M5250

Patients were instructed to take 20 mg of oral melatonin or placebo daily approximately 1 h before bedtime. Melatonin capsules were produced using crystalline melatonin with a certificate of purity (M-5250, Sigma Chemical, Saint Louis, MO, USA) by a compounding pharmacy. The tablets of melatonin and placebo were physically identical. Assessments to confirm adherence to treatment included: i) Pill counting during the study period. ii) Patient diaries were kept in order to record if they failed to use the medication. iii) Patients were encouraged to remain on melatonin throughout the ten days of treatment.

The mice were randomly divided into three groups: the sham group (n = 12), the MI group (MI, n = 12), and the melatonin treatment group (MI+MLT, n = 12). The MI model was generated with a method similar to that in previous studies [23 (link), 24 (link)]. Briefly, the mice were anaesthetized by avertin (0.2 g/kg) injection. The left anterior descending coronary artery was exposed and ligated with a 7-0 nylon suture for 24 h. The mice in the sham group underwent the same surgical procedures without coronary artery ligation. The mice in the MI+MLT group were administered 10 mg/kg melatonin (M5250, Sigma-Aldrich) per day by intragastric gavage for 14 days prior to MI surgery [25 (link)]. The mice in the sham and MI groups were given an equivalent volume of ddH₂O.

Human ovarian follicular fluid (FF) was collected from women (n=110) undergoing IVF treatment at the fertility center of AJUMS general hospital. The Ethics Committee of the Research Deputy of Ahvaz University of Medical Sciences (IRAJUMSABHC.REC1397.007) approved this study. During the procedure, an operator collected FF into sterile conical centrifuge tubes containing one drop of heparin. Subsequently, they were transported to the laboratory within 1 h. The hypo-osmotic technique described by Lobb & Younglai (2006) (link) was used to remove red blood cells. FF samples were centrifuged at 1400 rpm for 6 min; 9.0 mL of sterile distilled water was added to the buffy coat containing follicular fluid cells and the tube was capped and mixed. After 60s, 1.0mL of 10x concentrated phosphate buffer saline (PBS; pH≈7) was added and mixed again. The tubes were centrifuged at 800 rpm for 3 min for final collection. Isolated cells were cultured in 4-well plates with HG-DMEM (Dulbecco's Modified Eagle Medium) containing 15% FBS (fetal bovine serum) supplemented with amphotericin and penicillin plus streptomycin with different concentration of melatonin (Sigma-Aldrich, M5250) (10^-7,10^-9,10^-11 M); the samples and kept in a 5% CO₂ incubator at 37ºC for a maximum of 14 days, depending on the assay protocol.

Melatonin (≥ 99.8%, M5250), used as the standard for liquid chromatography tandem mass spectrometry (LC–MS/MS) detection, was purchased from Sigma Aldrich (St. Louis, MO, USA). Methanol (HPLC grade) was purchased from Thermo Fisher Scientific (Waltham, MA, USA). Acetonitrile (HPLC grade) was purchased from Thermo Fisher Scientific (Waltham, MA, USA). Hydrochloric acid was purchased from Shanghai Aladdin Biochemical Technology Co. (Shanghai, China). Formic acid (HPLC grade) was purchased from Shanghai Aladdin Biochemical Technology Co. (Shanghai, China). 2-Ethyl butyric acid was purchased from Shanghai Aladdin Biochemical Technology Co. (Shanghai, China). Ultrapure water with a resistance of 18.2 MΩ cm − 1 was purified using a Milli-Q system (Millipore, Bedford, USA).

All animal experiments were conducted following the experimental animal care and use guidelines of the National Institutes of Health (NIH, USA). Experiment protocols and procedures were approved by the Committee for Animal Experiments at Guangzhou University of Chinese Medicine in China (permit code: SYXK (YUE) 2017-0179). AD-modelled 3xTg AD mice and 5xFAD mice were kindly provided by Song lab in Guangzhou University of Chinese Medicine. Male and female mice were mated and bred in stable conditions in terms of temperature, humidity, and 12 h light/dark cycle. Standard laboratory diet and water were provided for the mice. The male age-matched wild type, 3xTg AD and 5xFAD mice at specific ages ranging from 2 months to 13 months were used in the present study for in vivo two-photon microscope imaging, immunofluorescence staining, and Western blot analysis. For melatonin treatment, mice at age of 12 months were administrated with melatonin (M5250, Sigma Aldrich) at a daily dose of 10 mg/kg for 4 weeks. melatonin was dissolved in ethanol and then diluted by water to 1% ethanol.