糖尿病小鼠肠道菌群移植对无菌小鼠肾脏病理结构及尿微量白蛋白的影响

Effect of gut microbiota transplantation from diabetic mice on the renal pathologic structure and microalbuminuria in germ-free mice

目的:探讨糖尿病小鼠肠道菌群移植对无菌小鼠肾脏病理结构及尿微量白蛋白的影响。方法:选取瘦素缺陷的ob/ob雄性小鼠(ob组,n=20)高脂喂养建立糖尿病模型,野生型雄性C57BL/6小鼠作为对照组(n=10)。糖尿病模型建模成功后,采用高通量测序方法对肠道菌群进行测序并进行生物信息学分析,以检测粪便菌群结构与多样性。选取无菌C57BL/6小鼠30只,根据随机数字表法分为无菌正常组(GF-NC组)、无菌对照组(GF-CON组)和无菌糖尿病组(GF-DM组),每组各10只,分别以灌胃针将等容量磷酸盐缓冲溶液(不含粪便)、磷酸盐缓冲溶液(含对照组野生型小鼠粪便)、磷酸盐缓冲溶液(含ob组小鼠粪便)灌胃,每次0.2 ml,每日1次。采用苏木精伊红染色观察各组小鼠肾脏病理变化,并检测尿微量白蛋白水平。两组比较采用t检验,多组比较采用单因素方差分析,组间两两比较采用LSD-t检验。结果:与对照组相比,造模成功的ob组小鼠体重、血糖浓度升高,差异具有统计学意义(均P<0.01),肠道菌群的Shannon、Simpson多样性指数降低,差异具有统计学意义(分别为3.66±0.16和4.40±0.13、0.028±0.002和0.046±0.005,t=3.19、2.57,均P<0.05)。与对照组相比,ob组小鼠肠道内厚壁菌门数量减少,拟杆菌门、变形菌门数量增加,差异均具有统计学意义(P<0.01或P<0.05);放线菌门数量虽有增加,但差异无统计学意义(P>0.05)。菌群移植8周时,GF-DM组小鼠肾脏肾小球细胞数增多、体积增大,肾小管萎缩。与GF-CON组相比,4周、8周时GF-DM组小鼠尿微量白蛋白显著升高,差异具有统计学意义[分别为(8.26±2.93)和(2.19±0.85)μmol/L,(11.04±3.32)和(3.13±0.74)μmol/L,t=2.80、3.65,均P<0.01]。结论:糖尿病小鼠菌群结构及多样性与正常小鼠菌群存在差异,肠道移植糖尿病小鼠菌群后的无菌小鼠肾脏病理结构出现异常,且尿微量白蛋白水平升高。

Objective To explore the effect of gut microbiota transplantation from diabetic mice on the renal pathologic structure and microalbuminuria in germ-free mice.Methods Leptin-deficient ob/ob male mice(n=20)were chosen as animal model of diabetes mellitus.Wild-type male C57BL/6 mice(n=10)were chosen as control group.The differences in bacterial species and diversity between diabetes mellitus and control group were analyzed through high-throughput sequencing technology and bioinformatics approaches.Thirty germ-free mice were selected and randomly divided into three groups:normal group(GF-CON),control group(GF-CON)and diabetic group(GF-DM),which was gavaged with equal volume phosphate buffer solution(absence of feces),phosphate buffer solution(including feces of wild-type mice in control group)and phosphate buffer solution(including feces of mice in ob group)at a single dose of 0.2 ml per day,respectively.Pathological changes in the kidney of germ-free mice were assessed using hematoxylin-eosin staining and concentrations of urine microalbumin were measured.The t test was used to compare the data between two groups.Statistical significance was performed by one-way ANOVA,followed by LSD-t test for pairwise comparison among multi-groups.Results Compared with control group,body weight and blood glucose concentration were significantly increased in diabetic mice(both P<0.01).A decrease in both Shannon′s and Simpson′s diversity indexes of gut microbiota was observed in diabetic mice(3.66±0.16 vs 4.40±0.13,0.028±0.002 vs 0.046±0.005,respectively,t=3.19,2.57,both P<0.05).The abundances of Firmicutes were significantly decreased,and that of Bacteroidetes and Proteobacteria were significantly increased in the diabetic mice compared with that of control group,respectively(P<0.01 or P<0.05).Although the abundances of Actinobacteria increased,there was no statistical difference between the two groups(P>0.05).The colonization of gut microbiota from diabetic mice induced glomerular cell proliferation and renal tubule atrophy in germ-free mice at week eight.The concentration of urine microalbumin was also significantly increased compared with control group after four weeks and eight weeks,respectively[(8.26±2.93)vs(2.19±0.85)μmol/L,(11.04±3.32)vs(3.13±0.74)μmol/L,t=2.80,3.65,both P<0.01].Conclusion There are differences in the gut microbiota composition between diabetes mellitus and control group.After gut microbiota transplantation from diabetic mice,pathological changes in kidney were observed,and urinary microalbumin increased in germ-free mice.