| The heavy metal lead(Pb)can come into contact with the human body through a variety of routes,including drinking water,electronic pollution and chemicals,and can be harmful to many human organs.Lead exposure is particularly harmful to the nervous system,causing delayed neurological development and cognitive impairment in individuals.Apoptosis is a genetically regulated process of autonomous cell death to maintain the stability of the internal environment and is important for the normal development of the nervous system.RB binding protein 4(RBBP4),one of the subunits that bind to core histones,regulates DNA replication and DNA repair processes and is closely related to the apoptotic process in neuronal cells.However,it is not known whether RBBP4 can regulate neuronal apoptosis in a lead-exposed environment.Furthermore,RBBP4 expression may be regulated by the transcription factor nuclear factor kappa B p65(NFκB p65).However,the existence of this regulatory mechanism in lead-exposed environment is unclear.Objective:Firstly,this experiment established PC12 cells and SD rats lead exposure model to observe the effect of lead exposure on neuronal apoptosis.Secondly,changes in RBBP4 expression were observed under lead exposure conditions to investigate the relationship between it and neuronal apoptosis.Finally,changes in the expression of NFκB p65 were observed under lead exposure conditions,and the relationship between NFκB p65 and RBBP4 was investigated.This work proposes a new molecular mechanism for the neurological dysfunction caused by lead exposure and provides a new target for the treatment of lead poisoning.Methods:1.Effect of lead exposure on apoptosis in PC12 cells and hippocampus of SD rats:Sexually mature SD rats were taken and caged together in the ratio of female: male = 2:1.The fertilized female rats were randomly divided into blank control group(CON),low concentration lead exposure group(LLG)and high concentration lead exposure group(HLG).Lead acetate at concentrations of 0,0.5 and 2.0 g/L was added to their drinking water for the establishment of the SD rat lead exposure model,respectively.For PC12 cells,media containing lead at concentrations of 0,1 and 100 μM were added after passaging and samples were collected 24 hours later.The expression levels of the apoptosis inhibitory protein Survivin in SD rat hippocampus and PC12 cells were determined by Reverse transcriptase polymerase chain reaction(RT-PCR)and protein blotting(Western blotting,WB).Apoptosis levels were measured in SD rat hippocampal tissue and PC12 cells using Td T-mediated d UTP nick end labeling(TUNEL).2.RBBP4 expression and the effect of RBBP4 on neuronal apoptosis in lead-exposed environment: SD rat and PC12 cell lead exposure models were established,and changes in RBBP4 m RNA and protein expression were observed using RT-PCR,WB and immunohistochemistry(IHC).Overexpress RBBP4 in lead-exposed environments,the cells were randomly divided into 10 μM Pb+pc DNA3.1 and 10 μM Pb+pc DNA3.1-RBBP4 groups to observe the changes in m RNA and protein expression of RBBP4 and Survivin.And Cytotoxicity Assay Kit8(CCK8)to determine PC12 cell viability and TUNEL to determine PC12 cell apoptosis.3.NFκB p65 expression in SD rat and PC12 cell lead exposure models: SD rat and PC12 cell lead exposure models were established and changes in NFκB p65 m RNA and protein expression were observed using RT-PCR,WB and IHC.4.Inhibition of NFκB p65 activity by pyrrolidinedithiocarbamate ammonium(PDTC)in PC12 cell lead exposure model to observe RBBP4 expression: Using phosphate buffer saline(PBS)to dissolve PDTC.Cells were randomly divided into three groups: control(0μM Pb+100 μM PBS),lead-exposed(10 μM Pb+100 μM PBS)and inhibitor(10 μM Pb+100 μM PDTC)group.Changes in m RNA and protein expression of NFκB p65 and RBBP4 were observed using RT-PCR,WB,and IHC.Results:1.Lead exposure induced apoptosis in SD rat hippocampal tissue and PC12 cells:The results of TUNEL assay found that apoptosis of neurons in DG,CA1 and CA3 regions of SD rat hippocampal tissue increased with increasing lead concentration;in the PC12 cell lead exposure model,apoptosis was found to increase with increasing lead concentration.The CCK8 results showed that the activity of PC12 cells decreased with increasing lead concentration in the medium(P<0.05).In addition,we found that m RNA and protein levels of the apoptosis inhibitory protein Survivin decreased with increasing lead concentrations(P<0.05).2.Lead exposure induced changes in RBBP4 expression in PC12 cells and hippocampus of SD rats: RT-PCR results showed that the m RNA expression of RBBP4 decreased with increasing lead concentration in hippocampus and PC12 cells(P<0.05).WB results showed that the protein expression of RBBP4 decreased with increasing lead concentration in rat hippocampal tissues and PC12 cells(P<0.05).The IHC results showed that the protein expression of RBBP4 decreased in PC12 cells and hippocampal tissue DG region,CA1 region and CA3 region with the increase of lead concentration(P<0.05).3.Overexpression of RBBP4 in lead-exposed environment increased Survivin expression and rescued neuronal apoptosis: RT-PCR results showed that m RNA expression of RBBP4 as well as Survivin was increased in the 10 μM Pb+pc DNA3.1-RBBP4 group compared to the 10 μM Pb+pc DNA3.1 group(P<0.05).WB results showed that the protein expression of RBBP4 and Survivin was increased in the 10μM Pb+pc DNA3.1-RBBP4 group compared to the 10μM Pb+pc DNA3.1 group(P<0.05).The CCK8 results showed a significant increase in cell activity in the 10 μM Pb+pc DNA3.1-RBBP4 group compared to the 10 μM Pb+pc DNA3.1 group(P<0.05).The TUNEL results showed that significantly fewer cells underwent apoptosis in the 10μM Pb+pc DNA3.1-RBBP4 group compared to the 10 μM Pb+pc DNA3.1 group.4.Lead exposure induced changes in NFκB p65 expression in PC12 cells and hippocampus of SD rats: RT-PCR results showed that the m RNA expression of NFκB p65 increased in hippocampus and PC12 cells(P<0.05).WB results showed that the protein expression of NFκB p65 increased with increasing lead concentration in rat hippocampal tissues and PC12 cells(P<0.05).IHC results showed that the protein expression of NFκB p65 in PC12 cells and hippocampal DG,CA1 and CA3 regions increased with increasing lead concentration(P<0.05).5.Inhibition of NFκB p65 activity in PC12 cells under lead exposure conditions increased RBBP4 expression: WB results showed that the protein expression of RBBP4 decreased and that of NFκB p65 increased in the lead-exposed group compared to the control group;whereas the protein expression of RBBP4 increased and that of NFκB p65 decreased in the inhibitor group compared to the lead-exposed group(P<0.05).The RT-PCR results showed that the m RNA expression of RBBP4 was decreased in the lead-exposed group compared to the control group,while the m RNA expression of RBBP4 was increased in the inhibitor group compared to the lead-exposed group(P<0.05).The IHC results showed that protein expression of RBBP4 decreased and NFκB p65 increased in the lead-exposed group compared to the control group,while protein expression of RBBP4 increased and NFκB p65 decreased in the inhibitor group compared to the lead-exposed group(P<0.05).Conclusion:Lead exposure inhibits RBBP4 expression by upregulating the expression level of NFκB p65,leading to a decrease in Survivin expression,which causes neuronal apoptosis and impairs learning memory.This provides new ideas and approaches to further elucidate the molecular mechanisms involved in the effects of lead on learning memory and to treat the neurotoxicity caused by chronic lead exposure. |
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