Ge Yun, Belcher Scott M, Pierce Dwight R, Light Kim E
Department of Pharmacology and Toxicology, College of Medicine, University of Arkansas for Medical Sciences, Little Rock, AR 72205, USA.
Brain Res Mol Brain Res. 2004 Oct 22;129(1-2):124-34. doi: 10.1016/j.molbrainres.2004.06.034.
Previous studies have demonstrated that ethanol exposure during the vulnerable postnatal (PN) day 4-6 period results in a dose-dependent loss of Purkinje neurons in rats by apoptosis. Although the mechanism of ethanol action and the reasons for Purkinje cell vulnerability are unknown, we hypothesize that during the PN4-6 vulnerable period Purkinje cells are dependent on active trophic factor suppression of apoptosis. Furthermore, ethanol acts to prevent the reception of this trophic signaling resulting in the execution of the apoptotic pathway that includes specific alterations of proteins in the Bcl2 gene family. Ethanol exposure that occurs after this vulnerable period (i.e. PN9) would not be expected to demonstrate alterations in these apoptotic proteins since the Purkinje cells no longer demonstrate vulnerability to ethanol. The current study was undertaken to identify the alterations in mRNA expression for members of the Bcl2-family within the initial hours following ethanol administration on PN4 or PN9. Semi-quantitative reverse transcriptase with polymerase chain reaction (PCR) techniques were used to determine the expression levels of pro-apoptotic factors Bad and Bax, and anti-apoptotic Bcl(2) mRNA. Ethanol was administered at four different doses (1.5, 3.0, 4.5, and 6.0 g/kg) on PN4 and analyses of whole cerebellar mRNA was conducted at 1, 4, 6, and 8 h after treatment. Doses greater than 1.5 g/kg produced significant decreases in Bcl(2) and significant increases in Bad and Bax mRNA during the 8-h period after treatment. In stark contrast, when ethanol was administered at 3.0 or 6.0 g/kg to PN9 pups, no significant alterations of these apoptotic factors were identified at either 1 or 4 h after treatment. These results are in agreement with and provide further support for our hypothesis that ethanol interrupts the active suppression of apoptosis that is a crucial feature of Purkinje cell vulnerability during this time period.
先前的研究表明，在出生后（PN）第4至6天这一易损期接触乙醇，会导致大鼠浦肯野神经元因凋亡而出现剂量依赖性损失。尽管乙醇作用机制以及浦肯野细胞易损性的原因尚不清楚，但我们推测，在PN4 - 6易损期，浦肯野细胞依赖于活性营养因子对凋亡的抑制。此外，乙醇会阻止这种营养信号的接收，从而导致凋亡途径的执行，这包括Bcl2基因家族中蛋白质的特定改变。在这个易损期之后（即PN9）接触乙醇，预计不会出现这些凋亡蛋白的改变，因为此时浦肯野细胞对乙醇不再敏感。本研究旨在确定在PN4或PN9给予乙醇后的最初几个小时内，Bcl2家族成员mRNA表达的变化。采用半定量逆转录聚合酶链反应（PCR）技术来测定促凋亡因子Bad和Bax以及抗凋亡Bcl(2) mRNA的表达水平。在PN4时，以四种不同剂量（1.5、3.0、4.5和6.0 g/kg）给予乙醇，并在处理后1、4、6和8小时对整个小脑mRNA进行分析。大于1.5 g/kg的剂量在处理后的8小时内导致Bcl(2)显著降低，Bad和Bax mRNA显著增加。与之形成鲜明对比的是，当以3.0或6.0 g/kg的剂量给PN9幼崽给予乙醇时，在处理后1小时或4小时均未发现这些凋亡因子有显著变化。这些结果与我们的假设一致，并为其提供了进一步支持，即乙醇会中断对凋亡的活性抑制，而这是此时间段内浦肯野细胞易损性的关键特征。
