利拉鲁肽对人胰岛淀粉样多肽聚集性及胰岛细胞毒性的影响

目的:探讨利拉鲁肽对人胰岛淀粉样多肽聚集性及细胞毒性的影响。方法:采用硫磺素T荧光法检测淀粉样纤维聚集的动力学,采用透射电镜检测淀粉样纤维的形态。分离Wistar大鼠胰岛,采用简单随机化分组的方法将大鼠胰岛随机分配成空白对照组、人胰淀素组、人胰淀素+利拉鲁肽组。24 h后采用Annexin-V/PI荧光染色及逆转录聚合酶链反应检测细胞凋亡及炎性因子白细胞介素（IL）-1β、肿瘤坏死因子α（TNFα） 、单核细胞趋化蛋白-1（CCL-2） mRNA的表达。结果:人胰淀素单独孵育时,荧光于1.6 h左右开始上升,并随时间延长逐渐增加,3.4 h后平稳,动力学曲线呈S型曲线;人胰淀素与利拉鲁肽（500 nmol/L）以10∶1分子浓度共同孵育时,荧光信号上升延迟缓慢;以1∶1分子浓度共同孵育时,荧光信号未见明显增加,动力学曲线呈直线型。透射电镜结果显示,人胰淀素单独孵育可见大量长条状纤维聚集,人胰淀素与利拉鲁肽（2 μmol）以10∶1浓度共同孵育后纤维数量明显减少,且短小模糊;1:1（20 μmol利拉鲁肽）浓度共同孵育后,无法找到纤维结构。与空白对照组相比,人胰淀素组IL-1、TNFα、CCL-2基因的mRNA表达均增加,而人胰淀素+利拉鲁肽组表达均下降（F=429.68、48.79、153.39, 均 P＜0.05）。人胰淀素组细胞凋亡率与空白对照组相比明显升高;人胰淀素＋利拉鲁肽组凋亡率比人胰淀素组降低（F=514.34、16.14、18.47,均P＜0.05）。结论:利拉鲁肽抑制胰淀素聚集,减少胰岛细胞炎症及凋亡。

Effect of liraglutide on the human islet amyloid polypeptide aggregation and cell toxicity

Objective: To explore the effect of liraglutide on human islet amyloid polypeptide（hIAPP）aggregation and cell toxicity. Methods: Thioflavin T（ThT） binding assays were used to test the real time characterization of hIAPP fibrils formation kinetics. The electron microscopy tests were used to test the morphology of hIAPP fibrils. Pancreatic islets were isolated from adult Wistar rats. The experiment groups included normal islets,hIAPP incubated islets,hIAPP and liraglutide co-incubated islets. The incubation time of three groups was 24 h. Annexin V/PI double-staining was used to detect islet cells apoptosis. The expression of inflammation-related and apoptosis-related genes was analyzed by reverse transcription-polymerase chain reaction. Results:ThT tests showed that 5 μmol/L hIAPP had undergone fibrillation after 1.6 h,reaching a maximum after 3.4 h,and the kinetic curve was S-shaped. When co-incubate with 500 nmol/L liraglutide (10:1),hIAPP fibrillation delayed. When the molecular ratio reached 1∶1,the fluorescence signal did not increase significantly,and the kinetic curve was linear. Electron microscopy tests showed that 20 μmol hIAPP aggregates into the abundant fibrils,whereas in the presence of 2 μmol liraglutide（10∶1）,the fibrils were much less and shorter. And when increasing the concentration of liraglutide（20 μmol,1∶1）,there were no fibrils. Compared with the blank control group,the mRNA expression of IL-1,TNF and CCL-2 in amylin group was increased,while the expression in liraglutide intervention group was decreased with statistical significance（F=429.68,48.79,153.39,all P<0.05）. The annexin V/PI double-staining tests and RT-PCR showed that when treated with hIAPP,the apoptosis increased,which was decreased by liraglutide（F=514.34,16.14,18.47,all P＜0.05）. Conclusion:Liraglutide inhibits the aggregation of hIAPP and protects islets from inflammation and apoptosis.