胰高血糖素样肽-1保护内质网应激诱导的胰岛β细胞凋亡  

Glucagon-like peptide-1 protects the apoptosis of pancreatic β cells induced by endoplasmic reticulum stress

作　　者：张菲菲[1] 许世清[3] 姜永玮[3] 张文健[3] 娄晋宁[3] 门秀丽[2] 

机构地区：[1]河北联合大学研究生院,唐山063000 [2]河北联合大学病理生理学教研室,唐山063000 [3]卫生部中日友好医院临床医学研究所

出　　处：《中华糖尿病杂志》2011年第5期425-428,共4页CHINESE JOURNAL OF DIABETES MELLITUS

基　　金：国家自然科学基金项目（30971410）

摘　　要：目的探讨胰高血糖素样肽-1（GLP-1）对毒胡萝卜素（TG）诱导的胰岛13细胞凋亡的保护作用及可能机制。方法体外培养大鼠胰岛β细胞系-19细胞，采用TG诱导内质网应激，并比较存在GLP-1和GLP-1受体激动剂exendin-4的条件下胰岛B细胞凋亡情况。实验分为正常对照组、TG组（100nmol／L）、GLP-1（10nmol／L）＋TG（100nmol／L）组和exendin-4（100nmol／L）＋TG（100nmol／L）组，通过DNA片段化、原位转移酶缺口末端标记法（TUNEL）染色、AnnexinV—PI（膜连蛋白V／碘化丙啶）双染流式细胞仪检测4组细胞凋亡率的差异。采用Westernblot检测细胞色素c的释放。通过荧光探针DCFH—DA检测细胞活性氧簇（ROS）水平。罗丹明-R110（Z—DEVD—R110）染色检测细胞Caspase-3活性。2组间均数比较采用t检验，4组间比较采用单因素方差分析。结果DNA片段化检测和TUNEL染色实验均显示后两组细胞凋亡较TG组明显减少（F=2．16；t值分别为6．13和6．73，均P〈0．01）。AnnexinV—PI双染流式细胞仪检测显示TG组细胞的凋亡率为26％，在GLP-1和exendin-4存在的条件下，TG诱导的细胞凋亡明显减至19．7％和19．2％。进-步实验表明GLP-1和exendin-4能降低内质网应激过程中细胞色素c释放、活性氧簇ROS的产生和Caspase-3活性。细胞浆蛋白的Westernblot检测结果显示，给予GLP-1和exendin-4能降低内质网应激过程中细胞色素c释放（F=0．875；t值为5．37和7．26，P〈0．01）。细胞浆中凋亡蛋白酶Caspase-3的检测结果显示，GLP-1和exendin-4能抑制Caspase-3的激活和释放（F=0．838，t值为3．46和4．52，P〈0．01）。细胞线粒体活性氧的释放检测结果显示，正常情况下，细胞浆中可检测到-定量的活性氧，给予TG后释放的活性氧明显增多（61．67±7．64，t=22．13，P〈0．01），给予GLP-1和exendin-4能减少活性氧的释放（32．3±3．21，t值分别为12．41和15．2Objective To explore the potential protective mechanism of glucagon-like peptide-1 （GLP-I） on thapsigargin （TG）induced pancreatic β cell apoptosis. Methods The rat pancreatic β cell line-r9 cells cultured in vitro were used to compare the pancreatic cell apoptosis with the presence of GLP-1 and GLP-I receptor agonists exendin-4 while endoplasmic reticulum （ER）stress was induced by thapsigargin. Further, we investigated the effects of GLP-1 and exendin-4 on the release of cytochrome c and generation of reactive oxygen species （ROS） during ER stress. This experiment was divided into four groups, normal group, TG group （ 100 nmol/L ） , GLP-1 （ 10 nmol/L ） ＋ TG （ 100 nmol/L ） group and Exendin-4（ 100 nmol/L） ＋ TG（ 100 nmol/L）group, and apoptosis rate of the four groups was detected by DNA fragmentation, TUNEL staining, AnnexinV-PI double dye flow cytometric analysis. We used the Western Blot to test the release of eytochrome e, used fluorescence probe DCFH-DA to test the reactive oxygen species （ROS） level and used rhodamine-110 （Z-DEVD-R110 ） dyeing to detect the cells Caspase3 ＇ s activity. We used t-testing to compare the difference between two groups and used one way analysis of variance to test the differences among the four groups. Results The apoptosis of the latter two groups which were tested by DNA fragmentation and TUNEL staining was significantly reduced compared with TG group （t =6.13 or 6.73, both P 〈 0.01 ）. The apoptosis rate of 13 ceils tested by AnnexinV-Pl double dye flow cytometry was 26% , which was reduced to 19.7% and 19.2% in the existence of GLP-1 and exendin-4, respectively. Further experiments showed that GLP-1 and exendin-4 reduced cytochrome c release, ROS generation and caspase3 activation. The Western blot of cytoplasm albumen showed that GLP-I and exendin- 4 can reduce the release of cytochrome c in ER stress （t =5.37 or 7.26,both P 〈0.01 ）. The detection of ROS in mitochondria showed that ROS, a certain amount of which i

关 键 词：胰高血糖素样肽-1 胰岛Β细胞 内质网应激 细胞凋亡 活性氧 

分 类 号：R587.1[医药卫生—内分泌]