All animal experiments were carried out in compliance with ICLAS guideline and Japanese regulations. The local institutional animal ethics board of Osaka Medical college approved all mouse experiments (approval number: 2019 − 138). Five week-old male C57BL/6 mice were purchased from Japan SLC, Inc. All mouse experiments were performed at the animal facility in Osaka Medical college, where the animals were housed in specific pathogen-free conditions. The mice were divided into four groups of 6 and fed either a control diet (CE-2; CLEA Japan) or an HFD (HFD32; CLEA Japan), for 12 weeks. For the IBATi and HFD + IBATi groups, the mice were treated with Elobixibat (EA Pharma Co. Ltd, Japan) (2.5 µmol/kg: corresponding to the clinically optimal dose for patients) by oral gavage in 0.2 ml of PBS daily, using animal feeding needles.
For some experiments, 6 week-old C57BL/6 mice were fed an HFD for 12 weeks and treated with oral IBATi from 6 to 12 weeks (IBATi-Tx group). For the control group, the equivalent mice were subjected to the same oral dose of PBS.
Metagenomic analysis of mouse feces.
Fresh feces were collected, placed into tubes, and kept at -80℃ until further use. Bacterial genomic DNA was extracted from fecal samples, as previously described . Briefly, whole bacterial DNA was extracted from the feces using a PureLinkTM Micobiome DNA Purification Kit (ThermoFisher Scientific K. K. Tokyo, Japan).
Fecal Microbiota Transplantation
Elobixibat has only 2% bioavailability and is excreted from the body 48 hours after oral administration. When absorbed, elobixibat is protein bound (> 99%) in plasma, with a half-life of less than 4 hours  . For fecal microbiota transplantation (FMT), feces were collected from the 12 week-HFD group or HFD + IBATi group of mice, 48 hours after the last oral administration of elobixibat, and stored at -80℃. Six week-old male C57/B6 mice were fed an HFD for 8 weeks. At 9 weeks of age, these mice were treated with an antibiotic solution (ATB) containing ampicillin (1 mg/ml), streptomycin ( 5 mg/ml) and colistin (1 mg/ml), added to their sterile drinking water. Solutions and bottles were changed 3 times per week. To investigate the efficacy of FMT, these 10 week-old “avatar mice” received ATB before FMT, and ATB treatment was discontinued 48 h before the first FMT. FMT was performed by thawing fecal materials and 500 µL of fecal suspension (50 mg/mouse) was then transferred by oral gavage, using animal needles, into each ATB pre-treated recipient three times every two days, for one week.
Library Preparation And Dna Sequencing
Preparation of the library for DNA sequencing was conducted as previously described  using ion-proton technology (ThermoFisher Scientific K. K. Tokyo, Japan). An Ion 16S™ Metagenomics Kit was used to amplify hypervariable regions from the polybacterial samples, according to the manufacturer’s protocol. Library preparation was performed from 50 ng pooled amplicons for each sample using an Ion Plus Fragment Library Kit. The StepOnePlus™ Real-time PCR system was used to perform the quantitative polymerase chain reaction amplification.
Sequence Data Analysis
Sequence data analysis was performed using QIIME v1.9.0, as previously described . Statistical differences (p < 0.05) in the relative abundance of bacterial phyla and genera between groups were evaluated using Student’s paired t test. The Shannon phylogenetic diversity indices were calculated using the R “phyloseq” package and statistically analyzed using a Kruskal-Wallis test followed by the Steel-Dwass post hoc test. The β-diversity was estimated using the Bray Curtis distances between the samples and visualized by principal coordinate analysis (PCoA); it was statistically examined using permutational analysis of variance (PERMANOVA). The final figures were generated using QIIME v1.9.0.
The liver tissue samples were fixed in 20% formalin and embedded in paraffin. Sections (4 µm thick) were cut and stained with hematoxylin-eosin (HE). Histological features and the fat area in liver tissue sections were determined using a BZ-X 710 fluorescence microscope (Keyence, Osaka, japan). Blinded histologic assessment of slides was performed by an experimented hepatopathologist. Hepatic steatosis was evaluated by NAFLD activity score (NAS)  and hepatic fibrosis was evaluated by fibrosis stage.
Serum Biochemical Analysis
Serum was obtained from each mouse. Concentrations of aspartate amino-transferase (AST), alanine amino-transferase (ALT), Total Bile Acid (TBA), total cholesterol (Tcho), low-density lipoprotein (LDL) and triacyl glyceride (TG) in serum were measured by ELISA. ELISA kits were purchased from Elabscience (TX., U.S.A.) and MyBiosource, Inc. (CA., U.S.A.).
Liver and terminal ileum samples were obtained from each group of mice. Hepatic mRNA expression (cholesterol 7-a-monoxygenase (Cyp7a1), cholesterol 8-a-monoxygenase (Cyp8a1) and farnesoid X receptor (Fxr)) and ileal mRNA expression (Fxr and fibroblast growth factor 15 (Fgf15)) were measured by real-time PCR using a RNeasy kit (Qiagen K. K, Tokyo, Japan) and CYBR Green real-time PCR master mix (ThermoFisher Scientific K. K. Tokyo, Japan).
All data are presented as mean SEM. Body weight gain curves were analyzed with an ANOVA, followed by Tukey’s multiple comparison tests. The Pearson’s rank correlation coefficient was used to analyze the correlation of gut microbiota. These statistical analyses were performed using statistical software (JMP institute, Inc., Cary, NC). All results were considered statistically significant at p < 0.05