心肾综合征中硫酸吲哚酚对巨噬细胞吞噬氧气型低密度脂蛋白的诱导作用的研究

Induction effect of indoxyl sulfate on uptake of ox-LDL by macrophages

目的探讨硫酸吲哚酚(IS)对RAW264.7巨噬细胞吞噬氧气型低密度脂蛋白(ox-LDL)的诱导作用及其作用机制。方法 0、10、50、100、200和400μmol/L IS处理RAW264.7细胞24 h,采用CCK-8法测定RAW264.7细胞存活率,流式细胞术测定RAW264.7细胞的Dil标记氧化型低密度脂蛋白(Dil-ox-LDL)吞噬量。200μmol/L IS处理RAW264.7细胞0、12、24、36和48 h,测定RAW264.7细胞存活率和Dil-ox-LDL吞噬量。IS组RAW264.7细胞用200μmol/L IS处理24 h,UO126+IS组RAW264.7细胞以5μmol/L UO126预处理2 h后再200μmol/L IS处理24 h,测定非处理组、IS组和UO126+IS组RAW264.7细胞存活率和Dil-ox-LDL吞噬量,并采用蛋白质印迹法(Western blot)检测在IS刺激15 min时的ERK1/1蛋白表达情况。结果 IS对RAW264.7细胞存活率的浓度效应和时间效应检测中,不同组间比较差异无统计学意义(P>0.05)。Dil-ox-LDL吞噬量与IS浸润浓度及处理时间均呈正相关(P<0.01)。IS组的Dil-ox-LDL吞噬量和磷酸化ERK1/2蛋白表达量均明显高于非处理组(P<0.01)。UO126+IS组的Dil-ox-LDL吞噬量略低于非处理组,但差异无统计学意义(P>0.05);磷酸化ERK1/2蛋白表达量大幅低于非处理组,差异有统计学意义(P<0.01)。UO126+IS组同IS组比较,Dil-ox-LDL吞噬量和磷酸化ERK1/2蛋白表达量均呈现大幅降低(P<0.01)。结论IS可在体外直接诱导RAW264.7巨噬细胞吞噬ox-LDL,该效应经由活跃MAPK信号转导通路实现,且随IS浓度升高和浸润时间延长而增强。

Objective To investigate the induction effect and mechanism of indoxyl sulfate（IS） on uptake of oxLDL by RAW264.7 macrophages.Methods RAW264.7 cells were treated with 0,10,50,100,200 and 400 μmol/L IS for 24 h.CCK-8 assay was employed to obtain the cell survival ratio of RAW264.7 cells,and flow cytometry was employed to detect uptake of ox-LDL by RAW264.7 cells.After RAW264.7 cells were treated with 200 μmol/L IS for 0,12,24,36 and 48 h,and the cell survival ratio and uptake of ox-LDL were obtained.RAW264.7 cells of the IS group were treated with 200 μmol/L IS for 24 h,while the cells of the UO126＋IS group were treated with 5 μmol/L UO126 for 2 h in advance and 200 μmol/L IS for 24 h.Then the survival ratio of RAW264.7 cells and uptake of ox-LDL were determined in the non-treated group,IS group and UO126 ＋IS group.And Western blot was used to detect the expression of ERK1/2 protein in the three groups.Results In the detection of dose-dependent effects and time-dependent effects of IS on RAW264.7 cell survival ratio,there was no significant difference between the groups（P 0.05）.Uptake of Dil-ox-LDL positively correlated with the concentration of IS and the action time（P 0.01）.Uptake of Dil-ox-LDL and the expression of p-ERK1/2 of the IS group were greatly higher than those of the nontreatment group（P 0.01）.Uptake of Dil-ox-LDL of the UO126＋IS group was slightly lower than that of the nontreatment group,and the difference was no statistically significant（P 0.05）.While the expression of p-ERK1/2 of the UO126 ＋IS group was obviously lower than that of the non-treatment group（P 0.01）.Uptake of Dil-ox-LDL and the expression of p- ERK1/2 of the UO126 ＋IS group were significantly lower than those of the IS group（P 0.01）.Conclusions IS can directly induce uptake of ox-LDL by RAW264.7 macrophages in vitro through activation of MAPK signaling pathway,and the effect is enhanced with increasing concentration and action time.