C57bl 6j

Six-week-old male C57BL/6J (24±0.5g) and KK-Ay mice (25 ± 0.4g) were obtained from Beijing HFK Bioscience Co. Ltd. (Beijing, China). They were housed at 24 ± 2°C. C57BL/6J mice were fed regular chow and there were considered as control group. KK-Ay mice were a kind of T2DM mouse model, and allowed free access to high-fat diet which consisted of 48.5% carbohydrates, 17.5% protein, 17.9% fat (Beijing HFK Bio-Technology Co. Ltd). KK-Ay mice were randomly assigned to 3 groups: diabetic control group (KK-Ay group, n=10), T2DM with HUA group (KK-Ay+HUA group, n=10), empagliflozin-treated KK-Ay mice with HUA group (KK-Ay+HUA+EMP group, n=10). The hyperuricemic model was induced by combination of peritoneal injection of PO at dose of 250mg/kg and intragastric administration of HX at dose of 300mg/kg daily, and mice in the KK-Ay+HUA+EMP group were orally administered with empagliflozin (10 mg/kg) in KK-Ay mice with HUA daily for 8 weeks. The dose of empagliflozin was determined based on previous studies 44 (link). The study was approved by the ethical committee of Tianjin Medical University, and all procedures involving mice were conducted according to the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health as well as the guidelines of the Animal Welfare Act.

Roswell Park Memorial Institute (RPMI) 1640 medium and Dulbecco’s modified Eagle’s medium (DMEM) containing 100 units/ml streptomycin and penicillin (PS) and 10% fetal bovine serum (FBS) were used to culture bone marrow-derived DCs (BMDCs), LL2 cells and EG7-OVA cells (American Type Culture Collection, Manassas, VA, USA). All cells were cultured in a cell incubator containing 5% CO₂ at 37 °C. RPMI 1640, DMEM, FBS and PS were all purchased from Thermo Fisher Scientific. Female six- to eight-week-old C57BL/6 J mice were purchased from HFK Bioscience (Beijing, China).
BMDCs were obtained from 4- to 6-week-old C57BL/6 J female mice according to a previously reported protocol [48 (link)]. Briefly, after treating bone marrow cells with red blood cell lysis buffer, fresh RPMI 1640 complete medium containing 20 ng/ml granulocyte-macrophage colony-stimulating factor (GM-CSF, PrimeGene Biotechnology, Shanghai, China) was added to 3 × 10⁶ mouse bone marrow cells. At day 8, the BMDCs were collected for further use.

Six-week-old male ApoE−/− mice with the C57BL/6J background were obtained from Beijing HFK Bioscience Co. (China) and were kept in specific pathogen-free facility. METH was dissolved in saline and the final concentrations were 0.8 mg/ml. Animals were randomly divided into saline control (NS, n = 5) and METH group (8 mg/kg/day through intraperitoneal injection, M8, n = 6) and treated for 24 weeks. During NS or METH treatment, mice were fed with a high-cholesterol diet containing 1.25% cholesterol and 20% lard. The mice were housed under conditions of controlled illumination (12:12-h light/dark cycle), humidity and temperature. They were allowed ad libitum access to water. All experimental procedures were performed under the guidelines of the Care and Use of Laboratory Animals (Science and Technology Department of Hubei Province, China, 2005) and approved by the Institutional Animal Research and Ethics Committee of Huazhong University of Science and Technology. All experiments were conducted in compliance with the ARRIVE guidelines and in accordance with the National Institutes of Health guide for the care and use of Laboratory animals (NIH Publications No. 8023, revised 1978).

Six-week old male ApoE^−/−mice (C57BL/6J) were purchased from HFK bioscience company (Beijing, China) were maintained at 22±2 °C, relative humidity 55%±5% with a 12hours light/dark cycle. After fed with a rodent chow diet (4.5%fat) for a week, ApoE^−/− mice were fed with western diet (21% fat, 1.25% cholesterol; HFK bioscience) for 12 weeks. Mice were then randomized into 2 groups (n=6 mice, respectively): antagomiR-19a-injected and control antagomiR-injected group. The mice received tail vein injections of 25mg/kg antagomiR-19a (GenePharma, Shanghai, China) or antagomiR once per week for 4 weeks^{40 (link),41 (link)}. After fasting for 6h, mice were euthanized and blood samples from the retro-orbital plexus, heart and aorta were collected for further analyses. All animal experiments were carried out in accordance with the protocol approved by the Institutional Animal Care and Use Committee of the Central South University.

C57Bl/6J (Beijing HFK Bioscience Co., China) and OT-1 mice were bred at Zhengzhou University in specific pathogen-free facility. All animal experiments were performed in accordance with national and institutional guidelines for animal care, and were approved by Ethics Committee of Zhengzhou University.

Ten-week-old male C57BL/6J and KK-Ay mice (license number: 2014-0004) were purchased from Beijing Huafukang Biotechnology Co., Ltd. The mice were bred adaptively for 2 weeks, and the temperature and humidity conditions maintained in the animal house were 25 ± 5°C and 55 ± 5%, respectively. All the animal experiments performed were approved by the Animal Ethics Committee of the Nanjing University of Traditional Chinese Medicine (code: 201903A019). C57BL/6J mice were fed a normal diet, while KK-Ay mice were fed a high-fat diet (458 kcal/100 g, containing 10% fat). Then, the KK-Ay mice were randomly divided into RR-CO, RR, and CO groups. The three groups were given RR-CO (3,000 mg/kg/d), RR (2,000 mg/kg/d), and CO (1,000 mg/kg/d), respectively, for 8 weeks. The KK-Ay mice in the model group were administrated saline. Seven C57BL/6J mice that were fed with a normal diet were also given saline. At the end of the experiment, the mice were sacrificed using the cervical dislocation technique, and blood was drawn from the orbit and serum was extracted. Testes were resected and weighed to calculate the ratio of the bilateral testis to body weight. The flow chart of the animal study is shown in Figure 1A.