吗啡诱导大鼠镇痛耐受和脊髓GIRK1-2表达减少

Morphine Induces Antinociceptive Tolerance and Down-regulation of GIRK 1-2 Expression in Rats

【目的】观察G蛋白门控内向整流钾离子通道(GIRK)亚单位GIRK1和GIRK2在吗啡耐受大鼠脊髓背角的表达变化并探讨其调控机制。【方法】成年雄性SD大鼠连续7 d鞘内注射吗啡(15μg/d)建立吗啡耐受模型。在每天吗啡注射前30 min,鞘内注射蛋白激酶C-ε(PKCε)选择性抑制剂εV1-2(10μg),观察对吗啡耐受以及脊髓背角GIRK1和GIRK2表达的影响。24只大鼠随机平均分成四组:生理盐水组(saline)、吗啡组(morphine)、吗啡+生理盐水组(morphine+saline)和吗啡+εV1-2组(morphine+εV1-2)。所有大鼠在1 d、3 d、5 d和7 d,药物注射前和吗啡注射后30 min后检测热痛阈值,在第7天行为学测试结束后,免疫荧光检测脊髓背角GIRK1和GIRK2的表达。【结果】荧光双染显示,GIRK1和GIRK2主要分布于大鼠脊髓背角I-Ⅱ板层,且与μ阿片受体(MOR)共染。与生理盐水组相比,连续7 d鞘内注射吗啡导致吗啡镇痛效应显著下降(P<0.001),同时导致脊髓背角GIRK1(22.45±10.58vs.62.83±20.80,P<0.001)和GIRK2(23.67±8.78 vs.50.17±11.05,P=0.001)表达显著降低。荧光双染显示,PKCε与GIRK1和GIRK2在脊髓背角共染。鞘内注射εV1-2可以有效抑制吗啡耐受的形成(P<0.001),且抑制吗啡诱导的GIRK1(54.50±10.37 vs.19.33±9.48,P<0.001)和GIRK2(39.83±6.24 vs.15.83±9.58,P=0.001)表达降低。【结论】脊髓背角GIRK1和GIRK2下调与吗啡耐受密切相关,PKCε是吗啡诱导GIRK1和GIRK2下调的重要原因。

【Objective】To observe the expression of spinal G protein-gated inwardly-rectifying potassium(GIRK)channel subunits 1 and 2 in spinal dorsal horn of morphine-tolerant rats and investigate the regulatory mechanism.【Methods】Twenty four rats were equally and randomly divided into 4 groups:saline,morphine,morphine+saline and morphine+εV1-2.The morphine-tolerant rat model was established by intrathecal administration of morphine(15μg/d)for 7 days.Thirty minutes before daily morphine administration,rats received protein kinase C-ε(PKCε)selective inhibitorεV1-2 to test its effect on morphine tolerance and GIRK1-2 expression.All rats received behavioral tests on days 1,3,5 and 7 and thereafter immunofluorescence.【Results】Double fluorescence staining showed that GIRK1 and GIRK2 were expressed primarily in the spinal laminae I-Ⅱand co-immunostained withμ-opioid receptor(MOR).Seven-day intrathecal administration of morphine induced antinociceptive tolerance and a significant reduction of the spinal GIRK 1(22.45±10.58 vs.62.83±20.80,P<0.001)and GIRK 2(23.67±8.78 vs.50.17±11.05,P=0.001)fluorescence intensity,as compared with saline control rats.In addition,pretreatment withεV 1-2 significantly delayed the reduction of morphine antinociception(P<0.001)and prevented the decrease of GIRK 1(54.50±10.37 vs.19.33±9.48,P<0.001)and GIRK 2(39.83±6.24 vs.15.83±9.58,P=0.001)expression induced by morphine treatment.【Conclusions】Morphine tolerance is closely related to down-regulation of GIRK 1-2 expression and PKCεplays a crucial regulatory role herein.