人胰岛淀粉样多肽对大鼠胰岛细胞瘤细胞自噬的影响及相关机制  

The effect of human islet amyloid polypeptide on autophagy in murine INS-1 cells and potential mechanisms

作　　者：夏广昊 靳育静 肖金凤[1] 李晓通[1] 朱铁虹[1] Xia Guanghao;Jin Yujing;Xiao Jinfeng;Li Xiaotong;Zhu Tiehong(Department of Endocrinology,the Tianjin Medical University General Hospital,Tianjin 300052,China)

机构地区：[1]天津医科大学总医院内分泌科,300052

出　　处：《中华内科杂志》2018年第9期667-673,共7页Chinese Journal of Internal Medicine

基　　金：国家自然科学基金项目（81241030）;天津市自然科学基金（M0102）

摘　　要：目的 研究人胰岛淀粉样多肽（hIAPP）对大鼠胰岛细胞瘤细胞（INS-1细胞）自噬的影响及相关机制.方法 hIAPP孵育大鼠INS-1细胞后,采用电镜观察细胞自噬体数量的变化,噻唑蓝（MTT）法检测细胞活力的变化,二氯二氢荧光素-乙酰乙酸酯（DCFH-DA）法检测细胞活性氧族（ROS）,蛋白质印迹检测磷酸腺苷依赖的蛋白激酶（AMPK）以及自噬标志物p62及微管相关蛋白1轻链3（LC3）的变化.结果 hIAPP组INS-1细胞自噬体的数量和LC3-Ⅱ/LC3-Ⅰ的表达明显增加（P值均＜0.05）.hIAPP组细胞ROS水平为对照组的1.76倍（P＜0.01）.抗氧化剂N-乙酰半胱氨酸（NAC）可抑制hIAPP所诱导的INS-1细胞ROS增加和抑制磷酸化AMPK及LC3-Ⅱ/LC3-Ⅰ表达升高（P值均＜0.05）.使用AMPK抑制剂compoundC预处理INS-1细胞可抑制hIAPP和AMPK激动剂5-氨基咪唑-4-甲酰胺-1-β-D-呋喃核糖苷（AICAR）所诱导的LC3-Ⅱ/LC3-1和磷酸化AMPK增加（P值均＜0.05）.自噬抑制剂3-甲基腺嘌呤（3-MA）和compound C加重hIAPP所诱导的INS-1细胞死亡和ROS升高（P值均＜0.05）.AICAR可明显缓解hIAPP的细胞毒性作用（P＜0.05）.结论 AMPK通路相关自噬减轻hIAPP对INS-1细胞的毒性作用和氧化损伤.Objective The aims of the study were to investigate the effects of human islet amyloid polypeptide （hIAPP） on autophagy in INS-1 cells and its underlying mechanism,and to explore the role of autophagy in hIAPP-induced cytotoxicity and oxidative stress.Methods INS-1 cells were treated with hIAPP （10 μmol/L） for 24 h in the presence or absence of N-acetyl-L-cysteine （NAC）,compound C,5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside （AICAR） and 3-methyladenine （3-MA）,respectively.Transmission electron microscopy was used to observe the number of autophagosome in cells.Cell viability was determined by methyl thiazolyl tetrazolium （MTT） test.2＇,7＇-dichlorofluorescin diacetate （DCFH-DA） assay was used to measure the relative levels of reactive oxygen species （ROS）.Western blot was used to detect expression of adenosine monophosphate-activated protein kinase （AMPK） and autophagic markers p62 and microtubule associated protein 1 light chain3 （LC3）.Results Treatment of INS-1 cells with hIAPP resulted in a significant increase in the number of autophagosomes and the expression of LC3-Ⅱ/LC3-Ⅰ （both P〈0.05）.Meanwhile,treatment of INS-1 cells with hIAPP enhanced the level of ROS to 1.76 times of control cells （P〈0.01）.Co-treatment with NAC,an antioxidant,inhibited hIAPP-induced ROS generation,and the expression of LC3-Ⅱ/LC3-Ⅰ and p-AMPK in the INS-1 cells （all P〈0.05）.Pretreatment of INS-1 cells with AMPK inhibitor compound C suppressed hIAPP and AICAR,an activator of AMPK,induced expression of LC3-Ⅱ/LC3-Ⅰ and p-AMPK （all P〈0.05）.Autophagic inhibitor 3-MA and compound C aggravated the hIAPP-induced cell death and ROS generation in INS-1 cells （All P〈0.05）.The cytotoxic effects of hIAPP were significantly attenuated by co-treatment with AICAR （P〈0.05）.Conclusion Autophagy may act as an adaptive mechanism to alleviate hIAPP-induced oxidative damage and toxicity in INS-1 cells.

关 键 词：胰岛淀粉样多肽 自噬 氧化应激 磷酸腺苷依赖的蛋白激酶 

分 类 号：R587.1[医药卫生—内分泌]