迷走神经刺激减轻内毒素致小鼠急性肺损伤时α7nAChR信号通路与调节性T细胞的关系

Relationship between α7nAChR signaling pathway and regulatory T cells during vagus nerve stimulation-induced reduction of endotoxin-caused acute lung injury in mice

目的评价迷走神经刺激对内毒素致小鼠急性肺损伤(ALI)时α7型烟碱乙酰胆碱受体(α7nAChR)信号通路与调节性T细胞(Treg细胞)的关系。方法清洁级健康雄性C57BL/6小鼠50只,6~8周龄,体重22~25g,采用随机数字表法分为5组(n=10):对照组(C组)、急性肺损伤组(ALI组)、迷走神经刺激组(VNS组)、α-银环蛇毒素组(α-BGT组)和迷走神经刺激+α-银环蛇毒素组(VNS+α-BGT组)。小鼠ALI模型制备成功后行迷走神经刺激,刺激强度0.5mA,频率20Hz,持续30s,每次电刺激脉冲间隔5min,共刺激60min。C组仅于气管内吹入等体积的无菌生理盐水,分离迷走神经但不行电刺激;ALI组制备ALI模型,分离迷走神经但不行电刺激;VNS组于ALI模型制备结束后1h行迷走神经刺激;α-BGT组腹腔注射α-银环蛇毒素1μg/kg,1h后制备ALI模型,分离迷走神经但不行电刺激;VNS+α-BGT组腹腔注射α-银环蛇毒素1μg/kg,1h后制备ALI模型,模型制备结束后1h行迷走神经刺激。于模型制备后72h,处死小鼠,取肺组织,测定肺组织含水量,流式细胞术检测Treg细胞比例,比色法检测MPO活性,Westernblot法检测α7nAChR的表达,ELISA法检测IL-10、TGF-β和IL-1β含量。结果与C组比较,余4组肺组织含水量、MPO活性和IL-1β含量升高,ALI组、α-BGT组和VNS+α-BGT组α7nAChR表达下调,VNS组Treg细胞比例升高,ALI组和α-BGT组IL-10含量降低,VNS组和VNS+α-BGT组IL-10含量升高,ALI组、α-BGT组和VNS+α-BGT组TGF-β含量降低,VNS组TGF-β含量升高(P<0.05);与ALI组比较,VNS组肺组织含水量、MPO活性和IL-1β含量降低,α7nAChR表达上调,Treg细胞比例、IL-10和TGF-β含量升高,α-BGT组TGF-β含量、VNS+α-BGT组IL-10和TGF-β含量升高(P<0.05);与VNS组比较,α-BGT组和VNS+α-BGT组肺组织含水量、MPO活性、IL-1β含量升高,α7nAChR表达下调,Treg细胞比例、IL-10含量和TGF-β含量降低(P<0.05);与α-BGT组比较,VNS+α-BGT组肺组织IL-10和TGF-β含量升高(P<0.05)。结论迷走神经刺激可激活α7nAChR通路升高肺组织Treg细胞比例,从而减轻小鼠内毒素性ALI。

Objective To evaluate the relationship between α7 nicotinic acetylcholine receptor (α7nAChR) signaling pathway and regulatory T cells (Tregs) during vagus nerve stimulation-induced reduction of endotoxin-caused acute lung injury (ALI) in mice. Methods Clean-grade healthy male C57BL/6 mice, aged 6-8 weeks, weighing 22-25 g, were divided into 5 groups (n=10 each) using a random number table method: control group (group C), group ALI, vagus nerve stimulation group (group VNS),α-BGT group (group α-BGT) and vagus nerve stimulation plus α-BGT group (group VNS +α-BGT). After successful establishment of the model, the vagus nerve was stimulated for 30 s with a stimulus intensity of 0.5 mA, frequency of 20 Hz, an interval of 5 min, 60 min in total.Sterile normal saline 100 μl was injected into the trachea, and the vagus nerve was only exposed but not stimulated in group C. In group ALI, the ALI model was established, and the vagus nerve was isolated but not stimulated.In group α-BGT,α-BGT 1 μg/kg was intraperitoneally injected, the ALI model was established 1 h later, and the vagus nerve was isolated but not stimulated.In group VNS+α-BGT,α-BGT 1 μg/kg was intraperitoneally injected, the ALI model was established 1 h later, and the vagus nerve was stimulated at 1 h after the end of establishment.Animals were sacrificed at 72 h after establishing the model, and lungs were removed for determination of lung water content, percentage of Tregs (using flow cytometry), myeloperoxidase (MPO) activity (by colorimetric assay), expression of α7nAChR (by Western blot) and contents of interleukin-10 (IL-10), transforming growth factor-β(TGF-β) and IL-1β(by enzyme-linked immunosorbent assay). Results Compared with group C, the lung water content, MPO activity and IL-1β content were significantly increased in the other four groups, the expression of α7nAChR was significantly down-regulated in ALI,α-BGT and VNS+α-BGT groups, the percentage of Tregs was significantly increased in group VNS, the IL-10 content was significantly decreased in ALI and α-BGT groups and increased in VNS and VNS+α-BGT groups, and TGF-β contents were significantly decreased in ALI,α-BGT and VNS+α-BGT groups and increased in group VNS (P<0.05). Compared with group ALI, the lung water content, MPO activity and IL-1β content were significantly decreased, the expression of α7nAChR was up-regulated, and the percentage of Tregs and contents of IL-10 and TGF-β were increased in group VNS, and the TGF-β content in group α-BGT and contents of IL-10 and TGF-β in group VNS+α-BGT were significantly increased (P<0.05). Compared with group VNS, the lung water content, MPO activity and IL-1β content were significantly increased, the expression of α7nAChR was down-regulated, and the percentage of Tregs and contents of IL-10 and TGF-β were decreased in α-BGT and VNS+α-BGT groups (P<0.05). The contents of IL-10 and TGF-β were significantly higher in group VNS+α-BGT than in group α-BGT (P<0.05). Conclusion Vagus nerve stimulation can activate α7nAChR signaling pathway and raise the percentage of Tregs, thus reducing ALI in mice.