糖基化终末产物对SH-SY5Y细胞内质网应激的影响

The effect of advanced glycation end products on endoplasmic reticulum stress of SH-SY5Y cell

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 endoplasmic reticulum stress（ERS） of SH-SY5Y cell,further to explore the possible role of AGEs in the pathogenesis Alzheimer＇ disease.Methods SH-SY5Y cells were treated with AGE-BSA for a 12、24、48h.Expression of ERS related protein GRP78、p-eIF2α、Caspase-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 immunofluorescence were adopted to estimate the expression of ERS related protein and the level of ROS were evaluated by the 2′,7′-dichlorofluorescein diacetate（DCFH-DA） method.Results After treated with AGE-BSA,the expression of GRP78 and p-eIF2α in SH-SY5Y cells reached their peak at 24h and the expression of Caspase-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（P0.01）,also the expression of ERS related protein decreased significantly（P0.01 or P0.05）.Conclusions AGEs could promote the production of ROS in SH-SY5Y cells and further to damage the cells through inducing oxidative stress and ERS.