大黄素对角膜基质细胞分泌细胞因子的干预作用

Inhibitory effect of emodin on cytokine secrete from cultured corneal fibroblasts

目的观察大黄素对体外培养的人眼角膜基质细胞分泌白细胞介素6(IL-6)的干预作用。方法四甲基偶氮唑蓝(MTT)法检测不同浓度大黄素对人眼角膜基质细胞活性的影响。绿脓杆菌脂多糖(LPS)刺激角膜基质细胞(LPS组),并用大黄素进行干预(大黄素预处理组)。分别于LPS刺激前(0h),刺激后1、2、4、8h收集各组细胞及细胞上清液。Western blot检测不同时点角膜基质细胞κB抑制因子-α(IκB-α)蛋白的表达及大黄素的作用,酶联免疫吸附实验(ELISA)检测不同时点角膜基质细胞IL-6蛋白的分泌及大黄素的作用。结果不同浓度大黄素对角膜基质细胞的活性没有影响。与0h相比,LPS刺激角膜基质细胞1h时,胞浆IκB-α蛋白表达出现下降,其电泳条带随着刺激时间的延长而逐渐变暗,4h时最暗。LPS刺激后各时点IκB-α蛋白表达较LPS刺激前均明显下降(P<0.01)。LPS刺激可引起人眼角膜基质细胞IL-6蛋白分泌增多,刺激后8h达峰值,LPS刺激细胞后各时点细胞分泌IL-6均较刺激前明显增加(P<0.01)。大黄素预处理后,与LPS组相比较,角膜基质细胞表达IκB-α蛋白下降、细胞分泌IL-6减少(P<0.01)。结论绿脓杆菌LPS可引起人眼角膜基质细胞IκB-α降解,引起细胞因子表达,在角膜炎的过程中可能起一定的作用。大黄素能有效抑制LPS诱导的人角膜基质细胞IκB-α降解,从而减少相应细胞因子的分泌。

Objective To investigate the inhibitory influence of emodin on interleukin-6（IL-6） released from cultured human cor- neal fibroblasts in vitro. Methods Cell viability insensitive to emodin on cultured human corneal fibroblasts was assessed using the IVrlT assay. Human corneal fibroblasts were randomly divided into three groups： the control group（group 0 h）, the lipopo- lysaccharide（LPS） group and the emodin pre-treatment group. Cells in the emodin pre-treatment group were incubated with emod- in for 30 min before being LPS challenged. Before and after 1,2,4 and 8 h treatment with LPS, expression of inhibitor of kB-α （IkB-α）was assayed by Westem blot, and secretion of IL-6 induced by LPS from cultured comeal fibroblasts was determined with enzyme-linked immunosorbent assays（ELISA）. Effects of emodin, an active component from the rhizome of Rheum palmatum, on expressions of IkB-α and IL-6 were also assessed in these cells. Results The concentration of emodin used in this study was safe for cultured human corneal fibroblasts. Compared with the Oh group, expression of IkB-α protein level was significantly decreased after being LPS challenged 1-8 h（ P 〈 0.01 ）, the lowest expression of IkB-α was observed at 4h after LPS challenged. Compared with cells in the Oh group, IkB-α level was significantly decreased in each group after treated with LPS, and degeneration of IkB- α induced by LPS was markedly inhibited by emodin（P〈0.01）. At the same time, the protein release of IL-6 in corneal fibro- blasts was significantly increased at each point after being challenged with LPS, which was significantly inhibited by pre-treatment with emodin（P〈0.01）. Conclusion These results suggest that LPS takes part in the degeneration of IkB-α in cultured human corneal fibroblasts in vitro, and emodin can partly inhibit this degeneration. By inhibiting IkB-α degeneration, emodin may sup- press LPS-induced cytokine release from these cells, which is one of the signal transduction mechanisms of emodin to prevent the occurrence of inflammation.