Palm oil-containing high-fat diet with LPS and CCl4 C57BL/6J mouse model induces the progression of NASH/liver fibrosis and remodels the gut microbiota and its function

Six-week-old male C57BL/6J mice were purchased from Taiwan National Laboratory Animal Center. After two weeks of adaptive feeding, the mice were randomly assigned to four groups: (1) control diet (Research Diets, Inc., NJ, USA; D12450K)+intraperitoneal injection of phosphate buffer saline (PBS); (2) Palm oil-containing high-fat diet (P-HFD; Gubra Amylin NASH (GAN) diet; Research Diets, Inc., NJ, USA; D09100310)+intraperitoneal injection of PBS; (3) P-HFD+intraperitoneal injection of LPS (500 ug/kg bw/week; LPS from Escherichia coli O55:B5; Sigma-Aldrich; L2880); and (4) P-HFD+intraperitoneal injection of CCl4 (320 ug/kg bw/week; Merck Millipore; code 1.02209.1000). Control diet comprises 10 kcal% fat and carbohydrates, mainly in the form of corn starch. P-HFD comprises 40 kcal% fat (mainly palm oil), 20% fructose, and 2% cholesterol. Mice (n=7-8) were sacrificed by carbon dioxide asphyxiation at 4, 8, 12, 16, 20, and 24 weeks.Mouse fecal samples were obtained from the colon when mice were killed. The samples were snap-frozen using liquid nitrogen and stored at -80 C before use. Fecal genomic DNA was extracted using the QIAmp Power Fecal Pro-DNA Kit (QIAGEN, Netherlands) and quantified using the NanoDrop ND-1000 spectrophotometer (Thermo Fischer Scientific).The V3-V4 hypervariable region of the 16S rRNA gene was amplified using the primer pair ((Forward=5-TCG TCG GCA GCG TCA GAT GTG TAT AAG AGA CAG CCT ACG GGN GGC WGC AG-3) and Reverse=5-GTC TCG TGG GCT CGG AGA TGT GTA TAA GAG ACA GGA CTA CHV GGG TAT CTA ATC C-3)). Polymerase chain reaction (PCR) amplification was conducted in a 25 uL reaction mixture containing 5 ng of DNA template, 0.2 uM forward and reverse primers, and 12.5 uL of 2xTaq Master Mix (KAPA HiFi HotStart ReadyMix, Roche, Switzerland). The PCR conditions included an initial step at 95 C for 3 min, followed by 25 cycles of 95 C, 55 C, and 72 C for 30 s each, and a final extension at 72 C for 5 min. Amplified products were subsequently visualized by using 2% agarose gel electrophoresis. Dual index and Illumina sequencing adapters were joined using a Nextera XT Index Kit via PCR. PCR product cleanup was conducted using AMPure XP beads to purify amplicon. The sizes of PCR products were validated using the Bioanalyzer DNA 1000 chip. Library quantification was carried out for quality control before sequencing using the Agilent Technologies 2100 Bioanalyzer. The pooled libraries were subjected to paired-end sequencing (2x300 bps) using the Illumina MiSeq platform.