Xia G H, Jin Y J, Xiao J F, Li X T, Zhu T H
Department of Endocrinology, the Tianjin Medical University General Hospital, Tianjin 300052, China.
Zhonghua Nei Ke Za Zhi. 2018 Sep 1;57(9):667-673. doi: 10.3760/cma.j.issn.0578-1426.2018.09.009.
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. 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). 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 0.05). Meanwhile, treatment of INS-1 cells with hIAPP enhanced the level of ROS to 1.76 times of control cells (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 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 0.05). Autophagic inhibitor 3-MA and compound C aggravated the hIAPP-induced cell death and ROS generation in INS-1 cells (All 0.05). The cytotoxic effects of hIAPP were significantly attenuated by co-treatment with AICAR (0.05). Autophagy may act as an adaptive mechanism to alleviate hIAPP-induced oxidative damage and toxicity in INS-1 cells.
本研究的目的是探讨人胰岛淀粉样多肽（hIAPP）对INS-1细胞自噬的影响及其潜在机制，并探究自噬在hIAPP诱导的细胞毒性和氧化应激中的作用。分别在存在或不存在N-乙酰-L-半胱氨酸（NAC）、化合物C、5-氨基咪唑-4-甲酰胺-1-β-D-呋喃核糖苷（AICAR）和3-甲基腺嘌呤（3-MA）的情况下，用hIAPP（10μmol/L）处理INS-1细胞24小时。采用透射电子显微镜观察细胞中自噬体的数量。通过甲基噻唑基四氮唑（MTT）试验测定细胞活力。用2',7'-二氯荧光素二乙酸酯（DCFH-DA）测定法测量活性氧（ROS）的相对水平。采用蛋白质免疫印迹法检测单磷酸腺苷激活蛋白激酶（AMPK）以及自噬标志物p62和微管相关蛋白1轻链3（LC3）的表达。用hIAPP处理INS-1细胞导致自噬体数量和LC3-Ⅱ/LC3-Ⅰ的表达显著增加（均P<0.05）。同时，用hIAPP处理INS-1细胞使ROS水平提高至对照细胞的1.76倍（P<0.01）。抗氧化剂NAC共同处理可抑制hIAPP诱导的ROS生成以及INS-1细胞中LC3-Ⅱ/LC3-Ⅰ和磷酸化AMPK的表达（均P<0.05）。用AMPK抑制剂化合物C预处理INS-1细胞可抑制hIAPP和AMPK激活剂AICAR诱导的LC3-Ⅱ/LC3-Ⅰ和磷酸化AMPK的表达（均P<0.05）。自噬抑制剂3-MA和化合物C加重了hIAPP诱导的INS-1细胞死亡和ROS生成（均P<0.05）。AICAR共同处理可显著减轻hIAPP的细胞毒性作用（P<0.05）。自噬可能作为一种适应性机制减轻hIAPP诱导的INS-1细胞氧化损伤和毒性。
