Wang Jing, Wang Jin, Wang Juan-Juan, Zhang Wei-Fang, Jiao Xiang-Ying
Department of Physiology, Shanxi Medical University, Taiyuan 030001, China.
Sheng Li Xue Bao. 2017 Aug 25;69(4):445-451.
Thioredoxin (Trx) interacting protein (TXNIP) is a Trx-binding protein that inhibits the antioxidative function of Trx and is highly expressed in the serum and tissue samples from diabetes patients. This study was to explore whether TXNIP overexpression could cause INS-1 cell autophagy under normal glucose and lipid concentrations, and to analyze the role of autophagy in the apoptosis of INS-1 cells. The INS-1 cells cultured under normal conditions were divided into three groups: normal control, empty adenovirus vector (Ad-eGFP) and TXNIP overexpression (Ad-TXNIP-eGFP) groups. Forty-eight hours after transfection, the expression levels of TXNIP mRNA and protein were measured. Western blot was used to examine the protein expression levels of Beclin-1 and P62, as well as LC3-II/LC3-I ratio, which are associated with autophagy. IF/ICC was used to measure the autophagosome. In addition, the cleaved caspase-3/caspase-3 ratio, the apoptosis marker, was also measured, and the apoptotic rates were detected by flow cytometry (FCM). The results showed that the TXNIP mRNA and protein levels were significantly up-regulated in Ad-TXNIP-eGFP group, suggesting that TXNIP overexpression model was successfully established. In Ad-TXNIP-eGFP group, the protein levels of Beclin-1 and LC3-II/LC3-I ratio were increased, while the protein expression of P62 was decreased, compared with those in Ad-eGFP group. Red fluorescent intensity, representing autophagy level, was higher in Ad-TXNIP-eGFP group than that in Ad-eGFP group. These results suggested that TXNIP overexpression can significantly promote INS-1 cell autophagy. Meanwhile, cleaved caspase 3/caspase 3 ratio and the number of apoptotic cells were significantly increased in Ad-TXNIP-eGFP group. The inhibitor of autophagy, 3-MA, reduced TXNIP overexpression-induced apoptosis in INS-1 cells. Taken together, our data demonstrate that autophagy appears to be an important pathway in TXNIP overexpression-induced apoptosis in INS-1 cells.
硫氧还蛋白（Trx）相互作用蛋白（TXNIP）是一种与Trx结合的蛋白，它会抑制Trx的抗氧化功能，且在糖尿病患者的血清和组织样本中高表达。本研究旨在探究在正常葡萄糖和脂质浓度下TXNIP过表达是否会导致INS-1细胞自噬，并分析自噬在INS-1细胞凋亡中的作用。将在正常条件下培养的INS-1细胞分为三组：正常对照组、空腺病毒载体（Ad-eGFP）组和TXNIP过表达（Ad-TXNIP-eGFP）组。转染48小时后，检测TXNIP mRNA和蛋白的表达水平。采用蛋白质免疫印迹法检测与自噬相关的Beclin-1和P62的蛋白表达水平，以及LC3-II/LC3-I比值。采用免疫荧光/免疫细胞化学法检测自噬体。此外，还检测了凋亡标志物裂解的caspase-3/caspase-3比值，并通过流式细胞术（FCM）检测凋亡率。结果显示，Ad-TXNIP-eGFP组中TXNIP mRNA和蛋白水平显著上调，表明成功建立了TXNIP过表达模型。与Ad-eGFP组相比，Ad-TXNIP-eGFP组中Beclin-1蛋白水平和LC3-II/LC3-I比值升高，而P62蛋白表达降低。代表自噬水平的红色荧光强度在Ad-TXNIP-eGFP组中高于Ad-eGFP组。这些结果表明TXNIP过表达可显著促进INS-1细胞自噬。同时，Ad-TXNIP-eGFP组中裂解的caspase 3/caspase 3比值和凋亡细胞数量显著增加。自噬抑制剂3-MA可降低TXNIP过表达诱导的INS-1细胞凋亡。综上所述，我们的数据表明自噬似乎是TXNIP过表达诱导INS-1细胞凋亡的重要途径。
