The main challenge of pain management with opioids is development of acute and chronic analgesic tolerance. Several studies on neuronal cells have focused on the molecular mechanisms involved in tolerance such as cyclic AMP (cAMP) activation, and nitric oxide (NO) pathway. However, the effects of opioids on non-neuronal cells and tolerance development have been poorly investigated. Lithium chloride is a glycogen synthase kinase 3β (GSK-3β) inhibitor and exert its effects through modulation of nitric oxide pathway. In this study we examined the effect of lithium on acute/chronic morphine and methadone administration in endothelial cells which express mu opioid receptors. Human umbilical vein endothelial cells (HUVECs) were treated with different doses of morphine, methadone, and lithium for six and 48 h. Then we evaluated cell viability, nitrite and cyclic AMP levels, as well as the expression of endothelial nitric oxide synthase (eNOS) protein using Immunocytochemistry (ICC) assay and phosphorylated GSK-3β enzyme by western blot analysis in cells. Both chronic morphine and methadone treatment increased NO level and eNOS expression in HUVECs. Morphine induced cAMP overproduction after 48 h exposure with cells. Lithium pretreatment (10 mM) in both morphine and methadone received groups significantly reduced nitrite and cAMP levels as well as eNOS expression as compared to the control. The decreased amount of phospho GSK-3β due to the opioid exposure was increased following lithium treatment. Tolerance like pattern may occur in non-neuronal cells with opioid receptors and this study clearly revealed the attenuation of morphine and methadone tolerance like behavior by lithium treatment in HUVECs.

阿片类药物疼痛管理的主要挑战是急性和慢性镇痛耐受的发展。一些关于神经元细胞的研究集中在参与耐受的分子机制，例如环磷酸腺苷 (cAMP) 激活和一氧化氮 (NO) 途径。然而，阿片类药物对非神经元细胞和耐受性发展的影响的研究很少。 Lithium Chloride 是一种糖原合成酶激酶 3β (GSK-3β) 抑制剂，通过调节一氧化氮途径发挥其作用。在这项研究中，我们研究了锂对表达μ阿片受体的内皮细胞的急性/慢性吗啡和美沙酮给药的影响。人脐静脉内皮细胞（HUVEC）用不同剂量的吗啡、美沙酮和锂处理 6 小时和 48 小时。然后，我们使用免疫细胞化学 (ICC) 测定法评估细胞活力、亚硝酸盐和环 AMP 水平，以及内皮一氧化氮合酶 (eNOS) 蛋白的表达，并通过蛋白质印迹分析评估细胞中磷酸化的 GSK-3β 酶。长期吗啡和美沙酮治疗均可增加 HUVEC 中的 NO 水平和 eNOS 表达。吗啡与细胞接触 48 小时后诱导 cAMP 过量产生。与对照组相比，吗啡和美沙酮接受锂预处理（10 mM）的组均显着降低了亚硝酸盐和 cAMP 水平以及 eNOS 表达。由于阿片类药物暴露而导致的磷酸化 GSK-3β 的减少量在锂治疗后增加。类耐受模式可能发生在具有阿片受体的非神经元细胞中，这项研究清楚地揭示了 HUVEC 中锂治疗可减弱吗啡和美沙酮类耐受行为。