咪达唑仑对氧葡萄糖剥夺后N2a/APP695细胞β淀粉样蛋白的抑制作用及机制

Inhibitory effects and mechanisms of midazolam on oxygen-glucose deprivation-induced amyloidβ-protein

目的探讨咪达唑仑对被剥夺氧和葡萄糖(oxygen-glucose deprivation,OGD)后的N2a/APP695细胞淀粉样前体蛋白(amyloid precursor protein,APP)代谢变化的影响及机制。方法将处于对数生长期的N2a/APP695细胞用平衡盐缓冲液冲洗后随机分为空白组、对照组及实验组。空白组未经OGD处理,在正常培养箱内培养;对照组行OGD处理后继续培养复氧;实验组在OGD处理后加入不同浓度(分别为70、100、125、150 ng/ml)的咪达唑仑继续培养复氧。24 h后用AnnexinV-FITC/双染法流式细胞术检测各组细胞凋亡比例;用Western blot方法检测各组细胞的Caspase-3、Aβ1-42、解整合素金属蛋白酶10(adisintegrin and metalloproteinase 10,ADAM10)、β-淀粉样前体蛋白水解酶1(β-site APP cleavage enzyme-1,BACE1)、早老素1(presenilin 1,PS1)蛋白水平。结果与空白组相比,对照组的细胞凋亡比例、Caspase-3、Aβ1-42、BACE1及PS1水平显著升高(P<0.05);APP及ADAM10水平无明显变化(P>0.05)。与对照组相比,实验组细胞凋亡比例、Caspase-3、Aβ1-42显著下降(P<0.05);咪达唑仑70 ng/ml组BACE1显著下降(P<0.05),而APP、ADAM10及PS1无明显变化(P>0.05)。结论OGD可通过上调BACE1及PS-1导致Aβ1-42表达增加促使细胞凋亡;而咪达唑仑可通过下调BACE1减少Aβ1-42表达进而减少神经细胞凋亡,发挥对OGD损伤后N2a/APP695细胞的保护作用。

Objective To study the inhibitory effects and mechanisms of midazolam on amyloid precursor protein(APP)in N2a/APP695 cells after oxygen-glucose deprivation(OGD).Method N2a/APP695 cells in logarithmic growth phase were rinsed with Earle’s Balanced Salt Buffer(EBSS)and randomly assigned into blank group,control group and experimental group.The blank group was cultured in a normal incubator without OGD treatment.The control group was reoxygenated after OGD treatment.The experimental group was reoxygenated and added different concentrations(70,100,125,150 ng/ml)of midazolam after OGD treatment.Twenty-four hours later,the apoptosis rate of the three groups was detected using AnnexinV-FITC/double staining flow cytometry.Western blot was used to detect Caspase-3,Aβ1-42,adisintegrin and metalloproteinase 10(ADAM10),β-site APP cleavage enzyme-1(BACE1),and presenilin 1(PS1)protein levels.Result Compared with the blank group,the percentage of apoptosis,the levels of Caspase-3,Aβ1-42,BACE1 and PS1 in the control group were significantly increased(P<0.05).No significant changes existed in APP and ADAM10 levels(P>0.05).Compared with the control group,the proportion of apoptosis,the levels of Caspase-3 and Aβ1-42 decreased significantly in the experimental group.The level of BACE1 decreased significantly in midazolam 70 ng/ml group(P<0.05),and no significant changes were found for APP,ADAM10 and PS1(P>0.05).Conclusion OGD can up-regulate the levels of BACE1 and PS-1 to increase Aβ1-42 expression to promote apoptosis.Midazolam can down-regulate BACE1 to reduce the expression of Aβ1-42 thus reducing the apoptosis of nerve cells,and play a protective role in N2a/APP695 cells after OGD injury.