GFRα3在神经病理性痛大鼠冷痛觉过敏时背根神经节TRPM8表达及膜转运中的作用

Role of GFRα3 in expression and membrane trafficking of TRPM8 in dorsal root ganglion during cold hyperalgesia in rats with neuropathic pain

目的 评价神经胶质细胞源性神经营养因子家族受体α3（GFRα3）在神经病理性痛大鼠冷痛觉过敏时背根神经节瞬时感受器电位M8（TRPM8）表达及膜转运中的作用.方法 鞘内置管成功的健康成年雄性SD大鼠32只,10～12周龄,体重250～280 g,采用随机数字表法分为4组（n=8）：假手术＋GFRα3 dsRNA组（Sham＋dsRNA组）、假手术＋GFRα3 siRNA组（Sham＋siRNA组）、神经病理性痛＋GFRα3 dsRNA组（NP＋dsRNA组）和神经病理性痛＋GFRα3 siRNA组（NP＋siRNA组）.采用坐骨神经慢性束缚性损伤法建立神经病理性痛模型.于术后10～13 d时,Sham＋dsRNA组和NP＋dsRNA组鞘内注射对照的GFRα3 dsRNA,Sham＋siRNA组和NP＋siRNA组鞘内注射正义链进行2′甲基化修饰和5′胆固醇修饰的GFRα3 siRNA,10 μg∕20 μl,每天1次,连续4 d.于术前1 d、术后10、11、12、13 d（鞘内注射前）及术后14 d时测定冷板抬足次数、机械缩足反应阈（MWT）和热缩足潜伏期（TWL）.最后一次痛行为学测试后处死大鼠,取术侧L4-6背根神经节,采用Western blot法测定GFRα3、总蛋白和膜蛋白TRPM8的表达水平,并计算膜蛋白与总蛋白TRPM8表达水平的比值（m∕t比值）.结果 与Sham＋dsRNA组比较,NP＋dsRNA组和NP＋siRNA组术后冷板抬足次数增多,MWT降低,TWL缩短,NP＋dsRNA组背根神经节GFRα3、总蛋白和膜蛋白TRPM8表达上调,m∕t比值升高,NP＋siRNA 组背根神经节GFRα3表达下调（P〈0.01）,总蛋白和膜蛋白TRPM8表达水平及m∕t比值差异无统计学意义（P〉0.05）;与NP＋dsRNA组比较,NP＋siRNA组术后冷板抬足次数减少,背根神经节GFRα3、总蛋白和膜蛋白TRPM8表达下调,m∕t比值降低（P〈0.01）,MWT和TWL差异无统计学意义（P〉0.05）.结论 背根神经节GFRα3可上调TRPM8表达及增强其膜转运,该作用可能参与了神经病理性痛大鼠冷痛觉过敏的维持机制.

Objective To evaluate the role of glial cell line-derived neurotrophic factor family re-ceptor alpha3（GFRα3）in the expression and membrane trafficking of transient receptor potential melasta-tin 8（TRPM8）in the dorsal root ganglion（DRG）during cold hyperalgesia in rats with neuropathic pain （NP）. Methods Thirty-two healthy adult male Sprague-Dawley rats, aged 10-12 weeks, weighing 250-280 g, in which intrathecal catheters were successfully implanted, were divided into 4 groups（n=8 each） using a random number table： sham operation plus GFRα3 dsRNA group（Sham＋dsRNA group）, sham operation plus GFRα3 siRNA group（group Sham＋siRNA）, NP plus GFRα3 dsRNA group（group NP＋dsRNA）and NP plus GFRα3 siRNA group（group NP＋siRNA）. NP was produced by chronic constriction injury to the sciatic nerve. At 10-30 days after operation, GFRα3 dsRNA 10 μg∕20 μl was intrathecally injected once a day for 4 consecutive days in Sham＋dsRNA and NP＋dsRNA groups, and 10 μg∕20 μl GFRα3 siRNA, of which the sense strand was modified with 2′-O-methyl and 5′-cholesterol, was intrathe-cally injected once a day for 4 consecutive days in Sham＋siRNA and NP＋siRNA groups. The number of paw lifts on the cold plate, mechanical paw withdrawal threshold（MWT）and thermal paw withdrawal latency （TWL）were measured on 1 day before operation and 10, 11, 12, 13（before intrathecal injection）and 14 days after operation. The rats were sacrificed after the last behavioral testing, and ipsilateral DRGs of the lumbar segment（L4-6）were dissected for detection of the expression of GFRα3 and TRPM8 in total and membrane proteins by Western blot, and the ratio of TRPM8 expression in the membrane protein to that in the total protein（m∕t ratio）was calculated. Results Compared with group Sham＋dsRNA, the number of paw lifts on the cold plate was significantly increased, the MWT was decreased, and TWL was shortened after operation in NP＋dsRNA and NP＋siRNA groups, the expression of GFRα3 and TRPM8 in total and membrane proteins was significantly up-regulated, and m∕t ratio was increased in group NP＋dsRNA, and the expression of GFRα3 in DRGs was significantly down-regulated（P〈0.01）, and no significant change was found in the expression of TRPM8 in total and membrane proteins or m∕t ratio in group NP＋siRNA（P〉0.05）. Compared with group NP＋dsRNA, the number of paw lifts on the cold plate was significantly de-creased, the expression of GFRα3 and TRPM8 in total and membrane proteins was down-regulated, m∕t ra-tio was decreased（P〈0.01）, and no significant change was found in MWT or TWL in group NP＋siRNA （P〉0.05）. Conclusion GFRα3 in DRGs can up-regulate the expression of TRPM8 and enhance the membrane trafficking of TRPM8, which may be involved in the maintenance mechanism of cold hyperalgesia in rats with NP.