Role of intestinal flora in the development of nonalcoholic fatty liver disease in children

Abstract

In China, 45% of adolescents with obesity develop fatty liver disease, a condition that increases the long-term risk of developing cirrhosis and liver cancer. Although the factors triggering nonalcoholic fatty liver disease (NAFLD) vary in children, the composition of intestinal microflora has been found to play an increasingly important role. However, evidence is limited on the prevalence of nonalcoholic fatty liver (NAFL) and nonalcoholic steatohepatitis (NASH) in Chinese children. Therefore, this study aimed to evaluate the fecal microbiome of Chinese children with NAFLD and further analyze the potential of flora in regulating NAFLD-related symptoms and metabolic functions. Specifically, the study applied a 16S rRNA and metagenomic sequencing to the fecal samples of pediatric patients with NAFLD, NASH, and NAFL, as well as healthy controls, to explore the correlation among NAFLD-related indexes, metabolic pathways, and gut flora. The findings showed that some fecal microbiota had a negative correlation with body mass index, and various NAFLD-related bacteria, including Lachnoclostridium, Escherichia-Shigella, and Faecalibacterium prausnitzii, were detected. Consequently, the study concluded that the variation in gut microbiota might be more important in improving NAFLD/NASH compared with single species, providing a microbiota diagnostic profile of NAFLD/NASH.IMPORTANCEThis study aims to characterize the gut microbiota in Chinese children with nonalcoholic fatty liver disease (NAFLD) through 16S rRNA and metagenomic sequencing. The results highlight the association between fecal microbiota and NAFLD in Chinese children, demonstrating distinct characteristics compared to adults and children from other countries. Based on the sequencing data from our cohort's fecal samples, we propose a microbiota model with a high area under the curve for distinguishing between NAFLD and healthy individuals. Furthermore, our follow-up study reveals that changes in the relative abundance of microbial biomarkers in this model are consistent with variations in patients' body mass index. These findings suggest the potential utility of the microbiota model and microbial biomarkers for diagnosing and treating NAFLD in children.

Keywords: BMI; NAFLD; NASH; children; gut microbiota; metabolism.

Fig 1

The histomorphological diagnosis of liver biopsy specimens in patients diagnosed with NAFL or NASH. The HE staining of the liver biopsy specimens of NAFL only shows steatosis. The HE staining of NASH shows steatosis, lobular inflammation, ballooning, and liver fibrosis. The black arrow, white arrow, black arrowhead, empty arrowhead, and empty arrow indicate macrovesicular steatosis, microvesicular steatosis, lobular inflammation, ballooning, and liver fibrosis, respectively. Scale bar, 250 µm. Images of all pathological sections were provided by the Pathological Department, Beijing Children’s Hospital.

Fig 2

Colony composition and diversity of fecal microbiota in pediatric patients with NAFLD and HCs. (A) Top 10 species at the phylum level. (B) Top 15 species at the genus level in each sample in pediatric patients with NAFLD and HCs. (C) Alpha-diversity group comparison of patients with NAFLD and HCs. (D) PCoA of patients with NAFLD and HCs (HC, n = 35 and NAFLD, n = 79).

Fig 3

Multiple bacterial species with significantly different abundance were found in the intestinal flora of patients with NAFLD and HCs (HC, n = 35 and NAFLD, n = 79, P < 0.05).

Fig 4

Differential diversity and multiple bacterial species with significantly different abundance were found in the intestinal flora of patients with NAFL and NASH. (A) Alpha-diversity group comparison of patients with NAFL and NASH. (B) NMDS of patients with NAFL and NASHs. (C) Genera with significant differences in abundance between patients with NAFL and those with NASH. (D) Species with significant differences in abundance between patients with NAFL and NASH (NAFL, n = 5 and NASH, n = 8, P < 0.05).

Fig 5

Abundance of species significantly correlated with NAFLD-related indicators at the species level (n = 79, *P < 0.05 and **P < 0.01).

Fig 6

Abundance of species significantly correlated with microbial metabolic pathways potentially related to NAFLD at the species level (n = 79, *P < 0.05 and **P < 0.01).

Fig 7

A prediction model of the identified bacterial taxa for the clinical discrimination of patients with NAFLD and HCs was built using the stochastic forest machine algorithm. (A) Prediction model constructed using the stochastic forest machine algorithm and (B) ROC curves for clinical discrimination.

Fig 8

Change in gut microbiota diversity and abundance of NAFLD-related bacteria was closely related to BMI decrease in pediatric patients with NAFLD after nonpharmacological treatment. (A) Observed species of bacteria in the flora of pediatric patients with NAFLD. (B) Enhanced bacterial abundance and (C) reduced bacterial abundance after nonpharmacological treatment.