自噬在电针改善小鼠脓毒症相关性脑病中的作用

Role of autophagy in electroacupuncture-induced improvement in sepsis-associated encephalopathy in mice

目的:评价自噬在电针改善小鼠脓毒症相关性脑病(SAE)中的作用。方法:健康雄性C57BL/6小鼠135只,8~12周龄,体质量22~25 g,随机选取10只小鼠麻醉后取盲肠内容物制备盲肠浆液;其余125只小鼠采用随机数字表法分为5组(n=25):假手术组(Sham组)、SAE组、SAE+电针组(SAE+EA组)、SAE+电针+自噬激动剂雷帕霉素组(SAE+EA+R组)和SAE+电针+自噬抑制剂3-甲基腺苷组(SAE+EA+MA组)。腹腔注射小鼠盲肠内容物浆液200μl建立脓毒症模型。SAE+EA组、SAE+EA+R组和SAE+EA+MA组分别于造模后2、24、48和72 h时在双侧足三里穴行电针刺激,SAE+EA+R组和SAE+EA+MA组于电针刺激前30 min时分别腹腔注射雷帕霉素10 mg/kg和3-甲基腺苷15 mg/kg。记录小鼠造模后7 d的生存情况。于造模后8~12 d时每组取10只小鼠进行Morris水迷宫实验检测学习记忆能力;于造模后12 d时每组取5只小鼠麻醉处死后取海马组织,采用ELISA法检测IL-1β、IL-18及TNF-α含量,采用Western blot法检测p62、自噬相关16样蛋白1(ATG16L1)和NOD样受体蛋白3(NLRP3)的表达水平。结果:与Sham组比较,其余4组造模后7 d生存率降低(P<0.01);与SAE组、SAE+EA组、SAE+EA+R组和SAE+EA+MA组造模后7 d生存率差异无统计学意义(P>0.05)。与Sham组比较,SAE组目标象限活动时间缩短,逃避潜伏期延长,穿越原平台位置次数减少,海马组织TNF-α、IL-1β及IL-18含量升高,ATG16L1表达下调,p62及NLRP3表达上调(P<0.05)。与SAE组比较,SAE+EA组逃避潜伏期缩短,目标象限活动时间延长,穿越原平台位置次数增多,海马组织TNF-α、IL-1β及IL-18含量降低,ATG16L1表达上调,p62及NLRP3表达下调(P<0.05)。与SAE+EA组比较,SAE+EA+R组水迷宫实验各指标差异无统计学意义(P>0.05),海马组织TNF-α、IL-1β及IL-18含量降低,ATG16L1表达上调,p62及NLRP3表达下调,SAE+EA+MA组逃避潜伏期延长,目标象限活动时间缩短,穿越平台次数减少,海马组织TNF-α、IL-1β及IL-18含量升高,ATG16L1表达下调,p62及NLRP3表达上调(P<0.05)。结论:电针改善小鼠SAE的机制与促进海马神经元自噬,抑制NLRP3炎症小体激活,减轻神经炎症反应有关。

Objective To evaluate the role of autophagy in electroacupuncture(EA)-induced improvement in sepsis-associated encephalopathy(SAE)in mice.MethodsA total of 135 healthy adult male mice,aged 8-12 weeks,weighing 22-25 g,were used in this study.Ten mice were randomly selected to prepare caecal slurry after anesthesia.The remaining 125 mice were divided into 5 groups(n=25 each)using a random number table method:sham operation group(group Sham),SAE group,SAE+EA group(group EA),SEA+EA+autophagy agonist rapamycin group(group SAE+EA+R),and SAE+EA+autophagy inhibitor 3-methyladenine group(group SAE+EA+MA).SAE was induced by intraperitoneal injection of cecal slurry 200μl.Bilateral Zusanli(ST36)acupoints were stimulated at 2,24,48 and 72 h after surgery in group SAE+EA,group SAE+EA+R and group SAE+EA+MA.Autophagy agonist rapamycin 10 mg/kg and autophagy inhibitor 3-methyladenine 15 mg/kg were intraperitoneally injected at 30 min before EA in SAE+EA+R group and SAE+EA+MA group,respectively.The survival of mice was recorded at 7 days after developing the model.Ten mice were selected from each group at 8-12 days after developing the model,and the learning and memory ability was assessed by Morris water maze test.Five mice from each group were sacrificed after anesthesia,brains were removed,and hippocampal tissues were obtained for determination of contents of interleukin-1beta(IL-1β),IL-18 and tumor necrosis factor-α(TNF-α)(by enzyme-linked immunosorbent assay)and expression of p62,autophagy-related protein 16 like protein 1(ATG16L1),and nucleotide like receptor protein 3(NLRP3)(by Western blot).ResultsCompared with Sham group,the survival rate at 7 days after developing the model was significantly decreased in the other 4 groups(P<0.01).There was no significant difference in the survival rate at 7 days after developing the model among SAE group,SAE+EA group,SAE+EA+R group and SAE+EA+MA group(P>0.05).Compared with Sham group,the activity time at the target quadrant was significantly shortened,the escape latency was prolonged,the number of crossing the original platform was reduced,the contents of TNF-α,IL-1βand IL-18 were increased,the expression of ATG16L1 was down-regulated,and the expression of p62 and NLRP3 was up-regulated in SAE group(P<0.05).Compared with SAE group,the escape latency was significantly shortened,the activity time at the target quadrant was prolonged,the number of crossing the original platform was increased,the contents of TNF-α,IL-1βand IL-18 were decreased,the expression of ATG16L1 was up-regulated,and the expression of p62 and NLRP3 was down-regulated in SAE+EA group(P<0.05).Compared with SAE+EA group,no significant change was found in the parameters of Morris water maze test(P>0.05),the contents of TNF-α,IL-1βand IL-18 were significantly decreased,the expression of ATG16L1 was up-regulated,and the expression of NLRP3 and P62 was down-regulated in SAE+EA+R group,and the expression of ATG16L1 was significantly down-regulated,and the expression of p62 and NLRP3 was up-regulated in SAE+EA+MA group(P<0.05).ConclusionsThe mechanism by which EA improves SAE is related to promotion of autophagy in hippocampal neurons,inhibition of NLRP3 inflammasome activation,and alleviation of neuroinflammatory responses in mice.