糖基化终末产物对SH-SY5Y细胞内质网应激的影响

摘要

目的 研究糖基化终末产物(AGEs)对体外培养的神经母细胞瘤细胞(SH-SY5Y)内质网应激的影响,探讨AGEs在AD发病中的可能机制.方法 以糖基化修饰的牛血清白蛋白(AGE-BSA)分别处理SH-SY5Y细胞0、12、24、48 h,蛋白质免疫印迹法检测内质网应激(ERS)相关蛋白GRP78、p-eIF2α、Caspase-12的表达水平.再将细胞随机分为正常对照组(NC组)、牛血清白蛋白对照组(BSA组)、AGE-BSA组、AGE-BSA+抗RAGE中和抗体组(RAGE-Ab组)、AGE-BSA+α-硫辛酸组(ALA组)、AGE-BSA+NADPH氧化酶抑制剂DPI组(DPI组).蛋白免疫印迹法半定量检测各组细胞ERS相关蛋白表达水平变化,同样处理后用免疫荧光染色观察GRP78、p-eIF2α表达水平,应用活性氧荧光探针DCFH-DA检测各组细胞活性氧 (ROS) 水平.结果 AGE-BSA处理细胞24 h后免疫蛋白印迹可见GRP78、p-eIF2α出现表达高峰,同时荧光染色可见两者高水平表达,48 h后Caspase-12表达水平显著升高,且ROS水平达到 NC 组的6.95倍.与AGE-BSA组相比,RAGE-Ab组、ALA组、DPI组的 ROS 水平都有不同程度的降低(P＜0.01),同时GRP78、p-eIF2α、Caspase-12的表达水平均有明显下调(P＜0.01或P＜0.05).结论 AGEs可诱导SH-SY5Y细胞ROS产生,并通过启动氧化应激及内质网应激对细胞造成损伤.%Objective To investigate the effect of advanced glycation end products(AGEs) on cndoplas-mic rcticulum strcss(ERS) of SH-SY5Y cell, further to explore the possible role of AGEs in the pathogencsis Alzheimer'disease. Methods SH-SY5Y cells were treated with AGE-BSA for a 12,24,48h. Expression of ERS related protein GRP78,p-cIF2a,Caspasc-12 were detected by Western blot at different time points. Then SH-SY5Y cells were randomly divided into normal control group (NC group) , BSA control group, AGE-BSA group, AGE-BSA + RAGE group, AGE-BSA + ALA group and AGE-BSA + DPI group. Western blot and im-munofluorcsccncc were adopted to estimate the expression of ERS related protein and the level of ROS were c-valuatcd by the 2', 7'-dichlorofluorcsccin diacctatc (DCFH-DA) method. Results After treated with AGE-BSA, the expression of GRP78 and p-cIF2a in SH-SY5Y cells reached their peak at 24h and the expression of Caspasc-12 reached its peak at 48h. The level of ROS in AGE-BSA group reached 6. 95 times of the NC group. Compared with AGE-BSA group, ROS level of the RAGE-Ab group, the ALA group and the DPI group decreased significantly at different degrees (P?). 01) , also the expression of ERS related protein decreased significantly (P<(). 01 or P<0. 05). Conclusions AGEs could promote the production of ROS in SH-SY5Y cells and further to damage the cells through inducing oxidativc stress and ERS.