Objective To analyze the composition and richness of intestinal microflora in children with non-alcoholic fatty liver disease (NAFLD) and the role of which in pathogenesis of NAFLD.Methods This was a prospective case-control study.From November 2015 to June 2017,19 children diagnosed with NAFLD according to the 2010 edition of diagnostic criteria were enrolled voluntarily in the Second and First Affiliated Hospitals of Zhejiang Chinese Medicine University.Twenty-two healthy children were enrolled in the control group.Among the patients,10 were males and 9 were females,at the mean age of (11.0± 1.0) years;10 males and 12 females in the control group,at the mean age of (9.0±1.2) years.The body mass index (BMI) and waist circumference were recorded,and the fasting blood glucose,total cholesterol,triglyceride,high-density lipoprotein and low-density lipoprotein were detected.Feces were collected and the fecal microorganisms were extracted with magnetic beads methods;the composition and the richness of intestinal microflora in the two groups were detected with 16S rDNA high throughput sequencing technology.The KO differential gene expression and KEGG signal pathway enrichment were analyzed with PICRUST software.The intestinal flora characteristics between the two groups were compared with t test or Mann-Whitney U test and Willcoxon W test.Results The BMI,waist circumference and triglyceride were higher in NAFLD group than those in the control group (BMI (25.1 ± 2.7) vs.(18.2 ± 1.5)kg/m2,t=9.912,P=0.000;waist circumference (88.6±6.6) vs.(71.5±6.3) cm,t=8.520,P--0.000;triglyceride (0.9±0.4) vs.(0.7±0.3)mmol/L,t=2.060,P=0.046).The abundance and diversity index of intestinal microflora were lower in the NAFLD group (Shannon index 3.99 (3.13,4.54) vs.4.63 (4.21,4.81),Z=-2.065,P=0.039;Simpson index 0.85 (0.70,0.89) vs.0.90 (0.88,0.93),Z=-2.431,P=0.015;ACE index 235.76 (205.26,361.94) vs.326.96 (275.34,368.65),Z=-2.092,P=0.036).At the level of phylum,the proportion of Actinomycetes was lower and the proportion of Thermus was higher in NAFLD group (Actinobacteri 29.807 (14.723,62.080) × 10-3 vs.63.212 (46.133,172.071) ×10-3,Z=-2.667,P=0.008;Thermus 0.033 (0.000,0.226) ×10-3 vs.0.000 (0.000,0.031) ×10-3,Z=-2.729,P=0.006).At the level of genus,the proportion of Bacteroides and Bifidobacterium in the NAFLD group were significantly lower (Bacteroides 78.757 (11.430,151.621) ×10-3 vs.356.821 (161.049,403.037) × 10-3,Z=-2.771,P=0.006;Bifidobacterium 19.680 (6.181,53.944)×10-3 vs.54.721 (31.911,146.410)×10-3,Z=-2.458,P=0.014);the proportion of Prevotella in NAFLD group was significantly higher (3.089 (0.165,63.502) × 10-3 vs.0.432 (0.029,2.257) × 10-3,Z=-2.112,P=0.035).Based on the KEGG database,78 differentially expressed genes and 26 differential metabolic pathways were found,among which the function genes of K01470,K01961 and K07258 were concentrated in the pathways of arginine and proline metabolism,fatty acid synthesis,and polysaccharides biosynthesis and metabolism.Besides,these three function genes were related to Bacteroides,Prevotella,Bifidobacterium and Ruminococcus.Conclusion NAFLD children have intestinal flora disturbances in both diversity and abundance,which may alter lipid metabolic pathways through differential gene expressions,contributing to the pathogenesis of NAFLD.%目的 观察非酒精性脂肪性肝病(NAFLD)患儿肠道菌群的组成和丰度变化,阐述NAFLD儿童的肠道菌群结构特征并探讨其参与NAFLD发生发展的可能机制.方法 前瞻性病例对照研究.选择2015年11月至2017年6月浙江中医药大学附属第二医院及第一医院收治的NAFLD患儿19例为研究对象,并以健康儿童22名为对照组,NAFLD组男10例,女9例,年龄(11.0±1.0)岁;对照组男10名,女12名,年龄(9.0±1.2)岁;测定两组儿童的体质指数(BMI)及腰围;检测空腹血糖、总胆固醇、甘油三酯、高密度脂蛋白、低密度脂蛋白;收集粪便,磁珠法提取粪便样品细菌DNA,采用16SrDNA高通量测序技术检测两组儿童粪便中肠道菌群的组成和丰度,并分析比较两组样本肠道菌群的结构特征.应用PICRUST软件对菌群的KO差异基因表达及KEGG信号通路富集分析进行预测.组间比较采用t检验或Mann-Whitney U检验及Willcoxon W检验.结果 NAFLD组BMI[(25.1±2.7)比(18.2±1.5)kg/m2,t=9.912,P=0.000],腰围[(88.6±6.6)比(71.5±6.3)cm,t=8.520,P=0.000],甘油三酯[(0.9±0.4)比(0.7±0.3)mmol/L,t=2.060,P=0.046]均高于对照组,差异均有统计学意义.NAFLD组香浓指数[3.99(3.13,4.54)比4.63(4.21,4.81),Z=-2.065,P=0.039],辛普森指数[0.85(0.70,0.89)比0.90(0.88,0.93),Z=-2.431,P=0.015],ACE指数[235.76(205.26,361.94)比326.96(275.34,368.65),Z=-2.092,P=0.036]均明显低于对照组,差异均有统计学意义.在门水平上,NAFLD组放线菌门比例低于对照组[29.807(14.723,62.080) ×10-3比63.212(46.133,172.071)×10-3,Z=-2.667,P=0.008],栖热菌门比例高于对照组[0.033(0.000,0.226) ×10-3比0.000(0.000,0.031) ×10-3,Z=-2.729,P=0.006],差异均有统计学意义;在属水平上,NAFLD组拟杆菌属比例[78.757(11.430,151.621) ×10-3比356.821(161.049,403.037)× 10-3,Z=-2.771,P=0.006]、双歧杆菌属比例明显低于对照组[19.680(6.181,53.944)×10-3比54.721(31.911,146.410)×10-3,Z=-2.458,P=0.014],普氏菌属比例显著高于对照组[3.089(0.165,63.502)×10-3比0.432(0.029,2.257) ×10-3,Z=-2.112,P=0.035],差异均有统计学意义.基于KEGG数据库,共发现78个差异表达基因及26条差异代谢途径,其中K01470、K01961、K07258这几种功能基因集中于精氨酸和脯氨酸的代谢、脂肪酸合成、多糖的生物合成和代谢这3条差异代谢途径上,且与拟杆菌属、普氏菌属、双歧杆菌属及瘤胃球菌属相关.结论 NAFLD患儿存在代谢异常及肠道菌群紊乱;差异菌属有可能通过功能基因的差异表达改变相关代谢途径进而影响脂代谢过程,参与NAFLD的发生发展.
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