Sixty 6-week-old, wild-type, male C57BL/6 SPF mice with a body mass of 20-22 g were purchased from Shanghai SLAC Laboratory Animal Co., Ltd. and kept, modeled and observed in a barrier-protected animal room in the animal research center at Shanghai University of Traditional Chinese Medicine. During model establishment, experimental animals had free access to food and water (SYXK (Shanghai) 2014-0008).
The diet for the high-fat and high-carbohydrate (HFHC) model group was a 58 kcal% fat w/sucrose Surwit Diet (D12331, Lot number: 17082101A10, Research Diets, USA), and the diet for the control (Con) group was an 11 kcal% fat w/cornstarch Surwit Diet (D12328, Lot number: 17100212A4, Research Diets, USA). Fructose and sucrose (F0001/S0001) were purchased from Trophic Animal Feed High-Tech Co., Ltd, China.
2.2.1 Animal grouping and model establishment
All animal experiment protocols were approved by Animal Experimental Ethics Committee of Shanghai University of Traditional Chinese Medicine. C57Bl/6 mice were housed in the animal research center at Shanghai University of Traditional Chinese Medicine. After one week of acclimation, the mice were randomly divided into the Con group (n=30) and the HFHC model group (n=30) according to their body weights. Mice in the HFHC model group were given free access to a high-fat (58% kcal fat, 25% kcal carbohydrate and 17% kcal protein) and high-carbohydrate (drinking water: 42 g/L, 55% fructose and 45% sucrose) diet, and the mice were equally divided at 0 (HFHC 0w), 20 (HFHC 20w) and 30 weeks (HFHC 30w), with 10 mice included at each time point. The Con group mice were fed a corresponding low-fat diet (10.5% fat, 73.1% kcal carbohydrate and 16.4% kcal protein) and normal drinking water; these mice were also equally divided at 0 (Con 0w), 20 (Con 20w) and 30 weeks (Con 30w), with 10 mice included at each time point.
2.2.2 Specimen collection
At the end of weeks 0, 20 and 30, mice were fasted for 12 h and anesthetized with 2% pentobarbital sodium at a dose of 3 mL/kg via intraperitoneal injection. Eyeballs were removed, and 1 mL of blood was collected from each mouse. A piece of liver tissue was extracted from the same lobe and position in each mouse and fixed in 10% neutral buffered formalin solution.
2.2.3 Biochemical analysis
① Hepatic enzymatic changes: Serum alanine aminotransferase (ALT) and serum aspartate aminotransferase (AST) were measured using ALT and AST assay kits (Lot number 20180628, Nanjing Jiancheng Bioengineering Institute, Nanjing, China). ② Glucose metabolism: Following 12 h of fasting, approximately 3 μl of blood was collected from the tail vein, and fasting blood glucose (FBG) was measured with a Roche blood glucose meter (Roche diagnostic GmbH, Germany). Fasting insulin (FINS) levels in the serum of mice from each group were measured with an enzyme-linked immunosorbent assay (ELISA) kit (Ultra Sensitive Mouse Insulin ELISA Kit, lot number: 90080, Crystal Chem, USA). The homeostatic model assessment-insulin resistance (HOMA-IR) index was calculated with the following formula: FBG (mM) x FIN (IU/L)/22.5. ④ Liver triglyceride (TG) content was measured with a kit (Lot number: 2018080029, Dong’ou Diagnostic Products Co. Ltd., Zhejiang, China). ⑤ Liver hydroxyproline (HYP) content was measured with a HYP assay kit (Lot number: 20180630, Nanjing Jiancheng Bioengineering Institute, Nanjing, China).
2.2.4 Hematoxylin and eosin (HE) and Sirius red staining
Fixed liver tissue was dehydrated and embedded using a tissue processor (Leica ASP300) and paraffin embedding station (Leica EG1160). Then, sections were then stained using an HE staining kit (Lot number 20180530, Nanjing Jiancheng Bioengineering) and a Sirius red staining kit (Lot number 20180528, Nanjing Jiancheng Bioengineering).
2.2.5 Oil red O staining
Liver tissue was fixed in liquid nitrogen, embedded in ornithine carbamoyl transferase (OCT) medium and sectioned at -20℃ at a thickness of 10 μm. The sections were stained using an oil red O staining kit (Lot number 20180528, Nanjing Jiancheng Bioengineering).
2.2.6 Western blot analysis
Antibodies targeting the following proteins were used in this study: α-smooth muscle actin (α-SMA, ab5694, 1:1,000), hepatic type I collagen (Col-I, ab34710, 1:1,000), and transforming growth factor-β1 (TGF-β1, Abcam, 1:1,000). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) protein expression was used as an internal control.
2.2.7 Immunohistochemistry (IHC)
Samples were first incubated in anti-α-SMA antibody at a dilution of 1:100 (α-SMA, 1:100, catalog number: ab5694, USA) overnight at 4℃ and then incubated with primary antibody at a dilution of 1:250. Some liver sections were also stained with anti-Col-I antibody (Col-I, Abcam, 1:100, ab34710, USA) using the same method.
2.2.8 Reverse transcription polymerase chain reaction (RT-PCR)
RNA extraction: RNA was extracted using a UNIQ-10 pillar Trizol total RNA extraction kit (Catalog number: E928KA9723, Sangon Biotech, Shanghai). The relative quantity (RQ) values of the PCR products were subjected to analysis using the △△CT method to assess the messenger RNA (mRNA) expression levels of Col-1, α-SMA, TGF-β1, Col-4 and Smad3.
2.2.9 Library preparation and Illumina Hiseq xten sequencing
Libraries were size selected for cDNA target fragments of 200–300 bp on 2% Low Range Ultra Agarose, followed by PCR amplification using Phusion DNA polymerase (NEB) for 15 PCR cycles. After quantification with a TBS380 fluorometer, the paired-end RNA-seq sequencing library was sequenced with an Illumina HiSeq xten sequencing system (2 × 150 bp read length).
The data were analyzed using the free online Majorbio I-Sanger Cloud Platform (www.i-sanger.com).
2.2.10 Statistical analysis
Statistical analysis of database data was performed using SPSS22.0 software for Mac OS. The measurement data in the statistical description are indicated by S and refer to the count data. When the normality and homogeneity of the variance were satisfied, a t test was applied for comparison of the two groups. The comparison of the hierarchical grouping data was calculated via Radit analysis.