三碘甲酰原氨酸（T3）对内皮细胞凋亡的影响及护骨素干预研究

Effect of triiodothyronine（ T3 ） on apoptosis of endothelial cells and interventional study with osteoprotegerin

目的探讨护骨素对不同浓度三碘甲酰原氨酸（L）下人脐静脉内皮细胞凋亡的影响及机制。方法将内皮细胞分组：对照组（0nmol／LT3）、低浓度组（0．1nmol／LT3）、生理浓度组（1nmol／LT，）和高浓度组（10nmoL／LL），护骨素干预48h后，Hoechst33258和流式细胞仪检测细胞凋亡情况；Western印迹分析各组细胞磷酸化NF．KB抑制蛋白激酶p（p-IKKl3）、NF．KB抑制蛋白激酶p（IKKl3）、磷酸化结节性硬化复合物2（p—TSC2）、结节性硬化复合物2（TSC2）、磷酸化核糖体蛋白S6激酶（p-S6K）、核糖体蛋白S6激酶（S6K）、磷酸化蛋白激酶B（p—AKT）、蛋白激酶B（AKT）、Bcl一2及Bax表达水平。结果（1）护骨素干预后的对照组、低浓度组、高浓度组细胞凋亡率低于未干预组（P〈0．05）；生理浓度组护骨素干预前后细胞凋亡率无明显差异（P〉0．05）。（2）与生理浓度组相比，对照组、低浓度组和高浓度组p-IKKl3、p-S6K、Bax表达显著增高（P〈0．05），p-TSC2、p-AKT、Bel-2表达显著降低（P〈0．05）；经护骨素干预后，对照组、低浓度组、高浓度组p-IKK[3、p-S6K、Bax低于未干预组（P〈0．05），而p-TSC2、p-AKT、Bel-2表达高于未干预组（P〈0．05）：生理浓度组护骨素干预前后上述各蛋白表达无明显差异（P〉0．05）。结论低、高浓度甲状腺激素激活IKK[3／mTOR信号通路引起内皮细胞凋亡，而护骨素抑制其IKKl3／mTOR活性，对内皮细胞具有保护作用。

Objective To explore the effect and mechanism of osteoprotegerin on apoptosis in human umbilical vein endothelial cells（HUVECs） induced by triiodothyronine （T3 ） with different concentrations. Methods HUVECs were cultured in control group （ 0 mnol/L T3 ） , low-concentration group （ 0.1 nmol/L T3 ） , physiological- concentration group（ I nmol/L T3 ） , high-concentration group（ i0 nmol/L T3 ） with OPG for 48 h. The cell apoptosis was measured by cytometry and Hoechst 33258. The expressions of p-IRKS, IKKI3, p-TSC2, TSC2, p-S6K, S6K, p-AKT, AKT, Bcl-2, and Bax were analyzed by Western blot. Results （ 1 ） After OPG pretreatment and being cultured in control group, low-concentration group and high-concentration group, the HUVEC apoptosis was decreased significantly（ P〈0.05 ） as compared with groups without OPG pretreatment. There was no difference in physiological concentration group before and after intervention of OPG. （2） The expression of p-IKK[3, p-S6K, and Bax in other groups were higher than those in physiological concentration group; however, the expressions of p-TSC2, p-AKT, and Bcl-2 were lower than those in physiological concentration group（ P〈0.05 ）. After OPG pretreatment in control group, low-concentration group, and high-concentration group, the expressions of p-IKK~, p-S6K, and Bax decreased significantly（ P〈0.05 ） and the expression of p-TSC2, p-AKT, and Bcl-2 increased significantly （ P〈0.05 ） ; there were no differences in the expressions of p-IKKI3, p-S6K, p-TSC2, p-AKT, Bcl-2, and Bax in physiological concentration group before and after intervention of OPG. Conclusions Low and high concentrations of T3 increase apoptosis of HUVECs by way of activation of IKKI3/mTOR pathway. Osteoprotegerin is able to protect HUVECs from apoptosis induced by low and high concentration of T3 via the way of inhibiting the IKKβ/mTOR pathway.