红霉素改善百草枯致损血管内皮细胞屏障功能的分子机制

Molecular Mechanism of Erythromycin in the Improvement of Paraquat-damaged Vascular Endothelial Cell Barrier Function

目的观察红霉素对百草枯所致血管内皮细胞屏障功能损伤的保护作用,并探讨可能的分子机制。方法采用"二室弥散装置"体外培养人脐静脉内皮细胞。设立百草枯组(P)、百草枯+红霉素组(P+E)、正常对照组(N),利用免疫荧光、放射免疫法分别检测各种因素下单层血管内皮细胞屏障对大分子物质的通透性变化;内皮细胞骨架蛋白F-actin形态分布以及含量变化;内皮细胞内cAMP浓度的变化。结果P组24 h后内皮细胞通透性较其它两组高(P<0.05或P<0.01),且随时间延长逐渐升高(P<0.01),引起细胞骨架F-actin重构和含量增加(P<0.05),同时伴内皮细胞内cAMP浓度降低(P<0.05);而P+E组细胞通透性24 h内升高,但36 h后明显改善(P<0.01),胞浆内F-actin逐渐减少,细胞周边增多,48 h细胞轮廓逐渐清晰,cAMP浓度也随时间延长而升高(P<0.01)。P组和P+E组内皮细胞通透率与细胞内cAMP浓度均呈负相关(r=-0.913,-0.648,P<0.05),而与F-actin含量变化均呈正相关(r=0.921,0.817,P<0.05)。结论红霉素能降低细胞通透性,升高内皮细胞cAMP浓度,稳定细胞骨架,这可能是改善百草枯致损血管内皮细胞屏障功能的机制。

Objective To find out the effects of erythromycin in protecting the vascular endothelial cell barrier against paraquat-induced damages and its underlying molecular and cellular mechanisms.Methods Human umbilical vein endothelial cells（HUVEC） was cultured in two compartment spreading apparatus in vitro.The cultured endothelial cells were divided into three groups： control group（N）,paraquat group（P）,paraquat＋erythromycin group（P＋E）.The cellular permeability,cAMP concentration,F-actin distribution and content in vascular endothelial cells were measured by immunofluorescence and immunoradiometric assay.Results Endothelial cell permeability in P group was significantly higher than that of the other two groups（P〈0.01）,which was associated with morphological changes,F-actin content elevation（P〈0.05）and the decreased endothelial cAMP concentration（P〈0.05）.While cellular permeability in P＋E group increased at 24 h,and dropped back at 36 h with cAMP elevation and F-actin redistribution from center to peripherals（P〈0.01）.There were all negative correlations between the permeability and cAMP concentration in P group and P＋E group（r =-0.913,-0.648,P〈0.05）,but positive correlation between the permeability and F-actin content in these two groups（r =0.921,0.817,P〈0.05）.Conclusion Erythromycin could decrease endothelial cell permeability with intracellular cAMP concentration increased and cytoskeleton stabilized,which might be the possible mechanisms for the protection of vascular endothelial cell barrier function impaired by paraquat.