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目的探讨非对称性二甲基精氨酸(ADMA)对单层内皮细胞通透性的影响,以及氧化应激、p38丝裂原活化蛋白激酶(MAPK)通路在此过程中的作用。方法利用Transwell小室建立单层内皮细胞屏障结构,设立实验组和对照组,实验组经浓度25、50、100、200μmol/L ADMA作用24 h和100μmol/L ADMA分别刺激细胞4、8、16、24 h,或分别经烟酰胺腺嘌呤二核苷酸磷酸(NADPH)氧化酶抑制剂、p38 MAPK抑制剂预处理细胞后再加入ADMA刺激。随后用异硫氰酸荧光素(FITC)标记的右旋糖酐漏出法测定单层内皮细胞的通透系数Pa值,并用免疫荧光染色显示细胞骨架及细胞间连接的形态学改变。结果 ADMA呈剂量及时间依赖性的增加单层内皮细胞的通透性,同时促进内皮细胞中应力纤维的形成并破坏细胞间连接。NADPH氧化酶抑制剂和p38 MAPK抑制剂均可对抗ADMA的上述作用。结论 ADMA通过引起氧化应激,激活p38 MAPK通路,改变细胞骨架及细胞间连接的结构,使单层内皮细胞通透性增高。
Objective To investigate the effect of asymmetric dimethylarginine (ADMA) on the permeability of monolayer endothelial cells and the role of oxidative stress and p38 mitogen-activated protein kinase (MAPK) pathway in this process. Methods The monolayer endothelial cell barrier was constructed by Transwell chamber. Experimental and control groups were established. The experimental groups were stimulated by 25, 50, 100 and 200μmol / L ADMA for 24 h and 100μmol / L ADMA, respectively. 24 h, or pretreated with nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibitor and p38 MAPK inhibitor, respectively, followed by ADMA stimulation. The permeability coefficient (Pa) of monolayer endothelial cells was measured by dextran-labeled dextran-fluorescein isothiocyanate (FITC), and the morphological changes of cytoskeleton and intercellular junctions were observed by immunofluorescence staining. Results ADMA increased the permeability of monolayer endothelial cells in a dose- and time-dependent manner, promoted the formation of stress fibers in endothelial cells and destroyed the intercellular junctions. Both NADPH oxidase inhibitors and p38 MAPK inhibitors can counteract these effects of ADMA. Conclusion ADMA can induce the oxidative stress, activate the p38 MAPK pathway, change the structure of cytoskeleton and intercellular junctions, and increase the permeability of monolayer endothelial cells.