Background

We aimed to determine sphingolipid levels and examine apoptotic pathways in human retinal pigment epithelial cells (ARPE-19) undergoing endoplasmic reticulum (ER) stress.

Methods

Cells were treated with tunicamycin (TM) to induce ER stress and tauroursodeoxycholic acid (TUDCA), an ER stress inhibitor, was administered to decrease cytotoxicity. Cell viability was measured by MTT assay. Levels of C16–C24 sphingomyelins (SM) and C16–C24 ceramides (CERs) were determined by LC-MS/MS. Glucose-regulated protein 78-kd (GRP78) and nuclear factor kappa-b subunit 1 (NFκB1) gene expressions were evaluated by quantitative PCR analysis, while GRP 78, NF-κB p65, cleaved caspase-3 and caspase-12 protein levels were assesed by immunofluorescence. Ceramide-1-phosphate (C1P) levels were determined by immunoassay, while caspase −3 and −12 activity in cell lysates were measured via a fluorometric method.

Results

Induction of ER stress in TM treated groups were confirmed by significantly increased mRNA and protein levels of GRP78. TM significantly decreased cell viability compared to controls. Treatment with TUDCA along with TM significantly increased cell viability compared to the TM group. A significant increase was observed in C22–C24 CERs, C1P, caspase-3, caspase-12, NFκB1 mRNA and NF-κB p65 protein levels in cells treated with TM compared to controls. Administration of TUDCA lead to a partial decrease in GRP78 expression, NFκB1 mRNA, NF-κB p65 protein, C22–C24 CERs and C1P levels along with a decrease in caspase-3 and -12 activity.

Conclusions

The results of this study reveal the presence of increased long chain CERs, C1P and apoptotic markers in retinal cells undergoing ER stress.

中文翻译：

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内质网应激对视网膜色素上皮细胞鞘脂水平和凋亡途径的影响。

背景

我们旨在确定鞘脂水平，并检查经历内质网（ER）应激的人类视网膜色素上皮细胞（ARPE-19）中的凋亡途径。

方法

用衣霉素（TM）处理细胞以诱导内质网应激，并给予内质网应激抑制剂tauroursodeoxycholic acid（TUDCA）以降低细胞毒性。细胞存活力通过MTT测定法测量。通过LC-MS / MS测定C16–C24鞘磷脂（SM）和C16–C24神经酰胺（CER）的水平。通过定量PCR分析评估葡萄糖调节蛋白78-kd（GRP78）和核因子κb亚基1（NFκB1）基因表达，而GRP 78，NF-κBp65，裂解的caspase-3和caspase-12蛋白水平通过免疫荧光评估。通过免疫测定法测定神经酰胺-1-磷酸（C1P）的水平，同时通过荧光法测量细胞裂解物中caspase -3和-12的活性。

结果

通过显着增加GRP78的mRNA和蛋白水平，证实了TM治疗组中ER应激的诱导。与对照相比，TM显着降低了细胞活力。与TM组相比，TUDCA和TM联合治疗显着提高了细胞活力。与对照组相比，经TM处理的细胞中C22–C24 CER，C1P，caspase-3，caspase-12，NFκB1mRNA和NF-κBp65蛋白水平显着增加。施用TUDCA会导致GRP78表达，NFκB1mRNA，NF-κBp65蛋白，C22–C24 CER和C1P水平部分降低，而caspase-3和-12活性降低。

结论

这项研究的结果揭示了在经历内质网应激的视网膜细胞中长链CER，C1P和凋亡标记物的存在。