Wang Wei-na, Xie Ke-liang, Chen Hong-guang, Han Huan-zhi, Wang Guo-lin, Yu Yong-hao
Department of Anesthesiology, Tianjin Medical University General Hospital, Tianjin 300052, China.
Department of Anesthesiology, Tianjin Medical University General Hospital, Tianjin 300052, China. Email:
Zhonghua Yi Xue Za Zhi. 2013 Nov 19;93(43):3467-9.
To explore the regulative effects of hydrogen-rich medium on lipopolysaccharide (LPS)-induced monocytes adhesion to human umbilical vein endothelial cells (HUVEC) and vascular endothelial permeability in vitro.
Endothelial cells were seeded in 6-well plates and randomly divided into 4 groups (n = 42 each):control (A), hydrogen-rich medium (B), LPS (C) and LPS+hydrogen-rich medium (D). Cells were cultured in plain culture medium in groups A and C or in hydrogen-saturated culture medium in groups B and D.LPS 1 µg/ml was added into groups C and D.When forming a monolayer, monocytes were added into each group after 6, 12 and 24 h respectively. After a 90-minute co-culturing, adhesion status was detected by Wright-Giemsa stain.Supernatants were collected to detect the concentrations of vascular cell adhesion molecule-1 (VCAM-1) and E-selectin by enzyme-linked immunosorbent assay (ELISA). The expression of VE-cadherin was measured by Western blot. Cells were stained with immunofluorescence to show the distribution of VE-cadherin after a 24-hour incubation.
Compared with group A, the adhesion of monocytes to endothelial cells increased (P < 0.05) in group C, the levels of E-selectin and VCAM-1 became elevated (P < 0.05) while the expression of VE-cadherin decreased significantly (P < 0.05). Compared with group C, adhesion decreased in group D (P < 0.05), the levels of E-selectin and VCAM-1 decreased (P < 0.05) while there was an increased expression of VE-cadherin (P < 0.05). Three timepoints showed the same tendency. The results of 24 h fluorescence indicated that, compared with group A, VE-cadherin was incomplete in cell-cell connections in group C.However it was complete and well-distributed in group D versus group C.
Hydrogen-rich medium may reduce the LPS-induced release of adhesion molecules, lessen monocytic adhesion to HUVEC and regulate the expression of VE-cadherin to protect vascular permeability.
探讨富氢培养基对脂多糖（LPS）诱导的单核细胞与人脐静脉内皮细胞（HUVEC）黏附及体外血管内皮通透性的调节作用。
将内皮细胞接种于6孔板，随机分为4组（每组n = 42）：对照组（A）、富氢培养基组（B）、LPS组（C）和LPS + 富氢培养基组（D）。A组和C组细胞在普通培养基中培养，B组和D组细胞在富氢饱和培养基中培养。C组和D组加入1 μg/ml LPS。细胞形成单层后，分别于6、12和24 h后向每组加入单核细胞。共培养90分钟后，采用瑞氏-吉姆萨染色检测黏附情况。收集上清液，采用酶联免疫吸附测定（ELISA）检测血管细胞黏附分子-1（VCAM-1）和E-选择素的浓度。采用蛋白质免疫印迹法检测VE-钙黏蛋白的表达。孵育24小时后，用免疫荧光法对细胞进行染色，以显示VE-钙黏蛋白的分布。
与A组相比，C组单核细胞与内皮细胞的黏附增加（P < 0.05），E-选择素和VCAM-1水平升高（P < 0.05），而VE-钙黏蛋白的表达显著降低（P < 0.05）。与C组相比，D组黏附减少（P < 0.05），E-选择素和VCAM-1水平降低（P < 0.05），而VE-钙黏蛋白的表达增加（P < 0.05）。三个时间点均显示相同趋势。24小时荧光结果表明，与A组相比，C组细胞间连接中VE-钙黏蛋白不完整。然而，与C组相比，D组中VE-钙黏蛋白完整且分布良好。
富氢培养基可能减少LPS诱导的黏附分子释放，减轻单核细胞对HUVEC 的黏附，并调节VE-钙黏蛋白的表达以保护血管通透性。
