Impairment of Autophagy Causes Superoxide Formation and Caspase Activation in 661 W Cells, a Cell Line for Cone Photoreceptors, under Hyperglycemic Conditions
Impairment of Autophagy Causes Superoxide Formation and Caspase Activation in 661 W Cells, a Cell Line for Cone Photoreceptors, under Hyperglycemic Conditions
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DOI：
10.3390/ijms21124240
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发表时间：
2020-06
影响因子：
5.6
通讯作者：
Koichiro Taki;Taeko Horie;T. Kida;Masashi Mimura;T. Ikeda;H. Oku
中科院分区：
文献类型：
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作者：
Koichiro Taki;Taeko Horie;T. Kida;Masashi Mimura;T. Ikeda;H. Oku
微血管改变是糖尿病视网膜病变最早的不良事件，但最近的研究表明，光感受器诱导的氧化应激与视网膜病变的发展有关。本研究的目的是确定在高血糖条件下光感受器形成的超氧化物对自噬的作用。为了实现这一点，我们培养661 W细胞，转化的鼠视锥细胞系，与5.5或25 mM葡萄糖在存在或不存在3甲基腺嘌呤（3 MA）或雷帕霉素。以氢乙啶为荧光探针，用流式细胞仪测定超氧化物。通过免疫印迹法测定LC 3B 2和P62表达的变化来确定自噬活性。线粒体自噬的程度取决于线粒体和溶酶体的积累。Caspase 3/7活性检测661 W细胞凋亡变化。我们的研究结果表明，在25 mM葡萄糖比在5.5 mM葡萄糖培养的细胞中的P62和超氧化物的水平更高。高糖培养的661 W细胞中加入3 MA后，P62、超氧化物歧化酶和caspase 3/7活性显著升高，而低糖培养的661 W细胞中则无此现象。这些发现表明，自噬是重要的功能和生存的661 W细胞在高血糖条件下。
Microvascular changes are the earliest adverse events in diabetic retinopathy, but recent studies have shown that oxidative stress induced by photoreceptors is associated with the development of the retinopathy. The purpose of this study was to determine the roles played by superoxides formed by photoreceptors under hyperglycemic conditions on autophagy. To accomplish this, we cultured 661 W cells, a transformed murine cone cell line, with 5.5 or 25 mM glucose in the presence or absence of 3 methyl adenine (3MA) or rapamycin. The superoxides were determined by flow cytometry using hydroethidine as a fluorescence probe. The autophagy activity was determined by changes in the expression of LC3B2 and P62 by immunoblotting. The degree of mitophagy was determined by the accumulation of mitochondria and lysosomes. Apoptotic changes of 661 W cells were determined by the caspase 3/7 activities. Our results showed higher levels of P62 and superoxides in cells cultured in 25 mM glucose than in 5.5 mM glucose. Addition of 3MA caused a significant increase of P62, superoxides, and caspase 3/7 activities in the 661 W cells cultured in high glucose but not in low glucose. These findings suggest that autophagy is important for the functioning and survival of 661 W cells under hyperglycemic conditions.