Comparative Analysis of Gelsemine and Gelsemium sempervirens Activity on Neurosteroid Allopregnanolone Formation in the Spinal Cord and Limbic System

Abstract

Centesimal dilutions (5, 9 and 15 cH) of Gelsemium sempervirens are claimed to be capable of exerting anxiolytic and analgesic effects. However, basic results supporting this assertion are rare, and the mechanism of action of G. sempervirens is completely unknown. To clarify the point, we performed a comparative analysis of the effects of dilutions 5, 9 and 15 cH of G. sempervirens or gelsemine (the major active principle of G. sempervirens) on allopregnanolone (3α,5α-THP) production in the rat limbic system (hippocampus and amygdala or H-A) and spinal cord (SC). Indeed, H-A and SC are two pivotal structures controlling, respectively, anxiety and pain that are also modulated by the neurosteroid 3α,5α-THP. At the dilution 5 cH, both G. sempervirens and gelsemine stimulated [(3)H]progesterone conversion into [(3)H]3α,5α-THP by H-A and SC slices, and the stimulatory effect was fully (100%) reproducible in all assays. The dilution 9 cH of G. sempervirens or gelsemine also stimulated 3α,5α-THP formation in H-A and SC but the reproducibility rate decreased to 75%. At 15 cH of G. sempervirens or gelsemine, no effect was observed on 3α,5α-THP neosynthesis in H-A and SC slices. The stimulatory action of G. sempervirens and gelsemine (5 cH) on 3α,5α-THP production was blocked by strychnine, the selective antagonist of glycine receptors. Altogether, these results, which constitute the first basic demonstration of cellular effects of G. sempervirens, also offer interesting possibilities for the improvement of G. sempervirens-based therapeutic strategies.

Figure 1

Characterization of [³H]-neurosteroids released in the incubation medium by: (a) lumbar SC slices after a 3-h incubation with [³H]PROG in the absence (b) or in presence of gelsemine 5 cH or (c) Gelsemium sempervirens 5 cH. Analyses were performed using a hexane/isopropanol gradient and a reverse-HPLC system coupled to a flow scintillation detector. The ordinate indicates the radioactivity measured in the HPLC eluent. The dashed line represents the gradient of secondary solvent (% isopropanol). The arrows indicate elution positions of standard steroids.

Figure 2

Effects of gelsemine (a) and G. sempervirens (b) at 9 or 5 cH on 3α,5α-THP production by SC slices. Each value was calculated as the relative amount of [³H]3α,5α-THP compared with the total [³H]-labeled compounds resolved by HPLC-Flo/One characterization (×100). The values were obtained from experiments similar to those presented in Figures 2(a). Results were then expressed as percentages of the amount of [³H]3α,5α-THP formed in absence of gelsemine or G. sempervirens (control). Each value is the mean (± SEM) of four independent experiments. **P < .01, ***P < .001 as compared to control (Student's t-test).

Figure fig3

Effects of gelsemine (a) and G. sempervirens (b) at 9 or 5 cH on 3α,5α-THP production by H-A slices. The values were obtained from experiments similar to those presented in Figure 1. Results were then expressed as percentages of the amount of [³H]3α,5α-THP formed in the control group. Each value is the mean (±SEM) of four independent experiments. *P < .05, ***P < .001 as compared to control (Student's t-test).

Figure 4

Comparative analysis of the effects of gelsemine and G. sempervirens at 9 or 5 cH on [³H]PROG conversion into [³H]3α,5α-THP by SC slices. The values were obtained from experiments similar to those presented in Figure 1. Results were then expressed as percentages of the amount of [³H]3α,5α-THP formed in the control group. Each value is the mean (±SEM) of four independent experiments. *P < .05, ***P < .001 as compared to control; ^# P < .05, ^## P < .01 as compared to gelsemine (ANOVA followed by Bonferroni's test).

Figure 5

Comparative analysis of the effects of gelsemine and G. sempervirens at 9 or 5 cH on [³H]PROG conversion into [³H]3α,5α-THP by H-A slices. The values were obtained from experiments similar to those presented in Figure 1. Results were then expressed as percentages of the amount of [³H]3α,5α-THP formed in absence of gelsemine and G. sempervirens (control). Each value is the mean (± SEM) of four independent experiments. *P < .05, ***P < .001 as compared to control; ^## P < .01 as compared to gelsemine (ANOVA followed by Bonferroni's test).

Figure 6

Effects of gelsemine (a) or G. sempervirens (b) at 5 cH on [³H]3α,5α-THP production by SC slices in the absence or presence of strychnine (10⁻⁵ M), the specific glycine receptor antagonist. The values were obtained from experiments similar to those presented in Figure 1. Results were then expressed as percentages of the amount of [³H]3α,5α-THP formed in the control group. Each value is the mean (±SEM) of four independent experiments. ***P < .001 as compared to gelsemine; ^## P < .01, ^### P < .001 as compared to G. sempervirens (ANOVA followed by Bonferroni's test). NS represents not statistically different.

Figure 7

Effects of (a) gelsemine or (b) G. sempervirens at 5 cH on 3α,5α-THP production by H-A slices in the absence or presence of strychnine (10⁻⁶ M). The values were obtained from experiments similar to those presented in Figure 1. Results were then expressed as percentages of the amount of [³H]3α,5α-THP formed in the control group. Each value is the mean (±SEM) of four independent experiments. ***P < .001 as compared to gelsemine; ^## P < .01, ^### P < .001 as compared to G. sempervirens (ANOVA followed by Bonferroni's test). NS represents not statistically different.

Figure 8

Effect of gelsemine (5 cH) or G. sempervirens (5 cH) on 3α,5α-THP production in the SC and H-A. (1) Gelsemine (5 cH) or G. sempervirens (5 cH) stimulated [³H]progesterone conversion into [³H]3,5-THP in SC and H-A slices. (2) Strychnine, the selective antagonist of glycine receptors (Gly-R), totally blocked the stimulatory effect of gelsemine (5 cH) or G. sempervirens (5 cH) on 3α,5α-THP production. (3) Further investigations are required for the identification of intracellular mechanisms triggered by gelsemine (5 cH) or G. sempervirens (5 cH) from Gly-R. However, the stimulatory action exerted by gelsemine (5 cH) or G. sempervirens (5 cH) on 3α,5α-THP production suggests that the intracellular cascade activated by these substances may increase the activity of 3α-HSOR which is the key 3α,5α-THP-synthesizing enzyme. [4, 5] Neurosteroid 3α,5α-THP newly synthesized by neurons or glial cells in the SC or H-A may modulate GABA_A-R or Gly-R through autocrine (4) or paracrine (5) mechanisms leading to analgesic and/or anxiolytic effects of G. sempervirens.