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《Chinese Journal of Critical Care Medicine》 2019-04
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The role of SIRT1/NF-κB pathway in injury of mouse hippocampal neurons induced by oxygen glucose deprivation and reperfusion

Yao Peng;Chen Yong;Xu Guo-hai;Department of Anesthesiology,Key Laboratory of Anesthesiology,Second Affiliated Hospital of Nanchang University;  
Objective To investigate the role of SIRT1/NF-κB pathway in the injury of mouse hippocampal neurons induced by oxygen glucose deprivation and reperfusion.Methods The model of oxygen glucose deprivation and reperfusion injury was established after regulation the expression of SIRT1 with overexpression of SIRT1 lentivirus and RNAi technology.The experiment was divided into seven groups:the control group,oxygen glucose deprivation and reperfusion injury model group(OGD group),OGD+SIRT1-siRNA group,OGD+NC-siRNA group,OGD+SIRT1 upregulation group,OGD+lentivirus vector group and OGD+SIRT1 upregulation + SIRT1-siRNA group.The control group was cultured at 37℃ under normoxic condition and the other six groups were treated with oxygen glucose deprivation for 9 h and reperfusion for 24 h after corresponding treatment.At the end of reperfusion,cell viability was measured by CCK-8 assay,LDH release was detected by chemical colorimetry,the apoptosis rate was determined by flow cytometry,and mRNA expression levels of SIRT1 and NF-κB were detected by qPCR,the expression of SIRT1,NF-κB,IκBa,Bcl-2,Bax,Cleaved caspase-3,β-actin and TBP were detected by Western blot.Results Compared with control group[cell viability:100%,SIRT1 protein expression:(0.62 ±0.04),IκBα protein expression:(1.01±0.05),Bcl-2/Bax ratio:(2.64 ±0.34),LDH release:(1.12 ±0.17)U/mL,apoptosis rate:(8.46±1.77)%,NF-κB protein expression:(0.23 ±0.03),cleaved caspase-3 protein expression:(0.19 ±0.03)],cell viability(53.83 ±1.88)%,SIRT1 expression(0.33 ±0.03),IKBα expression(0.42 ±0.03)and Bcl-2/Bax ratio(0.58 ±0.06)were significantly decreased in OGD group,the LDH release(2.76 ±0.23)U/mL,apoptosis rate(29.58 ±1.84)%,NF-κB expression(0.63 ±0.03)and Cleaved caspase-3 protein expression(0.82 ±0.04)were significantly increased(P0.05);Compared with OGD group and OGD + Vector group,cell viability(72.77 ± 1.80)%,SIRT1 expression(1.02 ±0.05),IκBα expression(0.62 ±0.04)and Bcl-2/Bax ratio(1.41 ±0.16)were significantly increased in OGD+SIRT1 up-regulated group,the LDH release(1.95 ±0.20)U/mL,apoptosis rate(20.61 ±2.81)%,NF-κB expression(0.35 ±0.04),and cleaved caspase-3 expression(0.46 ±0.04)were significantly decreased(P0.05); Compared with OGD group and OGD+NC-siRNA group,cell viability(34.86 ±3.17)%,SIRT1 expression(0.18 ±0.04),IKBαexpression(0.27 ±0.04)and Bcl-2/Bax ratio(0.15 ±0.02)were significantly decreased in OGD+SIRT1-siRN A group,the LDH release(3.51 ±0.31)U/mL,apoptosis rate(37.03 ±3.19)%,NF-κB expression(0.93 ±0.05)and cleaved caspase-3 expression(1.12 ±0.13)were significantly increased(P0.05).vThe role of SIRT1/NF-κB pathway is involved in the regulation of the injury induced by oxygen glucose deprivation and reperfusion in mouse hippocampal neurons.
【Fund】: 国家自然科学基金(81560193);; 南昌大学第二附属医院科技计划青年基金(2016YNQN12008)
【CateGory Index】: R743
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