Objective To explore the effect of ghrelin on the injury of human umbilical vein endothelial cells (HUVECs) induced by advanced glycation end products (AGE).Methods The HUVECs were isolated and cultured in vitro,exposed to AGE-bovine serum albumin (BSA) with a terminal concentration of 200 mg/L for 24 or 48h with or without pretreatment with ghrelin for 24 h.The cells were divided into serum free group,different concentrations of ghrelin group (0.01,0.1,1 and 10 μmol/L),different concentrations of ghrelin (0.01,0.1,1 and 10 μmol/L) + AGE-BSA group,and AGE-BSA group;48 h after treatment with AGE-BSA,the cell viabilities were measured by thiazolyl blue assays.The cells were divided into serum free group,different concentrations of ghrelin (0.01,0.1,1 and 10 μmol/L) + AGE-BSA group,and AGE-BSA group.48 h after treatment with AGE-BSA,the cell apoptosis was measured by Annexin V-FITC/PI binding assays.The cells were divided into serum free group,AGE-BSA group and ghrelin (1 μmol/L) + AGE-BSA group.24 h after treatment with AGE-BSA,the cell ultrastructure was observed by electron microscopy.The cells were divided into serum free group,ghrelin(1 μmol/L) group,AGE-BSA group and ghrelin(1 μmol/L) + AGE-BSA group;the level of reactive oxygen species (ROS) in cells was measured.Results In different concentrations of ghrelin group,ghrelin promoted cell proliferation in a dose-dependent manner,with remarkable differences of viability between 0.1,1,10 μmol/L ghrelin group and serum free group [(109 ± 18) ％,(113 ± 15) ％,(115 ± 14) ％ vs (100 ± 11) ％,all P ＜ 0.05].The reduction of cell viability induced by AGE-BSA was inhibited by ghrelin pretreatment in a dose-dependent manner,with significant differences of viability between 1,10 μmol/L ghrelin + AGE-BSA group and AGE-BSA group [(87 ± 18) ％,(97 ± 19) ％,vs (45 ± 10) ％,P ＜ 0.05].The quantity of apoptosis cells was significantly inhibited by 0.1,1,10 μmol/L ghrelin pretreatment in a dose-dependent manner [(14.7 ±9.5)％,(5.2±1.9)％,(4.5 ±2.1)％ vs (19.4±5.3)％,P＜0.05].Under electron microscopy,the number of apoptosis cells was increased with cell nucleus condensed or fragmented and with mitochondria flocculent in AGE-BSA group,which could be improved significantly in ghrelin + AGE-BSA group.The ROS level was lower in ghrelin group (0.75 ± 0.07) and was higher in AGE-BSA group (1.50 ± 0.17) compared with that in serum free group (1.00 ±0.10);the ROS level in ghrelin + AGE-BSA group (1.10 ±0.14) was significantly lower than that in AGE-BSA group (all P ＜0.01).Conclusion Ghrelin can inhibit the apoptosis and ROS induced by AGE in HUVECs.%目的 观察胃生长素对晚期糖基化终产物(AGE)致人脐静脉内皮细胞(HUVEC)损伤的影响.方法 体外分离及培养HUVEC细胞株,制备糖基化牛血清白蛋白(AGE-BSA).处理细胞时AGE-BSA终浓度为200 mg/L,胃生长素预作用时间为24h.将细胞分为无血清组、不同浓度胃生长素组(0.01,0.1,1和10 μmol/L)、AGE-BSA组和不同浓度胃生长素(0.01,0.1,1和10 μmol/L)+ AGE-BSA组,AGE-BSA处理48 h后采用噻唑蓝法检测细胞存活率;将细胞分无血清组、AGE-BSA组和不同浓度胃生长素(0.01,0.1,1和10 μmol/L)+ AGE-BSA组,AGE-BSA处理48 h后Annexin V/PI双染法检测细胞凋亡情况;将细胞分为无血清组、AGE-BSA组和胃生长素(1μmol/L)+AGE-BSA组,AGE-BSA处理24 h后,电镜观察细胞超微结构变化;将细胞分为无血清组、胃生长素组(1 μmol/L),AGE-BSA组和胃生长素(1μmol/L)+AGE-BSA组,检测细胞内活性氧簇(ROS)水平.结果 不同浓度胃生长素组中,胃生长素剂量依赖性促进细胞增殖,0.1,1和10 μmol/L胃生长素组与无血清组细胞存活率比较差异均有统计学意义[(109±18)％、(113±15)％、(115±14)％比(100±11)％,P＜0.05];胃生长素预处理抑制AGE-BSA所致HUVEC存活率下降,呈剂量依赖性,1和10 μmol/L胃生长素+AGE-BSA组与AGE-BSA组比较差异有统计学意义[(87±18)％、(97±19)％比(45±10)％,P＜0.05].胃生长素剂量依赖性抑制AGE-BSA所致细胞凋亡,0.1,1和10 μmol/L胃生长素+AGE-BSA组细胞凋亡数量与AGE-BSA组比较差异有统计学意义[(14.7±9.5)％、(5.2±1.9)％、(4.5±2.1)％比(19.4±5.3)％,P＜0.05].电镜下见AGE-BSA组凋亡细胞数量增多,细胞核呈致密浓染,或呈碎块状,线粒体呈絮状变;胃生长素+ AGE-BSA组细胞凋亡明显减轻.无血清组、胃生长素组、AGE-BSA组和胃生长素+AGE-BSA组ROS水平分别为(1.00±0.10)、(0.75±0.07)、(1.50±0.17)、(1.10±0.14),其中胃生长素组低于无血清组(P＜0.05),AGE-BSA组明显高于无血清组(P＜0.01),胃生长素+ AGE-BSA组明显低于AGE-BSA组(P＜0.01).结论 胃生长素能抑制AGE诱导脐静脉内皮细胞凋亡及ROS的增加.
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