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. mouse gut metagenome

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.

6周龄雄性C57BL/6J小鼠购自中国台湾地区国家实验动物中心。适应性饲养两周后，将小鼠随机分为4组：(1) 对照饲料（Research Diets, Inc., NJ, USA; 货号D12450K）+腹腔注射磷酸盐缓冲液（phosphate buffer saline, PBS）；(2) 含棕榈油的高脂饲料（Palm oil-containing high-fat diet, P-HFD；Gubra Amylin NASH (GAN) diet；Research Diets, Inc., NJ, USA; 货号D09100310）+腹腔注射PBS；(3) P-HFD+腹腔注射脂多糖（lipopolysaccharide, LPS，500 μg/kg体重/周；大肠杆菌O55:B5来源LPS；Sigma-Aldrich; 货号L2880）；(4) P-HFD+腹腔注射四氯化碳（carbon tetrachloride, CCl4，320 μg/kg体重/周；Merck Millipore; 货号1.02209.1000）。对照饲料脂肪与碳水化合物占比为10 kcal%，主要成分为玉米淀粉。P-HFD脂肪占比为40 kcal%（主要为棕榈油），添加20%果糖与2%胆固醇。分别于第4、8、12、16、20和24周，采用二氧化碳窒息法处死小鼠（每组n=7~8）。处死后从结肠中获取粪便样本，经液氮快速冷冻后于-80℃保存待用。采用QIAmp Power Fecal Pro-DNA试剂盒（QIAGEN, Netherlands）提取粪便基因组DNA，使用NanoDrop ND-1000分光光度计（Thermo Fischer Scientific）进行浓度定量。采用引物对（上游引物：5'-TCG TCG GCA GCG TCA GAT GTG TAT AAG AGA CAG CCT ACG GGN GGC WGC AG-3'；下游引物：5'-GTC TCG TGG GCT CGG AGA TGT GTA TAA GAG ACA GGA CTA CHV GGG TAT CTA ATC C-3'）扩增16S rRNA基因的V3-V4高变区。聚合酶链式反应（polymerase chain reaction, PCR）在25 μL反应体系中进行，体系包含5 ng DNA模板、0.2 μM上下游引物以及12.5 μL 2×Taq Master Mix（KAPA HiFi HotStart ReadyMix, Roche, Switzerland）。PCR反应程序为：95℃预变性3 min；随后25个循环，每个循环包含95℃变性30 s、55℃退火30 s、72℃延伸30 s；最后72℃终延伸5 min。扩增产物通过2%琼脂糖凝胶电泳进行可视化检测。采用Nextera XT Index试剂盒通过PCR反应添加双索引与Illumina测序接头，随后使用AMPure XP磁珠对扩增子进行纯化。采用Bioanalyzer DNA 1000芯片验证PCR产物片段大小，测序前使用Agilent 2100生物分析仪对文库进行定量以完成质量控制。混合后的文库采用Illumina MiSeq平台进行双端测序（2×300 bp）。