糖基化终末产物对SH-SY5Y细胞氧化应激及凋亡的影响

Effect of advanced glycation end products on oxidative stress and apoptosis of SH-SY5Y cells

目的研究糖基化终末产物(AGEs)对人神经母细胞瘤细胞(SH-SY5Y)凋亡的影响,探讨AGEs与阿尔茨海默病(AD)发病机制的关系。方法分别以不同浓度糖基化修饰的牛血清白蛋白(AGE-BSA)处理SH-SY5Y细胞48 h,选取AGE-BSA敏感浓度(200μg/mL)处理细胞不同时间,流式细胞仪(FCM)检测细胞凋亡率,确定AGE-BSA诱导SH-SY5Y凋亡最佳浓度及时间。将细胞随机分为对照组、牛血清白蛋白(BSA)对照组、AGE-BSA组、AGE-BSA+抗RAGE中和抗体组、AGE-BSA+α-硫辛酸组、AGE-BSA+DPI(NADPH氧化酶抑制剂)组。FCM检测细胞凋亡率变化,Hoechst 33258染色观察细胞形态改变,应用活性氧荧光探针DCFH-DA检测活性氧(ROS)水平,Western blot测定Caspase-12的表达。结果细胞凋亡率随AGE-BSA浓度增加及作用时间延长而升高,200μg/mL AGE-BSA作用48 h细胞凋亡率从对照组的(2.23±0.08)%增加到(16.8±1.27)%(P<0.05),同时,Hoechst染核后可见典型凋亡形态改变,细胞内ROS水平显著增高,Caspase-12蛋白表达明显上调,与对照组相比差异显著(P<0.05),而分别用抗RAGE中和抗体、α-硫辛酸、DPI预处理后再加入AGE-BSA,各组与AGE-BSA组比较,凋亡率明显下降,ROS水平、Caspase-12蛋白表达均明显降低(P<0.05)。结论糖基化终末产物可刺激SH-SY5Y细胞产生大量ROS及活化Caspase-12介导细胞凋亡,通过阻断其与特异性受体RAGE结合或减少细胞内ROS可减轻细胞凋亡的发生。

Objective To investigate the effect of advanced glycation end products（AGEs） on apoptosis of SH-SY5Y cells,and to further explore the relationship between AGEs and the mechanism of Alzheimer disease.Methods SH-SY5Y cells were treated with different concentrations of AGE-BSA for 48 h,or with AGE-BSA（200 μg/mL） for different times.Cell apoptosis was detected by flow cytometry（FCM） to determine the best concentration and time of AGE-BSA.SH-SY5Y cells were randomly divided into six groups： the normal control group,the BSA control group,the AGE-BSA group,the AGE-BSA＋RAGE antibody group,the AGE-BSA＋Alpha lipoic acid（ALA） group,and the AGE-BSA＋diphenyleneiodonium（DPI） group.Cell apoptosis was detected by FCM and Hoechst staining,the level of ROS was evaluated by the 2′,7′-dichlorofluorescein diacetate（DCFH-DA） method,and expression of Caspase-12 was analyzed by Western blot.Results AGE-BSA induced SH-SY5Y cells apoptosis in a time-and concentration-dependent manner.After treatment with 200 μg/mL of AGE-BSA for 48 hours,apoptosis of SH-SY5Y cells was significantly increased to（16.8±1.27） % from（2.23±0.08）%（P0.05）.Apoptosis-like cells could be found after Hoechst staining of nuclei,the level of ROS and expression of Caspase-12 statistically increased compared with the normal group（P0.05）.Compared with the AGE-BSA group,apoptosis of cells,level of ROS and expression of Caspase-12 in the AGE-BSA＋RAGE-Ab group,the AGE-BSA ＋ ALA group and the AGE-BSA ＋ DPI group were significantly decreased（P0.05）.Conclusion AGEs could induce the production of ROS and activation of Caspase-12,which may be involved in apoptosis of SH-SY5Y cells induced by AGEs.Blocking the combination of AGEs and its receptor（RAGE） or reducing production of ROS may protect against AGEs-induced SH-SY5Y apoptosis.