瑞芬太尼诱发切口痛大鼠痛觉过敏与脊髓背角TMEM16C和Slack通道的关系

Relationship between remifentanil-induced hyperalgesia and TMEM16C and Slack channel in spinal dorsal horn of rats with incisional pain

目的评价瑞芬太尼诱发切口痛大鼠痛觉过敏与脊髓背角Anotamins家族成员膜蛋白16C(TMEM16C)和Slack通道的关系。方法雄性SD大鼠48只,1月龄。实验Ⅰ取24只大鼠,采用随机数字表法分为4组(n=6):生理盐水组(S组)、病毒载体组(V组)、病毒载体+瑞芬太尼+切口痛组(VRI组)和AAV5-TMEM16C过表达+瑞芬太尼+切口痛组(ORI组)。经L4,5脊髓背角注射生理盐水(S组)、病毒载体(V组和VRI组)或AAV5-TMEM16C(ORI组) 1 μl,30 d时VRI组和ORI组尾静脉输注瑞芬太尼1 μg·kg-1·min-160 min,同时建立切口痛模型。于输注瑞芬太尼前24 h(T0)、输注停止后2、6、24和48 h (T1-4)时测定热缩足潜伏期(TWL)和机械缩足反应阈(MWT)。于行为学测定结束后,取脊髓背角L4,5节段,采用Western blot法测定总蛋白及膜蛋白TMEM16C和Slack的表达。实验Ⅱ 取24只大鼠,采用随机数字表法分为4组(n=6):生理盐水+人工脑脊液(ACSF)组(SA组)、病毒载体+ACSF(VA组)、病毒载体+瑞芬太尼组(VR组)和AAV5-TMEM16C过表达+瑞芬太尼组(OR组)。经L4,5脊髓背角注射生理盐水(SA组)、病毒载体(VA组和VR组)或AAV5-TMEM16C(OR组) 1 μl,30 d时取L4,5脊髓切片在ACSF(NSA组和VA组)或含有4 nmol/L瑞芬太尼的ACSF中(VR组和OR组)孵育60 min。各组孵育结束后应用全细胞膜片钳测定Slack通道电流的频率和振幅。结果实验Ⅰ与S组比较,VRI组T1-4时TWL缩短,MWT降低,脊髓背角总蛋白及膜蛋白TMEM16C和Slack表达下调(P<0.05);与VRI组比较,ORI组T1-4时TWL延长,MWT升高,脊髓背角总蛋白及膜蛋白TMEM16C和Slack表达上调(P<0.05)。实验Ⅱ与SA组比较,VR组脊髓背角Slack通道电流振幅和频率降低(P<0.05);与VR组比较,OR组Slack电流的振幅和频率升高(P<0.05)。结论瑞芬太尼诱发切口痛大鼠痛觉过敏形成的机制与下调脊髓背角MEM16C表达,进而下调Slack通道表达有关。

Objective To evaluate the relationship between remifentanil-induced hyperalgesia and Antamins family member membrane protein 16C (TMEM16C) and Slack channel in the spinal dorsal horn of rats with incisional pain. Methods Forty-eight male Sprague-Dawley rats, aged 1 month, were studied.The study was performed in two parts.Experiment Ⅰ Twenty-four rats were divided into 4 groups (n=6 each) using a random number table method: normal saline group (S group), virus vector group (V group), virus vector plus remifentanil plus incisional pain group (VRI group), and AAV5-TMEM16C over-expression plus remifentanil plus incisional pain group (ORI group). Normal saline (S group), virus vector (V group and VRI group) or AAV5-TMEM16C (ORI group) 1 μl was injected via the L4, 5 spinal dorsal horn.Remifentanil 1 μg·kg-1·min-1 was infused for 60 min via the caudal vein at 30 days, and the incisional pain model was simultaneously established in VRI and ORI groups.The thermal paw withdrawal latency (TWL) and mechanical paw withdrawal threshold (MWT) were measured at 24 h before remifentanil infusion (T0) and 2, 6, 24, and 48 h after stopping infusion (T1-4). The L4, 5 segments of spinal dorsal horns were obtained at the end of the behavioral testing, and the expression of TMEM16C and Slack in total and membrane proteins was determined by Western blot.Experiment Ⅱ Twenty-four rats were divided into 4 groups (n=6 each) using a random number table method: normal saline plus artificial cerebrospinal fluid (ACSF) group (SA group), virus vector plus ACSF group (VA group), virus vector plus remifentanil group (VR group), and AAV5-TMEM16C over-expression plus remifentanil group (OR group). Normal saline (SA group), virus vector (VA group and VR group) or AAV5-TMEM16C (OR group) 1 μl were injected via the spinal dorsal horn at the level of L4, 5.L4, 5 spinal cord slices were obtained at 30 days and incubated for 60 min in ACSF (SA and VA groups) or in ACSF containing 4 nmol/L remifentanil (VR group and OR group). The whole-cell patch-clamp was used to measure the frequency and amplitude of the Slack channel current after the end of incubation in each group. Results Experiment Ⅰ Compared with S group, the TWL was significantly shortened and the MWT was decreased at T1-4, and the expression of TMEM16C and Slack in total and membrane proteins was down-regulated in VRI group (P<0.05). Compared with VRI group, the TWL was significantly prolonged and the MWT was increased at T1-4, and the expression of TMEM16C and Slack in total and membrane proteins was up-regulated in ORI group (P<0.05). Experiment Ⅱ Compared with SA group, the amplitude and frequency of Slack channel current in spinal dorsal horns were significantly decreased in VR group (P<0.05). Compared with VR group, the amplitude and frequency of the Slack current were significantly increased in OR group (P<0.05). Conclusion The mechanism of remifentanil-induced hyperalgesia is related to down-regulating the expression of TMEM16C and further down-regulating the expression of the Slack channel in the spinal dorsal horn of rats with incisional pain.