电刺激迷走神经对脓毒症小鼠肠道通透性的作用及机制研究

Effect and mechanism of vagus nerve electrical stimulation on intestinal permeability in sepsis mice

目的探讨电刺激迷走神经对脓毒症小鼠肠道通透性的作用及机制。方法将50只C57BL/6小鼠随机分为对照组(CON组)、脓毒症组[脂多糖(LPS)组]、迷走神经切断组(VGX组)、电刺激迷走神经组(STM组)、α-银环蛇毒素(α-BGT)组,每组10只。LPS组、VGX组、STM组、α-BGT组均腹腔注射10 mg/kg LPS制备脓毒症模型,CON组则腹腔注射等体积生理盐水。CON组、LPS组仅暴露双侧颈迷走神经,VGX组、STM组、α-BGT组均切断双侧颈迷走神经,STM组电刺激左颈迷走神经,α-BGT组皮下注射α-BGT后再电刺激左颈迷走神经。处死各组小鼠后取回肠组织,光镜下进行组织病理学观察,评估肠道组织通透性,并检测紧密连接蛋白(ZO-1、claudin-2)、肌球蛋白轻链激酶(MLCK)和核因子-κB(NF-κB)蛋白相对表达量。结果LPS组小鼠回肠组织肠黏膜上皮细胞损伤严重,微绒毛坏死、脱落,大量炎性细胞浸润,VGX组回肠组织病理学改变程度较LPS组加重,STM组回肠组织病理学改变程度较LPS组、VGX组减轻,α-BGT组肠黏膜上皮细胞损伤最严重。与CON组比较,LPS组、VGX组小鼠肠上皮组织通透性均增加,差异有统计学意义(P<0.05);与LPS组、VGX组比较,STM组小鼠肠上皮组织通透性降低,差异有统计学意义(P<0.05);与STM组比较,α-BGT组小鼠肠上皮组织通透性增加,差异有统计学意义(P<0.05)。LPS组、VGX组小鼠肠上皮组织中ZO-1、claudin-2蛋白相对表达量低于CON组,STM组小鼠肠上皮组织中ZO-1、claudin-2蛋白相对表达量高于LPS组、VGX组,α-BGT组小鼠肠上皮组织中ZO-1、claudin-2蛋白相对表达量低于STM组,差异均有统计意义(P<0.05)。LPS组、VGX组小鼠肠上皮组织中MLCK、NF-κB蛋白相对表达量高于CON组,STM组小鼠肠上皮组织中MLCK、NF-κB蛋白相对表达量低于LPS组、VGX组,α-BGT组小鼠肠上皮组织中MLCK、NF-κB蛋白相对表达量高于STM组,差异均有统计意义(P<0.05)。结论电刺激迷走神经有助于保护脓毒症小鼠的肠道屏障功能,其潜在机制或与激活胆碱能抗炎通路,进而抑制炎症反应相关。

Objective To investigate the effect and mechanism of vagus nerve electrical stimulation on intestinal permeability in sepsis mice.Methods Fifty C57BL/6 mice were randomly divided into control group(CON group),sepsis group[lipopolysaccharide(LPS)group],vagus nerve transection group(VGX group),vagus nerve electrical stimulation group(STM group),andα-bungarotoxin(α-BGT)group,with 10 mice in each group.The LPS group,VGX group,STM group,andα-BGT group were injected intraperitoneally with 10 mg/kg LPS to prepare the sepsis model,while the CON group was injected intraperitoneally with an equal volume of normal saline.The bilateral cervical vagus nerves were exposed only in the CON and LPS groups,while they were transected in the VGX,STM,andα-BGT groups.In the STM group,the left cervical vagus nerve was electrically stimulated,andα-BGT was injected subcutaneously before electrical stimulation of the left cervical vagus nerve in theα-BGT group.The ileum tissues were collected from mice in each group after euthanasia,and were performed histopathological observations under a light microscope to evaluate intestinal tissue permeability and detect the relative expression levels of tight junction proteins(ZO-1,claudin-2),myosin light chain kinase(MLCK),and nuclear factor-κB(NF-κB)proteins.Results The ileal mucosa epithelial cells in the LPS group were severely damaged,with necrosis and shedding of microvilli and massive inflammatory cell infiltration.The degree of histopathological changes in the ileum tissue in the VGX group was more severe than in the LPS group,while was less severe in the STM group compared to the LPS and VGX groups.The intestinal mucosa epithelial cells were most severely damaged in theα-BGT group.Compared with the CON group,the intestinal epithelial tissue permeability was increased in the LPS and VGX groups(P<0.05).Compared with the LPS and VGX groups,the intestinal epithelial tissue permeability was decreased in the STM group(P<0.05).Compared with the STM group,the intestinal epithelial tissue permeability was increased in theα-BGT group(P<0.05).The relative expression levels of ZO-1 and claudin-2 proteins in the intestinal epithelial tissues were lower in the LPS and VGX groups than in the CON group,while they were higher in the STM group compared to the LPS and VGX groups,meanwhile,they were lower in theα-BGT group than in the STM group(P<0.05).The relative expression levels of MLCK and NF-κB proteins in the intestinal epithelial tissues were higher in the LPS and VGX groups than in the CON group,while they were lower in the STM group compared to the LPS and VGX groups,and were higher in the intestinal epithelial tissues in theα-BGT group than in the STM group(P<0.05).Conclusion Electrical stimulation of the vagus nerve helps protecting the intestinal barrier function in sepsis mice,and its potential mechanism may be related to the activation of the cholinergic anti-inflammatory pathway that further inhibits the inflammatory response.