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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 observe the effects of emodin on the secretion of interleukin-6 (IL-6) from cultured human corneal stromal cells. Methods The effect of different concentrations of emodin on the activity of human corneal stromal cells was detected by MTT assay. P. aeruginosa lipopolysaccharide (LPS) stimulated corneal stromal cells (LPS group) and was treated with emodin (emodin pretreatment group). The cells and cell supernatants were collected at 1, 2, 4, and 8 h after the LPS stimulation (0 h). The expression of κB-α (IκB-α) protein and the effect of emodin in corneal stromal cells were detected by Western blot. The secretion of IL-6 protein in corneal stromal cells was detected by enzyme-linked immunosorbent assay (ELISA) at different time points. The role of emodin. Results Different concentrations of emodin had no effect on the activity of corneal stromal cells. Compared with 0h, when LPS stimulated corneal stromal cells for 1h, the expression of cytoplasmic IκB-α protein decreased, and the electrophoresis bands gradually darkened with the prolongation of stimulation time, and the darkest at 4h. The expression of IκB-α protein at each time point after LPS stimulation was significantly lower than that before LPS stimulation (P<0.01). LPS stimulated the secretion of IL-6 protein in corneal stromal cells of human eyes, and reached the peak at 8h after stimulation. LPS stimulated the secretion of IL-6 from cells at all time points significantly (P<0.01). After emodin pretreatment, compared with LPS group, the expression of IκB-α protein in corneal stromal cells was decreased and IL-6 secretion was decreased (P<0.01). Conclusion Pseudomonas aeruginosa LPS can cause degradation of IκB-α in human corneal stromal cells and induce the expression of cytokines. It may play a role in the process of keratitis. Emodin can effectively inhibit LPS-induced degradation of IκB-α in human corneal stromal cells, thereby reducing the secretion of corresponding cytokines.