The Regulation Of SIRT1/autophagy In Lead-induced Neuronal Injury

ObjectiveSlient Information Regμlator 1(SIRT1)is a NAD~+-dependent deacetylase that is involved in gene transcription,energy metabolism and regulation of cellular senescence.SIRT1 has become an important therapeutic target for a range of diseases.Recent studies have shown that SIRT1 has neuroprotective effects in both in vivo and in vitro models.In this study,an in-vitro lead exposure model was used to observe the damage of lead to nerve cells,and to explore the changes and roles of nuclear transcription factor NF-κB and autophagy in the process of nerve injury during SIRT1 related regulation.MethodsThe human neuroblastoma SH-SY5Y cells were used as the research object,and the effects of different concentrations of lead acetate(0-125μM)solution on the morphology and cell viability of SH-SY5Y cells were observed.On this basis,a subsequent experiment of 25μM lead acetate exposure was selected.The cells were pretreated with SIRT1 activator SRT1720(2μM)and inhibitor EX527(2μM)for 2h,then treated with 25μM lead for 48h.The groups were divided into control group,lead-exposed group,SRT1720 group,SRT1720+Pb group,EX527 group and EX527+Pb group.The relative expression of SIRT1,NF-κB,BACE1,LC3II/I and Beclin-1 proteins were detected by Western blot.The relative expression of SIRT1,NF-κB,BACE1,LC3a and Beclin-1 mRNA was detected by qPCR.Results1.The CCK-8 result showed that the survival rate of SH-SY5Y cells treated with different concentrations of lead decreased significantly(P<0.001),which was concentration-dependent.The expression of SIRT1 protein decreased gradually at the lead concentration of 10-125uM(P<0.001).The relative expression of NF-κB protein did not change significantly under different concentrations of lead(P>0.05).When the lead concentration was in the range of 10-125μM,the relative expression of p-NF-κB,BACE1,LC3II/I and Beclin-1 proteins were significantly increased.2.The effect of lead exposure on the expression of SIRT1、NF-κB and BACE1molecules in SH-SY5Y cells.Compared with the control group,the relative expression levels of SIRT1 protein(P<0.01,P<0.05 and P<0.001)and mRNA(P<0.05,P<0.05 and P<0.001),in the lead-exposed group,EX527 and EX527pretreatment group were significantly decreased Compared with the lead-exposed group,the expression levels of SIRT1 protein(P<0.01,P<0.001)and mRNA(P<0.001,P<0.05)in SRT1720 group and SRT1720 pretreatment group were significantly increased.Compared with the control group,the relative expression of NF-κB protein was not significantly changed in the lead-treated group or the SRT1720 and EX527 pretreatment groups(P>0.05),but the relative expression of p-NF-κB protein were significantly increased in the lead-exposed group and the EX527 group as well as the SRT1720 and EX527 pretreatment groups(P<0.001,P<0.001,P<0.001 and P<0.001).The expression of NF-κB mRNA was significantly decreased in SRT1720 and SRT1720 pretreatment groups(P<0.01,P<0.05)Compared with lead-exposed group,the expression of p-NF-κB protein in SRT1720 group was significantly decreased(P<0.001).SRT1720 pretreatment inhibited the relative expression of p-NF-κB protein(P<0.001).In addition,the expression of p-NF-κB protein in EX527 pretreatment group also decreased(P<0.01),The relative mRNA expression of NF-κB was only significantly decreased in the SRT1720 group(P<0.05).Compared with the control group,the relative expression of BACE1 protein(P<0.001,P<0.001 and P<0.001)and mRNA(P<0.05,P<0.001 and P<0.001)were significantly increased in the lead-exposed group,EX527 group and EX527 pretreatment group.Compared with the lead-exposed group,the relative expression of BACE1 protein(P<0.001,P<0.001)and mRNA(P<0.001,P<0.01)decreased significantly in the SRT1720and SRT1720 pretreatment groups,and increased significantly in the EX527pretreatment group(P<0.001,P<0.01).3.Effect of lead exposure on the expression of autophagy-related molecules in SH-SY5Y cells.Compared with the control group,the relative expression of LC3II/I protein in other groups was significantly increased(P<0.001,P<0.01,P<0.001,P<0.001 and P<0.001).Compared with the lead-exposed group,the relative expression of LC3II/I protein was significantly decreased in the SRT1720group and the SRT1720 pretreatment group(P<0.001,P<0.001).However,there was no significant difference in the expression of LC3a mRNA between the groups(P>0.05).Compared with the control group,the relative expression levels of Beclin-1 protein(P<0.001,P<0.01 and P<0.001)and mRNA(P<0.01,P<0.01,P<0.001)in the lead-exposed group,EX527 group and EX527 pretreatment group were significantly increased.Compared with the lead-exposed group,the relative expression levels of Beclin-1 protein(P<0.001,P<0.01 and P<0.05)and mRNA(P<0.001,P<0.05 and P<0.01)were significantly decreased in the SRT1720,EX527 groups as well as the SRT1720 pretreatment group.Compared with the control group,the relative expression of P62 protein in lead-exposed or EX527pretreatment group was significantly increased(P<0.01,P<0.05).Compared with the lead-exposed group,the relative expression of P62 protein in SRT1720 and EX527 groups were decreased as well as SRT1720 pretreatment group(P all<0.01).Conclusions1.Lead-induced SH-SY5Y cell death is associated with decreased expression of SIRT1 and promotes nuclear transcription factors p-NF-κB and BACE1 as well as autophagy-related factors LC3II/I and Beclin-1 protein expression.2.Activation of SIRT1 protects neurons by inhibiting the nuclear transcription factors p-NF-κB and BACE1 as well as autophagy-related factors LC3II/I,Beclin-1 expression.It is suggested that lead-induced neuronal damage is associated with SIRT1/NF-κB/autophagy pathway.