目的 基于色氨酸（tryptophan,Trp）靶向代谢组学和16s rRNA高通量测序技术分析Trp对代谢相关脂肪性肝病（metabolic-associated fatty liver disease , MAFLD）小鼠Trp靶向代谢物及其肠道菌群的影响。方法 5~6 w龄雄性C57BL/6J小鼠随机分为对照组（control,n=15）、实验组（n=30）,对照组采用普通饲料喂养,实验组采用60%脂肪占比的高脂饲料（high-fat diet,HFD）喂养构建MAFLD小鼠模型,造模时间为16 w,模型构建成功后,将实验组随机分为HFD组（n=10）、Trp组（n=10）。对照组与HFD组灌胃等量0.9%生理盐水,Trp组灌胃Trp 50mg/（kg·d）,干预8 w。检测小鼠体重、摄食量、肝湿重、肝脏指数、肝脏病理改变、肝脏总胆固醇（total cholesterol,TC）、甘油三酯（triglyceride,TG）、血清转氨酶水平等指标,以及回肠病理和紧密连接蛋白mRNA表达水平,采用16s rRNA高通量测序与Trp靶向代谢组学技术检测各组小鼠肠道微生物以及血清代谢物,并通过Spearman相关性分析进行差异菌群与Trp靶向代谢物之间的关联分析。结果 与对照组相比,HFD组小鼠肝脏脂质沉积明显增加,体重、肝湿重、肝脏指数显著增加（P<0.01）;与HFD组相比,Trp组小鼠肝脏脂质沉积明显减轻,体重（P<0.01）、肝湿重（P<0.01）、肝脏指数均显著下降（P<0.05）,回肠紧密连接蛋白mRNA表达上调,乳酸杆菌属（Lactobacillus）、粪异杆菌属（Allobaculum）等有益菌丰度及肠道菌群多样性增加。Spearman相关性分析显示,Trp代谢物5-羟基吲哚乙酸（5-HIAA）水平与Allobaculum丰度呈显著负相关（P<0.001）。结论 Trp干预对小鼠MAFLD具有改善作用,可能与富集Lactobacillus、Allobaculum等有益菌、减少5-HIAA生成有关。
Abstract
Objective To analyze the effects of tryptophan (Trp) on the Trp-targeted metabolites and intestinal flora in mice with metabolic-associated fatty liver disease (MAFLD) based on Trp-targeted metabolomics and 16s rRNA high-throughput sequencing technology. Methods Male C57BL/6J mice aged 5-6 weeks were randomly divided into a control group (control, n=15) and an experimental group (n=30). The control group was fed a normal diet, while the experimental group was fed a high-fat diet (HFD) with a fat ratio of 60% to establish the MAFLD mouse model. The modeling time was 16 weeks. After the model was successfully constructed, the mice in the experimental group was randomly divided into the HFD group (n=10) and the Trp group (n=10). The HFD group was orally given the same amount of 0.9% normal saline, and the Trp group was given Trp at 50mg/ (kg·d). Both groups were intervened for 8 weeks. The body weight, food intake, liver wet weight, liver index, liver pathological changes, total cholesterol (TC), triglyceride (TG), and serum transaminase levels, as well as ileal pathology and mRNA expression levels of tight junction proteins were detected. The intestinal microbiota and serum metabolites were detected by 16s rRNA high-throughput sequencing and Trp-targeted metabolomics technology, and the association analysis between the differential microbiota and Trp-targeted metabolites was conducted through Spearman correlation analysis. Results Compared with the control group, the lipid deposition in the liver of mice in the HFD group increased significantly, and the body weight, liver wet weight and liver index increased significantly (P<0.01). Compared with the HFD group, the lipid deposition in the liver of mice in the Trp group was significantly reduced, and the body weight (P<0.01), liver wet weight (P<0.01), and liver index all decreased significantly (P<0.05). The mRNA expression of ileal tight junction proteins was up-regulated. The abundances of beneficial bacteria such as Lactobacillus and Allobaculum, as well as the diversity of intestinal flora were increased. Spearman correlation analysis showed that the level of the Trp metabolite 5-hydroxyindoleacetic acid (5-HIAA) was significantly negatively correlated with the abundance of Allobaculum (P<0.001). Conclusion Trp has an improving effect on MAFLD in mice, which is related to the enrichment of beneficial bacteria such as Lactobacillus, Allobaculum and the reduction of 5-HIAA production.
关键词
色氨酸 /
代谢相关性脂肪性肝病 /
肠道菌群 /
代谢组学
Key words
tryptophan /
metabolic associated fatty liver disease /
intestinal flora /
metabolomics
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