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表没食子儿茶素没食子酸酯抑制脂多糖诱导人视网膜内皮细胞中调节活化正常T细胞表达与分泌趋化因子的表达

张惠燕, 王剑勇, 姚航平

杭州职业技术学院，杭州 310018；浙江大学医学院附属第一医院眼科；感染病诊治国家重点实验室， 杭州 310003

摘要

本研究旨在探讨表没食子儿茶素没食子酸酯(epigallocatechin gallate, EGCG)对脂多糖(lipopolysaccharide, LPS)诱导人视网膜内皮细胞(human retinal endothelial cells, HRECs)炎症反应通路中调节活化正常T细胞表达与分泌趋化因子(regulated upon activation normal T cell expressed and secreted, RANTES)表达的影响及可能机制。将HRECs作为研究对象，分别用实时细胞计数法和非同位素细胞增殖法检测LPS (50~250 ng/mL)和EGCG (0~200 μmol/L)对HRECs的毒性作用，确定合适的实验药物浓度。再将细胞随机分为正常对照组、LPS组和LPS+不同浓度EGCG (100、50、25、12.5、6.25 μmol/L)共7组，用不同浓度EGCG预处理2 h，再加入LPS刺激24 h后，酶联免疫吸附法测定各组培养上清液中RANTES的表达水平，Western免疫印迹法检测蛋白激酶Akt及其磷酸化水平。结果显示，LPS可显著刺激诱导HRECs产生RANTES，EGCG抑制LPS诱导的RANTES表达，作用呈剂量依赖性。免疫印迹结果也显示，LPS对HRECs炎症过程中的Akt通路起重要作用，EGCG可显著抑制LPS诱导HRECs中Akt信号分子的蛋白磷酸化水平。以上结果提示，EGCG能有效抑制LPS诱导HRECs中RANTES的表达，其机制可能与抑制Akt信号通路有关。

关键词： 表没食子儿茶素没食子酸酯; 调节活化正常T细胞表达与分泌趋化因子; 人视网膜内皮细胞; Akt蛋白磷酸化

分类号：R774.1

[Epigallocatechin gallate attenuates the expression of regulated upon activation normal T cell expressed and secreted induced by lipopolysaccharide in human retinal endothelial cells.] [Article in Chinese]

ZHANG Hui-Yan, WANG Jian-Yong, YAO Hang-Ping

Hangzhou Vocational &； Technical College, Hangzhou 310018, China； Department of Ophthalmology

Abstract

The present study was undertaken to determine the effect of epigallocatechin gallate (EGCG) on lipopolysaccharide (LPS)-induced production of inflammatory chemokine regulated upon activation normal T cell expressed and secreted (RANTES) in human retinal endothelial cells (HRECs) and to explore the underlying regulatory mechanism. HRECs were stimulated with LPS in the presence or absence of EGCG at various concentrations (100, 50, 25, 12.5, 6.25 μmol/L). The optimum concentration of drug was determined by a real-time cell-electronic sensing (RT-CES) system, and MTS chromatometry was used to detect the toxicity of LPS and EGCG on HRECs. RANTES production in the culture supernatant was measured by ELISA. The expression levels of Akt and phosphorylated Akt were examined by Western blot assay. The result showed that LPS markedly stimulated RANTES secretion from HRECs. EGCG treatment significantly suppressed LPS-induced RANTES secretion in a dose-dependent manner. Furthermore, EGCG exhibited a dose-dependent inhibitory effect on LPS-induced phosphorylation of Akt. Taken together, our data suggest that EGCG suppresses LPS-induced RANTES secretion, possibly via inhibiting Akt phosphorylation in HRECs.

Key words: epigallocatechin gallate; RANTES; human retinal endothelial cells; Akt phosphorylation

收稿日期：2013-08-03　　录用日期：2013-12-24

通讯作者：王剑勇　　E-mail: wangjy4@zju.edu.cn

引用本文：

张惠燕, 王剑勇, 姚航平. 表没食子儿茶素没食子酸酯抑制脂多糖诱导人视网膜内皮细胞中调节活化正常T细胞表达与分泌趋化因子的表达[J]. 生理学报 2014; 66 (2): 145-150.

ZHANG Hui-Yan, WANG Jian-Yong, YAO Hang-Ping. [Epigallocatechin gallate attenuates the expression of regulated upon activation normal T cell expressed and secreted induced by lipopolysaccharide in human retinal endothelial cells.] [Article in Chinese]. Acta Physiol Sin 2014; 66 (2): 145-150 (in Chinese with English abstract).