卡巴胆碱对内毒素血症小鼠肠道屏障功能的影响

Protective effects of carbachol on lipopolysaccharide-induced intestinal barrier injury

目的观察卡巴胆碱对内毒素血症小鼠肠道屏障功能的保护作用及机制。方法将C57BL/6小鼠随机分为对照组、内毒素血症组、卡巴胆碱组、α银环蛇毒素组,每组10只。腹腔注射10 mg/kg内毒素建立内毒素血症模型。模型制备后15 min腹腔注射卡巴胆碱0.1 mg/kg或0.9%氯化钠溶液,内毒素注射后3 h处死动物。取距回盲瓣10 cm处回肠组织,光镜下观察回肠组织病理学改变,测定肠道组织通透性,检测紧密连接蛋白(Claudin-2)、肌球蛋白轻链激酶(MLCK)定位及蛋白含量等。结果FITC-葡聚糖水平:对照组、内毒素血症组、卡巴胆碱组、α银环蛇毒素组分别为(2.33±0.51)μg/ml、(55.25±5.41)μg/ml、(19.27±3.53)μg/ml、(48.45±9.50)μg/ml,4组总体差异有统计学意义(F=111.8,P<0.05),其中内毒素血症组与对照组、内毒素血症组与卡巴胆碱组、α银环蛇毒素组与卡巴胆碱组比较,差异均有统计学意义(t分别为22.52、15.31、12.42,P均<0.05)。Claudin-2蛋白含量:对照组、内毒素血症组、卡巴胆碱组、α银环蛇毒素组分别为(0.82±0.08)μg/ml、(0.52±0.09)μg/ml、(0.77±0.05)μg/ml、(0.53±0.09)μg/ml,4组总体差异有统计学意义(F=11.61,P<0.05),其中内毒素血症组与对照组、内毒素血症组与卡巴胆碱组、α银环蛇毒素组与卡巴胆碱组比较,差异均有统计学意义(t分别为6.518、5.366、5.167,P均<0.05)。MLCK蛋白含量:对照组、内毒素血症组、卡巴胆碱组、α银环蛇毒素组分别为(0.58±0.07)μg/ml、(1.07±0.17)μg/ml、(0.69±0.11)μg/ml、(0.94±0.05)μg/ml,4组总体差异有统计学意义(F=12.64,P<0.05),其中内毒素血症组与对照组、内毒素血症组与卡巴胆碱组、α银环蛇毒素组与卡巴胆碱组比较,差异均有统计学意义(t分别为7.79、5.881、3.892,P均<0.05)。结论卡巴胆碱对内毒素血症小鼠肠道屏障功能有保护作用,可降低肠道通透性,其机制可能与激活胆碱能抗炎通路有关。

Objective To investigate the effects of carbachol on lipopolysaccharide(LPS)-induced intestinal barrier breakdown.Methods C57BL/6 mice were randomly divided into four groups(n=10 per group):control group,lipopolysaccharide group,carbachol group,andα-bungarotoxin group.Endotoxemia was induced by administering 10 mg/kg lipopolysaccharide via intraperitoneal injection.The mice were intraperitoneally treated with 0.1 mg/kg carbachol 15 min after LPS administration.Mice were sacrificed at 3 h after LPS administration for biochemical studies and histological examination.The localization and expression of Claudin-2 and myosin light chain kinase(MLCK),and pathologic changes of the ileum were examined.Results The levels of FITC-glucan in the control group,endotoxemia group,carbachol group,andαbungarotoxin group were(2.33±0.51)μg/ml,(55.25±5.41)μg/ml,(19.27±3.53)μg/ml,and(48.45±9.50)μg,respectively;there was a significant difference among the four groups(F=111.8,P<0.05),as well as between the endotoxemia group and control group,between the endotoxemia group and carbachol group,and between theαbungarotoxin group and carbachol group(t=22.52,15.31,and 12.42,P<0.05).The contents of Claudin-2 protein in the control group,endotoxemia group,carbachol group,andαbungarotoxin group were(0.82±0.08)μg/ml,(0.52±0.09)μg/ml,(0.77±0.05)μg/ml,and(0.53±0.09)μg,respectively;there was a significant difference among the four groups(F=11.61,P<0.05),as well as between the endotoxemia group and control group,between the endotoxemia group and carbachol group,and between theαbungarotoxin group and carbachol group(t=6.518,5.366,and 5.167,respectively,P<0.05).The contents of MLCK protein in the control group,endotoxemia group,carbachol group,andαbungarotoxin group were(0.58±0.07)μg/ml,(1.07±0.17)μg/ml,(0.69±0.11)μg/ml,and(0.94±0.05)μg,respectively;there was a significant difference among the four groups(F=12.64,P<0.05),as well as between the endotoxemia group and control group,between the endotoxemia group and carbachol group,and between theαbungarotoxin group and carbachol group(t=7.79,5.881,and 3.892,respectively,P<0.05).Conclusion Carbachol treatment can protect against LPS-induced intestinal barrier dysfunction and the protective effects are associated with the activation of the cholinergic anti-inflammatory pathway.