小鼠肠缺血再灌注时肠黏膜肥大细胞与肠道菌群的关系

Relationship between intestinal mucosal mast cells and intestinal flora during intestinal ischemia-reperfusion in mice

目的评价小鼠肠缺血再灌注时肠黏膜肥大细胞与肠道菌群的关系。方法健康雄性C57BL/6小鼠48只,9~12周龄,体重20~25 g,采用随机数字表法分为4组(n=12):假手术组(Sham组)、假手术+肥大细胞膜稳定剂色甘酸钠组(Sham+CS组)、肠缺血再灌注组(I/R组)和肠缺血再灌注+色甘酸钠组(I/R+CS组)。采用夹闭肠系膜上动脉45 min再灌注4 h的方法制备肠缺血再灌注损伤模型。Sham+CS组和I/R+CS组于模型制备前2周腹腔注射色甘酸钠50 mg/kg,1次/d。再灌注结束后处死小鼠,取小肠组织,HE染色后光镜下观察肠黏膜病理学结果,采用Chiu评分法评估肠组织损伤程度。采用免疫组化SP染色法检测肥大细胞类胰蛋白酶的表达水平,并行肥大细胞计数。取肠内容物,采用1%琼脂糖凝胶电泳检测肠道菌群的细菌总量,并用16S rDNA测序法测定小鼠粪便中肠道细菌的多样性(Chao1指数和Shannon指数)和相对丰度。结果与Sham组比较,I/R组肠组织Chiu评分和肥大细胞计数升高,类胰蛋白酶表达上调,肠道细菌总量升高,Chao1指数和Shannon指数降低,拟杆菌门、变形菌门、拟杆菌科和肠杆菌科相对丰度增加,厚壁菌门、乳酸菌科和双歧杆菌科的相对丰度减少(P<0.05或0.01),Sham+CS组上述指标差异无统计学意义(P>0.05);与I/R组比较,I/R+CS组肠组织Chiu评分和肥大细胞计数降低,类胰蛋白酶表达下调,肠道细菌总量降低,Chao1指数和Shannon指数升高,拟杆菌门、变形菌门、拟杆菌科和肠杆菌科的相对丰度减少,厚壁菌门、乳酸菌科和双歧杆菌科的相对丰度增加(P<0.05或0.01)。结论肠黏膜肥大细胞激活可导致肠道菌群失调,降低益生菌数量,增加潜在致病菌数量,从而参与小鼠肠缺血再灌注损伤的病理生理机制。

Objective To evaluate the relationship between intestinal mucosal mast cells and intestinal flora during intestinal ischemia-reperfusion(I/R)in mice.Methods Forty-eight healthy male C57BL/6 mice,aged 9-12 weeks,weighing 20-25 g,were divided into 4 groups(n=12 each)using a random number table method:sham operation group(group Sham),sham operation plus mast cell membrane stabilizer cromolyn sodium(CS)group(group Sham+CS),I/R group and I/R+CS group.The intestinal I/R injury was induced by clamping the superior mesenteric artery for 45 min followed by 4 h of reperfusion in I/R and I/R+CS groups.The superior mesenteric artery was only exposed but not occluded in Sham and Sham+CS groups.CS 50 mg/kg was intraperitoneally injected once a day for two weeks before surgery in Sham+CS and I/R+CS groups.The equal volume of normal saline was intraperitoneally injected once a day for two weeks before surgery in Sham and I/R groups.Mice were then sacrificed and intestinal tissues were harvested for examination of the pathological changes of intestinal mucosa with a light microscope after HE staining,and the damage to intestinal tissues was assessed and scored according to Chiu.The expression of mast cell tryptase was detected using the immunohistochemical SP staining method,and the mast cells were counted.The intestinal contents were collected,the total amount of bacteria in intestinal flora was detected by 1%agarose gel electrophoresis,and the diversity(Chao1 index and Shannon index)and relative abundance of intestinal microbial bacteria were detected by 16S rDNA sequencing method.Results Compared with Sham group,the Chiu′s score and mast cell count were significantly increased,the expression of mast cell tryptase was up-regulated,the total amount of intestinal flora bacteria was increased,Chao1 index and Shannon index were decreased,the relative abundance of Bacteroidetes,Proteobacteria,Bacteroides and Enterobacteriaceae were increased,and the relative abundance of Firmicutes,Lactobacillus and Bifidobacteria was reduced in I/R group(P<0.05 or 0.01),and no significant change was found in the parameters mentioned above in Sham+CS group(P>0.05).Compared with I/R group,the Chiu′s score and mast cell count were significantly decreased,the expression of mast cell tryptase was down-regulated,the total amount of intestinal flora bacteria was decreased,Chao1 index and Shannon index were increased,the relative abundance of Bacteroidetes,Proteobacteria,Bacteroides and Enterobacteriaceae were reduced,and the relative abundance of Firmicutes,Lactobacillus and Bifidobacteria was increased in I/R+CS group(P<0.05 or 0.01).Conclusion The activation of intestinal mucosal mast cell can lead to imbalance of intestinal flora,decrease the number of probiotics and increase the number of potential pathogenic bacteria,and thus be involved in the pathophysiological mechanism of intestinal I/R injury in mice.