Institute of Pharmacology, Polish Academy of Sciences, Department of Pain Pharmacology, 12 Smetna Street, 31-343 Krakow, Poland.
Institute of Pharmacology, Polish Academy of Sciences, Department of Brain Biochemistry, 12 Smetna Street, 31-343 Krakow, Poland.
Int Immunopharmacol. 2017 Nov;52:261-271. doi: 10.1016/j.intimp.2017.09.021. Epub 2017 Oct 12.
Chemokine signaling has been implicated in the pathogenesis of diabetic neuropathy; however, the involvement of the chemokine CC motif ligand 1 (CCL1)-chemokine CC motif receptor 8 (CCR8) interaction remains unknown. The goal of this study was to examine the role of CCL1-CCR8 signaling interplay in the development of hypersensitivity and in opioid effectiveness in diabetic neuropathy.
Primary glial cell cultures and a streptozotocin (STZ; 200mg/kg, intraperitoneal)-induced mouse model of diabetic neuropathy were used. Analysis of mRNA/protein expression of glial markers and CCL1/CCR8 was performed by qRT-PCR, Western blotting and/or protein arrays. The co-localization of CCL1/CCR8 with neural/glial cells was visualized by immunofluorescence. The pharmacological tools were injected intrathecally, and pain behavior was evaluated by von Frey/cold plate tests.
Single STZ injection increased blood glucose levels and induced the development of hypersensitivity as measured on days 7-21. On day 7 after STZ, the protein levels of CCL1 and IBA1 but not of CCR8 or GFAP were elevated. Immunofluorescent staining revealed that CCR8 was predominantly localized in neurons, which are also the main source of spinal CCL1. Lipopolysaccharide stimulation of primary microglial cultures resulted in decreases in the levels of CCL1 and CCR8. Single intrathecal injection of CCL1 (10-500ng) induced the development of hypersensitivity, whereas on day 7 after STZ, a CCL1-neutralizing antibody dose-dependently (2-8μg) delayed pain behavior. Repeated administration of the CCL1-neutralizing antibody (4μg) also enhanced the effectiveness of morphine and buprenorphine (1μg).
These results reveal that CCL1/CCR8 neuronal signaling plays an important role in the development of diabetic neuropathy and the effectiveness of opioids.
趋化因子信号在糖尿病性神经病的发病机制中起作用；然而，趋化因子 CC 基序配体 1（CCL1）-趋化因子 CC 基序受体 8（CCR8）相互作用的参与仍不清楚。本研究的目的是研究 CCL1-CCR8 信号相互作用在糖尿病性神经病过敏和阿片类药物有效性发展中的作用。
使用原代神经胶质细胞培养物和链脲佐菌素（STZ；200mg/kg，腹腔内）诱导的糖尿病性神经病小鼠模型。通过 qRT-PCR、Western 印迹和/或蛋白质阵列分析神经胶质标志物和 CCL1/CCR8 的 mRNA/蛋白质表达。通过免疫荧光观察 CCL1/CCR8 与神经/胶质细胞的共定位。通过鞘内注射药理学工具，并通过 von Frey/cold plate 测试评估疼痛行为。
单次 STZ 注射增加血糖水平，并在第 7-21 天测量诱导过敏反应的发展。在 STZ 后第 7 天，CCL1 和 IBA1 的蛋白质水平升高，但 CCR8 或 GFAP 的蛋白质水平没有升高。免疫荧光染色显示 CCR8 主要定位于神经元，神经元也是脊髓 CCL1 的主要来源。原代小胶质细胞培养物的脂多糖刺激导致 CCL1 和 CCR8 水平降低。单次鞘内注射 CCL1（10-500ng）诱导过敏反应的发展，而在 STZ 后第 7 天，CCL1 中和抗体剂量依赖性（2-8μg）延迟疼痛行为。CCL1 中和抗体（4μg）的重复给药也增强了吗啡和丁丙诺啡（1μg）的有效性。
这些结果表明 CCL1/CCR8 神经元信号在糖尿病性神经病的发展和阿片类药物的有效性中起重要作用。
