CAJ | 학술논문

目的：观察大黄素对脂多糖（ lipopolysaccharide，LPS）诱导损伤的人脐静脉血管内皮细胞（ human umbilical vein endothelial cells，HUVECs）的干预作用，探讨其对内皮细胞细胞骨架及通透性的影响。方法：以0．2μg/ml LPS作用于HUVECs 24 h，造成血管内皮损伤模型。Y27632（ ROCK特异性抑制剂）和缬沙坦（西药对照，AngⅡ受体1拮抗剂，Rho/ROCK信号通路的间接抑制剂）作为阳性对照药物。观察了大黄素对LPS诱导的内皮损伤后的HUVECs的变化：流式细胞仪An-nexin V-FITC/PI染色观察凋亡率，transwell迁移实验观察细胞迁移能力，10 mg/ml波连蛋白预包被/无包被96孔板观察细胞黏附能力，硝酸还原酶法检测细胞培养上清中的cNOS、iNOS和NO浓度，免疫荧光法观察细胞骨架和黏着斑蛋白的结构和分布。结果：（1）LPS成功诱导构建了HUVECs细胞骨架损伤模型。HUVECs细胞增殖减弱、凋亡增加，细胞骨架肌动蛋白皱缩、黏着斑蛋白聚集，LPS活化内皮、诱导肌球蛋白收缩，导致细胞间隙开放、内皮通透性增加、细胞迁移能力和黏附能力下降。LPS作用24 h后，cNOS减低，tNOS、iNOS、NO升高，呈现内皮损伤和炎症状态。（2）大黄素诱导HUVECs细胞凋亡、提高细胞迁移能力和黏附能力、cNOS升高、iNOS减低，对细胞骨架和黏着斑蛋白形态无明显影响。结论：LPS诱导了HUVECs细胞骨架损伤，大黄素通过调节细胞的凋亡率、调节细胞合成和利用NO的能力、改变细胞迁移和黏附能力，而调节血管内皮通透性、改善内皮屏障功能。同时大黄素促进HUVECs凋亡，抑制内皮细胞的过度增殖，对内皮细胞有一定的保护作用。
목적：관찰대황소대지다당（ lipopolysaccharide，LPS）유도손상적인제정맥혈관내피세포（ human umbilical vein endothelial cells，HUVECs）적간예작용，탐토기대내피세포세포골가급통투성적영향。방법：이0．2μg/ml LPS작용우HUVECs 24 h，조성혈관내피손상모형。Y27632（ ROCK특이성억제제）화힐사탄（서약대조，AngⅡ수체1길항제，Rho/ROCK신호통로적간접억제제）작위양성대조약물。관찰료대황소대LPS유도적내피손상후적HUVECs적변화：류식세포의An-nexin V-FITC/PI염색관찰조망솔，transwell천이실험관찰세포천이능력，10 mg/ml파련단백예포피/무포피96공판관찰세포점부능력，초산환원매법검측세포배양상청중적cNOS、iNOS화NO농도，면역형광법관찰세포골가화점착반단백적결구화분포。결과：（1）LPS성공유도구건료HUVECs세포골가손상모형。HUVECs세포증식감약、조망증가，세포골가기동단백추축、점착반단백취집，LPS활화내피、유도기구단백수축，도치세포간극개방、내피통투성증가、세포천이능력화점부능력하강。LPS작용24 h후，cNOS감저，tNOS、iNOS、NO승고，정현내피손상화염증상태。（2）대황소유도HUVECs세포조망、제고세포천이능력화점부능력、cNOS승고、iNOS감저，대세포골가화점착반단백형태무명현영향。결론：LPS유도료HUVECs세포골가손상，대황소통과조절세포적조망솔、조절세포합성화이용NO적능력、개변세포천이화점부능력，이조절혈관내피통투성、개선내피병장공능。동시대황소촉진HUVECs조망，억제내피세포적과도증식，대내피세포유일정적보호작용。
Objective:To observe the protective effect of emodin on LPS- induced endothelial cell injury and to discuss the effect of the monomers on cytoskeleton and endothelial permeability. Methods:Human umbilical vein endothelial cells( HUVECs)in-jury was induced by treatment with 0. 2 μg/ml LPS for 24 h. Y27632 and valsartan were taken as positive controls. We studied the effect of emodin on LPS-induced cell viability of HUVECs;apoptosis rate with Annexin V-FITC/PI by flow cytometry;cell migra-tion assay with transwell insert system;adhesion assay with 96-well plate with or without the pre-coat with 10 mg/mL vitronectin;cNOS,iNOS and NO in culture supernatant with nitrate reductase assay;and cytoskeleton reorganization and vinculin distribution with immunofluorescence assay. Emodin demonstrated superiorly protective effects on LPS-induced endothelial cell injury in the study. Results:(1)LPS succeeded inducing cytoskeleton injury of HUVECs. The proliferation of HUVECs decreased,apoptosis rate in-creased,actin cytoskeleton polymerizated and vinculin contracted. LPS activated the endothelium and induce actomyosin contraction, and resulting in the opening of intercellular gaps,making the endothelium permeable to fluid and molecules,and cell migratory and adhensive capability was decreased significantly. CNOS decreased and tNOS,iNOS and NO increased after LPS treatment for 24 h, showing vascular endothelial injury and in inflammatory state.(2)Emodin induced HUVECs apoptosis,migration and adhesion capa-bility increased,beneficial cNOS levels increased and harmful iNOS decreased. But emodin performed poor in improving cytoskeleton remolding. Conclusion:LPS induced cytoskeleton damage in HUVECs. Emodin regulated the endothelial permeability and improved endothelial barrier function by regulating apoptosis,regulated NO synthesis and utilization and changed migration and adhesion capa-bility. Emodin demonstrated the protective effect on endothelial cells.