Lu Jiajun, Sheng Minjie, Yao Panpan, Ran Chaochao, Liu Hao, Chen Li, Liu Rui, Li Bing
Department of Ophthalmology, Yangpu Hospital, Tongji University School of Medicine, Shanghai 200090, China.
J Ophthalmol. 2018 Jul 8;2018:8234906. doi: 10.1155/2018/8234906. eCollection 2018.
To investigate the effect of high concentration of glucose on reactive oxygen species (ROS) production in rabbit corneal epithelial cells (RECEs) and explore whether the increased ROS initiates the apoptosis process of RECEs through oxidative stress and endoplasmic reticulum (ER) stress pathway.
RECEs were treated by different concentrations of glucose for a while, and then the production of ROS was detected by flow cytometry. The expressions of PERK, p-PERK, Akt, p-Akt, and CHOP were determined by western blot, and the cell viability was measured by Cell Counting Kit-8 (CCK-8). Flow cytometry was used to detect the early apoptosis rate. Meanwhile, the effects of -acetyl-L-cysteine (NAC), an active oxygen inhibitor, on the experimental results were observed.
Compared with the normal glucose concentration group, the fluorescence intensity of ROS in the high concentration (1 mM glucose) of glucose group was significantly increased ( < 0.05). NAC-inhibited ROS production was induced by high concentration of glucose ( < 0.05).Western blot demonstrated that the expressions of the p-PERK and CHOP increased significantly ( < 0.05), the p-Akt expression decreased ( < 0.05), and the PERK and Akt expressions did not change significantly in the high concentration of glucose group compared to the normal concentration group. CCK-8 results revealed that compared with the normal concentration of glucose group, the cell activity of the high concentration of glucose group decreased. For the cells in the high concentration of glucose group, the cell survival rate of NAC-treated cells was higher than that of untreated ( < 0.05). The flow cytometry results indicated that the early apoptosis rate of the cells in the high concentration of glucose group increased in contrast with that in the normal concentration of glucose group ( < 0.05). Treating the cells in the high concentration of glucose group with NAC could reduce the cell apoptosis resulted from high glucose ( < 0.05).
High concentration of glucose may induce the formation of ROS which leads to oxidative stress and ER stress in RECEs and even leads to cell apoptosis. The reactive oxygen inhibitor, NAC, can play a protective character in the high concentration of glucose environment. These results might provide theoretical basis for the study of the diabetes-related dry eye.
探讨高浓度葡萄糖对兔角膜上皮细胞（RECEs）活性氧（ROS）生成的影响，并探究ROS增加是否通过氧化应激和内质网（ER）应激途径引发RECEs的凋亡过程。
用不同浓度的葡萄糖处理RECEs一段时间，然后通过流式细胞术检测ROS的生成。采用蛋白质免疫印迹法检测PERK、p-PERK、Akt、p-Akt和CHOP的表达，并用细胞计数试剂盒-8（CCK-8）检测细胞活力。采用流式细胞术检测早期凋亡率。同时，观察活性氧抑制剂N-乙酰-L-半胱氨酸（NAC）对实验结果的影响。
与正常葡萄糖浓度组相比，高浓度（1 mM葡萄糖）葡萄糖组中ROS的荧光强度显著增加（P<0.05）。NAC抑制了高浓度葡萄糖诱导的ROS生成（P<0.05）。蛋白质免疫印迹法表明，与正常浓度组相比，高浓度葡萄糖组中p-PERK和CHOP的表达显著增加（P<0.05），p-Akt表达降低（P<0.05），而PERK和Akt的表达无显著变化。CCK-8结果显示，与正常浓度葡萄糖组相比，高浓度葡萄糖组的细胞活性降低。对于高浓度葡萄糖组的细胞，NAC处理组的细胞存活率高于未处理组（P<0.05）。流式细胞术结果表明，与正常浓度葡萄糖组相比，高浓度葡萄糖组细胞的早期凋亡率增加（P<0.05）。用NAC处理高浓度葡萄糖组细胞可减少高糖诱导的细胞凋亡（P<0.05）。
高浓度葡萄糖可能诱导RECEs中ROS的形成，导致氧化应激和ER应激，甚至导致细胞凋亡。活性氧抑制剂NAC在高浓度葡萄糖环境中可发挥保护作用。这些结果可能为糖尿病相关性干眼的研究提供理论依据。
