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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(0～200μmol/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、6.25μmol/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。
This study aimed to investigate the effect of epigallocatechin gallate (EGCG) on the regulation of the expression of activated normal T cells in the inflammatory response pathway of human retinal endothelial cells (HRECs) induced by lipopolysaccharide (LPS) And regulated upon activation of normal T cell expressed and secreted (RANTES) expression and its possible mechanism. HRECs were selected as experimental subjects. Toxicity of LPS (50 ~ 250 ng / mL) and EGCG (0 ~ 200 μmol / L) to HRECs were detected by real-time cytometry and non-isotope cell proliferation assay. The cells were randomly divided into normal control group, LPS group and LPS + EGCG (100,50,25,12.5,6.25μmol / L) for 7 hours, pretreated with different concentrations of EGCG for 2 hours and then treated with LPS for 24 hours Afterwards, the expression of RANTES in each culture supernatant was determined by enzyme-linked immunosorbent assay (ELISA), and the protein kinase Akt and its phosphorylation were detected by Western blotting. The results showed that LPS significantly stimulated the production of RANTES in HRECs, and EGCG inhibited LPS-induced RANTES expression in a dose-dependent manner. Immunoblotting also showed that LPS plays an important role in the Akt pathway in HRECs inflammatory process. EGCG can significantly inhibit the phosphorylation of Akt signaling molecules in HRECs induced by LPS. These results suggest that EGCG can effectively inhibit LPS-induced RANTES expression in HRECs, its mechanism may be related to the inhibition of Akt signaling pathway.