Mann Val, Huber Christene, Kogianni Giolanta, Collins Frances, Noble Brendon
Musculoskeletal Tissue Engineering Collaboration, Level 1, University of Edinburgh Medical School, The Chancellor's Building, 49 Little France Crescent, Edinburgh, UK.
Bone. 2007 Mar;40(3):674-84. doi: 10.1016/j.bone.2006.10.014. Epub 2006 Dec 13.
Withdrawal of estrogen represents the primary factor determining post menopausal bone loss and has been associated with negative indicators of bone quality including the apoptotic death of osteocytes in vivo. While hormone replacement therapy in the form of Estrogen or Selective Estrogen Receptor Modulators (SERMs) demonstrates clear estrogen receptor (ER)-mediated benefits to bone mass, less is known regarding the mechanism of action of these compounds in the maintenance of bone cell populations. We have investigated the potential antioxidant effects of estrogen, estrogen derivatives and the SERMs Raloxifene and LY117018 in the prevention of oxidative stress induced apoptosis in the osteocyte like cell line MLO-Y4. Treatment of MLO-Y4 with 0.3 mM H(2)O(2) induced apoptosis that was significantly inhibited (p< or =0.002) when the cells were pre-treated for 1 h with either 17beta-estradiol, Raloxifene or LY117018 (10 nM). The stereoisomer 17alpha-estradiol also prevented H(2)O(2) induced apoptosis in MLO-Y4. Importantly, pre-treatment of ER-negative HEK293 cells with either 1 microM, 100 nM or 10 nM 17beta-estradiol, Raloxifene or LY117018 significantly inhibited H(2)O(2) induced apoptosis in these cells (p< or =4.2x10(-5)) indicating an estrogen receptor-independent effect of these compounds. Comparisons of 17beta-estradiol and similar molecules containing the putative free radical scavenger C3-OH moiety on the steroid A-ring (17alpha-estradiol, 17alpha-ethinylestradiol; 10 nM) with structurally related molecules lacking the C3-OH grouping (Mestranol and Quinestrol; 10 nM) demonstrated that only compounds containing the C3-OH moiety showed anti-apoptotic behavior in these studies (p< or =0.0033). Similarly the identification of the presence of reactive oxygen species (ROS) in cells as evidenced by the free radical indicator 2'7'-dichlorodihydrofluorescein diacetate demonstrated that 17beta-estradiol, SERMs and related molecules with C3-OH moiety were capable of blocking ROS generated in cells by H(2)O(2) (p< or =0.002) while Mestranol and Quinestrol showed no such blockade. It is possible that the loss of osteocytes during estrogen insufficiency may occur through a failure to suppress the activity of naturally occurring or disease associated oxidant molecules. These data suggest that the osteocyte protective effects of estrogen and SERMs may operate through a common receptor-independent mechanism which may be related to the antioxidant activity of these molecules.
雌激素撤减是决定绝经后骨质流失的主要因素，且与骨质质量的负面指标相关，包括体内骨细胞的凋亡死亡。虽然以雌激素或选择性雌激素受体调节剂（SERM）形式进行的激素替代疗法对骨量有明显的雌激素受体（ER）介导的益处，但关于这些化合物在维持骨细胞群体中的作用机制知之甚少。我们研究了雌激素、雌激素衍生物以及SERM雷洛昔芬和LY117018在预防氧化应激诱导的骨细胞样细胞系MLO - Y4凋亡中的潜在抗氧化作用。用0.3 mM过氧化氢处理MLO - Y4会诱导凋亡，当细胞用17β - 雌二醇、雷洛昔芬或LY117018（10 nM）预处理1小时时，凋亡受到显著抑制（p≤0.002）。立体异构体17α - 雌二醇也能预防过氧化氢诱导的MLO - Y4细胞凋亡。重要的是，用1 μM、100 nM或10 nM的17β - 雌二醇、雷洛昔芬或LY117018预处理ER阴性的HEK293细胞能显著抑制这些细胞中过氧化氢诱导的凋亡（p≤4.2×10⁻⁵），表明这些化合物具有雌激素受体非依赖性作用。将17β - 雌二醇和类固醇A环上含有假定自由基清除剂C3 - OH部分的类似分子（17α - 雌二醇、17α - 炔雌醇；10 nM）与缺乏C3 - OH基团的结构相关分子（炔雌醚和炔诺酮；10 nM）进行比较，结果表明在这些研究中只有含有C3 - OH部分的化合物表现出抗凋亡行为（p≤0.0033）。同样，自由基指示剂2',7' - 二氯二氢荧光素二乙酸酯证明细胞中存在活性氧（ROS），这表明17β - 雌二醇、SERM和具有C3 - OH部分的相关分子能够阻断过氧化氢在细胞中产生的ROS（p≤0.002），而炔雌醚和炔诺酮则没有这种阻断作用。雌激素不足期间骨细胞的丢失可能是由于未能抑制天然存在的或与疾病相关的氧化分子的活性。这些数据表明，雌激素和SERM对骨细胞的保护作用可能通过一种共同的受体非依赖性机制发挥作用，这可能与这些分子的抗氧化活性有关。
