亚低温后处理对小鼠神经母细胞瘤细胞氧糖剥夺复糖复氧模型胰岛素降解酶的影响

Effects of mild hypothermia postconditioning on IDE in oxygen-glucose deprivation/reperfusion model of mouse neuroblastoma cells

目的观察亚低温后处理对小鼠神经母细胞瘤(N2a)细胞氧糖剥夺/复糖复氧(OGD/R)模型胰岛素降解酶(IDE)的影响。方法采用随机数字表法将生长状态良好的N2a细胞分为对照组、模型组和低温组(n=60),对照组在正常条件下培养,其余2组先氧糖剥夺培养3 h后,模型组在正常条件下复糖复氧,低温组于32℃低温下复糖复氧24 h后再在正常条件下复糖复氧。各组于复糖复氧24 h和48 h时,用流式细胞学检测细胞凋亡率,乳酸脱氢酶检测细胞毒性,分光光度法检测半胱氨酸天冬氨酸蛋白酶3(Caspase-3)活性,ELISA法检测β淀粉样蛋白(Aβ)浓度,Western blot和qRT-PCR法检测IDE蛋白和mRNA相对表达。结果与对照组比较,模型组和低温组24 h和48 h细胞凋亡率、细胞毒性和Caspase-3活性明显升高(P<0.05),IDE表达明显降低(P<0.05),模型组Aβ浓度明显升高(P<0.05);与模型组相比,低温组24 h和48 h细胞凋亡率、细胞毒性、Caspase-3活性和Aβ浓度明显降低(P<0.05),IDE蛋白(0.24±0.01 vs 0.12±0.00,0.33±0.01 vs 0.17±0.01)和mRNA(0.67±0.02 vs 0.48±0.01,0.63±0.02 vs 0.41±0.01)相对表达明显升高(P<0.05)。结论亚低温后处理可以减轻小鼠N2a细胞OGD/R模型损伤,该机制可能与上调IDE的表达,减少Aβ的生成,减少细胞凋亡率有关。

Objective To determine the effects of mild hypothermia postconditioning on insulin-degrading enzyme(IDE)in mouse N2 a cells induced by OGD/R.Methods Well growing N2 a cells were randomly divided into 3 groups:control group,model group and hypothermia group,60 in each group.The cells in control group were cultured under normal condition,and those in the other 2 groups were cultured with oxygen-glucose deprivation for 3 h.Then the cells in the model group were cultured under normal condition for oxygen-glucose reperfusion,those in hypothermia group were cultured for reperfusion at 32℃for 24 h followed by normal condition.At 24 and 48 h after oxygen-glucose reperfusion,the apoptotic rate was detected by flow cytometry,cell cytotoxicity was detected by measuring the release of LDH,activity of Caspase-3 was measured by spectrophotomery,Aβconcentration was tested with ELISA,and the expression of IDE at mRNA and protein levels was detected by qRT-PCR and Western blot.Results Compared with the control group,the apoptotic rate,cell cytotoxicity and Caspase-3 activity were significantly increased(P<0.05),the IDE expression level was obviously decreased(P<0.05)in the other 2 groups at 24 and 48 h after oxygen-glucose reperfusion,and elevated Aβconcentration only seen in the model group(P<0.05).The hypothermia group had notably decreased apoptostic rate,cell cytotoxicity and Caspase-3 activity as well as Aβconcentration(P<0.05),and enhanced IDE expression levels(P<0.05)than the model group at 24 and 48 h after oxygen-glucose reperfusion(0.24±0.01 vs 0.12±0.00,0.33±0.01 vs 0.17±0.01;0.67±0.02 vs 0.48±0.01,0.63±0.02 vs 0.41±0.01,P<0.05).Conclusion Mild hypothermia postconditioning attenuates the injury of N2 a cells induced by OGD/R,which may be associated with up-regulation of IDE,reduced production of Aβof the cells.