雌激素对人脐静脉血管内皮细胞凋亡的影响

Effects of estrogen on the apoptosis of human umbilical vein endothelial cells

目的目前认为内质网是心血管疾病治疗的新靶点,其应激反应在高血压心血管重塑中具有重要作用。文中通过建立衣霉素及二硫苏糖醇诱导人脐静脉血管内皮细胞(human umbilical vein endothelial cells,HUVECs)内质网应激(endoplasmic reticulum stress,ERS)凋亡模型,探讨17-β雌二醇对衣霉素/二硫苏糖醇诱导HUVECs ERS凋亡的影响。方法实验前期已确定10μmol/L衣霉素处理细胞10 h或2 mmol/L二硫苏糖醇处理细胞8 h为诱导细胞ERS凋亡最佳浓度和时间。浓度为10-8mol/L的17-β雌二醇对ERS的保护作用最明显。根据是否用衣霉素/二硫苏糖醇,17-β雌二醇,雌激素受体拮抗剂(ICI182,780和G15)处理进行如下分组:1空白对照A、B组;2衣霉素/二硫苏糖醇组;3衣霉素/二硫苏糖醇+17-β雌二醇组;4衣霉素/二硫苏糖醇+17-β雌二醇+ICI组;5衣霉素/二硫苏糖醇+17-β雌二醇+G15组;6衣霉素/二硫苏糖醇+17-β雌二醇+ICI+G15组。Western blot检测ERS诱导凋亡的标志分子GRP78、剪切的caspase 12及CHOP的表达,及Hochest染色检测凋亡细胞数的变化。结果与空白对照A、B组比较,衣霉素/二硫苏糖醇组GRP78、caspase-12和CHOP的表达升高(P<0.05);与衣霉素组比较,衣霉素+17-β雌二醇组GRP78、caspase-12和CHOP的表达均下降(6.80±1.07 vs4.01±0.46,1.54±0.32 vs 0.88±0.10,1.91±0.37 vs 0.91±0.02,P<0.05);与衣霉素+17-β雌二醇组比较,衣霉素+17-β雌二醇+ICI组CHOP表达升高(0.91±0.02 vs 0.96±0.02,P<0.05),衣霉素+17-β雌二醇+G15组GRP78、caspase-12表达升高(4.01±0.46 vs 5.25±0.80,0.88±0.10 vs 1.02±0.07,P<0.05),衣霉素+17-β雌二醇+ICI+G15组GRP78、caspase-12和CHOP的表达升高(P<0.05)。与二硫苏糖醇组比较,二硫苏糖醇+17-β雌二醇组GRP78和CHOP表达均下降(1.81±0.08 vs 1.30±0.14,1.00±0.13 vs 0.51±0.01,P<0.05);与二硫苏糖醇+17-β雌二醇组比较,二硫苏糖醇+17-β雌二醇+ICI组GRP78和CHOP的表达下降(P<0.05),二硫苏糖醇+17-β雌二醇+G15组GRP78表达升高(P<0.05),二硫苏糖醇+17-β雌二醇+ICI+G15组GRP78、caspase-12和CHOP的表达升高(P<0.05)。Hochest染色检测细胞凋亡数目的变化与Western blot的结果一致。结论 17-β雌二醇可抑制衣霉素/二硫苏糖醇诱导的人脐静脉内皮细胞ERS引起的凋亡,雌激素通过调节雌激素受体保持ERS的稳态,进而保护心血管。

Objective Recent researches find that endoplasmic reticulum is a new target for the treatment of cardiovascular disease and its stress response plays an important role in the hypertension-induced cardiovascular remodeling. The study built up the apoptosis model of endoplasmic reticulum stress （ERS） in human umbilical vein endothelial cells （HUVECs） and investigated the impact of 1713-estradiol （ E2 ） on the ERS apoptosis of HUVECs induced by tunicamycinand （TM）/ dithiothreitol （DTT）. Methods HUVECs incubated for 10 h in 10 μmol/L TM or 8 h in 2 mmol/L UIT were the best concentration and time for ERS apoptosis. E2 of 10^-8 mol/L was prominent in protecting ERS. According to the application of TM/DTT, E2, estrogen antagonists （ICI182, 780 and G15）, there were 6 groups： control group, TM/DTT group, TM/DTT ＋ E2 group, TM/ DTT E2 ＋G15 group, TM/DTT ＋E2 ＋ICI ＋G15 group. Western blot was applied to detect the expressions of biomarkers for ERS induced apoptosis （ GRP78, cleaved caspase-12 and CHOP）. Hochest staining was used to observe the changes of the apoptosis cell number. Results Compared with control group, the expressions of GRP78, caspase-12 and CHOP significantly increased（P 〈0.05）. In comparison with TM group, the expressions of GRP78, caspase-12 and CHOP in TM ＋ E2 group significantly decreased （6.80 ± 1.07 vs 4.01±0.46, 1.54 ±0.32 vs 0. 88 ±0.10, 1.91±0.37 vs 0. 91 ± 0.02, P〈0.05）. In comparison with TM ＋ E2group, the CHOP expression in TM ＋ E2 ＋ ICI group increased（0.91 ±0.02 vs 0.96 ± 0.02, P 〈 0.05 ）, and the expressions of GRP78 and caspase-12 in TM ＋ E2 ＋ G15 group increased（4.01 ± 0.46 vs 5.25 ± 0.80, 0.88 ±0.10 vs 1.02 ±0.07, P 〈0.05）, and the expressions of GRP78, caspase-12 and CHOP in TM ＋ E2 ＋ ICI ＋ G15 group increased （P 〈 0.05）. Compared with DTI＂ group, the expressions of GRP78 and CHOP in DTr ＋ E2group decreased （1.81 ± 0.08 vs 1.30± 0.14, 1.00 ± 0.13 vs 0.51 ± 0.01, P 〈 0.05 ）. In comparison with DTT ＋ E2 group, the expressions of GRP78 and CHOP in DTT ＋ E2 ＋ ICI group decreased（ P 〈 0.05 ）, and the expression of GPR78 in DTT ＋ E2 ＋ G15 group increased（ P 〈 0.05 ）, and the expressions of GRP78, caspase-12 and CHOP in DTT ＋ E2 ＋ ICI ＋ G15 group increased（P 〈 0.05 ）. The change of the apop- tosis cell number observed by hochest staining was in consistence with the result of western blot. Conclusion E2can protect human endothelial cells from ERS induced apoptosis caused by TM and DTr. Estrogen maintains homeostasis by regulating ERS estrogen receptors, thereby protecting the cardiovascular system.