Hearts from Sprague-Dawley rats aging one to 3 days were excised and rapidly taken off using sterilized surgical scissors. After rinsing three times in cold phosphate buffered saline (PBS) solution, the hearts were cut into approximately 1–3 mm3 cubes and transferred into a 50 mL of conical bottle. About 1.5–2.0 mL of Dulbecco’s modified eagle medium (DMEM, Wisent Inc., Montreal, QC, Canada) having trypsin was added into the conical bottle placed on an incubator with shaking for 5 min at 37°C to start digestion. The first digestion’s supernatant was discarded. Then, the precipitate underwent further digestion for 3 min at a time and repeated for about 10 times. All digested supernatants were collected into a beaker containing DMEM with 10% fetal bovine serum (FBS, Gibco, Thornton, NSW, Australia). The cell suspension after filtering with a cell sieve was centrifuged in a centrifuge tube for 5 min at 1,200 r/min. Following removing the supernatant, the cells in the precipitate were re-suspended and inoculated into a new culture dish with DMEM having 10% FBS. The cells were placed at 37°C in 5% CO2 incubator, and the differential adhesion method to acquire cardiac fibroblasts was performed. In detail, culture medium containing cardiomyocytes was removed after cells had adhered to the plate for 180 min. The cardiac fibroblasts that remained attached to the plate were digested and cultured in fresh DMEM having 10% FBS. The cardiac fibroblasts were sub-cultured basing on their growth conditions, and the cardiac fibroblasts of 3rd to 4th generation were seeded into plates in the present study. After starvation for 12 h, the cardiac fibroblasts were pre-administrated with RORα antagonist SR3335 (5, 10, 20 and 40 μM, MedChemexpress, Rahway, NJ, United States) or RORα agonist SR1078 (2.5, 5, 10 and 20 μM, MedChemexpress) followed by 48 h of stimulation with normal glucose (NG) or high glucose (HG) respectively (Liang et al., 2021 (link); Chen D. et al., 2024 (link); Wahyuni et al., 2021 (link); Zhang Y. et al., 2022 (link); Xiong et al., 2020 (link); Shen et al., 2023 (link)). Cardiac fibroblasts under the normal glucose (5.5 mM) group and high glucose (11.1 mM, 22.2 mM and 33.3 mM) group were exposed to 27.8 mM, 22.2 mM, 11.1 mM mannitol and 0 mM mannitol respectively to balance the osmotic pressure (Gong et al., 2022 (link); Zhang S. et al., 2023 (link); Tian et al., 2021 (link); Lu et al., 2023 (link)).
The study was conducted according to National Institutes of Health guidelines for the Care and Use of Laboratory Animals, and approved by Committee of Nantong University (approval no. S20210227-011 on 27 February 2021). The study was conducted in accordance with the local legislation and institutional requirements.
The study was conducted according to National Institutes of Health guidelines for the Care and Use of Laboratory Animals, and approved by Committee of Nantong University (approval no. S20210227-011 on 27 February 2021). The study was conducted in accordance with the local legislation and institutional requirements.
