Neuroprotective Potential of Astragaloside IV in SH-SY5Y Cells

SH-SY5Y cells purchased from BeNa Culture Collection (Suzhou, Jiangsu, China) were seeded in culture flasks containing complete medium [90% basic Dulbecco’s modified Eagle’s medium (DMEM, Invitrogen, Carlsbad, CA, United States), 10% heat inactivated fetal bovine serum, 100 μg/mL streptomycin and 100 U/mL penicillin (Sangon Biotech Co., Ltd, Shanghai, China)] at 37°C and 5% CO₂. The medium was refreshed every other day and cells were passaged every 2–3 days. The cells at passage 3 were used for the subsequent experiments.
SH-SY5Y cells in good conditions were treated with different concentrations (10, 50, 100, 150, and 200 μM) of 6-OHDA (Sigma-Aldrich, Merck KGaA, Darmstadt, Germany) for different time (0, 3, 6, 12, 24, and 48 h) (Chen et al., 2020 (link)). The concentration and time of 6-OHDA intervention at 50% cell activity were selected as the experimental conditions. Different concentrations (25, 50, 100, 150, and 200 μM) of AS-IV (National Institute for the Control of Pharmaceutical and Biological Products, Beijing, China) was added to the medium 2 h before 6-OHDA treatment, or 15 μL JAK2/STAT3 pathway inhibitor SC99 [2-(2-(3-chloro-4-fluorophenyl)hydrazono)-3-(4-chlorophenyl)-3-oxo-propanenitrile, 2-(2-(2-(3-4-fluorophenyl)hydrazine)-3-(4-chlorophenyl)-3-oxo-acrylonitrile)] (15 mM, Sigma-Aldrich) was added to interfere with the pathway (Zhang et al., 2016 (link)).