下调SRR可缓解Aβ对PC12细胞的神经毒性和突触损伤

Alleviation of amyloid beta-protein induced neurotixicity and synaptic damage in PC12 cells by downregulation of serine racemase

目的研究下调丝氨酸消旋酶(SRR)缓解β-淀粉样蛋白(Aβ)对PC12细胞的神经毒性、突触损伤作用及其可能机制。方法(1)将体外培养的PC12细胞分为0、20、40、80μmol/L Aβ_(25-35)组,分别加入0、20、40、80μmol/L Aβ_(25-35)作用24 h,采用细胞计数试剂盒-8(CCK-8)法检测细胞存活率,采用Western blotting实验检测细胞SRR蛋白的表达。用40μmol/L Aβ_(25-35)分别处理PC12细胞0、12、24、48 h,采用CCK-8法、Western blotting实验分别检测细胞存活率、SRR蛋白的表达。(2)将PC12细胞分为对照组、无义序列组、SRR小干扰RNA(siRNA)1组、SRR siRNA 2组、SRR siRNA 3组,后4组细胞分别转染SRR无义序列或不同SRR siRNA序列,48 h后采用Western blotting实验检测细胞SRR蛋白的表达,选择效果最佳的SRR siRNA用于后续实验。(3)将PC12细胞分为对照组、AD组、AD+无义序列组、AD+SRR siRNA组,后2组细胞分别瞬时转染无义序列或SRR siRNA,作用48 h,后3组细胞均加入40μmol/L Aβ_(25-35),对照组加入等量溶剂。处理24 h后采用Western blotting实验检测细胞SRR蛋白的表达,CCK-8法检测细胞存活率,Hoechst 33258染色检测细胞凋亡,ELISA法检测细胞半胱氨酸蛋白酶3(Caspase 3)活性,Western blotting实验检测细胞活化Caspase 3、N-甲基-D-天冬氨酸(NMDA)受体相关蛋白、突触后致密蛋白95(PSD95)的表达。结果(1)0、20、40、80μmol/L Aβ_(25-35)组细胞存活率依次降低,SRR蛋白的表达依次增高,差异均有统计学意义(P<0.05)。40μmol/L Aβ_(25-35)处理PC12细胞0、12、24、48 h后细胞存活率依次降低,SRR蛋白的表达依次增高,差异均有统计学意义(P<0.05)。(2)SRR siRNA 1组、SRR siRNA 2组、SRR siRNA 3组细胞SRR蛋白的表达均低于对照组和无义序列组,差异均有统计学意义(P<0.05),其中SRR siRNA 2组降低最明显。(3)与对照组比较,AD组细胞SRR蛋白的表达、细胞凋亡率升高,细胞存活率降低,Caspase 3活性和活化Caspase 3蛋白表达升高,NMDA受体2A(NMDAR2A)和NMDA受体2B(NMDAR2B)蛋白的表达升高,PSD95蛋白的表达降低,差异均有统计学意义(P<0.05)。与AD组比较,AD+SRR siRNA组细胞SRR蛋白的表达、细胞凋亡率降低,细胞存活率升高,Caspase 3活性和活化Caspase 3蛋白表达降低,NMDAR2A蛋白的表达降低,PSD95蛋白的表达升高,差异均有统计学意义(P<0.05)。结论下调SRR可通过降低PC12细胞内NMDAR2A蛋白表达,缓解细胞NMDA受体的过度激活,减少细胞凋亡,提高细胞存活率,保护Aβ_(25-35)损伤的神经细胞,还可以升高PSD95蛋白的表达,缓解突触损伤。

Objective To investigate the role of down-regulating serine racemase(SRR)in alleviating theβ-amyloid peptide(Aβ)induced neurotoxicity and synaptic damage and possible mechanism in PC12 cells.Methods(1)PC12 cells cultured in vitro were divided into 0,20,40 and 80µmol/L Aβ_(25-35)treatment groups;they were treated with 0,20,40 and 80µmol/L Aβ_(25-35)for 24 h,respectively;cell counting kit(CCK)-8 was used to detect the survival rate of cells in each group,and Western blotting was used to detect the SRR protein expression.PC12 cells were treated with 40µmol/L Aβ_(25-35)for 0,12,24 and 48 h,respectively;cell survival and SRR protein expression were detected by CCK-8 and Western blotting,respectively.(2)PC12 cells were divided into control group,nonsense sequence group,SRR small interfering RNA(siRNA)group 1,SRR siRNA group 2,and SRR siRNA group 3;cells in the later three groups were transfected with SRR nonsense sequence or different SRR siRNA sequences,respectively;48 h after that,Western blotting was used to detect the SRR protein expression of cells in each group,and SRR siRNA with best effect was selected for subsequent experiments.(3)PC12 cells were divided into control group,AD group,AD+nonsense sequence group,and AD+SRR siRNA group;cells in the latter two groups were transfected with nonsense sequence or SRR siRNA for 48 h,respectively;cells in the latter three groups were added 40µmol/L Aβ_(25-35),and cells in the control group were added same amount of solvent;24 h after treatment,the SRR protein expression was detected by Western blotting,cell survival was detected by CCK-8,cell apoptosis was detected by Hoechst 33258 fluorescent staining,Caspase 3 activity was detected by enzyme linked immunosorbent assay,and the expressions of activated Caspase 3,N-methyl-D aspartate(NMDA)receptor-associated proteins and postsynaptic dense protein 95(PSD95)were detected by Western blotting.Results(1)The survival rate of cells in 0,20,40 and 80µmol/L Aβ_(25-35)treatment groups was successively decreased and the SRR protein expression was successively increased,with significant differences(P<0.05);PC12 cells treated with 40µmol/L Aβ_(25-35)for 0,12,24 and 48 h had successively decreased survival rate and successively increased SRR protein expression,with significant differences(P<0.05).(2)The SRR protein expressions in the SRR siRNA group 1,SRR siRNA group 2 and SRR siRNA3 group 3 were significantly decreased as compared with those in the control group and nonsense sequence group(P<0.05),and the decrease in the SRR siRNA group 2 was the most obvious.(3)As compared with the control group,the cells in the AD group had significantly increased SRR protein expression and apoptosis rate,statistically decreased cell survival rate,significantly increased Caspase 3 activity and activated Caspase 3 protein expression,significantly increased protein expressions of NMDA receptor 2A(NMDAR2A)and NMDA receptor 2B(NMDAR2B),and statistically decreased PSD95 protein expression(P<0.05);as compared with cells in the AD group,cells in the AD+SRR siRNA group had significantly decreased SRR protein expression and apoptosis rate,statistically increased cell survival rate,significantly decreased Caspase 3 activity and activated Caspase 3 protein expression,significantly decreased NMDAR2A protein expression,and statistically increased PSD95 protein expression(P<0.05).Conclusion Down-regulation of SRR expression can reduce the NMDAR2A protein expression,alleviate the over-activation of NMDA receptor,reduce the cell apoptosis,improve cell survival rate,protect nerve cells,increase PSD95 protein expression,and alleviate synaptic damage in PC12 cells.