阿尔茨海默病合并2型糖尿病与海马损伤——从应激反应到自噬产生

Alzheimer＇s disease complicated with type 2 diabetes and hippocampal injury： from oxidative stress to autophagy

目的 研究氧化应激、细胞增殖、自噬在阿尔茨海默病合并2型糖尿病（AD＋ T2DM）中的相互关系,探讨自噬在AD＋ T2DM中的作用.方法 建立T2DM、AD及AD＋ T2DM小鼠模型,实验共分为4组：对照组、T2DM组、AD组和AD＋ T2DM组,每组20只小鼠,共计80只小鼠.通过水迷宫实验观察小鼠的学习记忆能力;测定小鼠空腹血糖值,用酶学法检测血清胰岛素浓度来计算小鼠胰岛素抵抗指数;应用免疫荧光染色法检测胰岛素受体底物2、氧化应激、细胞增殖及自噬变化情况,同时采用透射电子显微镜观察自噬发生的超微结构,Western印迹技术半定量分析自噬发生程度.结果 与对照组小鼠相比,在第4天时,T2DM组、AD组及AD＋ T2DM组在水迷宫实验中找到平台的时间延长[分别为（26.08±4.93）s、（38.46 ±4.07）s、（47.32 ±5.86）s、（53.01 ±6.12）s,F=2.454,P=0.025],AD± T2DM组用时比AD组更长（t=-3.624,P=0.033）;与对照组相比,T2DM组、AD组及AD± T2DM组分别出现了不同程度胰岛素抵抗（4.35 ±0.48、16.12 ±3.57、7.03±3.11、18.78±5.06,F=5.602,P=0.009）;各组间通过免疫组织化学染色及Western印迹半定量分析比较,T2DM、AD及AD＋ T2DM组胰岛素受体底物2表达减少、氧化应激表达增多、细胞增殖受到抑制、自噬表达上调（分别为F=418.344、222.514、436.250、113.934、23.772、35.469,均P＜0.05）,而AD＋ T2DM组比AD组趋势更明显（分别为t=-2.796、21.723、-8.041、9.037、-4.403、-32.011、-26.593,均P ＜0.05）.结论 AD＋ T2DM组认知功能障碍比AD组及T2DM组严重;AD＋ T2DM诱导氧化应激损伤发生,氧化应激上调抑制了细胞增殖,进一步诱导自噬发生.

Objective To study the pathological alterations,such as oxidative stress,cell proliferation and insulin resistance,especially autophagy,in Alzheimer＇ s disease （AD） complicated with type 2 diabetes （AD ＋ T2DM）.Methods The mouse models of T2DM,AD and AD ＋ T2DM were used in the study,and totally 80 mice were divided into four groups：control group,T2DM group,AD group and AD ＋ T2DM group.Morris water maze was applied to test the ability of learning and memory among the above mentioned groups.In the meantime,insulin resistance index,the expression of insulin receptor substrate 2,oxidative stress,cell proliferation and autophagy were observed with chemical analysis,immunofluorescent labeling,transmission electron microscopy and Western blotting.Results On day 4,the difference of time to find Morris water maze in control group,T2DM group,AD group and AD ＋ T2DM group （（26.08 ±4.93） s,（38.46 ± 4.07） s,（47.32 ± 5.86） s,（53.01 ± 6.12） s,F =2.454,P =0.025） was statistically significant,and the time in AD ＋ T2DM group was longer than that in AD group （t =-3.624,P =0.033）.Compared with control group,insulin resistance occurred in T2DM group,AD group and AD ＋ T2DM group （4.35 ± 0.48,16.12 ± 3.57,7.03 ± 3.11,18.78 ± 5.06,F =5.602,P =0.009）,and the reduction of insulin receptor substrate 2 expression,the oxidative stress reaction,neural proliferative suppression and autophagy （F =418.344,222.514,436.250,113.934,23.772,35.469,all P ＜ 0.05） were induced in T2DM group,AD group and AD ＋ T2DM group,which were more serious in AD ＋ T2DM group than in AD group （t =-2.796,21.723,-8.041,9.037,-4.403,-32.011,-26.593,all P ＜0.05）.Conclusion AD ＋ T2DM mice suffered more serious cognitive impairment than AD and T2DM mice.The oxidative stress levels of AD ＋ T2DM mice were upregulated,and thus led to the inhibition of cell proliferation,eventually leading to promotion of autophagy.