Bhavnani Bhagu R, Berco Mauricio, Binkley Julie
Department of Obstetrics and Gynecology, Institute of Medical Sciences, University of Toronto, and St. Michael's Hospital, Toronto, Ontario, Canada.
J Soc Gynecol Investig. 2003 Jul;10(5):302-8. doi: 10.1016/s1071-5576(03)00087-x.
In the present study, neuronal PC12 cells and hippocampal HT22 cells maintained in culture were used to test the neuroprotective effect of equine estrogens estrone, 17beta-estradiol, 17alpha-estradiol, equilin, 17beta-dihydroequilin, 17alpha-dihydroequilin, equilenin, 17beta-dihydroequilenin, 17alpha-dihydroequilenin, Delta(8)-estrone(,) and Delta(8),17beta-estradiol against glutamate toxicity.
The HT22 and PC12 cells were grown in Dulbecco modified Eagle medium supplemented with 5% horse serum, 10% fetal bovine serum, and 10 mM HEPES. The undifferentiated PC12 cells were plated on collagen-coated, 96-well plastic plates at 10,000 cells per well, and the HT22 cells were plated on uncoated 96-well plates at 2500 cells per well. Twenty-four hours after plating, various concentrations of estrogens (0.1-40 microM) and glutamate (1-10 mM) were added in a total volume of 100 microL. After 24 hours, cell viability was determined using the MTS cell proliferation assay. Results were verified in some experiments by using the lactate dehydrogenase cytotoxicity assay.
The results indicate that cell toxicity in both cell lines was directly proportional to the concentration of glutamate. The lowest dose of glutamate that reduced cell viability by 50% under these conditions was 1.8 mM for HT22 cells and 3 mM for PC12 cells. All estrogens tested were neuroprotective against glutamate-induced cell death in a typical dose-related manner. However, these estrogens differed extensively with respect to their neuroprotective potencies. In both cell lines, the Delta(8)-ring B unsaturated estrogens were the most neuroprotective, whereas the classic estrogens 17beta-estradiol, estrone, and 17alpha-estradiol were the least potent. The order of potency was Delta(8),17beta-estradiol > Delta(8)-estrone > 17beta-dihydroequilenin > 17alpha-dihydroequilenin > equilenin > 17beta-dihydroequilin = equilin > 17alpha-dihydroequilin > 17beta-estradiol > estrone > 17alpha-estradiol in PC12 cells and Delta(8),17beta-estradiol > Delta(8)-estrone > equilenin = 17beta-dihydroequilenin > 17beta-dihydroequilin > equilin > 17alpha-dihydroequilenin > 17alpha-dihydroequilin > 17alpha-estradiol = 17beta-estradiol > estrone in HT22 cells.
Our data indicate that the neurotoxic effects of glutamate can be inhibited differentially by various equine estrogens. The less estrogenic (uterotropic) Delta(8) estrogens were the most effective neuroprotectors, and further chemical modifications of these estrogens may provide compounds that are useful for preventing neurodegenerative diseases in both women and men.
在本研究中，使用培养的神经元PC12细胞和海马HT22细胞来测试马雌激素雌酮、17β-雌二醇、17α-雌二醇、马萘雌酮、17β-二氢马萘雌酮、17α-二氢马萘雌酮、马烯雌酮、17β-二氢马烯雌酮、17α-二氢马烯雌酮、Δ⁸-雌酮和Δ⁸,17β-雌二醇对谷氨酸毒性的神经保护作用。
HT22和PC12细胞在补充有5%马血清、10%胎牛血清和10 mM HEPES的杜尔贝科改良 Eagle培养基中生长。未分化的PC12细胞以每孔10000个细胞接种在胶原蛋白包被的96孔塑料板上，HT22细胞以每孔2500个细胞接种在未包被的96孔板上。接种24小时后，加入各种浓度的雌激素（0.1 - 40 μM）和谷氨酸（1 - 10 mM），总体积为100 μL。24小时后，使用MTS细胞增殖测定法测定细胞活力。在一些实验中通过使用乳酸脱氢酶细胞毒性测定法验证结果。
结果表明，两种细胞系中的细胞毒性与谷氨酸浓度直接相关。在这些条件下使细胞活力降低50%的最低谷氨酸剂量，HT22细胞为1.8 mM，PC12细胞为3 mM。所有测试的雌激素均以典型的剂量相关方式对谷氨酸诱导的细胞死亡具有神经保护作用。然而，这些雌激素在神经保护效力方面差异很大。在两种细胞系中，Δ⁸-环B不饱和雌激素的神经保护作用最强，而经典雌激素17β-雌二醇、雌酮和17α-雌二醇的效力最低。在PC12细胞中效力顺序为：Δ⁸,17β-雌二醇 > Δ⁸-雌酮 > 17β-二氢马烯雌酮 > 17α-二氢马烯雌酮 > 马烯雌酮 > 17β-二氢马萘雌酮 = 马萘雌酮 > 17α-二氢马萘雌酮 > 17β-雌二醇 > 雌酮 > 17α-雌二醇；在HT22细胞中效力顺序为：Δ⁸,17β-雌二醇 > Δ⁸-雌酮 > 马烯雌酮 = 17β-二氢马烯雌酮 > 17β-二氢马萘雌酮 > 马萘雌酮 > 17α-二氢马萘雌酮 > 17α-二氢马烯雌酮 > 17α-雌二醇 = 17β-雌二醇 > 雌酮。
我们的数据表明，各种马雌激素可不同程度地抑制谷氨酸的神经毒性作用。雌激素活性较低（子宫促生长作用较弱）的Δ⁸雌激素是最有效的神经保护剂，对这些雌激素进行进一步的化学修饰可能会提供对预防男性和女性神经退行性疾病有用的化合物。
