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. 2014 Mar 19;14:104.
doi: 10.1186/1472-6882-14-104.

Extreme Sensitivity of Gene Expression in Human SH-SY5Y Neurocytes to Ultra-Low Doses of Gelsemium Sempervirens

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Free PMC article

Extreme Sensitivity of Gene Expression in Human SH-SY5Y Neurocytes to Ultra-Low Doses of Gelsemium Sempervirens

Marta Marzotto et al. BMC Complement Altern Med. .
Free PMC article

Abstract

Background: Gelsemium sempervirens L. (Gelsemium s.) is a traditional medicinal plant, employed as an anxiolytic at ultra-low doses and animal models recently confirmed this activity. However the mechanisms by which it might operate on the nervous system are largely unknown. This work investigates the gene expression of a human neurocyte cell line treated with increasing dilutions of Gelsemium s. extract.

Methods: Starting from the crude extract, six 100 × (centesimal, c) dilutions of Gelsemium s. (2c, 3c, 4c, 5c, 9c and 30c) were prepared according to the French homeopathic pharmacopoeia. Human SH-SY5Y neuroblastoma cells were exposed for 24 h to test dilutions, and their transcriptome compared by microarray to that of cells treated with control vehicle solutions.

Results: Exposure to the Gelsemium s. 2c dilution (the highest dose employed, corresponding to a gelsemine concentration of 6.5 × 10(-9) M) significantly changed the expression of 56 genes, of which 49 were down-regulated and 7 were overexpressed. Several of the down-regulated genes belonged to G-protein coupled receptor signaling pathways, calcium homeostasis, inflammatory response and neuropeptide receptors. Fisher exact test, applied to the group of 49 genes down-regulated by Gelsemium s. 2c, showed that the direction of effects was significantly maintained across the treatment with high homeopathic dilutions, even though the size of the differences was distributed in a small range.

Conclusions: The study shows that Gelsemium s., a medicinal plant used in traditional remedies and homeopathy, modulates a series of genes involved in neuronal function. A small, but statistically significant, response was detected even to very low doses/high dilutions (up to 30c), indicating that the human neurocyte genome is extremely sensitive to this regulation.

Figures

Figure 1
Figure 1
Morphological and functional properties of SH-SY5Y neuroblastoma cells used in the assay. A. Phase contrast micrograph of adherent cells cultured in Petri dishes. Bar, 10 μm. B. Spectrofluorometric measurement of intracellular calcium changes induced by carbachol at the specified doses.
Figure 2
Figure 2
UV–vis spectra of representative Gelsemium s. solutions. A: Gelsemium s. 1c dilution supplied by the manufacturer, B: Gelsemium s. 2c dilution supplied, C: Gelsemium s. 3c dilution supplied, D: Gelsemium s. 2c dilution prepared by 100 × dilution of solution A and used in the experiments.
Figure 3
Figure 3
Effects of Gelsemium s. on SH-SY5Y cell viability. Cell viability was determined by WST assay after 24 h treatment with Gelsemium s. or Control dilutions. Values in abscissa are mean absorbance values ± SD (n = 6).
Figure 4
Figure 4
Differential effect of Gelsemium s. on two cell lines. Fold changes of the 56 selected genes in SH-SY5Y (red bars) and IMR32 (blue bars) cells after 24 h treatment with Gelsemium s. 2c.
Figure 5
Figure 5
Frequency of fold change values in the down-regulated gene-set after Gelsemium s. treatments. In this analysis the 49 genes whose expression was down-regulated by Gelsemium s. 2c were considered. Mean Log2 fluorescence values from Gelsemium s.-treated samples (Gnc) and those from controls (Ctnc) were obtained from 4 microarray experiments and their difference was considered as fold change attributable to Gelsemium s. effect (see Methods). Absolute fold changes less than or equal to 0.05 were considered null. Blue bars: frequencies of genes with negative fold change (< -0.05); grey bars: frequency of unaffected genes (from -0.05 to 0.05); pink bars: frequencies of genes with positive fold change (> 0.05). Fisher exact p values are reported in each panel except the G2c-Ct2c that are significant by definition.
Figure 6
Figure 6
Number of genes modulated by Gelsemium s. dilutions in the panel of up-regulated genes. In this analysis the 7 genes whose expression was up-regulated by Gelsemium s. 2c were considered. Differences less than or equal to 0.05 were considered null. Blue bars: number of genes with negative fold change (< -0.05); grey bars: number of unaffected genes (from -0.05 to 0.05); pink bars: number of genes with positive fold change (> 0.05). Fisher exact test is not significant in any dilution except in the 2c dilution that is significant by definition.
Figure 7
Figure 7
K-mean clustering of the genes modulated upon exposure to Gelsemium s. dilutions. The expression profile of 56 genes significantly modulated by Gelsemium s. 2c was evaluated also upon exposure to increasing Gelsemium s. (G) dilutions. Fold change was calculated as the difference between Log2 fluorescence values of each Gelsemium s. dilution and the mean Log2 fluorescence of the controls. Data are means of 4 replicate experiments. A. K-mean clusters (KMC) visualized as a colour-coded heat map. The down-regulated genes (green) with similar expression profiles were grouped in 4 clusters and the up-regulated genes (red) in one cluster. B. Centroid graphs of the mean fold change of genes in the 5 clusters obtained in KMC analysis.

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