促肾上腺皮质激素释放因子2型受体参与慢性偏头痛小鼠痛觉敏化及焦虑的机制研究

Corticotropin releasing factor receptor 2 involves in pain sensitization and anxiety of chronic migraine mice

目的探讨促肾上腺皮质激素释放因子2型受体(CRFR2)对慢性偏头痛小鼠痛觉敏化及焦虑的调控作用及潜在机制。方法将48只C57BL/6J小鼠按随机数字表法分为对照组、模型组、NBI35965组、K41498组,每组12只。后3组小鼠于第1、3、5、7、9天腹腔注射10 mg/kg硝酸甘油建立慢性偏头痛模型,NBI35965组及K41498组小鼠于第2、4、6、8天双侧三叉神经脊束尾核分别注射100 nL NBI35965、K41498溶液,对照组小鼠注射同体积生理盐水。第1、3、5、7、9天腹腔注射2 h后及第10天上午11时采用Von frey纤维丝检测小鼠眶额部机械痛阈值。于第11天上午11时采用高架十字迷宫实验检测小鼠的焦虑样行为。采用Western blotting实验检测小鼠三叉神经脊束尾核中促肾上腺皮质激素释放因子(CRF)、促肾上腺皮质激素释放因子1型受体(CRFR1)、CRFR2蛋白的表达。采用实时荧光定量聚合酶链式反应(RT-qPCR)检测小鼠三叉神经脊束尾核中CRFR1及CRFR2 mRNA的表达。采用免疫荧光染色检测小鼠三叉神经脊束尾核中降钙素基因相关肽(CGRP)、即刻早期基因(c-fos)、胶质纤维酸性蛋白(GFAP)及离子钙结合适配器分子1(Iba-1)蛋白的表达。结果 (1)与对照组比较,模型组、NBI35965组、K41498组小鼠第3、5、7、9、10天的眶额部机械痛阈值较低,差异均有统计学意义(P<0.05)。与模型组比较,K41498组小鼠第7、9、10天的眶额部机械痛阈值较高,差异均有统计学意义(P<0.05)。(2)与对照组比较,模型组、NBI35965组、K41498组小鼠的开臂进入次数较少,开臂停留时间较短,差异均有统计学意义(P<0.05)。与模型组比较,K41498组小鼠的开臂进入次数较多,开臂停留时间较长,差异均有统计学意义(P<0.05)。(3)与对照组比较,模型组、NBI35965组、K41498组小鼠三叉神经脊束尾核中CRF和CRFR2蛋白的表达较高,差异均有统计学意义(P<0.05)。与模型组比较,K41498组小鼠三叉神经脊束尾核中CRF蛋白的表达较低,差异有统计学意义(P<0.05)。(4)与对照组比较,模型组、NBI35965组、K41498组小鼠三叉神经脊束尾核中CRFR2 mRNA的表达较高,差异均有统计学意义(P<0.05)。(5)与对照组比较,模型组、NBI35965组、K41498组小鼠三叉神经脊束尾核中CGRP、c-fos、Iba-1、GFAP蛋白的表达均较高,差异均有统计学意义(P<0.05)。与模型组比较,K41498组小鼠三叉神经脊束尾核中CGRP及c-fos蛋白的表达较低,差异均有统计学意义(P<0.05)。结论 CRFR2通过调控三叉神经脊束尾核的神经元活化及CGRP释放而影响慢性偏头痛小鼠眶额部痛觉敏化及焦虑样行为的发生发展。

Objective To explore the role of corticotrophin releasing factor receptor 2(CRFR2)in regulating pain sensitization and anxiety and its mechanism in chronic migraine mice.Methods Forty-eight C57BL/6J mice were randomly divided into control group,model group,NBI35965 group and K41498 group(n=12);chronic migraine models in the later 3 groups were established by intraperitoneally administrating 10 mg/kg nitroglycerin on the 1st,3rd,5th,7th and 9th d;mice in the NBI35965 group and K41498 group were injected with 100 nL NBI35965 or K41498 solution into the bilateral trigeminal nucleus caudalis on the 2nd,4th,6th and 8th d,and mice in the control group were injected with same volume of normal saline.Von frey fiber was used to detect the orbitofrontal mechanical pain threshold 2 h after intraperitoneal injection on the 1st,3rd,5th,7th and 9th d,and at 11 a.m.on the 10th d.Elevated plus maze was used to detect the anxiety-like behaviors at 11 a.m.on the 11th d.Western blotting was performed to detect the protein expressions of corticotrophin releasing factor(CRF),corticotrophin releasing factor receptor 1(CRFR1),CRFR2 in the trigeminal nucleus caudalis.Real-time quantitative PCR(RT-qPCR)was used to detect the CRFR1 and CRFR2 mRNA expressions in the trigeminal nucleus caudalis.Immunofluorescent staining was used to detect the protein expressions of calcitonin gene-related peptide(CGRP),immediate-early gene c-fos,glial fibrillary acidic protein(GFAP)and ionized calcium-binding adapter molecule 1(Iba-1)in the trigeminal nucleus caudalis.Results Compared with the control group,the model group,NBI35965 group and K41498 group had significantly decreased orbitofrontal mechanical pain thresholds 3,5,7,9,and 10 d after intraperitoneal injection(P<0.05);compared with model group,the K41498 group had significantly increased orbitofrontal mechanical pain thresholds 7,9,and 10 d after intraperitoneal injection(P<0.05).Compared with control group,the model group,NBI35965 group and K41498 group had significantly decreased entries and shorter time in opened arms(P<0.05);compared with the model group,the K41498 group had significantly increased entries and shorter time in opened arms(P<0.05).Compared with the control group,the model group,NBI35965 group and K41498 group had significantly higher CRF and CRFR2 protein expressions in the trigeminal nucleus caudalis(P<0.05);compared with the model group,the K41498 group had statistically lower CRF protein expression in the trigeminal nucleus caudalis(P<0.05).Compared with the control group,the model group,NBI35965 group and K41498 group had significantly higher CRFR2 mRNA expression in the trigeminal nucleus caudalis(P<0.05).Compard with the control group,the model group,NBI35965 group and K41498 group had significantly increased CGRP,c-fos,Iba-1 and GFAP protein expressions in the trigeminal nucleus caudalis(P<0.05);compared with the model group,the K41498 group had significantly decreased CGRP and c-fos protein expressions in the trigeminal nucleus caudalis(P<0.05).Conclusion CRFR2 can alter the orbitofrontal pain sensitization and anxiety-like behaviors in chronic migraine mice by regulating neuronal activation and CGRP release in the trigeminal nucleus caudalis.