同型半胱氨酸诱导人单核细胞分泌趋化因子的氧化应激机制的实验研究

Folic acid attenuates homocysteine induced human monocytes chemokine secretion via reducing NADPH oxidase activity

目的探讨同型半胱氨酸(Hcy)诱导人单核细胞分泌趋化因子的致炎症作用及其氧化应激的机制,明确大剂量叶酸在这一过程中的潜在作用。方法用 Hcy(100 μmol/L)和叶酸(5 μmol/L)处理人单核细胞24 h。使用酶联免疫吸附实验检测单核细胞趋化蛋白-1(MCP-1)、IL-8水平(n=8)。应用激光共聚焦显微镜探测单核细胞内氧自由基(ROS)的表达。使用细胞色素 C 还原法评价单核细胞内 NADPH 氧化酶活性。结果 Hcy 显著增加了单核细胞对趋化因子 MCP-1的分泌[对照组(1.88±0.51)ng/ml 比 Hcy 处理组(4.36±0.72)ng/ml,P<0.05],而叶酸显著抑制了Hcy 诱导的单核细胞分泌 MCP-1[Hcy 处理组(4.36±0.72)ng/ml 比 Hcy 与叶酸共同处理组(2.40±0.60)ng/ml,P<0.05]。此外,Hcy 显著增加了单核细胞对趋化因子 IL-8的分泌[对照组(4.9±1.9)ng/ml 比 Hcy 处理组(12.7±1.5),P<0.05],而叶酸明显抑制了这一过程[Hcy 处理组(12.7±1.5)比 Hcy 与叶酸共同处理组(7.2±1.9)ng/ml,P<0.05]。Hcy(100 μmol/L)显著增加了单核细胞 ROS 产生[对照组100%比 Hcy 处理组(443.0±60.9)%,P<0.05],叶酸(5 μmol/L)抑制了 Hcy诱导的单核细胞 ROS 产生[Hcy 处理组(443.0±60.9)%比 Hcy 与叶酸共同处理组(133.0±34.7)%,P<0.05]。Hcy 显著增加了单核细胞 NADPH 氧化酶的活力[对照组100%比 Hcy 处理组(520±80)%,P<0.05],叶酸(5μmoL/L)及 NADPH 氧化酶的抑制剂 DPI 明显降低了 Hcy 诱导的人单核细胞 NADPH 氧化酶的活性[Hcy 处理组(520±80)%比 Hcy 与叶酸共同处理组(310±70)%比Hcy 与 DPI 共同处理组(280±40)%,P<0.05]。结论 Hcy 可能通过活化 NADPH 氧化酶的途径,诱导 ROS 的产生及分泌趋化因子,叶酸可能通过降低 NADPH 氧化酶活性这一途径抑制 Hcy 诱导的ROS 的产生及趋化因子的分泌。提示 Hcy 可能通过诱导炎症的机制促进动脉粥样硬化的发展,叶酸可能通过降低 Hcy 水平以外的抗氧化机制,对动脉粥样硬化发挥潜在的有益作用。

Objective To investigate the effect of folio acid on homocysteine （Hey）-induced chemokine secretion and NADPH oxidase activity in human monocytes. Methods Human monocytes from healthy volunteers were incubated with Hey （ 100μmol/L） with or without folio acid （5 μmol/L） for 24 h; MCP-1 and IL-8 were assessed by ELISA. DCFH-DA was added to monitor intracellular ROS production on confocal microscopy. A cytochrome c reduction assay was used to measure NADPH oxidase activity. Results The Hey-induced secretion of MCP-1 and IL-8 was significantly reduced by folio acid [ （1.88 ± 0. 51）ng/ml vs. （4.36 ±0.72）ng/ml vs. （2.40±0.60）ng/ml and （4.9 ±1.9） ng/ml vs. （12.7 ±1.5） ng/ml vs. （ 7.2 ± 1.9） ng/ml, all P 〈 0. 05 ]. The Hey-induced production of ROS was also significantly attenuated by folio acid. Moreover, the Hey-induced NADPH oxidase activity increase was significantly inhibited by cotreatment with folio acid. Conclusion Folio acid may attenuate oxidative stress induced by Hey by reducing NADPH oxidase activity in monocytes.