Endocrine Program, Department of Animal Sciences, Graduate Program of Neuroscience, Rutgers, The State University of New Jersey, New Brunswick, NJ, USA.
Alcohol Clin Exp Res. 2013 Aug;37(8):1370-9. doi: 10.1111/acer.12104. Epub 2013 Mar 29.
We have previously shown that ethanol (EtOH) increases cellular apoptosis to developing neurons via the effects on oxidative stress of neurons directly and via increasing production of microglia-derived factors. To study further the mechanism of EtOH action on neuronal apoptosis, we determined the effects of 2 well-known PKA activators, dibutyryl cAMP (dbcAMP) and brain-derived neurotrophic factor (BDNF), on EtOH-activated oxidative stress and apoptotic processes in the hypothalamic neurons in the presence and absence of microglial cells' influence.
In enriched neuronal cells from fetal rat hypothalami treated with EtOH or with conditioned medium from EtOH-treated microglia, we measured cellular apoptosis by the free nucleosome assay and the levels of cAMP, BDNF, O²⁻, reactive oxygen species (ROS), nitrite, glutathione (GSH), and catalase following treatment with EtOH or EtOH-treated microglial culture conditioned medium. Additionally, we tested the effectiveness of dbcAMP and BDNF in preventing EtOH or EtOH-treated microglial conditioned medium on cellular apoptosis and oxidative stress in enriched hypothalamic neuronal cell in primary cultures.
Neuronal cell cultures following treatment with EtOH or EtOH-activated microglial conditioned medium showed decreased production levels of cAMP and BDNF. EtOH also increased apoptotic death as well as oxidative status, as demonstrated by higher cellular levels of oxidants but lower levels of antioxidants, in neuronal cells. These effects of EtOH on oxidative stress and cell death were enhanced by the presence of microglia. Treatment with BDNF or dbcAMP decreased EtOH or EtOH-activated microglial conditioned medium-induced changes in the levels of intracellular free radicals, ROS and O²⁻, nitrite, GSH, and catalase.
These data support the possibility that EtOH by acting directly and via increasing the production of microglial-derived factors reduces cellular levels of cAMP and BDNF to increase cellular oxidative status and apoptosis in hypothalamic neuronal cells in primary cultures.
我们之前已经表明，乙醇（EtOH）通过直接影响神经元的氧化应激以及增加小胶质细胞衍生因子的产生，增加发育中神经元的细胞凋亡。为了进一步研究 EtOH 对神经元凋亡的作用机制，我们测定了 2 种已知的蛋白激酶 A（PKA）激活剂，二丁酰环磷腺苷（dbcAMP）和脑源性神经营养因子（BDNF），对小胶质细胞存在和不存在时 EtOH 激活的氧化应激和下丘脑神经元凋亡过程的影响。
在经 EtOH 或 EtOH 处理的小胶质细胞条件培养基处理的胎鼠下丘脑富含神经元细胞中，我们通过游离核小体测定法测量细胞凋亡，并测量 cAMP、BDNF、O²⁻、活性氧（ROS）、亚硝酸盐、谷胱甘肽（GSH）和过氧化氢酶的水平，用 EtOH 或 EtOH 处理的小胶质细胞培养条件培养基处理后。此外，我们还测试了 dbcAMP 和 BDNF 在预防 EtOH 或 EtOH 处理的小胶质细胞条件培养基对原代培养的富含下丘脑神经元细胞的细胞凋亡和氧化应激中的作用。
用 EtOH 或 EtOH 激活的小胶质细胞条件培养基处理后的神经元细胞培养物显示 cAMP 和 BDNF 的产生水平降低。EtOH 还增加了细胞凋亡和氧化应激，如神经元细胞中氧化剂的细胞水平升高，但抗氧化剂的水平降低。这些 EtOH 对氧化应激和细胞死亡的影响因小胶质细胞的存在而增强。BDNF 或 dbcAMP 的治疗降低了 EtOH 或 EtOH 激活的小胶质细胞条件培养基诱导的细胞内自由基、ROS 和 O²⁻、亚硝酸盐、GSH 和过氧化氢酶水平的变化。
这些数据支持这样一种可能性，即 EtOH 通过直接作用和增加小胶质细胞衍生因子的产生，降低原代培养的下丘脑神经元细胞中细胞内 cAMP 和 BDNF 的水平，增加细胞氧化应激和凋亡。
