Department of Anesthesiology, Affiliated Shanghai Sixth People's Hospital, Shanghai Jiao Tong University, Shanghai, 200233, China.
Department of Physiology, Konyang University College of Medicine, Seo-gu, Daejeon, 35365, Republic of Korea.
Eur J Pharmacol. 2020 Jul 5;878:173118. doi: 10.1016/j.ejphar.2020.173118. Epub 2020 Apr 19.
This study investigated the effect of DAMGO-induced μ opioid receptor (MOR) internalization on morphine tolerance. Male Sprague-Dawley rats (200-250 g) aged 6-8 weeks were administered morphine via intrathecal (i.t.) injection (15 μg/10 μl twice daily for 6 days) to induce antinociceptive tolerance, which was evaluated using the tail-flick and paw-withdrawal tests. Response latency was calculated as the percentage of maximum possible effect (%MPE). A bolus of DAMGO was administered by i.t. injection on day 6, and the tail-flick and paw-withdrawal tests were carried out 24, 48, and 72 h later. Membrane and cytosolic MOR expression was assessed by western blotting. HEK293 cells were transfected with MOR-FLAG plasmid and after 6 days of morphine treatment (10 μM), the cells were treated with 1 μM DAMGO, and MOR localization was examined by immunofluorescence analysis 30 and 60 min later. Repeated morphine treatment induced tolerance after 5 days; however, i.t. DAMGO administration restored morphine sensitivity and enhanced acute morphine-induced antinociception after 24, 48, and 72 h. In HEK293 cells, DAMGO treatment stimulated MOR internalization after 30 min and MOR recycling to the membrane after 1 h. Membrane and cytoplasmic MOR expression in vivo was unchanged 24, 48, and 72 h after i.t. DAMGO injection. Morphine does not cause significant MOR internalization or downregulation, and can readily induce tolerance. DAMGO counters this effect by enhancing receptor endocytosis, thereby reversing morphine-induced antinociceptive tolerance and restoring its analgesic efficacy.
本研究探讨了 DAMGO 诱导 μ 阿片受体 (MOR) 内化对吗啡耐受的影响。6-8 周龄雄性 Sprague-Dawley 大鼠（体重 200-250g）通过鞘内（i.t.）注射吗啡（15 μg/10 μl，每日两次，连续 6 天）诱导抗伤害性耐受，采用甩尾和缩足反射试验进行评估。反应潜伏期计算为最大可能效应的百分比（%MPE）。在第 6 天通过 i.t.注射给予 DAMGO 冲击，然后在 24、48 和 72 小时后进行甩尾和缩足反射试验。通过 Western blot 评估膜和胞质 MOR 表达。将 MOR-FLAG 质粒转染到 HEK293 细胞中，在 10 μM 吗啡处理 6 天后，用 1 μM DAMGO 处理细胞，30 和 60 分钟后通过免疫荧光分析检查 MOR 定位。重复吗啡处理 5 天后诱导耐受；然而，i.t. DAMGO 给药恢复了吗啡敏感性，并增强了 24、48 和 72 小时后的急性吗啡诱导的抗伤害性作用。在 HEK293 细胞中，DAMGO 处理刺激 MOR 在 30 分钟内化，在 1 小时后刺激 MOR 再循环到膜上。i.t. DAMGO 注射后 24、48 和 72 小时，体内膜和胞质 MOR 表达没有变化。吗啡不会导致明显的 MOR 内化或下调，并且可以轻易诱导耐受。DAMGO 通过增强受体内吞作用来对抗这种作用，从而逆转吗啡诱导的抗伤害性耐受并恢复其镇痛功效。
