Au480

The serum levels of AST, ALT, TG, TC, HDL, LDL, and GLU were detected by an automatic biochemical analyzer (AU480, Olympus, Japan). Serum cytokines, including tumor necrosis factor-α (TNF-α) (ERC102a, NeoBioscience, China), monocyte chemotactic protein-1 (MCP-1) (ERC113, NeoBioscience, China), and interleukin- 6 (IL-6) (ERC003, NeoBioscience, China), as well as 25(OH)D₃ (E-EL-0015c, Elabscience, China) were measured by enzyme-linked immunosorbent assay (ELISA) kits according to the manufacturer’s instructions. Liver tissues were homogenized in a working solution and centrifuged at 12000 × g for 20 min. The TG contents (A110-1-1, Jiancheng Bio, China) and FFA contents (A042-2-1, Jiancheng Bio, China) of the liver tissue were detected according to manufacturer’s instructions. The lipid level was normalized with the respective protein concentration.

Body weights were measured and blood samples were collected at the baseline (9-month-age) and after HFHSD induction. After 16 h of fasting, venous blood was collected from the anterior vena cava and centrifuged at 4 °C, 1000 g for 10 min to isolate plasma. Samples for the glucagon-like peptide 1 (GLP-1) test were stored in a BDtm p800 blood collection tube at −80 °C in the refrigerator until the assay. Plasma biochemical parameters including blood glucose, insulin, and GLP-1 were tested using an automatic biochemical analyzer (AU480, Olympus Co., Tokyo, Japan).

After a 12-h overnight fast and 48-h alcohol abstinence, all participants underwent a physical examination in the morning. The height, weight, waist circumstance (WC), systolic pressure (SBP), and diastolic blood pressure (DBP) of the participants were measured and recorded by a professional nurse. Fasting venous blood was collected and centrifuged to obtain serum for detection of the following parameters using an automatic biochemical analyzer (AU480, Olympus, Japan): aspartate aminotransferase (AST), ALT, TG, TC, HDL-C, LDL-C, γ-glutamine transferase (GGT), alkaline phosphatase (ALP), albumin (ALB), plasma glucose (GLU), fasting insulin (INS), and blood platelet (PLT). Serum 25(OH)D₃ was detected by a chemiluminescence immunoassay (CLIA) (VD-T, Mindray, China). Homeostatic Model Assessment for Insulin Resistance (HOMA-IR) was estimated as previously described (50 (link)). Body mass index (BMI) was calculated using the following equation: BMI (kg/m²) = body weight (kg)/height² (m). Participants with a BMI greater than 25 but less than 30 were defined as overweight, and participants with a BMI greater than 30 were defined as obese (51 (link)). According to the Vitamin D Deficiency Screening Method of China in 2020, VD deficiency was defined as circulating 25(OH)D₃ less than 12 ng/mL, and VD insufficiency was defined as serum 25(OH)D₃ levels between 12 and 20 ng/mL (52 ).

Use the fully automated biochemical analyzer AU480 from Japan’s Olympus company to detect serum biochemical indicators aspartate transaminase (AST), alanine transaminase (ALT), alkaline phosphatase (ALP), r-glutamyl transferase (r-GT), total bilirubin (TBIL), and glucose (GLU). The TGF-β1, IL-1β, TNF-α and α-SMA in liver tissues and cells were assayed using ELISA method as instructed by the manufacturer.

The levels of ALT, AST in serum were detected using an AU480 automatic biochemistry analyzer (Olympus, Tokyo, Japan).

The analyzed biochemical indices included alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), total bilirubin (TBIL), superoxide dismutase (SOD), and malondialdehyde (MDA). Serum biochemical analyses were performed using a clinical biochemistry analyzer (AU480, Olympus, Japan) at Beijing De–Yi Biotechnology Co., Ltd. (China). Liver oxidative stress biomarkers were examined using visible spectrophotometers (Nanjing Jiancheng Bioengineering Institute, China).