Supplementary Material for: Microbial Intervention as a Novel Target in Treatment of Non-Alcoholic Fatty Liver Disease Progression

<b><i>Background/Aims:</i></b> Emerging evidence suggests a close link between gut microbiota and non-alcoholic fatty liver disease (NAFLD). In this study, we aimed to investigate the association between gut microbiota and the DNA methylation of adiponectin (an adipocyte-specific adipocytokine) in rats, following diet-induced NAFLD. <b><i>Methods:</i></b> 50 male SD rats were randomly divided into five groups with or without a high fat diet (HFD), antibiotics, and probiotics, in order to establish an imbalanced gut microbiota and probiotic treatment model in NAFLD rats. After 13 weeks of treatment, blood, liver, and cecal tissue samples were collected. Serum lipids, liver function indexes by biochemical analyzers, and changes in liver pathology with hematoxylin-eosin (HE) and masson staining were detected. Furthermore, the serum adiponectin by enzyme-linked immunosorbent assay (ELISA) and liver adiponectin methylation levels in the promoter regions by pyrophosphate sequencing were determined. High throughput Illumina sequencing targeted microbial 16S genes, bioinformatics and statistical analysis identified cecal-associated gut microbiota. <b><i>Results:</i></b> HFD with antibiotic exposure showed the most severe steatohepatitis and a severe gut microbiota alteration. Reduced bacterial diversity was also seen and the abundances of Firmicutes, <i>Lactobacillus</i>, Cyanobacteria, Acidobacteria, Chlamydiae, Chlamydiales, Rubrobacteria, Verrucomicrobia, <i>Blautia, Shewanella, Bacteroides, Bacteroides acidifaciens</i>, and <i>Bacteroides uniformis</i>, were shown to be partly reversed by probiotic treatment. Decreased serum adiponectin levels and increased DNA methylation levels of adiponectin promoter regions were also markedly associated with the NAFLD progression during gut microbiota alteration. <b><i>Conclusion:</i></b> Our results suggested that both gut microbiota alteration and adiponectin variability may be drivers of NAFLD progression and that targeting the gut microbiota, such as via administration of a probiotic, may delay NAFLD progression via adiponectin.