Chemix 180

Glucose was measured by a glucometer (Accu-check, Roche; Indianapolis, IN, USA). Blood samples were kept on ice until plasma preparation (centrifugation at 3000 rpm for 15 min at 4 °C). Plasma was immediately separated and stored at −80 °C until analysis. TG, total-CHOL, HDL-CHOL, and LDL-CHOL from plasma and intestinal supernatant were measured using Chemix-180 (Sysmex Corp.; Mundelein, IL, USA).

Alanine aminotransferase (ALT), aspartate aminotransferase (AST), and high-density lipoprotein (HDL-C) in the serum were quantitated using an automated analyzer (Sysmex CHEMIX-180, Japan). The concentrations of serum triglycerides (TG) and total cholesterol (TC) were separately quantitated using triglyceride and cholesterol assay kits (Applygen Technologies Inc., Beijing, China) according to the manufacturer’s instructions. Additionally, the oxidative stress was elucidated by measuring the malondialdehyde (MDA) and superoxide dismutase (SOD) enzymes in the serum samples following ELISA-based standard protocols.

Total serum cholesterol (TC), triglyceride (TG), alanine aminotransferase (ALT), aspartate aminotransferase (AST), fasting blood glucose (FBG), low-density lipoprotein cholesteryl ester (LDL-C), and high-density lipoprotein cholesteryl ester (HDL-C) were estimated using an automatic biochemistry analyzer (Sysmex CHEMIX-180, Japan). The fasting insulin levels were determined using a commercial insulin enzyme-linked immunosorbent assay kit (Crystalchem, USA). All procedures were conducted according to the manufacturer’s instructions.

Six-week old male C57BL/6 mice (21–25 g) were obtained from Slac Laboratory Animal Co., Ltd. The mice were housed in cages at 24 ± 2 °C, humidity of 40 ± 5%, under a 12-hour light/dark cycle, and received standard diet and water ad libitum. Mice were adapted for 2 weeks, then fed with a high-cholesterol diet (prepared by and purchased from Biomodel Organism Science & Technology Development Co., Ltd., Shanghai, China) for 8 weeks, and subsequently randomly assigned to the following groups: sham, vehicle (saline), YXS-1 (60 mg·kg⁻¹·d⁻¹, equivalent to clinical dosage), or YXS-2 (120 mg·kg⁻¹·d⁻¹) by gavage for 1 week. At the end of vehicle or YXS treatment, tail vein blood was collected and centrifuged at 4 °C to determine plasma triglyceride (TG), total cholesterol (TC), and low-density lipoprotein-cholesterol (LDL-C) levels using an auto-biochemical analysis system (Chemix-180, Sysmex, Japan). The protocols for in vivo study with mice were approved by the Animal Ethics Committee of Shanghai Jiao Tong University, and the methods for in vivo study were carried out in accordance with the approved guidelines.

Following 24 h of reperfusion, blood samples were taken immediately after the eyeballs of the experimental mice were removed. The serum was obtained by centrifugation at 4,000 × g for 5 min. The levels of serum creatinine (Scr) and blood urea nitrogen (BUN) were determined by a biochemical auto-analyzer (Sysmex CHEMIX-180) using the commercial diagnostic kits.

Serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), and fasting blood glucose were measured using an automated analyzer (Sysmex CHEMIX-180, Japan). Insulin (Rat/Mouse Insulin ELISA Kit, Merck-Millipore) and GLP-1 (Glucagon Quantikine ELISA Kit, R&D Systems) in the serum were measured using an enzyme-linked immunosorbent assay. A homeostatic model assessment of insulin resistance (HOMA-IR) and the insulin sensitive index (ISI) were calculated.