Beclin1及Atg7在维格列汀保护db/db小鼠胰岛β细胞中的作用

The role of Beclin1 and Atg7 in the protective effect of vildagliptin on islet β cells of db/db mice

目的 探讨维格列汀对糖尿病小鼠胰岛β细胞自噬相关因子表达及β细胞存活的影响.方法 16只db/db小鼠按血糖水平分为糖尿病组（DM组,n=8）及维格列汀治疗组（VT组,n=8）,同龄野生型小鼠作为正常对照组（NC组,n=8）,其中DM组及VT组间血糖水平无显著差异（t=1.918,P＞ 0.05）.VT组小鼠给予维格列汀（35 mg＆#183;kg-1＆#183;d-1）灌胃,DM组及NC组小鼠给予生理盐水灌胃.干预6周后,经心脏穿刺取血检测随机HbA1c水平；采用免疫荧光技术分析胰岛形态及结构；TUNEL法检测β细胞凋亡；RT-PCR技术分析胰岛β细胞自噬相关基因6（Beclin1）及自噬相关基因7（Atg7）mRNA表达水平；免疫组化技术分析β细胞Beclin1蛋白表达水平.结果 DM组小鼠血HbA1c水平明显高于NC组；VT组小鼠血HbA1c水平较DM组降低（F=579.19,P＜0.01）.免疫荧光及细胞凋亡检测发现,与NC组相比,DM组胰岛α及β细胞分布紊乱,凋亡增加（P＜0.001）；与DM组相比,VT组胰岛α及β细胞分布趋于正常,β细胞凋亡减少（F =37.25,P＜0.01）.RT-PCR结果显示,DM组小鼠胰岛β细胞Beclin1及Atg7 mRNA表达显著高于NC组（P ＜0.001）;VT组中上述基因mRNA表达较DM组降低（F=61.35,41.43,P均＜0.001）.免疫组化分析结果显示,DM组Beclin1蛋白表达高于NC组,而VT组中表达较DM组降低（F=15.92,P＜0.05）.结论 维格列汀治疗可下调Beclin1及Atg7在糖尿病小鼠β细胞中的表达,该作用可能与其改善胰岛结构及促进β细胞存活有关.

Objective To investigate the influence of vildagliptin on the expressions of autophagy related genes in islet β cells and β cell survival in diabetic mice.Methods Sixteen db/db mice were allocated into two groups according to the blood glucose levels： diabetic group（DM group,n =8） and vildagliptin treatment group（VTgroup,n=8）; the difference of blood glucose levels in DM and VT group was not significant（t=1.918,P 〉0.05）.Wildtype mice with the same age were used as normal control group（NC group,n =8）.Mice in VT group were administered with vildagliptin （35 mg＆#183; kg-1＆#183; d-1） by oral garage and mice in DM and NC group were administered with saline.After six weeks of treatment,blood samples collected by cardiac puncture were used to examine plasma HbA1c levels.The morphology of islet was analyzed by immunofluorescence.Apoptosis of islet cells were analyzed by TUNEL method.The mRNA expressions of Beclin1 and Atg7 were determined by RT-PCR.The protein expressions of Beclin1 were examined by immunohistochemistry method.Results Plasma HbA 1c levels in DM group were higher than those in NC group,plasma HbA 1c levels in VT group were lower than that in DM group （F =579.19,P 〈0.01）.In contrast to NC group,DM group showed abnormal islet morphology and higher apoptosis rate of β cells （P 〈0.001）.In contrast to DM group,VT group showed near normal islet morphology and lower apoptosis rate of β cells（F=37.25,P 〈0.01）.RT-PCR analysis demonstrated the expressions of Beclin1 and Atg7 mRNA increased markedly inDMgroupcomparedwithNC group（P〈0.001） and decreased in VT group in comparison with DM group （F =61.35,41.43,all P 〈0.001）.Beclin1 protein expressions increased significantly in DM group compared to NC group （P〈0.01）and decreased in VT group,compared to DM group （F =15.92,P 〈0.05）.Conclusion Treatment with vildag liptin downregulates expressions of Beclin1 and Atg7 in islet β cells of diabetic mice,which may contribute to improved islet architecture and β cells survival.