1-甲基色氨酸对脂多糖诱导的人脐静脉内皮细胞通透性及凋亡的影响

目的探讨1-甲基色氨酸(1-methyltryptophan,1-MT)对脂多糖(lipopolysaccharide,LPS)诱导的人脐静脉内皮细胞(human umbilical vein endothelial cells,HUVECs)通透性及细胞凋亡的影响。方法将HUVECs分为三组,即磷酸缓冲盐溶液(phosphate buffered saline,PBS对照组)、1μg/mL LPS(LPS组)、含有LPS及1 mmol/L 1-MT(1-MT组)。采用蛋白免疫印迹试验(Western blot)检测孵育8 h后的p120连环(p120 concatemer,p120ctn)、血管内皮钙粘蛋白(vascular endothelial cadherin,VE-cadherin)、caspase-3及DNA修复酶多聚二磷酸腺苷核糖聚合酶-1(DNA repair enzyme polyadenylate ribose polymerase-1,PARP-1)的表达。采用高效液相色谱法(high performance liquid chromatography,HPLC)分别检测孵育2、4、6和8 h的犬尿氨酸(kynurenine,Kyn)浓度,并计算吲哚胺2,3-双加氧酶(indoleamine 2,3-dioxygenase,IDO)活性。组间两两均数比较用LSD-t检验。结果与PBS对照组相比较,LPS组caspase-3(74.01±7.91)%vs(157.14±7.63)%,P<0.01]、Kyn表达显著增高,而p120ctn(49.12±2.15)%vs(37.61±1.80)%,P<0.01]、VE-cadherin(107.70±7.01)%vs(90.66±2.58)%,P=0.027]及抗凋亡蛋白PARP-1(67.95±3.08)%vs(57.93±5.26)%,P=0.038]水平显著降低,IDO活性升高(P<0.05)。与LPS组比较,1-MT组caspase-3(157.14±7.63)%vs(110.74±7.89)%,P<0.01]表达显著降低,然而p120ctn(37.61±1.80)%vs(47.19±0.82)%,P<0.01]、VE-cadherin(90.66±2.58)%vs(107.27±9.89)%,P=0.029]及PARP-1(57.93±5.26)%vs(74.12±4.90)%,P=0.005]表达显著升高,不同时间点IDO活性均降低(P<0.05)。PBS组和1-MT组p120ctn、VE-cadherin、PARP-1蛋白水平、Kyn浓度及IDO活性差异无统计学意义(P>0.05),1-MT组caspase-3蛋白水平较PBS组升高(P=0.001)。结论LPS刺激HUVECs通透性增加,而1-MT可通过抑制IDO活性和降低Kyn水平拮抗LPS的刺激作用,此外,1-MT还可降低细胞凋亡,其机制可能是通过增加PARP-1表达,降低caspase-3水平,从而发挥内皮细胞的保护作用。

Effect of 1-methyltryptophan on lipopolysaccharide-induced permeability and apoptosis in human umbilical vein endothelial cells

Objective:To explore the effect of 1-methyltryptophan (1-MT) on lipopolysaccharide (LPS)-induced permeability and apoptosis of human umbilical vein endothelial cells (HUVECs).Methods:HUVECs were treated with phosphate buffer saline (PBS, control group), 1 μg/mL LPS (LPS group), and LPS combined with 1 mmol/L 1-MT (1-MT group). The expression levels of the p120 concatemer (p120ctn), vascular endothelial (VE) cadherin, caspase-3, and DNA repair enzyme polyadenylate ribose polymerase-1 (PARP) after incubation at 8 h were detected using Western blot. The concentrations of kynurenine (Kyn) after incubation at 2, 4, 6, and 8 h were measured by high-performance liquid chromatography, and indoleamine2, 3-dioxygenase (IDO) activity was calculated. Comparisons among groups were performed using the LSD- t test. Results:Compared with the control group, the expression of caspase-3 (74.01±7.91)% vs (157.14±7.63)%, P<0.01] and the concentration of Kyn were significantly up-regulated, while the expression of p120ctn (49.12±2.15)% vs (37.61±1.80)%, P<0.01], VE-cadherin (107.70±7.01)% vs (90.66±2.58)%, P=0.027], and PARP-1 (67.95± 3.08)% vs (57.93±5.26)%, P=0.038] were significantly down-regulated, and IDO activity was significantly increased in the LPS group ( P<0.05). Compared with the LPS group, the expression of caspase-3 (157.14±7.63)% vs (110.74±7.89)%, P<0.01] was significantly down-regulated, while the expression of p120ctn (37.61±1.80)% vs (47.19±0.82)%, P<0.01], VE-cadherin (90.66±2.58)% vs (107.27±9.89)%, P=0.029], and PARP-1 (57.93±5.26)% vs (74.12±4.90)%, P=0.005] were significantly up-regulated, and the activity of IDO was significantly decreased over time in the 1-MT group ( P<0.05). No significant differences were observed between the PBS and 1-MT groups in the protein levels of p120ctn, VE-cadherin, and PARP-1 protein as well as Kyn concentration and IDO activity ( P>0.05), while the expression of caspase-3 was increased in 1-MT group ( P=0.001). Conclusions:LPS aggravates the permeability of HUVECs, which can be reversed by 1-MT via inhibiting IDO activity and reducing Kyn concentrations. Moreover, 1-MT can also reduce apoptosis, which may be via increasing the expression of PARP-1 and reducing the expression of caspase-3, thus protecting endothelial cells.