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目的研究蛋白结合硫酸吲哚酚(indoxyl sulfate,IS)对单核细胞趋化功能的影响及其可能机制。方法以4%人血白蛋白(human serum albumin,HSA)结合游离的IS制备蛋白结合IS,高效液相色谱法(HPLC)检测其蛋白结合率。CCK-8法检测人单核细胞(THP-1)经不同浓度的游离IS和蛋白结合IS分别作用24 h后细胞增殖能力的变化。200μmol/L游离IS和蛋白结合IS分别孵育THP-1细胞24 h后,Transwell小室法检测THP-1细胞趋化能力;ELISA法检测细胞培养上清中单核细胞趋化蛋白-1(MCP-1)浓度;流式细胞术检测THP-1细胞上CC类趋化因子受体2(CCR2)的表达水平。结果成功制备蛋白结合IS,其蛋白结合率约为90%;蛋白结合IS和游离IS均可抑制THP-1细胞增殖,且蛋白结合IS对THP-1细胞的增殖抑制作用具有浓度依赖性(P<0.05);蛋白结合IS和游离IS均可增强THP-1细胞的趋化活性(P<0.05);蛋白结合IS和游离IS均可使THP-1细胞的MCP-1和CCR2表达上调(P<0.05)。结论蛋白结合IS同游离IS一样,可通过上调MCP-1及CCR2的表达增强单核细胞的趋化功能。
Objective To study the effect of protein-binding indoxyl sulfate (IS) on monocyte chemotactic function and its possible mechanism. Methods The protein binding rate of 4% human serum albumin (HSA) combined with free IS protein was determined by IS and high performance liquid chromatography (HPLC). CCK-8 method was used to detect the proliferation of human mononuclear cells (THP-1) after 24 h treatment with different concentrations of free IS and protein-bound IS respectively. THP-1 cells were incubated with 200 μmol / L free IS and protein-bound IS for 24 h, and the chemotactic ability of THP-1 cells was detected by Transwell chamber assay. The levels of monocyte chemoattractant protein-1 (MCP- 1). The expression of CCR2 on THP-1 cells was detected by flow cytometry. Results The protein binding rate of IS was about 90%. Protein-bound IS and free IS inhibited the proliferation of THP-1 cells, and protein-bound IS had a concentration-dependent inhibition on the proliferation of THP-1 cells <0.05). Protein-binding IS and free IS both enhanced the chemotactic activity of THP-1 cells (P <0.05). Protein-binding IS and free IS upregulated the expression of MCP-1 and CCR2 in THP- <0.05). Conclusions Protein-binding IS, like IS, can enhance the chemotactic function of monocytes by up-regulating the expression of MCP-1 and CCR2.