自噬在二甲双胍缓解小鼠切口痛中的作用

Role of autophagy in metformin relieving incisional pain in a mouse model

目的探讨自噬在二甲双胍缓解小鼠切口痛中的作用。方法选择4～6周清洁级健康成年雄性ICR小鼠,体重18～22 g。采用随机数字表法分为5组:对照组(C组)、二甲双胍组(M组)、切口痛组(I组)、切口痛+二甲双胍组(IM组)、切口痛+二甲双胍+自噬抑制剂三甲基腺嘌呤组(MA组),每组8只。C组、M组不造模。I组、IM组及MA组小鼠建立切口痛模型。M组、IM组、MA组从造模当天开始腹腔注射二甲双胍200 mg/kg,每日重复给药至造模后7 d。C组每日同一时点腹腔注射等容量生理盐水。MA组每日于腹腔注射二甲双胍30 min前预给予30 mg/kg三甲基腺嘌呤。分别于造模前1 d,造模后1、3、5、7 d测定机械缩足阈值(MWT);造模后30 d,深麻醉下处死小鼠,取L_4—L_6脊髓膨大节段组织,采用Western blot法测定促炎细胞因子IL-1β和TNF-α浓度,自噬相关蛋白LC3Ⅱ和p62蛋白含量。结果与造模前1 d比较,造模后1、3、5 dⅠ组、IM组和MA组MWT明显降低(P<0.05)。造模后1、3、5 dⅠ组、IM组、MA组MWT明显低于C组(P<0.05);IM组MWT明显高于Ⅰ组(P<0.05);MA组MWT明显低于IM组(P<0.05)。Ⅰ组IL-1β和TNF-α浓度明显高于C组(P<0.05);IM组IL-1β和TNF-α浓度明显低于I组(P<0.05);MA组IL-1β和TNF-α浓度明显高于IM组(P<0.05)。M组和I组LC3Ⅱ蛋白含量明显高于C组,p62蛋白含量明显低于C组(P<0.05);IM组LC3Ⅱ蛋白含量明显高于I组,p62蛋白含量明显低于I组(P<0.05);MA组LC3Ⅱ蛋白含量明显低于IM组,p62蛋白含量明显高于IM组(P<0.05)。结论二甲双胍可抑制脊髓的神经炎症反应,缓解小鼠切口痛,其作用机理可能是通过激活自噬改善神经炎症。

Objective To evaluate the effects of autophagy on metformin relieving incisional pain in a mouse model.Methods SPF adult male ICR mice(aged 4-6 weeks,weighing 18-22 g)were divided into 5 groups by using random number table(n=8):control group(group C),metformin group(group M),plantar incision group(group I),plantar incision+metformin group(group IM),and plantar incision+metformin+autophagy inhibitor 3-methyladenine group(group MA).Group C and group M did not make models,while group I,group IM and group MA established incision pain model.Group M,group IM,and group MA were administered with metformin 200 mg/kg by intraperitoneal injection,while group C were administered with the same volume of normal saline by intraperitoneal injection.Group MA was pre-administered with 30 mg/kg 3-methyladenine 30 minutes before intraperitoneal injection of metformin.The above administration was repeated daily until the 7th day after modeling from the day of modeling.The mechanical withdraw threshold(MWT)was measured 1 day before and 1,3,5 and 7 d after the modeling.3 days after modeling,the mice were executed under deep anesthesia,and the L4-L6 spinal cord enlarged segments were taken out.Western blot was used to detect expression quantity of IL-1β,TNF-α,LC3II and p62.Results Compared with 1 d before modeling,the MWT of group I,group IM and group MA decreased significantly after modeling1,3,5 d(P<0.05);At 1,3,5 d after modeling,the MWT of group I,group IM and group MA was significantly lower than that of group C(P<0.05);The MWT of group IM was significantly higher than that of group I(P<0.05);The MWT of MA group was significantly lower than that of group IM(P<0.05).The contents of IL-1βand TNF-αin group I were significantly higher than those in group C(P<0.05);The contents of IL-1βand TNF-αin group IM were significantly lower than those in group I(P<0.05);The content of IL-1βand TNF-αin group MA was significantly higher than that in group IM(P<0.05).In group M and group I,the content of LC3II was significantly higher than that in group C,and the content of p62 was significantly lower than that in group C(P<0.05).In group IM,the content of LC3II was significantly higher than that in group I,and the content of p62 was significantly lower than that in group I(P<0.05);In group MA,the content of LC3II was significantly lower than that in group IM,and the content of p62 was significantly higher than that in group IM(P<0.05).Conclusion The mechanism of metformin in relieving incisional pain may be that it reduces the neuroinflammation by activating autophagy.