Department of Molecular Pharmacology & Therapeutics , Stritch School of Medicine, Loyola University Chicago, Maywood, Illinois.
Alcohol Clin Exp Res. 2014 Jan;38(1):161-9. doi: 10.1111/acer.12221. Epub 2013 Aug 1.
Brain neurodamage from chronic binge ethanol (EtOH) exposure is linked to neuroinflammation and associated oxidative stress. Using rat organotypic hippocampal-entorhinal cortical (HEC) slice cultures of developing brain age, we reported that binge EtOH promotes release of a neuroinflammatory instigator, arachidonic acid (AA), concomitant with neurodegeneration, and that mepacrine, a global inhibitor of phospholipase A2 (PLA2) enzymes mobilizing AA from phospholipids, is neuroprotective. Here, we sought with binge EtOH-treated HEC cultures to establish that PLA2 activity is responsible in part for significant oxidative stress and to ascertain the PLA2 families responsible for AA release and neurodegeneration.
HEC slices, prepared from 1-week-old rats and cultured 2 to 2.5 weeks, were exposed to 100 mM EtOH over 6 successive days, with 4 daytime "withdrawals" (no EtOH). Brain 3-nitrotyrosinated (3-NT)- and 4-hydroxy-2-nonenal (4-HNE)-adducted proteins, oxidative stress footprints, were immunoassayed on days 3 through 6, and mepacrine's effect was determined on day 6. The effects of specific PLA2 inhibitors on neurodegeneration (propidium iodide staining) and AA release (ELISA levels in media) in the cultures were then determined. Also, the effect of JZL184, an inhibitor of monoacylglycerol lipase (MAGL) which is reported to mobilize AA from endocannabinoids during neuroinflammatory insults, was examined.
3-NT- and 4-HNE-adducted proteins were significantly increased by the binge EtOH exposure, consistent with oxidative stress, and mepacrine prevented the increases. The PLA2 inhibitor results implicated secretory PLA2 (group II sPLA2) and to some extent Ca(2+) -independent cytosolic PLA2 (group VI iPLA2) in binge EtOH-induced neurotoxicity and in AA release, but surprisingly, Ca(2+) -dependent cytosolic PLA2 (group IV cPLA2) did not appear important. Furthermore, unlike PLA2 inhibition, MAGL inhibition failed to prevent the neurodegeneration.
In these developing HEC slice cultures, pro-oxidative signaling via sPLA2 and iPLA2, but not necessarily cPLA2 or MAGL, is involved in EtOH neurotoxicity. This study provides further insights into neuroinflammatory phospholipase signaling and oxidative stress underlying binge EtOH-induced neurodegeneration in developing (adolescent age) brain in vitro.
慢性 binge 乙醇（EtOH）暴露导致的大脑神经损伤与神经炎症有关，并伴有氧化应激。我们使用发育中的大脑年龄的大鼠器官型海马-内嗅皮质（HEC）切片培养物报告称， binge EtOH 促进神经炎症引发剂花生四烯酸（AA）的释放，同时伴有神经退行性变，而 mepacrine 是一种从磷脂中动员 AA 的磷脂酶 A2（PLA2）酶的全球抑制剂，具有神经保护作用。在这里，我们在 binge EtOH 处理的 HEC 培养物中寻求确定 PLA2 活性部分负责显著的氧化应激，并确定负责 AA 释放和神经退行性变的 PLA2 家族。
从 1 周龄大鼠中制备 HEC 切片，并培养 2 至 2.5 周，然后用 100mM EtOH 连续处理 6 天，每天进行 4 次白天“撤药”（无 EtOH）。在第 3 天至第 6 天，对脑 3-硝基酪氨酸（3-NT）和 4-羟基-2-壬烯醛（4-HNE）加合物蛋白进行免疫测定，并测定第 6 天 mepacrine 的作用。然后确定特定 PLA2 抑制剂对培养物中神经退行性变（碘化丙啶染色）和 AA 释放（培养基中的 ELISA 水平）的影响。此外，还研究了抑制剂 JZL184 的作用，JZL184 是一种单酰甘油脂肪酶（MAGL）抑制剂，据报道在神经炎症损伤期间可从内源性大麻素中动员 AA。
binge EtOH 暴露显著增加了 3-NT-和 4-HNE 加合物蛋白，与氧化应激一致，而 mepacrine 可防止其增加。PLA2 抑制剂的结果表明，分泌型 PLA2（II 组 sPLA2）和在某种程度上钙非依赖性胞浆型 PLA2（VI 组 iPLA2）参与了 binge EtOH 诱导的神经毒性和 AA 释放，但令人惊讶的是，钙依赖性胞浆型 PLA2（IV 组 cPLA2）似乎并不重要。此外，与 PLA2 抑制不同，MAGL 抑制未能防止神经退行性变。
在这些发育中的 HEC 切片培养物中，通过 sPLA2 和 iPLA2 产生的促氧化信号，而不是 cPLA2 或 MAGL，参与了 EtOH 神经毒性。这项研究为 binge EtOH 诱导的发育中（青春期）脑内神经退行性变的神经炎症磷脂酶信号和氧化应激提供了进一步的见解。
