HFD/STZ-induced diabetic mouse models

C57BL/6J mice (body weight, 18±2 g; 4-weeks-old; male) were purchased from Guangzhou Medical Animal Centre (Guangzhou, China) and housed under controlled conditions (constant temperature: 22±2°C; constant humidity: 60±5%; 12 h dark/light cycle). The study was performed in strict accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and the protocol was approved by the Bioethics Committee of Shenzhen International Graduate School, Tsinghua University, China (Ethics issue [2020] No. 9). The diabetic mouse models were fed with HFD (41% energy from fat; Beijing HFK Bioscience, Beijing, China, {"type":"entrez-nucleotide","attrs":{"text":"H10141","term_id":"874963","term_text":"H10141"}}H10141). The model group and drug treated group were intraperitoneally injected with STZ (40 mg/kg once before drug administration; Sangon Biotech Co., Ltd., Shanghai, China), which was freshly dissolved in ice-cold 0.1 M citrate solution (pH 4.5), according to a previous study (Xie et al., 2007). After one week, the diabetic mice with fasting blood glucose more than 11.1 mmol/L were divided into two groups: An untreated HFD/STZ control group and a CAN (Biochempartner, Shanghai, China)-treated group (25 mg/kg/d; dissolved in 0.5% sodium salt of carboxymethyl cellulose [CMC-Na; Sangon Biotech, Shanghai, China]). The intragastric dose of CAN administered to the mice was converted according to the clinical dose in humans (100 mg/kg/day). The normal control and HFD/STZ control groups were orally administered with an equal volume of 0.5% CMC-Na daily, CAN group were normal mice only treated with CAN (25 mg/kg/day) alone. The body weight of the mice, and food and water uptake were monitored once a week. After 12 weeks of treatment, the heart function was assayed by echocardiographic method. Briefly, the mice were subjected to an anesthesia with isopentane. Then, the left ventricular ejection fraction (LVEF) and left ventricular diastolic internal diameter (LVID) was analyzed by high-resolution small animal imaging system (Vevo 2100, VisualSonics, Toronto, Canada) equipped with 22–55 MHz transducer at a frame rate of 233 Hz. Finally, the mice were subjected to fasting for 6 h, anesthetized with urethane, which was dissolved in saline (intraperitoneal injection of urethane at a dose of 1000 mg kg-1; Sangon Biotech Co., Ltd., Shanghai, China), then sacrificed while under anesthesia. The blood was collected from the orbital plexus veins, and the serum was extracted from the blood samples using centrifugation (1000 rpm for 10 min at 4°C) and stored at -20°C for further analysis. Simultaneously, unrecovered anesthetized animals were sacrificed using cervical dislocation by skillful well-trained investigators, and the hearts, abdominal adipose tissues and livers were removed and weighed. A portion of the heart tissue was immersed in 4% paraformaldehyde solution for regular pathological slicing, and hematoxylin and eosin (H&E), wheat germ agglutinin (WGA) and Masson staining. The remaining samples were instantly frozen by using liquid nitrogen and stored at -80°C for further analysis.