乳腺炎小鼠肠道菌群改变及与炎性因子的相关性分析

Changes of intestinal flora and its correlation with inflammatory factors in mastitis mice

目的采用高通量测序技术分析乳腺炎小鼠肠道菌群的丰度特征与组织炎性因子的关系。方法选用24只泌乳10d的昆明雌鼠,随机分成4组(每组6只):健康对照组、低剂量攻菌组(含金黄色葡萄球菌103CFU/25μL)、中剂量攻菌组(含金黄色葡萄球菌104CFU/25μL)和高剂量攻菌组(含金黄色葡萄球菌105CFU/25μL)。用非损伤方法经乳头管分别注入不同浓度的金黄色葡萄球菌悬浊液于小鼠的第4对(腹部)乳腺内。观察小鼠、组织病理学变化以及乳腺组织中TNF-α和Cav-1含量变化;收集小鼠粪便,采用高通量测序技术对小鼠粪便菌群进行分析。结果(1)与健康对照组小鼠相比,高剂量攻菌组乳腺组织呈明显的炎性病理变化,TNF-α表达明显升高(P<0.05),Cav-1表达明显降低(P<0.05);(2)不同组小鼠肠道菌群物种丰度不同,对照组肠道菌群以拟杆菌门和厚壁菌门为主;与对照组相比,高剂量攻菌组乳腺炎模型小鼠厚壁菌门丰度增高,拟杆菌门、疣微菌门丰度降低,变形菌门无显著差异;进一步比较后发现,从属以上水平看,后者拟杆菌属、另枝菌属、普雷沃菌属丰度相对较低。结论提示乳腺炎发生和进展与肠道菌群的平衡有密切的关系,结果为研究乳腺炎的免疫学、病理学提供实验依据。

Objective To analyze the relationship between the abundance of intestinal flora and tissue inflammatory factors in mastitis mice by high-throughput sequencing. Methods Twenty-four female Kunming mice were randomly divided into the healthy control group, low-dose challenge group(containing Staphylococcus aureus 10~3 CFU/25 μL), medium-dose challenge group(Staphylococcus aureus 10~4 CFU/25 μL) or high-dose challenge group(Staphylococcus aureus 10~5 CFU/25 μL). Different concentrations of S. aureus suspension were injected into the 4 th(abdominal) mammary gland of the mice with papillary tube using non-invasive methods. The changes of TNF-α and Cav-1 in mouse tissues, histopathological changes and mammary gland tissues were observed. The feces of mice were collected and the fecal microbial community was analyzed using high-throughput sequencing technology. Results(1) Compared with the healthy control group, the mammary gland of the mice in high-dose challenge group showed obvious inflammatory pathological changes;the expression of TNF-α significantly increased(P<0.05), and that of Cav-1 significantly decreased(P<0.05).(2) The abundances of intestinal flora were different in different groups. The intestinal flora in healthy control group consisted mainly of Bacteroidetes and Firmicutes. In mouse model of mastitis, Firmicutes increased while Bacteroides and Verrucomicrobia decreased significantly(all P<0.05);there was no significant difference in Proteobacteria. Further comparison found that, at levels above genus, the ratios of Bacteroides, Alistipes and Prevotella were relatively low in mastitis mice. Conclusion The occurrence and progression of mastitis are closely related to the balance of intestinal flora. The results may provide experimental basis for the immunological and pathological research on mastitis.