Mechanism Of Sirt-1 On Oxidative Stress-induced Apoptosis Of SK-N-SH Cells

ObjectiveStudies have found that oxidative stress is closely related to major depression(MDD)and Alzheimer’s disease(AD).In this study,we intend to investigate whether Sirt-1may protect neurons from oxidative stress by regulating FOXO or p53.SK-N-SH cells were used to study the effect of Sirt-1 expression on the physiological function of nerve cells.This study is the first to confirm that sirt-1 may regulate p53 and eliminate ROS induced by oxidative stress through its deacetylation activity,proving the importance of Sirt-1 and ROS induced by oxidative stress in the progression of AD and related diseases.MethodsSK-N-SH cells were transfected with Sirt-1 gene silencing lentiviral vector to detect the expression of Sirt-1.The effects of Sirt-1 on the proliferation,cell cycle progression and apoptosis of nerve cells were explored.RT-PCR,Western blotting,luciferase reporter assay,soft agar colony formation assay and edu staining were used to determine the relationship between Sirt-1 and p53,and whether p53 changes can change the expression of downstream genes.The effects of these changes on the growth and cell cycle progression of SK-N-SH cells were analyzed.To further confirm whether Sirt-1 affects cell proliferation by regulating p53,SK-N-AS(p53 deficient)cells were selected to analyze the effect of Sirt-1 on the proliferation of SK-N-AS cells.200 μM H2O2 was used to induce oxidative stress to determine whether oxidative stress could inhibit the transcriptional activity of p53 and induce Sirt-1 overexpression.The inhibitory effect and mechanism of Sirt-1 overexpression on oxidative stress-induced apoptosis were explored.ResultsSirt-1 gene silencing could significantly reduce the number of cells in G1/G0 phase,increase the number of cells in S phase and block the G2/M phase of SK-N-SH cells compared with scrambled group(P<0.05);on the contrary,Sirt-1 gene overexpression could significantly increase the number of cells in G1/G0 phase of SK-N-SH cells.Edu results showed that compared with scrambled group,the number of edu positive cells in Sirt-1 gene silenced group(shSirt-1 group)was significantly decreased(P<0.05),and cell proliferation was inhibited;while Sirt-1 gene overexpression could significantly increase the number of EDU positive cells in SK-N-SH cells,which further confirmed the effect of Sirt-1 on cell proliferation.Western blotting showed that the protein expression levels of p53 and FoxO1 in SK-N-SH cells were not affected by Sirt-1 gene silencing or overexpression.Double Luciferase Report showed that Sirt-1 gene silencing transfection enhanced the luciferase activity(P<0.05).After Sirt-1 gene overexpression transfection,the luciferase activity of Sirt-1 decreased significantly.After treatment with 30 μM pifithrin-α(p53 specific inhibitor),luciferase activity decreased significantly,indicating that Sirt-1 is a negative regulator of p53 transcription activityRT qPCR results showed that Sirt-1 gene silencing could significantly increase p21 mRNA level(P<0.05),but there was not significantly different for p53,Bax and bcl-2 mRNA levels,indicating that Sirt-1 could selectively inhibit the p53-p21 pathway in SK-N-SH cells,and the binding activity of p53 and p21 was increased,indicating that Sirt-1 could inhibit the binding of p53 and p21 promoter.Western blotting showed that the level of Sirt-1 had not affect to the expression of p53 protein,but could significantly inhibit the deacetylation of p53 lysine 382.Sirt-1 attenuates its transcriptional activity by deacetylating p53,thus regulating cell cycle progression.Compared with SK-N-SH cells,Sirt-1 modification had no significant effect on the proliferation of SK-N-AS cells(P>0.05).In conclusion,Sirt-1 mainly affects cell proliferation and cell cycle distribution through the presence of p53.Sirt-1 gene silencing transfection significantly reduced Sirt-1 expression,and significantly reduced the number of cell membrane penetration and migration,thus significantly weakened the invasion and migration of SK-N-SH cells.On the contrary,Sirt-1 overexpression and transfection significantly enhanced the invasion and migration of SK-N-SH cells.After Sirt-1 gene overexpression/silencing,SK-N-SH cells were transfected with 200μM H2O2 to induce oxidative stress.The results showed that Sirt-1 overexpression could eliminate ROS production induced by H2O2 in SK-N-SH cells.The addition of 10 mm EX527 significantly increased the ROS level of SK-N-SH cells,indicating that the deacetylation activity of Sirt-1 is crucial for its ROS inhibitory activity.Western blotting results showed that H2O2 treatment could significantly increase the level of Sirt-1 protein,but had no effect on the level of p53 protein,but could reduce the transcriptional activity of p53.After adding EX527,the low transcriptional activity of p53 induced by H2O2 was significantly inhibited.These results indicate that oxidative stress can induce the increase of Sirt-1,deacetylation of Sirt-1 and inhibit the transcriptional activity of p53.After adding EX527,the expression of p21,Bax and bcl-2 mRNA increased significantly,which indicated that the increase of Sirt-1 induced by H2O2 could affect the expression of downstream genes p21,Bax and Bcl-2,and then regulate the proliferation and apoptosis of SK-N-SH cells.ConclusionSirt-1 is a positive regulator of nerve cell proliferation.Sirt-1 can promote cell cycle progression and inhibit apoptosis.Sirt-1 can affect the transcriptional activity of p53 through deacetylation,and may selectively inhibit the expression of p21 mediated by p53,so as to affect the expression of Bax、Bcl-2,regulate cell cycle progression and promote the growth of SK-N-SH cells.In neurons,oxidative stress inhibits the transcriptional activity of p53 and induces Sirt-1,which can protect them from oxidative stress.The deacetylation activity of Sirt-1 is crucial for its ROS inhibitory activity.Overexpression of Sirt-1 inhibits oxidative stress-induced apoptosis through its deacetylation activity.