| ObjectiveEnvironmental lead(Pb)pollution could induce neurotoxicity by entering the organism through various pathways.Abnormal synaptic plasticity is the main pathological mechanism of learning and memory impairment induced by Pb in hippocampus,but its molecular mechanism has not been clearly elucidated.SIRT1 has been found to be involved in various biological functions of the brain and has neuroprotective effects.Our group showed that activation of SIRT1 reversed the expression of synaptic proteins in hippocampus of Pb-exposed rats,thereby attenuating the impairment of learning and memory ability in Pb-exposed rats.In this study,we established in vivo and in vitro Pb exposure models to observe the effects of activating SIRT1 on synaptic plasticity-related genes and their DNA methylation levels,and to analyze the role of SIRT1 in relation to DNA methylation,which provided potential intervention and therapeutic targets for the prevention and treatment of cognitive impairment caused by Pb exposure.MethodsAnimal experiment: Healthy Sprague-Dawley(SD)rats of 190-220 g were selected and randomly divided into five groups: control,exposure groups(0.05%,0.1%and 0.2% exposure groups)and treatment group(0.2% Pb exposure + resveratrol group(0.2% + R group)).Afterwards,female and male rats were mated according to the ratio of 3:1,and female rats continued to be exposed to different concentrations of Pb,and male pups were selected for subsequent experiments at postnatal day 21.Graphite furnace atomic absorption spectrometry was used to detect the Pb concentration of hippocampal tissue,and Western blot and RT-qPCR were used to examine the relative expression of protein expression and mRNA of SIRT1,DNMT1,DNMT3 a,DNMT3b,MeCP2,BDNF,RELN,PP1 and cleaved caspase-3 in the hippocampus of SD rats.Immunohistochemistry was selected to analyze the effects of Pb exposure and resveratrol treatment on RELN expression in the CA1 region of the hippocampus,and Nissl and HE staining were used to observe the number of neuronal cells and pathological changes in the CA1 region of the hippocampus.Methylation-specific PCR(MSP)was used to detect changes in methylation levels of BDNF,RELN and PP1 promoters.In vitro experiments: PC12 cells were selected to establish an in vitro Pb exposure model,and the effects of different concentrations of Pb,SRT1720 and different pretreatment times on cell viability were detected by the CCK-8 to determine the subsequent exposure concentrations and pretreatment times.Cells were treated with SRT1720(1μM)for 2h and then treated with Pb acetate solution(10μM)for 24 h.Western blot and RT-qPCR were used to detect the relative expression of protein expression and mRNA of SIRT1,DNMT1,DNMT3 a,DNMT3b,MeCP2,BDNF,RELN,PP1 and cleaved caspase-3 in PC12 cells,and cellular immunohistochemistry was used to analyze the expression level of RELN.Methylation levels of BDNF,RELN and PP1 promoters were detected by MSP,and the apoptosis rate of each treatment group was analyzed using flow cytometry.Results1.Effects of Pb exposure and resveratrol treatment on Pb concentration in hippocampusCompared with the control group,the Pb concentration in the hippocampus of rats in each Pb exposure group increased with the increase of Pb concentration(P < 0.001).Compared with the 0.2% Pb-exposed group,Pb concentration of hippocampal was decreased in the 0.2%+R group(P < 0.001).2.Effects of Pb exposure and SIRT1 activation on the relative expression of protein expression and mRNA of target genesRT-qPCR results showed that the relative mRNA expression levels of SIRT1,BDNF and RELN were decreased(P < 0.001;P < 0.001;P < 0.01)as well as the relative mRNA expression levels of DNMT1,DNMT3 a,DNMT3b,MeCP2 and PP1 were increased in the 0.2% Pb-exposed group of the rat hippocampal tissue compared with the control group;Compared with the control group,the relative mRNA expression levels of SIRT1,BDNF and RELN were decreased(P < 0.01;P < 0.05;P< 0.001),and the relative mRNA expression levels of DNMT1,DNMT3 a,DNMT3b,MeCP2 and PP1 were also increased in the Pb group.Resveratrol and SRT1720 treatment reversed the changes in the relative mRNA expression levels of the above molecules induced by Pb.The results of Western blot showed that the protein expression of SIRT and BDNF was decreased(P < 0.01;P < 0.05)and the protein expression of DNMT1,DNMT3 a,DNMT3b,MeCP2,PP1 and cleaved caspase-3 was increased in the 0.2% Pb-exposed group of the rat hippocampal tissue compared with the control group;Compared with the control group,the protein expression of SIRT1 and BDNF was decreased(P < 0.01;P < 0.05),and the protein expression of DNMT1,DNMT3 a,DNMT3b,MeCP2,PP1 and cleaved caspase-3 was significantly increased in the Pb group.Furthermore,resveratrol and SRT1720 treatment reversed the effects of Pb exposure on protein expression of the above molecules.3.Effects of Pb exposure and SIRT1 activation on promoter methylation levels of genes related to synaptic plasticityThe MSP results of animal experiments showed that BDNF and RELN promoter methylation levels were increased(P < 0.05;P < 0.01)while PP1 promoter methylation levels were decreased(P < 0.05)in the 0.2% Pb-exposure compared to the control group.Moreover,resveratrol treatment significantly reversed the modification of BDNF,RELN and PP1 promoter methylation levels induced by Pb exposure in rat hippocampal tissue.In vitro experiments showed that BDNF and RELN promoter methylation levels were increased(P < 0.05;P < 0.05)and PP1 promoter methylation levels were decreased(P < 0.001)in the Pb group compared to the control group.Furthermore,the treatment with SRT1720 alleviated the effects of BDNF,RELN and PP1 promoter methylation levels induced by Pb exposure.4.Effects of Pb exposure and resveratrol treatment on neuronal cell number and pathological changes in CA1 region of hippocampusThe results of Nissl staining showed that the number of neuronal cells was decreased in the 0.2% Pb-exposure group compared to the control group(P < 0.001).The number of neuronal cells was increased in the 0.2%+R group compared with the0.2% Pb-exposure group(P < 0.01).The results of HE staining showed that the neuronal cells were tightly arranged and morphologically intact in an oval shape in the CA1 area of the hippocampus of the control group,while the neuronal cells of the 0.2%Pb-exposure group were loosely arranged,with abnormal cell morphology and the presence of polygonal and ruptured cells.After resveratrol treatment,the neuronal cells were tightly arranged and had a normal morphological structure compared to the 0.2%Pb-exposure group.Conclusion1.Pb exposure causes modification of synaptic plasticity-related gene expression by regulating DNA methylation levels.2.Activation of SIRT1 reverses the DNA methylation of BDNF,RELN and PP1 molecules induced by Pb exposure and ameliorates neuropathic damage in Pb-exposed rats. |
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