Yoon Byung Koo, Kang Young Hee, Oh Won Jong, Roh Cheong Rae, Kim Duk Kyung, Kang Chi Dug
Department of Obstetrics, Gynecology and Women's Health, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Korea.
Samsung Biomedical Research Institute, Samsung Medical Center, Seoul, Korea.
J Menopausal Med. 2020 Apr;26(1):1-8. doi: 10.6118/jmm.19019.
Coronary heart disease (CHD) risk increases in women after menopause, but menopausal hormone therapy (MHT) helps prevent CHD if started early after menopause. To explore the mechanism underlying the direct vascular actions of estrogen, the effects of 17β-estradiol (E₂) on apoptosis of vascular smooth muscle cells (VSMCs) induced with lysophosphatidylcholine (lysoPC), an active component of oxidized low-density lipoprotein, were investigated in the present study.
VSMCs were isolated from rat aortas. Apoptosis and protein expression of caspases were assessed using propidium iodide staining and Western blot analysis, respectively. Intracellular formation of reactive oxygen species (ROS) was examined using dichlorofluorescein diacetate, a cell-permeable oxidation-sensitive probe, and quantitated with flow cytometry. Nuclear factor-κB (NF-κB) activation was determined after transfection with a reporter plasmid containing the luciferase reporter gene.
After pre-treatment for 24 hours, 17β-E₂ suppressed lysoPC-induced (15 μM) apoptotic cell death in a dose-dependent manner with statistical significance at near physiological concentration. 17β-E₂ (10⁻⁶ M) also increased protein levels of caspase-9 and -8 precursors and decreased the active form of caspase-3. Western blot analysis using subcellular fractions showed that 17β-E₂ decreased mitochondrial Bax levels and concomitantly increased cytosolic Bax expression. Furthermore, intracellular production of ROS and NF-κB-mediated transcriptional activity were reduced with 17β-E₂. In addition, estrogen effects on apoptosis were partially blocked by ICI 182,780, a specific estrogen receptor antagonist.
In cultured VSMCs treated with lysoPC, 17β-E₂ reduced apoptotic cell death by down-regulating both extrinsic and intrinsic apoptosis pathways, contributing to the preventive action of MHT against CHD.
绝经后女性患冠心病（CHD）的风险增加，但绝经后激素治疗（MHT）如果在绝经后早期开始，则有助于预防CHD。为了探究雌激素直接血管作用的潜在机制，本研究调查了17β-雌二醇（E₂）对由氧化型低密度脂蛋白的活性成分溶血磷脂酰胆碱（lysoPC）诱导的血管平滑肌细胞（VSMC）凋亡的影响。
从大鼠主动脉分离VSMC。分别使用碘化丙啶染色和蛋白质印迹分析评估凋亡和半胱天冬酶的蛋白表达。使用二氯荧光素二乙酸酯（一种细胞可渗透的氧化敏感探针）检查细胞内活性氧（ROS）的形成，并用流式细胞术进行定量。在用含有荧光素酶报告基因的报告质粒转染后，测定核因子κB（NF-κB）的激活情况。
预处理24小时后，17β-E₂以剂量依赖方式抑制lysoPC诱导（15 μM）的凋亡细胞死亡，在接近生理浓度时具有统计学意义。17β-E₂（10⁻⁶ M）还增加了半胱天冬酶-9和-8前体的蛋白水平，并降低了半胱天冬酶-3的活性形式。使用亚细胞组分的蛋白质印迹分析表明，17β-E₂降低了线粒体Bax水平，并同时增加了胞质Bax表达。此外，17β-E₂降低了细胞内ROS产生和NF-κB介导的转录活性。此外，雌激素对凋亡的作用被特异性雌激素受体拮抗剂ICI 182,780部分阻断。
在用lysoPC处理的培养VSMC中，17β-E₂通过下调外源性和内源性凋亡途径减少凋亡细胞死亡，这有助于MHT对CHD的预防作用。
