| Objective: In this study,we conducted lead exposure and PAS-Na intervention experiments in vitro and vivo to observe the occurrence of cortical inflammatory response and the changes of SIRT1/ NF-κB pathway related proteins in rats after lead exposure.In addition,the intervention effect of PAS-Na on lead-induced cortical inflammatory response and SIRT1/ NF-κB pathway related protein changes in rats was further investigated,so as to provide scientific basis or clues for the prevention and treatment of lead poisoning.Methods: In vitro study : The primary neurons of lead injury model group were identified and morphological observation after 0,25,50 and 100 μmol/L lead exposure,and then the contents of HMGB1 and SIRT1 were tested by WB.Primary neurons of PAS-Na intervention group were divided into control group,50 μmol/L lead-treated group,50 μmol/L lead +100 μmol/L PAS-Na(L-PAS group),50 μmol/L lead +200 μmol/L PAS-Na(M-PAS group),and 50 μmol/L lead+400 μmol/L PAS-Na intervention group(H-PAS group),400 μmol/L PAS-Na control group,50 μmol/L lead +15 μmol/L EX527(SIRT1 inhibitor),15 μmol/L EX527 control group randomly.BDNF was detected by immunofluorescence after intervention.The protein expression levels of NF-κB,SIRT1 and HMGB1 were detected by WB.The contents of inflammatory cytokines TNF-α and IL-1βwere detected by ELISA.In vivo studies:72 SPF healthy male SD rats were randomly divided into control group,6 mg/kg lead exposure group,lead +100mg/kg PAS-Na group(L-PAS),lead +200 mg/kg PAS-Na group(M-PAS),and lead +300 mg/kg PAS-Na group(H-PAS),300 mg/kg PAS-Na control group.The growth and development of the rats were observed,and the organ coefficients were calculated after the intervention.Water maze test was applied to mesure the change of spatial learning and memory ability of rats.The expression of BDNF protein in the cortex was detected by immunofluorescence.The protein expressions of CD11,CREB,SIRT1,HMGB1 and NF-κB in cortex were detected by immunohistochemistry or WB.The levels of inflammatory cytokines TNF-αand IL-1β in the cortex were detected by ELISA.Results:(1)In vitro study:(1)It was observed that lead exposure had obvious damage effect on neurons under microscope.Compared with the control group,50 and 100 μmol/L lead exposure decreased the expression of SIRT1 protein,and increased the expression of HMGB1(P<0.05).(2)After EX527 pretreatment and lead exposure,the content of SIRT1 protein in cortical neurons was decreased(P<0.05),while that in PAS-Na treatment was increased(P<0.05).The contents of inflammatory proteins HMGB1 and p-p65 in neurons were increased after lead exposure and Ex527 pretreatment,and decreased after PASNa treatment(P<0.05).(3)The content of TNF-α and IL-1β increased to 80.6pg/mg and 12.6 pg/mg protein,respectively,after lead exposure,while the content of TNF-α decreased after PAS-Na treatment(P<0.05).(2)In vivo study:(1)Compared with the control group,weight loss and liver coefficient increased in rats after lead exposure,and PAS-Na treatment could antagonize the above changes(P<0.05).(2)Compared with the control group,the escape latency of lead-treated rats on the first and second days and the mean swimming distance on the second day were longer(P<0.05),and the escape latency on the second day was shortened after PAS-Na treatment(P<0.05).(3)Lead exposure decreased the protein contents of CREB and BDNF,while PAS-Na treatment increased the protein contents of CREB and BDNF.(4)Compared with the control group,CD11 b positive cells in the cortex were significantly increased after lead exposure(P <0.05).Immunohistochemistry and WB assay showed that the content of SIRT1 protein decreased and the content of NF-κB and HMGB1 increased after lead exposure,and PAS-Na treatment antagonized the above changes(P<0.05).(5)Compared with the control group,the contents of TNF-α and IL-1β increased to50.9 pg/mg and 49.5 pg/mg protein after lead exposure,respectively,and the contents of inflammatory factors decreased after PAS-Na treatment(P<0.05).Conclusion:(1)Lead exposure can affect the growth,development,learning and memory ability of rats,and PAS-Na has a certain antagonistic effect on the growth,development,learning and memory damage of rats induced by lead.(2)Lead exposure can inhibit the CREB/BDNF pathway,while PAS-Na therapy can improve the learning and memory impairment induced by lead via activating the CREB/BDNF pathway.(3)Lead exposure can induce cortical inflammation in rats through the SIRT1/NF-κB pathway,and PAS-Na can regulate the SIRT1/NF-κB pathway to inhibit the neuroinflammatory response,thereby improving the learning and memory impairment of rats induced by lead. |
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