.RT-PCR and WB were used to detect the mRNA and protein levels of NPAS4 to determine HDAC5 and NPAS4 relationship.Results:1.Pb exposure impaired the learning and memory ability of rats,and up-regulated the apoptosis of hippocampus and HT22 cells.The ICP-AES test results showed that the lead concentration in the whole blood and hippocampus increased with the increase of Pb exposure concentration,and there was a dose dependence(p<0.05).These meaned the animal model was successfully established.The experimental results of Morris Water Maze show that the escaping latency of Pb exposure group rats was longer than the control group with the increase of lead concentration,while the times across the platform were less(p<0.05).These indicating that Pb exposure caused a decline in the learning and memory ability of rats.The results of CCK-8 and flow cytometry showed that at the lead concentration of 0μM,1μM and 100μM,with the increase of lead concentration,the vitality of the cell gradually decreases and the apoptosis gradually increases.And the results of RT-PCR and WB experiments showed that lead exposure induced the reduction of Bcl-2 and the rise of Caspas-3.2.Pb exposure decreased the expression of NPAS4,and the apoptosis level decreased after overexpression of NPAS4.The results of RT-PCR,WB and IHC experiments showed that the mRNA and protein levels of NPAS4 in rat hippocampus and HT22 cells decreased with the increase of lead concentration,showing a dose-dependent relationship(p<0.05).In HT22 cell models exposed to lead,cell activity increased and apoptosis level decreased after overexpression of NPAS4,and the changes of Bcl-2 and Caspas-3 expression induced by lead were partially reversed.These results indicated that overexpression of NPAS4 inhibited lead induced apoptosis of HT22 cells.3.Pb exposure increased HDAC5 expression,and NPAS4 expression increased after HDAC5 silencing.The results of RT-PCR,WB and IHC experiments showed that the levels of HDAC5 mRNA and protein in rat hippocampus and HT22 cell increased with the increase of lead concentration,showing dose dependence.RT-PCR and WB experimental results showed that after the silence of HDAC5 in the lead exposure HT22 cell model,the level of NPAS4 mRNA and protein increased significantly.Conclusion:Lead exposure inhibits NPAS4 expression through up-regulation of HDAC5,thus inducing neuronal apoptosis and causing learning and memory impairment in rats.This study provides a new idea for the mechanism of Pb exposure affecting learning and memory and the treatment of central nervous system damage caused by Pb exposure.