Hypoxia induces reversible κ-opioid receptor (KOR) internalization similar to the internalization that is induced by KOR agonists. In the current study, we demonstrate that this KOR internalization is a protective mechanism via the β-arrestin specific pathway in an oxygen-glucose deprivation (OGD) model. Mouse neuroblastoma Neuro2A cells were stably transfected with mouse KOR-tdTomato fusion protein (N2A-mKOR-tdT cells). Various concentrations of salvinorin A (SA), a highly selective KOR agonist, were given in the presence and absence of norbinaltorphimine (norBNI), which is a KOR antagonist, or Dyngo-4a (internalization inhibitor) or API-2 (Akt/Protein kinase B signaling inhibitor-2). Various concentrations of SA and RB-64 (22-thiocyanatosalvinorin A, selective for the G protein signaling pathway) were administered both in normoxic and hypoxic conditions. Autophagosomes and ultrastructural components of cells were observed using transmission electron microscopy (TEM). Cell viability, severity of cell injury, and levels of proteins related to the Akt signaling pathway were evaluated using live cell counting (by Cell Counting Kit-8), the lactic acid dehydrogenase (LDH) release rate, and Western blot analysis, respectively. SA promoted cell survival and attenuated OGD-induced cell injury. The Akt signaling pathway is activated by SA. KOR internalization, when blocked by norBNI or Dyngo-4a, increased LDH release and decreased cell viability under OGD. Treatment with SA significantly inhibited autophagy, and the effects of SA on autophagy were reversed by API-2 pretreatment. RB-64 in a low concentration without β-arrestin recruitment did not reduce LDH release and increase cell viability as observed with SA. KOR internalization through β-arrestin activation is a protective mechanism against OGD. The Akt pathway might play a critical role in modulating these protective effects by inhibiting autophagy.

中文翻译：

---

Kappa阿片受体内在化通过β-arrestin激活和Akt介导的信号传导通路来保护氧-葡萄糖的剥夺。

缺氧诱导可逆性κ阿片受体（KOR）内在化，类似于KOR激动剂诱导的内在化。在当前的研究中，我们证明了这种KOR内在化是通过氧-葡萄糖剥夺（OGD）模型中的β-arrestin特异性途径的一种保护机制。小鼠神经母细胞瘤Neuro2A细胞用小鼠KOR-tdTomato融合蛋白（N2A-mKOR-tdT细胞）稳定转染。在存在和不存在作为KOR拮抗剂的norbinaltorphimine（norBNI）或Dyngo-4a（内在化抑制剂）或API-2（Akt /蛋白质）的情况下，给予不同浓度的高选择性KOR激动剂Salvinorin A（SA）。激酶B信号抑制剂2）。各种浓度的SA和RB-64（22-硫氰基阿维诺林A，在高氧和低氧条件下都可以选择G蛋白信号通路。使用透射电子显微镜（TEM）观察细胞的自噬体和超微结构成分。分别使用活细胞计数（通过Cell Counting Kit-8），乳酸脱氢酶（LDH）释放速率和Western印迹分析来评估细胞活力，细胞损伤的严重性以及与Akt信号通路相关的蛋白质的水平。SA促进细胞存活并减轻OGD诱导的细胞损伤。Akt信号通路被SA激活。当被norBNI或Dyngo-4a阻断时，KOR内在化会增加ODH下LDH的释放并降低细胞活力。用SA处理可显着抑制自噬，API-2预处理可逆转SA对自噬的影响。如用SA观察到的那样，低浓度的RB-64在没有β-arrestin募集的情况下不会减少LDH的释放并增加细胞活力。通过β-arrestin激活进行的KOR内在化是针对OGD的保护机制。Akt途径可能通过抑制自噬而在调节这些保护作用中起关键作用。