自噬在富氢液减轻瑞芬太尼诱发切口痛大鼠痛觉过敏中的作用

Role of autophagy in hydrogen-rich solution-induced reduction of remifentanil-induced hyperalgesia in rats

目的评价自噬在富氢液减轻瑞芬太尼诱发切口痛大鼠痛觉过敏中的作用。方法清洁级健康雄性SD大鼠32只,2~3月龄,体质量240~260 g,采用随机数字表法分为4组(n=8):切口痛组(I组)、瑞芬太尼+切口痛组(RI组)、富氢液组+瑞芬太尼+切口痛组(HRI组)和富氢液+自噬抑制剂+瑞芬太尼+切口痛组(MHRI组)。大鼠经尾静脉穿刺置管,I组建立切口痛模型的同时静脉输注等容量生理盐水60 min;RI组、HRI组和MHRI组建立切口痛模型的同时静脉输注瑞芬太尼1μg·kg^(-1)·min^(-1)60 min;HRI组于造模前10 min时腹腔注射富氢液10 ml/kg;MHRI组于造模前1 h腹腔注射自噬抑制剂三甲基腺嘌呤15 mg/kg,余处理同HRI组。分别于输注瑞芬太尼或生理盐水前24 h(T0)、输注结束后2、6、24和48 h(T_(1-4))时测定机械缩足反应阈(MWT)及热缩足潜伏期(TWL)。行为学测试结束后麻醉状态下处死大鼠,取脊髓腰膨大节段,采用Western blot法测定微管相关蛋白1轻链3Ⅱ(LC3Ⅱ)、Beclin-1和P62的表达。结果与T0时比较,4组T_(1-4)时MWT降低,TWL缩短(P<0.05);与I组比较,RI组和HRI组T_(1-4)时MWT降低,TWL缩短,脊髓LC3Ⅱ和Beclin-1表达上调,P62表达下调(P<0.05);与RI组比较,HRI组和MHRI组T_(1-4)时MWT升高,TWL延长,HRI组脊髓LC3Ⅱ和Beclin-1表达上调,P62表达下调,MHRI组脊髓LC3Ⅱ和Beclin-1表达下调,P62表达上调(P<0.05);与HRI组比较,MHRI组T_(1-4)时MWT降低,TWL缩短,脊髓LC3Ⅱ和Beclin-1表达下调,P62表达上调(P<0.05)。结论富氢液减轻瑞芬太尼诱发切口痛大鼠痛觉过敏的机制可能与增强脊髓自噬水平有关。

Objective To evaluate the role of autophagy in hydrogen-rich solution-induced reduction of remifentanil-induced hyperalgesia in rats.Methods Thirty-two clean-grade healthy male Sprague-Dawley rats,aged 2-3 months,weighing 240-260 g,were divided into 4 groups(n=8 each)by a random number table method:incisional pain group(group Ⅰ),remifentanil+incisional pain group(group RI),hydrogen-rich solution+remifentanil+incisional pain group(group HRI),and hydrogen-rich solution+autophagy inhibitor+remifentanil+incisional pain group(MHRI group).The tail vein was catheterized,the equal volume of normal saline was intravenously infused for 60 min while the incisional pain model was developed in group Ⅰ,and remifentanil was intravenously infused at a rate of 1μg·kg^(-1)·min^(-1) for 60 min while the incisional pain model was developed in RI,HRI and MHRI groups,hydrogen-rich solution 10 ml/kg was intraperitoneally injected at 10 min before preparing the model in group HRI,and 3-MA 15 mg/kg was intraperitoneally injected at 1 h before preparing the model in MHRI group,and the other treatments were similar to those previously described in group HRI.The mechanical paw withdrawal threshold(MWT)and thermal paw withdrawal latency(TWL)were determined at 24 h before and 2,6,24 and 48 h after the end of infusion.The rats were sacrificed under anesthesia after the behavioral testing,and the lumbar enlargement segment of the spinal cord was removed for determination of the expression of microtubule-associated protein 1 light chain 3Ⅱ(LC3Ⅱ),Beclin-1 and P62 by Western blot.Results Compared with the baseline at T0,the MWT was significantly decreased and TWL was shortened at T_(1-4) in the four groups(P<0.05).Compared with group Ⅰ,the MWT was significantly decreased and TWL was shortened at T_(1-4),the expression of LC3Ⅱ and Beclin-1 was up-regulated,and the expression of P62 was down-regulated in group RI and group HRI(P<0.05).Compared with group RI,the MWT was significantly increased and TWL was prolonged at T_(1-4) in group HRI and group MHRI,the expression of LC3Ⅱ and Beclin-1 was significantly up-regulated,and the expression of P62 was down-regulated in group HRI,and the expression of LC3Ⅱ and Beclin-1 was significantly down-regulated,and the expression of P62 was up-regulated in group MHRI(P<0.05).Compared with group HRI,the MWT was significantly decreased and TWL was shortened at T_(1-4),the expression of LC3Ⅱ and Beclin-1 was down-regulated,and the expression of P62 was up-regulated in group MHRI(P<0.05).Conclusions The mechanism by which hydrogen-rich solution alleviates hyperalgesia may be related to enhancing the level of autophagy in the spinal cord of rats with incisional pain induced by remifentanil.