Shrivastava Pallavi, Cabrera Miguel A, Chastain Lucy G, Boyadjieva Nadka I, Jabbar Shaima, Franklin Tina, Sarkar Dipak K
The Endocrine Program, Department of Animal Sciences, Rutgers, The State University of New Jersey, 67 Poultry Lane, New Brunswick, NJ, 08901, USA.
J Neuroinflammation. 2017 Apr 14;14(1):83. doi: 10.1186/s12974-017-0844-3.
Opioid receptors are known to control neurotransmission of various peptidergic neurons, but their potential role in regulation of microglia and neuronal cell communications is unknown. We investigated the role of mu-opioid receptors (MOR) and delta-opioid receptors (DOR) on microglia in the regulation of apoptosis in proopiomelanocortin (POMC) neurons induced by neonatal ethanol in the hypothalamus.
Neonatal rat pups were fed a milk formula containing ethanol or control diets between postnatal days 2-6. Some of the alcohol-fed rats additionally received pretreatment of a microglia activation blocker minocycline. Two hours after the last feeding, some of the pups were sacrificed and processed for histochemical detection of microglial cell functions or confocal microscopy for detection of cellular physical interaction or used for gene and protein expression analysis. The rest of the pups were dissected for microglia separation by differential gradient centrifugation and characterization by measuring production of various activation markers and cytokines. In addition, primary cultures of microglial cells were prepared using hypothalamic tissues of neonatal rats and used for determination of cytokine production/secretion and apoptotic activity of neurons.
In the hypothalamus, neonatal alcohol feeding elevated cytokine receptor levels, increased the number of microglial cells with amoeboid-type circularity, enhanced POMC and microglial cell physical interaction, and decreased POMC cell numbers. Minocycline reversed these cellular effects of alcohol. Alcohol feeding also increased levels of microglia MOR protein and pro-inflammatory signaling molecules in the hypothalamus, and MOR receptor antagonist naltrexone prevented these effects of alcohol. In primary cultures of hypothalamic microglia, both MOR agonist [D-Ala 2, N-MePhe 4, Gly-ol]-enkephalin (DAMGO) and ethanol increased microglial cellular levels and secretion of pro-inflammatory cell signaling proteins. However, a DOR agonist [D-Pen2,5]enkephalin (DPDPE) increased microglial secretion of anti-inflammatory cytokines and suppressed ethanol's ability to increase microglial production of inflammatory signaling proteins and secretion of pro-inflammatory cytokines. In addition, MOR-activated inflammation promoted while DOR-suppressed inflammation inhibited the apoptotic effect of ethanol on POMC neurons.
These results suggest that ethanol's neurotoxic action on POMC neurons results from MOR-activated neuroinflammatory signaling. Additionally, these results identify a protective effect of a DOR agonist against the pro-inflammatory and neurotoxic action of ethanol.
已知阿片受体可控制各种肽能神经元的神经传递，但其在小胶质细胞和神经元细胞通讯调节中的潜在作用尚不清楚。我们研究了μ-阿片受体（MOR）和δ-阿片受体（DOR）在小胶质细胞对下丘脑新生乙醇诱导的促肾上腺皮质激素原（POMC）神经元凋亡调节中的作用。
新生大鼠幼崽在出生后第2至6天喂食含乙醇的奶粉或对照饮食。一些喂食酒精的大鼠额外接受了小胶质细胞激活阻滞剂米诺环素的预处理。最后一次喂食后两小时，处死部分幼崽并进行处理，用于小胶质细胞功能的组织化学检测或共聚焦显微镜检测细胞物理相互作用，或用于基因和蛋白质表达分析。其余幼崽解剖后通过差速梯度离心分离小胶质细胞，并通过测量各种激活标志物和细胞因子的产生进行表征。此外，使用新生大鼠下丘脑组织制备小胶质细胞原代培养物，并用于测定细胞因子的产生/分泌和神经元的凋亡活性。
在下丘脑中，新生期喂食酒精会提高细胞因子受体水平，增加具有阿米巴样圆形的小胶质细胞数量，增强POMC与小胶质细胞的物理相互作用，并减少POMC细胞数量。米诺环素逆转了酒精对这些细胞的影响。喂食酒精还会增加下丘脑中MOR蛋白和促炎信号分子的水平，而MOR受体拮抗剂纳曲酮可预防酒精的这些作用。在下丘脑小胶质细胞原代培养物中，MOR激动剂[D-Ala 2，N-MePhe 4，Gly-ol]-脑啡肽（DAMGO）和乙醇均会增加小胶质细胞中促炎细胞信号蛋白的水平和分泌。然而，DOR激动剂[D-Pen2,5]脑啡肽（DPDPE）会增加小胶质细胞抗炎细胞因子的分泌，并抑制乙醇增加小胶质细胞产生炎症信号蛋白和分泌促炎细胞因子的能力。此外，MOR激活的炎症促进而DOR抑制的炎症抑制了乙醇对POMC神经元的凋亡作用。
这些结果表明，乙醇对POMC神经元的神经毒性作用源于MOR激活的神经炎症信号传导。此外，这些结果确定了DOR激动剂对乙醇的促炎和神经毒性作用具有保护作用。
