Department of Dermatology & Cutaneous Biology Research Institute, Yonsei University College of Medicine, Seoul, South Korea.
J Dermatol Sci. 2012 Feb;65(2):118-25. doi: 10.1016/j.jdermsci.2011.12.007. Epub 2011 Dec 22.
Cytosolic NADP(+)-dependent ICDH (IDPc) has an antioxidant effect as a supplier of NADPH to the cytosol, which is needed for the production of glutathione.
To evaluate the expression of IDPc in melanocytes and to elucidate its role as an antioxidant.
The knock-down of IDPc expression in immortalized mouse melanocyte cell lines (melan-a) was performed using the short interfering RNA (siRNA)-targeted gene silencing method. After confirming the silencing of IDPc expression with mRNA and protein levels, viability, apoptosis and necrosis, as well as ROS production in IDPc-silenced melanocytes were monitored under conditions of oxidative stress and non-stress. Also, the ratio of oxidized glutathione to total glutathione was examined, and whether the addition of glutathione recovered cell viability, decreased by oxidant stress, was checked.
The expression of IDPc in both primary human melanocytes and melan-a cells was confirmed by Western blot and RT-PCR. The silencing of IDPc expression by transfecting IDPc siRNA in melan-a cells was observed by Western blotting and real-time RT-PCR. IDPc knock-down cells showed significantly decreased cell viability and an increased number of cells under apoptosis and necrosis. IDPc siRNA-treated melanocytes demonstrated a higher intensity of DCFDA after the addition of H(2)O(2) compared with scrambled siRNA-treated melanocytes, and a lower ratio of reduced glutathione to oxidized glutathione were observed in IDPc siRNA transfected melanocytes. In addition, the addition of glutathione recovered cell viability, which was previously decreased after incubation with H(2)O(2).
This study suggests that decreased IDPc expression renders melanocytes more vulnerable to oxidative stress, and IDPc plays an important antioxidant function in melanocytes.
细胞质 NADP(+)-依赖性 IDH（IDPc）作为 NADPH 的供应源具有抗氧化作用，细胞质中的 NADPH 是产生谷胱甘肽所必需的。
评估 IDPc 在黑素细胞中的表达，并阐明其作为抗氧化剂的作用。
使用短发夹 RNA（siRNA）靶向基因沉默法敲低永生化小鼠黑素细胞系（melan-a）中的 IDPc 表达。在用 mRNA 和蛋白质水平证实 IDPc 表达沉默后，在氧化应激和非应激条件下监测 IDPc 沉默的黑素细胞中的活力、凋亡和坏死以及 ROS 产生。此外，还检查了氧化型谷胱甘肽与总谷胱甘肽的比值，并检查了添加谷胱甘肽是否可以恢复因氧化剂应激而降低的细胞活力。
通过 Western blot 和 RT-PCR 证实了 IDPc 在原代人黑素细胞和 melan-a 细胞中的表达。通过转染 IDPc siRNA 在 melan-a 细胞中观察到 IDPc 表达的沉默，通过 Western blot 和实时 RT-PCR 证实。IDPc 敲低细胞表现出明显降低的细胞活力，以及在凋亡和坏死下增加的细胞数量。与用 scrambled siRNA 处理的黑素细胞相比，用 IDPc siRNA 处理的黑素细胞在用 H(2)O(2)处理后显示出更高强度的 DCFDA，并且在用 IDPc siRNA 转染的黑素细胞中观察到还原型谷胱甘肽与氧化型谷胱甘肽的比值降低。此外，添加谷胱甘肽恢复了先前在用 H(2)O(2)孵育后降低的细胞活力。
本研究表明，IDPc 表达降低使黑素细胞更容易受到氧化应激的影响，并且 IDPc 在黑素细胞中发挥重要的抗氧化功能。
