SIRT1/NF-κB通路在氧糖剥夺再灌注诱导小鼠海马神经元损伤中的作用

The role of SIRT1/NF-κB pathway in injury of mouse hippocampal neurons induced by oxygen glucose deprivation and reperfusion

目的探讨沉默信号调节因子1(SIRT1)/细胞核因子-κB(NF-κB)信号通路在氧糖剥夺再灌注诱导海马神经元损伤中的作用。方法通过SIRT1过表达慢病毒和RNAi技术调控小鼠海马神经元SIRT1的表达后建立氧糖剥夺再灌注损伤模型,实验设置七组:对照组(Control组)、氧糖剥夺再灌注损伤模型组(OGD组)、OGD+转染SIRT1-siRNA质粒组(OGD+SIRT1-siRNA组)、OGD+转染空载质粒组(OGD+NC-siRNA组)、OGD+感染过表达SIRT1慢病毒组(OGD+SIRT1上调组)、OGD+感染空载慢病毒组(OGD+Vector组)及OGD+SIRT1上调+SIRT1-siRNA组。对照组在37℃常氧条件下培养,其余六组在经过相应处理后给予氧糖剥夺处理9 h及再灌注处理24 h,再灌注处理结束后采用CCK-8法测定细胞活力、化学比色法测定乳酸脱氢酶(LDH)释放量、流式细胞术检测细胞凋亡率,荧光定量PCR法检测SIRT1、NF-κB的mRNA表达量,Western blot检测SIRT1、NF-κB、IκBα、Bcl-2、Bax、Cleaved caspase-3、β-actin、TBP的表达量。结果与Control组比较,OGD组细胞活力(%:100 vs.53.83±1.88)、SIRT1蛋白表达量(0.62±0.06 vs.0.33±0.03),IκBα蛋白表达量(1.01±0.06 vs.0.42±0.03)、Bcl-2/Bax比值(2.64±0.34 vs.0.58±0.06)明显降低(P<0.05),LDH释放量(U/mL:1.12±0.17 vs.2.76±0.23),细胞凋亡率(%:8.46±1.77 vs.29.58±1.84),NF-κB蛋白表达量(0.23±0.03 vs.0.63±0.03),Cleaved caspase-3蛋白表达量(0.19±0.03 vs.0.82±0.04)明显升高(P<0.05);与OGD组和OGD+Vector组比较,OGD+SIRT1上调组细胞活力(%:53.83±1.88、50.33±3.83 vs.72.77±1.80)、SIRT1表达量(0.63±0.03、0.58±0.04 vs.1.02±0.05)、IκBα表达量(0.42±0.03、0.38±0.04 vs.0.62±0.04)及Bcl-2/Bax比值(0.58±0.06、0.55±0.04 vs.1.41±0.16)明显升高(P<0.05),LDH释放量(U/mL:2.76±0.23、2.91±0.25 vs.1.95±0.20)、细胞凋亡率(%:29.58±1.84、28.87±2.91 vs.20.61±2.81)%、NF-κB表达量(0.63±0.03、0.58±0.04 vs.0.35±0.04)、Cleaved caspase-3表达量(0.82±0.04、0.71±0.05 vs.0.46±0.04)明显降低(P<0.05);与OGD组和OGD+NC-siRNA组比较,OGD+SIRT1-siRNA组细胞活力(%:53.83±1.88、49.23±3.12 vs.34.86±3.17)、SIRT1表达量(0.33±0.03、0.32±0.04 vs.0.18±0.04)、IκBα表达量(0.42±0.03、0.46±0.05 vs.0.27±0.04)及Bcl-2/Bax比值(0.580.06、0.62±0.05 vs.0.15±0.02)明显降低(P<0.05),LDH释放量(U/mL:2.76±0.23、2.78±0.24 vs.3.51±0.31)、细胞凋亡率(%:29.58±1.84、28.52±2.08 vs.37.03±3.19)、NF-κB表达量(0.63±0.03、0.64±0.04 vs.0.93±0.05)、Cleaved caspase-3表达量(0.82±0.04、0.85±0.05 vs.1.12±0.13)明显升高(P<0.05)。结论SIRT1/NF-κB通路参与了氧糖剥夺再灌注诱导小鼠海马神经元损伤过程。

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,IkBol,Bel-2,Bax,Cleaved caspase-3,P-actin and TBP were detected by Western blot.Results Compared with control group[cell viability:100%,SIRT1 protein expression:(0.62±0.04),IkBck protein expression:(1.01±0.05),Bel-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),IkBol expression(0.42±0.03)and Bel-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(P<0.05);Compared with OGD group and OGD+Vector group,cell viability(72.77±1.80)%,SIRT1 expression(1.02±0.05),IkBcx expression(0.62±0.04)and Bel-2/Bax ratio(1.41±0.16)were significantly increased in OGD 4-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(P<0.05);Compared with OGD group and OGD+NC-siRNA group,cell viability(34.86±3.17)%,SIRT1 expression(0.18±)were significantly decreased in OGD+SIRT1-siRNA group,the LDH release(3.51±0.0.04),IkB(x expression(0.27±0.04)and Bel-2/Bax ratio(0.15±0.0231)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(P<0.05).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.