氢气对脂多糖诱导人脐静脉内皮细胞损伤的影响

Effects of hydrogen gas on lipopolysaccharide-induced damage to human umbilical vein endothelial cells

目的 评价氢气对脂多糖（LPS）诱导人脐静脉内皮细胞损伤的影响.方法 人脐静脉血管内皮细胞培养于含10％胎牛血清的DMEM培养基,以2×106个/ml的密度接种于6孔培养板,2ml/孔.采用随机数字表法,将细胞分为4组（n=35）：对照组（C组）、氢气组（H2组）、LPS组和L胳＋H2组.C组和LPS组用正常培养基培养,H2组和LPS＋ H2组用含饱和氢气培养基培养.LPS组和LPS＋H2组分别加入LPS1 μg/ml孵育,C组和H2组加入等容量生理盐水.分别于孵育6、12和24h时,取5孔细胞,采用瑞姬氏染色观察细胞损伤情况;分别于孵育6、12和24h时,取5孔细胞,采用Western blot法测定血管内皮细胞钙粘连蛋白和β-连环蛋白的表达.于孵育24h时,取5孔细胞,采用免疫荧光法测定血管内皮细胞钙粘连蛋白和p连环蛋白的表达.结果 LPS组内皮细胞出现病理学损伤;与LPS组比较,LPS＋ H2组内皮细胞病理学损伤减轻.与C组比较,LPS组和LPS＋ H2组血管内皮细胞钙粘连蛋白表达下调（P＜0.05）,β-连环蛋白表达差异无统计学意义（P＞0.05）;与LPS组比较,LPS＋ H2组血管内皮细胞钙粘连蛋白表达上调（P＜0.05）,p连环蛋白表达差异无统计学意义（P＞0.05）.结论 氢气可有效减轻LPS诱导人脐静脉内皮细胞损伤,与抑制血管内皮细胞钙粘连蛋白表达下调有关.

Objective To evaluate the effects of hydrogen gas （H2） on lipopolysaccharide （LPS）-induced damage to human umbilical vein endothelial cells （HUVECs） in vitro.Methods HUVECs were cultured in DMEM culture medium containing 10％ fetal bovine serum.The cells were seeded in 6-well plates （2 ml/hole） at a density of 2 × 106 cells/ml and randomly divided into 4 groups （n =35 each） using a random number table：control group （group C）,group H2,LPS group and LPS ＋ H2 group.The cells were cultured in the plain culture medium in C and LPS groups or in hydrogen-saturated culture medium in H2 and LPS ＋ H2 groups.In addition,LPS 1 μg/ml was added simultaneously in LPS and LPS ＋ H2 groups,and the equal volume of normal saline was added instead in C and H2 groups.The cells were incubated for 24 h.At 6,12 and 24 h of incubation,the cells of 5 wells were chosen and stained with Wright-Giemsa to observe the destruction of HUVEC barrier.At 6,12 and 24 h of incubation,the cells of 5 wells were chosen to detect the expression of VE-cadherin and β-catenin using Western blot.At 24 h of incubation,the cells of 5 wells were chosen to detect the expression of VE-cadherin and βcatenin using immunofluorescence.Results Pathological changes of endothelial cells were observed in LPS group.The pathological changes of the cells were lighter in LPS ＋ H2 group than in LPS group.Compared with group C,VE-cadherin expression was significantly down-regulated （P ＜ 0.05）,while no significant change was found in β-catenin expression in LPS and LPS ＋ H2 groups （P ＞ 0.05）.Compared with group LPS,VE-cadherin expression was significantly up-regulated （P ＜ 0.05）,while no significant change was found in ~catenin expression in group LPS ＋ H2 （P ＞ 0.05）.Conclusion H2 can effectively reduce LPS-induced damage to HUVECs through inhibiting down-regulation of VE-cadherin expression.