Homeopathy 2014; 103(01): 74-75
DOI: 10.1016/j.homp.2013.10.029
Abstracts - Oral Presentation
Copyright © The Faculty of Homeopathy 2013

Study of Gelsemium sempervirens in a neurocyte model. An update

Debora Olioso
Marta Marzotto
Paolo Bellavite

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24. Januar 2018 (online)

Previous investigations showed significant anxiolytic-like activities of Gelsemium sempervirens L. (Gelsemium s.) in mice models. To provide new insights into the neural substrates of anxiety and to identify drug targets, we decided to investigate the Gelsemium s. mechanism of action in neuronal models by assessing the genome expression changes. The SH-SY5Y and IMR-32 human neuroblastoma cells were used since are widely employed in neuropharmacology and well characterized. The drugs were produced by Boiron Laboratoires (Lyon), starting from a whole-plant-hydroalcoholic extract and the cells were treated with 6 increasing dilutions 2c, 3c, 4c, 5c, 9c, 30c. We compared the drug effects with those of control solutions prepared by the same procedure, but with the solvent vehicle without the plant extract. All dilution steps were followed by strong succussion. Final ethanol concentration was 0.03% v/v. After having ruled out possible toxic effects of any test solution on cell viability, we evaluated gene expression firstly by using a microarray designed for the whole human transcriptome (Nimblegen, Roche). We used the Limma statistics approach (n=4 biological replicates) to select a set of differentially expressed genes and Friedman test followed by Wilcoxon signed-rank test to check the null hypothesis that high dilutions have no effect in this model. The exposure to 2c dilution promoted a small (fold changes between 0.5 and 1.0) but significant down expression of 49 genes as compared with untreated controls. With higher dilutions, most of the genes down-regulated in the 2c-treated samples were also under-expressed in 3c and, to a varying extent, even in higher dilutions. No changes of housekeeping genes were recorded, confirming the specificity of drug action. The changes in the 49 selected genes of SH-SY5Y cells were in the same direction in the IMR32 cells, showing that the expression of the same gene set was also modified in a second type of neurocyte. Afterword we performed the RT-qPCR on a subgroup of relevant genes modulated in 2c treatment (i.e. transcription factors, G-protein coupled receptors or neuropeptides) and we confirmed the down-regulation for the genes DDI1, EN2, GALR2, GPR25, OR5C1, Klkbl4 and TAC4. In the Wilcoxon analysis, applied to the 49 genes, the number of down-regulated ones was systematically higher than the number of genes with positive fold change over all dilutions (p<0.0001). No significant differences between treatments and controls in a randomly chosen gene set of 49 genes were observed, suggesting that the Gelsemium s. effects are not due to chance. In parallel we adopted, for the dilutions 2c and 9c, an RT-PCR Array approach (SABioscience, Qiagen) containing 84 genes, including receptors and regulators of neuronal function. In this further investigation we observed a trend to down-regulation for DRD2, CHRN4B, CHRNG, PROKR2 and PHOX2A genes in 2c and BRS3, GRPR genes in 9c dilution. In particular the down-regulation of DRD2 and PROKR2, effective in the inhibition of Gabaergic neurotransmission, suggests a specific effect of Gelsemium s. in increasing the endogenous GABA activity. Overall, the genes modulated in this experimental model outline new working hypotheses on the anxiolytic and analgesic action of this plant. In conclusion this study provides evidence that Gelsemium s. exerts a prevalently inhibitory effect on a series of genes, in particular involved in G-protein coupled transduction systems, in olfactory transduction, in calcium signaling and in neurotransmission. Furthermore the whole genome expression analysis (microarray and real-time PCR), indicates that the “omics” molecular biology is a suitable approach to study the effects of highly diluted natural compounds.