三磷酸腺苷对脂多糖诱导内皮祖细胞表达炎症因子的影响及其机制

Effects of ATP on expression of inflammatory factors in endothelial progenitor cells induced by LPS and the mechanisms

目的:分析三磷酸腺苷(adenosine triphosphate,ATP)对脂多糖(lipopolysaccharide,LPS)诱导内皮祖细胞(endothelial progenitor cells,EPCs)表达炎症细胞因子的影响,并探讨其机制。方法:采用密度梯度离心法分离人脐血单个核细胞,用RT-PCR检测LPS(1 mg/mL)诱导EPCs表达炎症细胞因子的情况,低浓度的ATP(5μmol/L)对LPS诱导EPCs表达细胞炎症因子的影响,不同浓度(5,50μmol/L)的ATP对EPCs中TLR4,MyD88和CD14 mRNA表达的影响;Western印迹检测LPS(1 mg/mL)对EPCs表达TLR4调节蛋白MyD88和CD14的影响以及信号通路的活化情况,低浓度ATP(1,5μmol/L)对LPS诱导EPCs表达TLR4,MyD88和CD14的影响以及信号通路的活化情况。结果:EPCs高表达TLR4,其配体LPS(1 mg/mL)显著上调IL-1β,MCP-1和ICAM-1的mRNA表达(均P<0.01),并呈时间依赖性上调My D88和CD14蛋白的表达,同时活化ERK和NF-κB信号通路。低浓度ATP抑制LPS诱导的IL-1β,MCP-1和ICAM-1 mRNA的表达(均P<0.05),同时也下调LPS诱导的TLR4,MyD88和CD14蛋白的表达(P<0.01或P<0.05),并抑制LPS诱导的NF-κB信号通路的活化(P<0.05)。结论:ATP在低浓度时通过负性调节TLR4信号通路而抑制LPS诱导的炎症细胞因子在EPCs中的表达。

Objective: To investigate the effects of adenosine triphosphate (ATP) on expression of infl ammatory factors induced by lipopolysaccharide (LPS) in endothelial progenitor cells (EPCs),and to elucidate the possible mechanisms. Methods: Mononuclear cells were isolated from human umbilical cord blood by density gradient centrifugation, RT-PCR was performed to detect the expression of inflammatory factors induced by LPS (1 mg/mL) in EPCs, the effect of low concentration (5 μmol/L) of ATP on expression of IL-1β, MCP-1 and ICAM-1, and the effect of different concentrations (5, 50 μmol/L) of ATP on the expression of Toll-like receptor (TLR) 4, myeloid differentiation primary response protein 88 (MyD88) and CD14. Western blot was performed to detect expression of TLR4 regulated proteins MyD88 and CD14 or to detect the low concentration (1, 5 μmol/L) of ATP on the expression of TLR4, MyD88 and CD14 and the NF-κB signaling pathway. Results: EPCs highly expressed TLR4, and its ligand LPS (1 mg/mL) significantly upregulated mRNA expression of IL-1β, MCP-1 and ICAM-1 and protein expression of MyD88 and CD14 in a time-dependent manner (P<0.01), accompanied by activation of ERK and NF-κB signal pathway. ATP at low concentration (5 μmol/L) significantly inhibited LPS-induced mRNA expression of IL-1β, MCP-1 and ICAM-1(P<0.05), downregulated the LPS-induced protein expression of TLR4, MyD88 and CD14 in EPCs (P<0.05), and suppressed LPS-induced activation of NF-κB signaling pathway (P<0.05). Conclusion: ATP at low concentration may suppress LPS-induced expression of inflammatory factors in EPCs through negative regulation of the TLR4 signaling pathway.