Biochemical kit

The concentrations of total cholesterol (TC), triacylglycerol (TG), low-density lipoprotein cholesterol (LDL), high-density lipoprotein cholesterol (HDL), superoxide dismutase (SOD), malondialdehyde (MDA), glutathione peroxidase (GPX), reduced glutathione (GSH), glutathione S-transferase (GST) and glucose in serum or liver homogenates were examined by biochemical kits (Nanjing Jiancheng, China). The content of insulin, tumor necrosis factor-α (TNF-α), C-reactive protein (CRP), and interleukin-6 (IL-6) was analyzed by enzyme-linked immunoassay (ELISA) kits (Nanjing Jiancheng). The content of ATP was examined with bioluminescent kits (Sigma, MO, USA).

Blood was collected from the inner canthus after 90 days of administration and left still at room temperature for over 30 min, from which serum was separated by centrifugation at 1500 rpm for 10 min. Afterward, 100 µL of serum was collected from every sample. The serum TG, TC, LDL, and HDL levels were tested using biochemical kits (Jiancheng Bioengineering Institute, Nanjing, China) by HITACHI 7020 Chemistry Analyzer.^{54 (link)}

Triglycerides and cholesterol in serum samples were measured using the biochemical kits (Jiancheng Bioengineering Institute, Nanjing, China). SAM and acetyl-CoA were measured by using ELISA kits (Meimian Biotechnology, Jiangsu, China).

Hepatic levels of O^.−₂ were measured using the chemiluminescence method^{43 (link)}. First, the mouse liver tissues were weighed and homogenized in pH 7.4 lysis buffer containing 10 mM ethylenediaminetetraacetic acid (EDTA) and 20 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES). Samples were centrifuged at 1000 g for 10 min. Then, an aliquot of each sample was incubated with Krebs-HEPES buffer, pH 7.4, containing 5 mM lucigenin (Sigma, Shanghai, China) for ~ 2 min at 37 °C. Next, light emission data were obtained using a M200 PRO multifunctional microplate reader (TECAN, Switzerland), and the results are reported as the mean light units min/mg protein. Levels of O^.−₂ were measured by adding superoxide dismutase (SOD) (350 U/mL) to the medium according to the manufacturer’s instruction (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). In addition, liver tissues were homogenized in normal saline, and the samples were treated with an equal volume of cold methanol for 60 min in the 4 °C icebox. Then, samples were centrifuged for at 10,000 g for 30 min, and we obtained the supernatant to measure H₂O₂ levels using biochemical kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). Protein concentrations were measured using the Bradford method, and bovine serum albumin (BSA) was employed as the standard.

As indicators of liver function, serum, and liver levels of glutathione (GSH), alanine transaminase (ALT), alkaline phosphatase (ALP), and aspartate transaminase (AST) were analysed by employing biochemical kits from Nanjing Jiancheng Bioengineering Institute (Nanjing, China).

Livers (0.1 g) were homogenized in 0.9 mL of saline solution in an ice-water bath and centrifuged (3,000 rpm for 10 min), after that, the supernatant was taken for assaying various indicators of oxidative stress. The concentrations of proteins in the liver homogenates were determined using Bradford assays (Beijing Solarbio Science Technology Co., Ltd., Beijing, China). The MDA content in the liver homogenates, as well as the activities of the antioxidant enzymes SOD, GSH-Px, and CAT, were evaluated using biochemical kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, Jiangsu, China) according to the manufacturer's instructions.

Serum levels of TC, TG, HDL-C, and LDL-C were measured using biochemical kits [obtained from Nanjing Jiancheng Bioengineering Institute (Nanjing, Jiangsu, China)].