Capozzi Megan E, McCollum Gary W, Cousins David B, Penn John S
Departments of Molecular Physiology and Biophysics and Ophthalmology and Visual Sciences at Vanderbilt University School of Medicine, Nashville, TN 37232-8808, USA.
J Diabetes Metab. 2016 Dec;7(12). doi: 10.4172/2155-6156.1000718. Epub 2016 Nov 30.
To determine the effect of oleic acid and linoleic acid on the production and secretion of specific diabetic retinopathy- (DR-) related cytokines: vascular endothelial growth factor (VEGF), interleukin-6 (IL-6), and interleukin-8 (IL-8) by human retinal glial cells, retinal endothelial cells, and retinal pigment epithelial cells. These expression profiles will be compared to those obtained by treatment of the same cell types with elevated D-glucose, a diabetes-relevant stimulus often used in retinal cell culture experiments.
Primary cultures of human retinal Müller cells, astrocytes, and microvascular endothelial cells (RMEC) and a human retinal pigment epithelial cell line (ARPE-19) were treated with oleic acid, linoleic acid, elevated D-glucose, or L-glucose as an osmotic control. VEGF, IL-6, and IL-8 concentrations in conditioned media were determined by colorimetric ELISA and normalized to total cellular protein.
In the conditioned medium of human Müller cells, linoleic and oleic acid increased VEGF production by 6.4-fold and 9.9-fold, respectively. Linoleic acid also significantly increased IL-6 by 2.9-fold and IL-8 by 5.7-fold. L-glucose and D-glucose both increased VEGF by 3.1-fold in Müller cell conditioned medium. Linoleic acid increased IL-8 concentrations by 56% in human RMEC conditioned medium. Human retinal astrocytes and ARPE-19 were unaffected by all stimuli.
Linoleic and oleic acid induce inflammatory mediators believed to be involved in the pathogenesis of diabetic retinopathy (DR). In culture, the free fatty acid insults, particularly linoleic acid, significantly increased cytokine production by Müller cells. In summary, these data identified Müller cells as the primary producer of these inflammatory mediators when treated with unsaturated fatty acids. This study also demonstrates that elevated glucose is an inadequate stimulus for assessing the production of inflammatory mediators. Therefore this study provides a novel in vitro model system of the dyslipidemia-induced inflammation occurring in DR.
确定油酸和亚油酸对人视网膜神经胶质细胞、视网膜内皮细胞和视网膜色素上皮细胞产生和分泌特定糖尿病视网膜病变（DR）相关细胞因子的影响，这些细胞因子包括血管内皮生长因子（VEGF）、白细胞介素-6（IL-6）和白细胞介素-8（IL-8）。将这些表达谱与用高浓度D-葡萄糖处理相同细胞类型所获得的表达谱进行比较，高浓度D-葡萄糖是视网膜细胞培养实验中常用的与糖尿病相关的刺激物。
用人视网膜Müller细胞、星形胶质细胞和微血管内皮细胞（RMEC）的原代培养物以及人视网膜色素上皮细胞系（ARPE-19）分别用油酸、亚油酸、高浓度D-葡萄糖或L-葡萄糖作为渗透对照进行处理。通过比色ELISA法测定条件培养基中VEGF、IL-6和IL-8的浓度，并将其标准化为总细胞蛋白。
在人Müller细胞的条件培养基中，亚油酸和油酸分别使VEGF的产生增加6.4倍和9.9倍。亚油酸还使IL-6显著增加2.9倍，IL-8增加5.7倍。在Müller细胞条件培养基中，L-葡萄糖和D-葡萄糖均使VEGF增加3.1倍。亚油酸使人类RMEC条件培养基中的IL-8浓度增加56%。人视网膜星形胶质细胞和ARPE-19对所有刺激均无反应。
亚油酸和油酸诱导了被认为参与糖尿病视网膜病变（DR）发病机制的炎症介质。在培养中，游离脂肪酸损伤，特别是亚油酸，显著增加了Müller细胞的细胞因子产生。总之，这些数据确定Müller细胞是用不饱和脂肪酸处理时这些炎症介质的主要产生者。本研究还表明，高糖是评估炎症介质产生的不充分刺激物。因此，本研究提供了一种新型的体外模型系统，用于研究DR中血脂异常诱导的炎症。
