护骨素对高糖环境人脐静脉内皮细胞的保护作用及机制研究

Protective effect and its mechanism of osteoprotegerin on human umbilical vein endothelial cells cultured with high glucose

目的探讨护骨素对高糖环境人脐静脉内皮细胞（HUVECs）的影响及其机制。方法（1）HUVECs分别在正糖（5．5mmol／L葡萄糖）、高糖（33mmol／L葡萄糖）、高糖＋护骨素（0．5、1、2μg／m1）、高渗（5．5mmol／L葡萄糖＋27．5mmol／L甘露醇）环境培养48h，以流式细胞术及Hoechst33258核染色检测各组细胞凋亡情况，Western印迹分析各组Bcl-2、Bax蛋白表达水平：（2）HUVECs分别在正糖、高糖、高糖＋护骨素（2μg/ml）、高糖＋雷帕霉素（10ng／mL）、高糖＋雷帕霉素（10ng／m1）＋护骨素（2μg/ml）环境培养24h，Western印迹分析各组mTORCl下游核糖体蛋白s6激酶（S6K）、p-S6K、真核细胞翻译起始因子4E结合蛋白1（4EBP1）及p-4EBPl蛋白表达水平；（3）HUVECs分别在正糖、高糖、高糖＋护骨素（2μg/ml）环境培养24h，Western印迹分析各组mTORC1上游马铃薯球蛋白（tuberin）、p-tuberin蛋白表达水平。结果（1）与正糖组比较，高糖组细胞凋亡率、Bax表达水平显著增加（P〈0．05），Bcl-2表达显著降低（P〈0．05）；高糖＋护骨素组细胞凋亡率、Bax表达水平明显低于高糖组，而又高于正糖组（P〈0．05），Bcl-2表达明显高于高糖组，而又低于正糖组（P〈0．05）；高渗组与正糖组比较无统计学差异；（2）与正糖组比较，高糖组p-S6K、p-4EBPl表达水平显著增高（P〈0．05）；高糖＋护骨素组p-S6K、p-4EBP1表达水平明显低于高糖组，而又高于正糖组（P〈0．05）；高糖＋雷帕霉素组与高糖＋护骨素组比较无统计学差异；（3）与正糖组比较，高糖组p-tuberin表达水平显著增高（P〈0．05）；高糖＋护骨素组p-tuberin表达水平明显低于高糖组，而又高于正糖组（P〈0．05）。结论护骨素对高糖环境HUVECs具有保护作用．其机制可能通过下调tuberin—mTORC1活性，从而实现对高糖损伤的血管内皮细胞的保护效应。

Objective To explore the effect and its mechism of osteoprotegerin （OPG） on human umbilical vein endothelial cells（HUVECs） induced by high glucose. Methods （ 1 ） The cultured HUVECs were treated with normal glucose（5.5 mmol/L）, high glucose（33 mmol/L）, high glucose ＋ OPG（0.5, 1, and 2 μg/ml） as well as mannitol（5.5 mmoL/L glucose＋27.5 mmol/L nlannitol）for 48 h, respectively. Flow cytometry assay and Hoechst 33258 staining were used to detect the cell apoptosis. The expression levels of Bcl-2 and Bax protein were measured by western blot analysis. （ 2 ） The cultured HUVECs were treated with normal glucose, high glucose, high glucose ＋ OPG （2 μg/ml OPG）, high glucose ＋ rapamycin（ 10 ng/ml rapamycin） as well as high glucose ＋ rapamycin ＋ OPG for 24h, respectively. The expression levels of S6K, p-S6K, 4EBP1 and p-4EBP1 protein were measured by western blot analysis. （3）The cultured HUVECs were treated with normal glucose, high glucose, high glucose ＋ OPG（ 2 μg/ml） for 24 h, respectively. The expression levels of tuberin and p-tnberin protein were measured by western blot analysis. Results （ 1 ） Compared with normal glucose group, the apoptosis of HUVECs and the expression level of Bax was dramatically increased, and the expression of Bcl-2 was decreased significantly in high glucose group （P〈 0.05 ）. The apoptosis of HUVECs and the expression level of Bax in high glucose ＋ OPG group was significantly lower than that in high glucose group, which was still higher than that in nomml glucose group （ P〈 0.05 ）. and the expression of Bcl-2 in high glucose ＋ OPG group was significantly higher than that in high glucose group, which was still lower than that in normal glucose group （ P〈0.05 ）. There was no statistically difference between hyperosmolar control group and normal glucose group. （2） Compared with normal glucose group, the expression levels of p-S6K and p-4EBP1 were increased markedly in high glucose group（ P〈0.05 ）. The expression levels of p-S6K and p-4EBP1 inhigh glucose ＋ OPG group were significantly lower than that in high glucose group, which were still higher than that in normal glucose group（ P〈0.05 ）. No significant difference was found between high glucose ＋ OPG group and high glucose ＋ rapamycin group. （ 3 ） Compared with normal glucose group, the expression level of p-tuberin was increased markedly in high glucose group （P〈0.05）. The expression level of p-tuberin in high glucose ＋ OPG group was significantly lower than that in high glucose group, which was still higher than that in normal glucose group （ P 〈 0.05 ）. Conclusions It suggests that the protective effect and mechanism of OPG on HUVECs cultured with high glucose may be association with tuberin/mTORC1 pathway.