College of Medicine, Hunan Normal University, Changsha, Hunan 410006, PR China.
Int J Mol Med. 2012 Nov;30(5):1159-65. doi: 10.3892/ijmm.2012.1109. Epub 2012 Aug 23.
7-difluoromethoxy-5,4'-Di-hydroxyl isoflavone (dFGEN), prepared by the difluoromethylation of genistein, is an active chemical entity. In this study, our main purpose was to investigate whether dFGEN had an effect on glutamate-induced apoptosis in cultured PC12 cells. The PC12 cells were treated with different glutamate concentrations for 24 h in vitro. The PC12 cells impaired by glutamate were used as the cell model of excitability. Cells were incubated for 30 min with genistein, dFGEN, vitamin E, and exposed to 10 mM glutamate for 24 h. Cell morphology was observed by light microscopy. The growth and proliferation of PC12 cells were detected by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay. Cell apoptosis was determined by flow cytome-try (FCM) with propidium iodide (PI) staining. The activity of lactate dehydrogenase (LDH), superoxide dismutase (SOD) and the content of malondialdelyde (MDA) were measured by kits, respectively. Acridine orange (AO) staining was used to detect characteristics of cell apoptosis. When PC12 cells were incubated with glutamate for 24 h, cells appeared to have significant changes in shape. The cellular viability was reduced and the apoptotic rate was increased. The levels of LDH and the content of MDA were increased. The activity of SOD was decreased. After PC12 cells were pretreated with dFGEN, dFGEN significantly improved cell morphology, cell growth and proliferation, suppressed apoptosis of cells, reduced the release of LDH, improving SOD activity and decreased MDA content in a concentration-dependent manner. AO staining displayed that apoptosis was decreased. These results suggested that dFGEN has a protective effect against glutamate-induced damage in PC12 cells. dFGEN seemed to have a better protective effect than the lead compound genistein in a concentration-dependent manner. The mechanism of protective effect of dFGEN may be mainly related to its antioxidative activity.
7-二氟甲氧基-5,4'-二羟基异黄酮（dFGEN）是通过染料木黄酮的二氟甲基化制备的，是一种活性化学实体。在这项研究中，我们的主要目的是研究 dFGEN 是否对培养的 PC12 细胞中的谷氨酸诱导的细胞凋亡有影响。体外将不同浓度的谷氨酸处理 PC12 细胞 24 小时。用谷氨酸损伤的 PC12 细胞作为兴奋性细胞模型。将细胞用染料木黄酮、dFGEN、维生素 E 孵育 30 分钟，然后暴露于 10 mM 谷氨酸 24 小时。通过相差显微镜观察细胞形态。通过 3-[4,5-二甲基噻唑-2-基]-2,5-二苯基四氮唑溴盐（MTT）测定法检测 PC12 细胞的生长和增殖。通过碘化丙啶（PI）染色的流式细胞术（FCM）测定细胞凋亡。通过试剂盒分别测定乳酸脱氢酶（LDH）、超氧化物歧化酶（SOD）的活性和丙二醛（MDA）的含量。吖啶橙（AO）染色用于检测细胞凋亡的特征。当 PC12 细胞用谷氨酸孵育 24 小时时，细胞形态发生明显变化。细胞活力降低，凋亡率增加。LDH 水平和 MDA 含量增加。SOD 活性降低。用 dFGEN 预处理 PC12 细胞后，dFGEN 显著改善细胞形态、细胞生长和增殖，抑制细胞凋亡，减少 LDH 释放，提高 SOD 活性，降低 MDA 含量，呈浓度依赖性。AO 染色显示凋亡减少。这些结果表明 dFGEN 对 PC12 细胞中的谷氨酸诱导损伤具有保护作用。dFGEN 似乎比先导化合物染料木黄酮具有更好的保护作用，呈浓度依赖性。dFGEN 的保护作用机制可能主要与其抗氧化活性有关。
