Objective: To investigate the effects of fecal microbiota transplantation (FMT) on non-obese diabetic (NOD) mice of type 1 diabetes mellitus (T1DM) and its underlying mechanisms. Methods: A total of 8-9 week-female NOD mice were randomly divided into control (n=36) and FMT groups (n=36) according to the random number table. Fecal microbiota from C57BL/6 mice were transplanted into FMT group, and control group were transplanted with microbiota from themselves, once every two days for 5 times. The insulitis score and incidence of T1DM were compared between two groups;16S rRNA gene sequencing was used to evaluate the structure of fecal bacteria in NOD mice. The expressions of intestinal barrier related genes were detected by real-time quantitative PCR. The proportions of regulatory T cells (Tregs), helper T cell (Th)-1 and Th17 in the enteric-pancreatic immune axis were detected by flow cytometry. Amino acid in serum was measured by amino acid metabolomics. Results: Incidence of T1DM in NOD mice from FMT group was 40.9% (9/22), lower than 72.7% (13/22) from control group at 26 weeks of age (P=0.034). FMT promoted colonization of probiotics such as Lactobacillus, Clostridium_sp_ND2, Candidatus_Arthromitus and Clostridiaceae_1; mRNA of intestinal barrier related genes were up-regulated in FMT group [ mucins(Muc)-1: 0.93±0.29 vs 2.97±0.79, P=0.036; Muc2: 0.72±0.39 vs 10.70±3.54, P=0.019;Muc3: 1.79±0.69 vs 10.97±2.78, P=0.009;Muc4: 1.01±0.23 vs 2.42±0.49, P=0.029;Occludin(Ocln): 0.96±0.08 vs 1.81±0.36, P=0.045; Claudin(Cldn)-1:0.94±0.17 vs 2.20±0.43, P=0.022] compared to control. The proportions of Treg in mesenteric lymphoid node, pancreatic lymph node and peyer's patches of FMT group [(6.10±0.49)% vs (7.54±0.27)%, P=0.020;(5.28±0.39)% vs (6.42±0.34)%, P=0.048;(6.78±0.42)% vs (7.88±0.13)%, P=0.029] were increased compared to control,while proportions of Th1 [(1.02±0.06)% vs (0.83±0.06)%, P=0.040;(0.82±0.10)% vs (0.56±0.05)%, P=0.038;(1.28±0.12) vs (0.85±0.07), P=0.012] and proportions of Th17 [(0.40±0.01)% vs (0.30±0.02)%, P=0.004;(0.40±0.02)% vs (0.31±0.02)%, P=0.008;(0.51±0.06) vs (0.36±0.02), P=0.027] were decreased. The contents of leucine [(92.86±7.32) vs (91.87±12.62) μmol/L, P=0.027], valine [(162.74±15.97) vs (155.89±25.70) μmol/L, P=0.046] and isoleucine [(75.65±5.59) vs (73.61±9.67) μmol/L, P=0.048] in serum were decreased in FMT group. Conclusions: FMT can alleviate insulitis and T1DM occurrence in NOD mice, of which mechanism may be related to remodeling gut microbiota and improving intestinal barrier function, affecting immune response of enteric-pancreatic immune axis, correcting amino acid metabolism disorder and reducing the accumulation of branch chain amino acids in NOD mice.
目的： 探讨粪菌移植（FMT）对非肥胖糖尿病（NOD）小鼠发生1型糖尿病（T1DM）的影响及相关机制。 方法： 8~9周龄的雌性NOD小鼠72只，随机数字表法分为对照组（n=36）和FMT组（n=36）。FMT组移植C57BL/6鼠的粪菌，对照组移植自身粪菌，隔日1次，共5次。比较两组小鼠的胰岛炎评分和T1DM发病率；通过16S rRNA基因测序评估NOD小鼠粪菌组成；实时定量PCR检测肠屏障相关基因的表达水平；流式细胞术检测肠-胰腺免疫轴中调节性T细胞（Treg）、辅助性T细胞（Th）-1及Th17的比例；通过氨基酸代谢组学检测血清中氨基酸的水平。 结果： 26周龄时FMT组的T1DM发病率为40.9%（9/22），低于对照组的72.7%（13/22）（P=0.034）。FMT促进了肠道中有益菌，如Lactobacillus、Clostridium_sp_ND2、Candidatus_Arthromitus和Clostridiaceae_1的定植。与对照组相比，FMT组肠屏障相关基因的mRNA表达上调［黏蛋白（Muc）-1：0.93±0.29比2.97±0.79，P=0.036；Muc2：0.72±0.39比10.70±3.54，P=0.019；Muc3：1.79±0.69比10.97±2.78，P=0.009；Muc4：1.01±0.23比2.42±0.49，P=0.029；封闭蛋白（Ocln）：0.96±0.08比1.81±0.36，P=0.045；闭合蛋白（Cldn）-1∶0.94±0.17比2.20±0.43，P=0.022］。与对照组相比，FMT组肠系膜淋巴结、胰腺淋巴结、派伊尔结中Treg比例增加［（6.10±0.49）%比（7.54±0.27）%，P=0.020；（5.28±0.39）%比（6.42±0.34）%，P=0.048；（6.78±0.42）%比（7.88±0.13）%，P=0.029］；Th1比例减少［（1.02±0.06）%比（0.83±0.06）%，P=0.040；（0.82±0.10）%比（0.56±0.05）%，P=0.038；（1.28±0.12）比（0.85±0.07），P=0.012］，Th17比例减少［（0.40±0.01）%比（0.30±0.02）%，P=0.004；（0.40±0.02）%比（0.31±0.02）%，P=0.008；（0.51±0.06）比（0.36±0.02），P=0.027）］。与对照组相比，FMT组小鼠血清中亮氨酸［（92.86±7.32）比（91.87±12.62）μmol/L，P=0.027）］、缬氨酸［（162.74±15.97）比（155.89±25.70）μmol/L，P=0.046）］、异亮氨酸［（75.65±5.59）比（73.61±9.67）μmol/L，P=0.048）］含量减少。 结论： FMT可减轻NOD小鼠的胰岛炎并减缓T1DM的发生，其机制可能与重塑NOD小鼠肠道菌群并改善肠屏障功能、影响肠-胰腺免疫轴的免疫反应、同时纠正胰岛炎期NOD小鼠的氨基酸代谢紊乱、减少支链氨基酸的蓄积有关。.