Healthy diet intervention reverses the progression of NASH through gut microbiota modulation

Eight-week-old C57BL/6J male mice were then randomly divided into two groups: one group was fed a control diet (CON) (10 kcal% fat and carbohydrate mainly as corn starch, Research Diets, Inc., NJ, USA; D12450K), and the other a GAN diet (Gubra-Amylin NASH (GAN) diet; 40% total fat kcal, 20% fructose, 2% cholesterol; Research Diets, Inc., NJ, USA; D09100310) for 12 weeks to induce NASH. At the end of 12 weeks, the CON group were fed the control diet, whereas the GAN group was randomly divided into four subgroups: (1) GAN group, which continued to receive the GAN diet; (2) G-C group, which was switched to the control diet; (3) G-C + ginger essential oil (GEO) group, which was switched to the control diet and oral administration of GEO supplementation by gavaging (125 mg/kg bw/day); (4) G-C + OCA group, which was switched to the control diet along with oral obeticholic acid (OCA) supplementation (30 mg/kg bw/day). This intervention was conducted for an additional 3 weeks. DNA was extracted from the feces using the QIAmp Power Fecal Pro DNA Kit (QIAGEN, Netherlands) following the manufacturer's instructions. The V3-V4 hypervariable region of the 16S rRNA gene was amplified with forward and reverse primers (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) through PCR. The PCR products were cleaned up and quantified before undergoing paired-end sequencing (2 x 300 bp) on the Illumina MiSeq platform.