胰高血糖素样肽1通过调控葡萄糖调节蛋白78表达改善高糖及波动性高糖诱导的内皮细胞内质网应激及凋亡

Glucagon like peptide-1 protects human umbilical vein endothelial cells against endoplasmic reticulum stress and apoptosis induced by constant or fluctuating high glucose via regulating the expression level of glucose regulated proteins 78

目的探讨胰高血糖素样肽1（GLP-1）对持续性高糖和波动性高糖诱导的内皮细胞内质网应激及凋亡的保护作用和机制。方法将体外培养的人脐静脉内皮细胞（HUVECS）分成6组：正常葡萄糖对照组（5．5mmol／L葡萄糖培养）、持续性高糖tN30mmol／L葡萄糖培养）、波动性高糖组（5．5mmol／L和30mmol／L葡萄糖培养，每12h交替1次）、正常葡萄糖＋10nmol／LGLP-1组、持续性高糖＋10nmol／LGLP-1组、波动性高糖＋10nmol／LGLP．1组。后3组均先加入10nmol／LGLP-1预处理半小时，后以相应浓度葡萄糖培养。各组细胞经处理48h后，流式细胞仪测定细胞凋亡率，Westernblotting法检测葡萄糖调节蛋白78（GRP78）表达水平的变化。应用30mmol／L高糖处理HUVECS不同时间（0、3、6、12、24、48h），Westernblotting法检测加入GLP-1前后GRP78蛋白表达水平的变化。两组问比较采用LSD—t检验，多组比较采用单因素方差分析。结果将正常对照组的细胞凋亡率设定为1，波动性高糖组细胞凋亡率增至1．20＋0．05，与对照组差异有统计学意义（F=45．086，P〈0．05）；波动性高糖＋GLP-1组细胞凋亡率降至0．87-＋0．01，与波动性高糖组差异有统计学意义（t=17．400，P〈0．05）。与对照组（0h组，设定为1）相比，高糖处理HUVECS3hGRP78的表达量升高至1．62±0．48，差异有统计学意义（t=2．584，P〈0．05）；6h达到高峰（2．82±0．59），与对照组差异有统计学意义（t=6．123，P〈0．05）；12h后开始下降，高糖作用48h下降至0．98±0．59，与对照组相比无统计学差异（t=0．378，P〉0．05）。波动性高糖组GRP78的表达水平降至（0．77±0．04），与持续性高糖组（0．96±0．10）相比，差异有统计学意义（t=3．134，P〈0．05）。结论持续性高糖和波动性高糖均可引起内皮细胞发生内质网应激和凋亡，后者通过进一步抑制防御蛋白GRP78的表达，加重内质网应激；GLP-1可通过增加细胞内GRP78的表达改善内质网应激进而改善凋亡。

Objective To investigate the effect and mechanism of glucagon like peptide-l（GLP-1） on endothelial cell endoplasmic reticulum stress （ER stress） and apoptosis induced by constant or fluctuating high glucose. Methods Cuhured human umbilical vein endothelial cells （HUVECs） were divided into 6 groups： normal glucose control （cultured with 5.5 mmol/L glucose）, constant high glucose （cultured with 30 mmol/L glucose）, fluctuating high glucose （cultured with high glucose and normal glucose, changed every 12 hours）, normal glucose control＋ 10 nmol/L GLP-1, constant high glucose＋ 10 nmol/L GLP-1, fluctuating high glucose＋ 10 nmol/L GLP-1. The later 3 groups were pretreated with 10 nmol/L GLP-1 for 30 minutes, thencultured with corresponding glucose. After treatment for 48 h, the apoptosis rate of HUVECs was measured by flow cytometer, the glucose regulated proteins 78（GRP78） expression level was measured by Western blotting methods. HUVECS were treated by 30 mmol/L high glucose plus GLP-1 or not, then GRP78 expression level at different times （0, 3, 6, 12, 24, 48 h） was measured by using Western blotting methods. Differences between two groups were compared by LSD-t test, differences among more than two groups were compared by one-way analysis of variance. Results Assuming apoptosis rate of normal control group was 1, the apoptosis rate in fluctuating high glucose group was （1.2±0.05）, which was significantly higher than that of control group（F=45.086, P〈0.05）; After GLP- 1 was added, the apoptosis rate of cells in fluctuating high glucose group decreased to 0.87±0.01, which was significantly lower than that in fluctuating high glucose group （t=17.4, P〈0.05）. After treatment of 30 mmol/L high glucose for 3 hours, GRP78 expression in HUVECS increased to （1.62±0.48）, which was significantly higher than that of in control group （treated with high glucose for 0 h, GRP78 expression assumed as 1, t=2.584, P〈0.05）; after 6 hours, GRP78 expression peaked to 2.82±0.59, and was significantly higher than that in control group（t=6.123,P〈0.05）; 12 hours later, GRP78 expression began to decline and it decreased to 0.98±0.59 after treated with high glucose for 48 hours, no significant difference was found between it and that in control group（t=0.378, P〉0.05）. The GRP78 expression in constant high glucose group was 0.96±0.10, while it was 0.77±0.04 in fluctuating high glucose group（t=3.134, P〈0.05）. Conclusions Both constant and fluctuating high glucose can induce ER stress and apoptosis in HUVECS, the latter has stronger effect by further inhibition of GRP78 expression, GLP- 1 can alleviate the ER stress and apoptosis by inducing the expression of GRP78.