Slit2/Robo4信号通路在输血相关急性肺损伤体外模型中的表达及作用

Expression and effect of Slit2/Robo4 signaling pathway in vitro model of transfusion related acute lung injury

目的探讨Slit2/Robo4信号通路在输血相关急性肺损伤(TRALI)体外模型中的表达及作用。方法采用两次打击多形核中性粒细胞(PMNs)介导的人肺微血管内皮细胞(HMVECs)损伤模型作为TRALI体外模型,培养0、0.5、1、2、4、6 h后,采用Western-blotting法检测Robo4和血管内皮细胞钙粘连蛋白(VE-cadherin)表达,反转录酶PCR(RT-PCR)检测Robo4和Slit2信使RNA(m RNA)表达,体外内皮细胞通透性实验测定HMVECs通透性。加入不同浓度Slit2-N(0、0.5、1、4、10、20μg/L)与HMVECs一起温孵24 h后,检测HMVECs完整性和通透性。结果在TRALI体外模型中,Robo4和Slit2 m RNA的表达在各时间点的比较,差异均有统计学意义(F=12.880、11.060,P均<0.001);两者在2 h(0.72±0.04、0.78±0.05)、4 h(0.49±0.04、0.49±0.06)和6 h(0.34±0.03、0.43±0.11)均显著低于0 h(1.29±0.06、1.40±0.09),差异均有统计学意义(P均<0.05)。Robo4蛋白表达在各时间点的比较,差异有统计学意义(F=11.560,P<0.001);其在2、4和6 h(0.99±0.04、0.66±0.03、0.45±0.04)均显著低于0 h(1.44±0.04),差异均有统计学意义(P均<0.05)。同时发现,VE-cadherin蛋白表达在各时间点的比较,差异也有统计学意义(F=9.667,P<0.001);与0 h(1.46±0.09)比较,VE-cadherin的表达在2、4和6 h(0.91±0.08、0.78±0.05、0.50±0.04)也均有减少,差异均有统计学意义(P均<0.05)。体外内皮细胞渗透性试验提示,HMVECs通透性在各时间点的比较,差异有统计学意义(F=21.940,P<0.001);与0 h(1.42±0.16)比较,HMVECs通透性在2、4、6 h(4.00±0.35、5.70±1.71、10.02±2.24)均有增加,差异均有统计学意义(P均<0.05)。加入外源性Slit2-N后,VE-cadherin蛋白表达的比较,差异有统计学意义(F=13.220,P<0.001);与0μg/L Slit2-N组(0.41±0.08)相比,VE-cadherin在4、10、20μg/L Slit2-N组表达(1.19±0.35、1.49±0.13、2.12±0.21)均有增加,差异均有统计学意义(P均<0.05)。体外内皮细胞渗透性实验提示,HMVECs通透性比较,差异有统计学意义(F=19.430,P<0.001);与0μg/L Slit2-N组(10.0±2.2)相比,HMVECs通透性在4、10、20μg/L Slit2-N组(4.2±1.1、2.1±0.7、1.8±0.8)均有降低,差异均有统计学意义(P均<0.05)。结论 Slit2/Robo4信号通路可能参与TRALI的病理生理过程;使用外源性Slit2-N能调节TRALI体外模型中HMVECs的完整性和通透性,为治疗TRALI提供新的思路。

Objective To investigate the expression and effect of Slit2/Robo4 signaling pathway in vitro model of transfusion related acute lung injury（TRALI）. Methods A two-hit model of polymorphonuclear neutrophils（PMNs）-mediated human pulmonary microvascular endothelial cells（HMVECs） damage was used as TRALI in vitro model. After the HMVECs were incubated for 0, 0.5, 1, 2, 4, and 6 h, the protein expressions of Robo4 and vascular endothelial cadherin（VE-cadherin） were detected by Western-blotting, and the m RNA expressions of Robo4 and Slit2 were analyzed by reverse transcription PCR（RT-PCR）. The permeability of HMVECs was detected by in vitro experiment of endothelial cell permeability. Then the HMVECs were incubated with 0, 0.5, 1, 4, 10, and 20 μg/L Slit2-N for 24 h. The integrity and permeability of HMVECs were detected. Results In vitro model of TRALI, the m RNA expressions of Robo4 and Slit2 both showed significant differences at different time points（F =12.880, 11.060; both P〈0.001）; they were much lower at 2 h（0.72 ± 0.04, 0.78 ± 0.05）, 4 h（0.49 ± 0.04, 0.49 ± 0.06）, and 6 h（0.34 ± 0.03, 0.43 ± 0.11） than those at 0 h（1.29 ± 0.06, 1.40 ±0.09; all P〈0.05）. The protein expression of Robo4 showed significant difference at each time point（F = 11.560, P〈0.001）; it decreased at 2, 4, 6 h（0.99 ± 0.04, 0.66 ± 0.03, 0.45 ± 0.04） in comparing with 0 h（1.44 ± 0.04, all P〈0.05）. In addition, the protein expression of VE-cadherin at each time point was significantly different（F = 9.667, P〈0.001）; its expression decreased significantly at 2, 4, 6 h（0.91 ± 0.08, 0.78 ± 0.05, 0.50 ± 0.04） in comparing with 0 h（1.46 ±0.09, all P〈0.05）. The vitro experiment of endothelial cell permeability revealed that the permeability of HMVECs was significant different at each time point（F = 21.940, P〈0.001）; as comparing with 0 h（1.42 ± 0.16）, the permeability of HMVECs was significantly higher at 2, 4,6 h（4.00 ± 0.35, 5.70 ± 1.71, 10.02 ± 2.24; all P〈0.05）. After adding the exogenous Slit2-N, the expression of VE-cadherin protein was statistically significantly different（F = 13.220, P〈0.001）; it increased in the 4, 10, 20 μg/L Slit2-N groups（1.19 ± 0.35, 1.49 ± 0.13, 2.12 ± 0.21） as comparing with the 0 μg/L Slit2-N group（0.41 ± 0.08, all P〈0.05）. The vitro experiment of endothelial cell permeability revealed that the permeability of HMVECs was significant different（F = 19.430, P〈0.001）; the permeability of HMVECs was significantly lower in the 4, 10, 20 μg/L Slit2-N groups（4.2 ± 1.1, 2.1 ± 0.7, 1.8 ± 0.8） as comparing with the 0 μg/L Slit2-N group（10.0 ± 2.2,all P〈0.05）. Conclusion The Slit2/Robo4 signaling pathway may be involved in the pathophysiological process of TRALI. Exogenous Slit2-N may modulate the integrity and permeability of HMVECs in vitro TRALI model, and become a promising candidate for developing novel therapies against TRALI.