Cellular and transcriptional responses of SH-SY5Y human neurocytes following in vitro exposure to Gelsemium sempervirens

Background: Gelsemium sempervirens (Gelsemium s.) is a highly toxic plant but is employed at low doses and/or high dilutions as an anxiolytic and antidepressant. Previous investigations in our laboratory [1,2] have shown a significant anxiolytic-like activity of Gelsemium s., using emotional response models in laboratory mice. Although there is some biochemical evidence of a possible role of neurosteroid metabolism [3], the cellular and molecular mechanisms involved in the effects of Gelsemium s. at the level of nervous system are largely unknown. To help determine these pathways, we used human neurocytes (SH-SY5Y cell line) treated in vitro with different dilutions of Gelsemium s. and evaluated their vitality and gene expression changes. Methods: The drugs were produced by Boiron Laboratoires, Lyon (F), starting from a whole-plant-hydroalcoholic extract of Gelsemium s. Solutions 1C, 2C, 3C, 4C, 8C and 29C (C= centesimal dilution/dynamization prepared in 30% ethanol/distilled water) were provided in 30-ml glass bottles, wrapped in aluminium foil and were stored in the dark at room temperature in a metal cupboard. Control solutions ( ￠a “placebo ￠a ) were serially diluted/dynamized 30% ethanol/distilled water. Before each experiment, 0.05 ml samples of Gelsemium s. and placebo were added to 5 ml of distilled sterile and apyrogenic water in a 15 ml Falcon polystyrene plastic tube, closed and shaken in mechanical shaker DinaA for 7.5 sec (150 strokes) to obtain the final 2C, 3C, 4C, 5C, 9C and 30C succussed dilutions, with ethanol concentration of 0.3% (v/v) (final 0.03% in the assay system). Human neuroblastoma cell line SHSY5Y was grown in DMEM-F12 medium (Lonza), with 10% fetal bovine serum (FBS), penicillin (100 units/ml) and streptomycin (100 mg/ml). To assess cell viability and metabolism, 20,000 cells per well were seeded in 96 microplate wells in 200 l of medium. After overnight incubation, 22 l of drug or placebo were added and the plate was incubated at 37 °C with 5% CO2 in a humidified atmosphere for 72 hours. Then the viability test with reagent WST-1 (Roche) was performed for 3 hours and the absorbance was detected with multiplate reader. A total of 17 experiments, each done with six replicate microwells. To make a relative measurement of protein, the cells were lysed and a Bradford assay was done directly in the plate. The Student t-test and the sign rank test for paired data were utilized for data analysis. To obtain a profile of gene expression, cells were pre preconditioned with Gelsemium s./placebo dilutions for 24 h, then R