Berco M, Bhavnani B R
Department of Obstetrics and Gynecology, Institute of Medical Sciences, University of Toronto and St. Michael's Hospital, 30 Bond Street, Toronto, Ontario, Canada M5B 1W8.
J Soc Gynecol Investig. 2001 Jul-Aug;8(4):245-54. doi: 10.1016/s1071-5576(01)00111-3.
In the present study, the neurotoxic effect of oxidized low density lipoprotein on PC-12 neuronal cells maintained in culture was used to test the neuroprotective effect of several equine estrogens, such as estrone (E(1)), 17beta-estradiol (17beta-E(2)), 17alpha-estradiol (17alpha-E(2)), equilin (Eq), 17beta-dihydroequilin (17beta-Eq), 17alpha-dihydroequilin (17alpha-Eq), equilenin (Eqn), 17beta-dihydroequilenin (17beta-Eqn), 17alpha-dihydroequilenin (17alpha-Eqn), Delta(8)-estrone (Delta(8)-E(1)), and Delta(8),17beta-estradiol (Delta(8),17beta-E(2)).
The PC-12 cells (10,000 cells/well) were grown on collagen-coated 96-well plates in Dulbecco's Modified Eagle Medium supplemented with 10% horse serum, 5% fetal bovine serum, and 10 mM HEPES. In culture, the cells displayed normal PC-12 morphology and behavior, exhibiting increased dendritic growth and cessation of cell division upon exposure to nerve growth factor. Twenty-four hours after plating, various concentrations (0.1-50 microM) of estrogens were added followed by addition of oxidized low density lipoprotein (5-12.5 microg/well) in a total volume of 100 microL. After 24 hours, cell viability was determined using the MTS (3-[4,5-dimethylthiazol-2-yl]-5-[3-carboxymethoxyphenyl]-2H-tetrazolium, inner salt) cell proliferation assay.
The results indicate that the extent of low density lipoprotein oxidation and concentration of oxidized low density lipoprotein is directly proportional to cell toxicity. The mean +/- standard deviation cell death obtained using 10.0 microg/well of oxidized low density lipoprotein was 53.6% +/- 8.7%. With the exception of 17alpha-estradiol, all estrogens tested were found to be neuroprotective against oxidized low density lipoprotein toxicity in a typical dose-dependent manner. The order of neuroprotective potency was Delta(8)-E(1) (1.2 microM), Delta(8),17beta-E(2) (1.3 microM), Eqn (5.3 microM), 17beta-Eqn (5.3 microM), Eq (6 microM), 17beta-Eq (8.5 microM), E(1) (11 microM), 17beta-E(2) (11 microM), 17alpha-Eq (12 microM), and 17alpha-Eqn (16 microM) followed by 17alpha-E(2) which provided less than 50% protection.
Our data indicate that the neurotoxic effects of oxidized low density lipoprotein can be inhibited differentially by various estrogens, with the Delta(8) estrogens being the most potent neuroprotectors. These novel findings further suggest that some of the neuroprotective benefits associated with estrogen therapy might occur by the suppression of oxidized low density lipoprotein neurotoxicity. Because estrogens such as Delta(8)-E(1) are relatively less uterotropic and potentially less feminizing than the classic estrogen 17beta-E(2), they may be useful in the prevention of Alzheimer disease and other neurodegenerative diseases in both women and men.
在本研究中，利用氧化型低密度脂蛋白对培养的PC-12神经元细胞的神经毒性作用，来测试几种马雌激素的神经保护作用，这些雌激素包括雌酮（E(1)）、17β-雌二醇（17β-E(2)）、17α-雌二醇（17α-E(2)）、马萘雌酮（Eq）、17β-二氢马萘雌酮（17β-Eq）、17α-二氢马萘雌酮（17α-Eq）、马烯雌酮（Eqn）、17β-二氢马烯雌酮（17β-Eqn）、17α-二氢马烯雌酮（17α-Eqn）、Δ(8)-雌酮（Δ(8)-E(1)）以及Δ(8),17β-雌二醇（Δ(8),17β-E(2)）。
将PC-12细胞（10,000个细胞/孔）接种于包被有胶原蛋白的96孔板中，培养基为补充了10%马血清、5%胎牛血清和10 mM HEPES的杜氏改良伊格尔培养基。在培养过程中，细胞呈现出正常的PC-12形态和行为，在暴露于神经生长因子时，树突生长增加且细胞分裂停止。接种24小时后，加入不同浓度（0.1 - 50 μM）的雌激素，随后加入氧化型低密度脂蛋白（5 - 12.5 μg/孔），总体积为100 μL。24小时后，使用MTS（3-[4,5-二甲基噻唑-2-基]-5-[3-羧甲氧基苯基]-2H-四唑鎓，内盐）细胞增殖测定法测定细胞活力。
结果表明，低密度脂蛋白的氧化程度和氧化型低密度脂蛋白的浓度与细胞毒性直接相关。使用10.0 μg/孔氧化型低密度脂蛋白时，平均 ± 标准差的细胞死亡率为53.6% ± 8.7%。除17α-雌二醇外，所有测试的雌激素均以典型的剂量依赖性方式对氧化型低密度脂蛋白毒性具有神经保护作用。神经保护效力的顺序为Δ(8)-E(1)（1.2 μM）、Δ(8),17β-E(2)（1.3 μM）、Eqn（5.3 μM）、17β-Eqn（5.
