Premanand Chinnaraj, Rema Mohan, Sameer Mahmood Z, Sujatha Mohan, Balasubramanyam Muthusamy
Madras Diabetes Research Foundation, Gopalapuram, Chennai, India.
Invest Ophthalmol Vis Sci. 2006 May;47(5):2179-84. doi: 10.1167/iovs.05-0580.
To investigate the effect of high glucose on the proliferation of human retinal endothelial cells (HRECs) and to elucidate the possible mechanisms of antiangiogenic activity of curcumin, a diferuloylmethane.
Human retinal endothelial cells were isolated from the retinal tissue obtained from human donors and the culture system was established. The effect of curcumin on the proliferation of primary HRECs in the presence of low and high glucose was measured by MTT and thymidine uptake assays. Apoptosis was assessed by TUNEL assay and other adjuvant tools. Effect of curcumin on phorbol ester stimulated intracellular reactive oxygen species (ROS) generation in high glucose conditions was assessed by fluorescence assay. Finally, semiquantitative RT-PCR and Western blot analysis was performed to measure VEGF mRNA production and VEGF induced PKC-betaII translocation, respectively in the presence and absence of curcumin.
HREC culture was established successfully at passages 3 and 4 at 80% confluence. Curcumin effectively inhibited endothelial cell proliferation in a dose-dependent manner. At a concentration of 10 microM, curcumin significantly inhibited HREC proliferation in high-glucose-treated cells, as verified by both MTT and thymidine uptake assay. Curcumin also showed a significant (P = 0.03) reduction of intracellular ROS generation in HRECs. RNA expression studies showed that curcumin had an inhibitory effect on the glucose-induced VEGF mRNA expression. In addition, VEGF-mediated, membrane-associated changes in the PKC-betaII translocation in HRECs was inhibited by 31% on treatment with 10 microM curcumin.
These data suggest an underlying mechanism whereby curcumin induces the apoptosis in HRECs by the regulation of intracellular ROS generation, VEGF expression and release, and VEGF-mediated PKC-betaII translocation.
研究高糖对人视网膜内皮细胞（HRECs）增殖的影响，并阐明姜黄素（一种二阿魏酰甲烷）抗血管生成活性的可能机制。
从人类供体的视网膜组织中分离出人视网膜内皮细胞并建立培养体系。通过MTT和胸腺嘧啶核苷摄取试验测定姜黄素在低糖和高糖条件下对原代HRECs增殖的影响。通过TUNEL试验和其他辅助工具评估细胞凋亡。通过荧光试验评估姜黄素在高糖条件下对佛波酯刺激的细胞内活性氧（ROS）生成的影响。最后，分别在存在和不存在姜黄素的情况下进行半定量RT-PCR和蛋白质印迹分析，以测量VEGF mRNA的产生和VEGF诱导的PKC-βII易位。
在第3和第4代以80%汇合度成功建立了HREC培养物。姜黄素以剂量依赖性方式有效抑制内皮细胞增殖。MTT和胸腺嘧啶核苷摄取试验均证实，在10 microM浓度下，姜黄素显著抑制高糖处理细胞中的HREC增殖。姜黄素还使HRECs中的细胞内ROS生成显著减少（P = 0.03）。RNA表达研究表明，姜黄素对葡萄糖诱导的VEGF mRNA表达具有抑制作用。此外，用10 microM姜黄素处理后，HRECs中VEGF介导的PKC-βII膜相关易位变化受到31%的抑制。
这些数据提示了一种潜在机制，即姜黄素通过调节细胞内ROS生成、VEGF表达和释放以及VEGF介导的PKC-βII易位来诱导HRECs凋亡。
