不同剂量胰岛素对高糖环境下牛视网膜微血管内皮细胞 ROS 生成和 VEGF 表达的不同影响。
Different effects of low- and high-dose insulin on ROS production and VEGF expression in bovine retinal microvascular endothelial cells in the presence of high glucose.
机构信息
Department of Ophthalmology, Eye Ear Nose and Throat Hospital of Fudan University, #83 Fenyang Road, Shanghai 200031, China.
出版信息
Graefes Arch Clin Exp Ophthalmol. 2011 Sep;249(9):1303-10. doi: 10.1007/s00417-011-1677-x. Epub 2011 Apr 15.
BACKGROUND
Clinical trials have demonstrated that acute intensive insulin therapy may cause transient worsening of retinopathy in type 1 and type 2 diabetes patients. However, the related mechanism still remains controversial. The purpose of the present study was to investigate the effect of insulin on the mitochondrial membrane potential (△Ψm), reactive oxygen species (ROS) production, UCP-2 and VEGF expression in bovine retinal microvascular endothelial cells (BRECs) in the presence of normal or high glucose and the related mechanisms.
METHODS
BRECs were isolated as primary cultures and identified by immunostaining. Passage BRECs were initially exposed to normal (5 mM) or high glucose (30 mM) for 3 days, with equimolar L: -glucose supplemented for osmotic equation. Then the cells were treated with 1 nM, 10 nM, or 100 nM insulin for 24 h: △Ψm and ROS production were determined by JC-1 and CM-H2DCFDA, respectively. Expression of UCP-2 and VEGF mRNA was determined by real-time RT-PCR; expression UCP-2 and VEGF protein was determined by Western-blotting analysis. A general ROS scavenger N-acetylcysteine (NAC, 10 mM) and an NADPH oxidase inhibitor apocynin (1 mmol/l) were added 1 h before treatment with 100 nM insulin.
RESULTS
Insulin increased △Ψm, ROS production, and expression of UCP-2 and VEGF in BRECs at normal glucose (5 mM) in a dose-dependent manner. Low-dose insulin (1 nM) decreased △Ψm, ROS production, and UCP-2, VEGF expression in BRECs at high glucose (30 mM); and high-dose insulin (10 nM, 100nM) recovered △Ψm, ROS production, and UCP-2, VEGF expression. Pretreatment of cells with NADPH oxidase inhibitor apocynin significantly suppressed 100 nM insulin-induced ROS production (p < 0.01, one-way ANOVA). Pretreatment of cells with ROS scavenger N-acetylcysteine completely blocked insulin-induced UCP-2 expression (p < 0.01, one-way ANOVA) and significantly suppressed VEGF expression (p < 0.01, one-way ANOVA).
CONCLUSIONS
High-dose insulin-induced ROS production and VEGF expression in BRECs in the presence of high glucose might be one of the reasons for the transient worsening of diabetic retinopathy during intensive insulin treatment.
背景
临床试验已经证明，急性强化胰岛素治疗可能导致 1 型和 2 型糖尿病患者的视网膜病变暂时恶化。然而，相关机制仍存在争议。本研究的目的是探讨胰岛素对正常或高葡萄糖存在下牛视网膜微血管内皮细胞（BRECs）线粒体膜电位（△Ψm）、活性氧（ROS）产生、UCP-2 和 VEGF 表达的影响及其相关机制。
方法
BRECs 作为原代培养物进行分离并通过免疫染色进行鉴定。传代 BRECs 最初在正常（5 mM）或高葡萄糖（30 mM）中孵育 3 天，用等摩尔 L：-葡萄糖补充渗透压方程。然后，用 1 nM、10 nM 或 100 nM 胰岛素处理 24 小时：通过 JC-1 和 CM-H2DCFDA 分别测定△Ψm 和 ROS 产生。通过实时 RT-PCR 测定 UCP-2 和 VEGF mRNA 的表达；通过 Western-blotting 分析测定 UCP-2 和 VEGF 蛋白的表达。在 100 nM 胰岛素处理前 1 小时加入通用 ROS 清除剂 N-乙酰半胱氨酸（NAC，10 mM）和 NADPH 氧化酶抑制剂 apocynin（1 mmol/L）。
结果
胰岛素在正常葡萄糖（5 mM）下以剂量依赖性方式增加 BRECs 的△Ψm、ROS 产生以及 UCP-2 和 VEGF 的表达。低剂量胰岛素（1 nM）降低了高葡萄糖（30 mM）下 BRECs 的△Ψm、ROS 产生和 UCP-2、VEGF 表达；而高剂量胰岛素（10 nM、100 nM）恢复了△Ψm、ROS 产生和 UCP-2、VEGF 表达。细胞用 NADPH 氧化酶抑制剂 apocynin 预处理显著抑制了 100 nM 胰岛素诱导的 ROS 产生（p<0.01，单因素方差分析）。细胞用 ROS 清除剂 N-乙酰半胱氨酸预处理完全阻断了胰岛素诱导的 UCP-2 表达（p<0.01，单因素方差分析）并显著抑制了 VEGF 表达（p<0.01，单因素方差分析）。
结论
高葡萄糖存在下高剂量胰岛素诱导的 BRECs 中 ROS 产生和 VEGF 表达可能是强化胰岛素治疗期间糖尿病性视网膜病变暂时恶化的原因之一。
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