On the other hand we

On the other hand, we have previously demonstrated that gelsemine displaced the H3-strychnine from membrane fractions of rat spinal cord homogenates. In addition, spinal gelsemine antinociception was blocked by intrathecal injection of the specific glycine receptor antagonist strychnine or gene silencer of the glycine receptor α3 subunit. These results suggested that gelsemine produced antinociception via activation of spinal α3 glycine receptors [10]. In superficial dorsal horn pain processing circuitry, ionotropic inhibition is mediated via glycine receptors and GABAA receptors. Blockade of glycine receptors or GABA receptors exacerbates painful behaviors in the models of neuropathic and inflammatory pain [17], [18], [19]. Endogenous neuroactive steroids are continuously expressed in the superficial dorsal horn to enhance GABAergic inhibition, which is enhanced by pathological pain states [20], [21]. Pharmacologic interventions that enhance endogenous neuroactive steroid production can be considered as potential approach for neuropathic pain management [21]. Activation of glycine receptors by gelsemine and glycine was reported to stimulate the endogenous neurosteroid allopregnanolone (or 3α,5α-tetrahydroprogesterone) biosynthesis from 5α-dihydroprogesterone in the spinal cord and hippocampus, via activation of 3α-hydroxysteroid oxidoreductase (3α-HSOR) [22], [23]. Subcutaneous injection of koumine was also reported to increase the NSC228155 and activity of 3α-HSOR and stimulate allopregnanolone biosynthesis in the spinal cords of inflammatory and neuropathic rats [8], [12]. It is therefore postulate that gelsemine and koumine produce antinociception via neuronal 3α-HSOR/allopregnanolone pathway following activation of spinal glycine receptors. In this study, we used glycine as a positive control and explored the molecular mechanisms underlying the mechanical antiallodynic effects of gelsemine and koumine, two representative alkaloids of Gelsemium, in neuropathic pain. We first employed the radioligand binding and displacement assays to characterize the glycine receptor binding patterns of gelsemine and koumine. We further measured the stimulatory effects of gelsemine and koumine on 3α-HSOR expression in primary cultures of neurons, microglia and astrocytes and the spinal cords of neuropathic rats. Moreover, we tested whether the mechanical antiallodynic effects of gelsemine and koumine were blocked by the intrathecal injection of strychnine, the specific 3α-HSOR inhibitor MPA, gene silencer siRNA/3α-HSOR and GABAA receptor antagonist isoallopregnanolone. Our data indicated that gelsemine and koumine, likewise glycine, were the orthosteric agonists of glycine receptors and produced mechanical antiallodynia through spinal neuronal glycine receptor/3α-HSOR/allopregnanolone pathway.
Materials and methods
Results
Discussion We have previously demonstrated that gelsemine inhibited H3-strychnine binding to the membrane fractions of the rat spinal cord homogenates, with a maximal 100% inhibition. In addition, intrathecal injection of gelsemine produced potent antinociception in formalin-induced tonic pain, bone cancer-induced mechanical allodynia, and spinal nerve ligation-induced painful neuropathy. The antinociceptive effects of gelsemine were blocked by intrathecal injection of the glycine receptor antagonist strychnine and α3 glycine receptor gene silencer siRNA/α3 glycine receptor, indicating that gelsemine exhibited antinociception through activation of spinal α3 glycine receptors [10]. Our current study further revealed that gelsemine and glycine concentration dependently displaced the concentration-binding of H3-strychnine to the right, without reducing the maximal binding, indicating a competitive manner. The results suggest that gelsemine, likewise glycine, is an orthosteric agonist of glycine receptors with full efficacy and probably acts at the same binding site as strychnine. The notion was supported by a recent study that gelsemine specifically modulated glycine receptor functions by electrophysiological techniques [46]. Koumine, an indole analog of gelsemine included in this study, was also confirmed to be the orthosteric agonist of the glycine receptor although with less potency, by exhibiting the same pattern to displace the concentration-binding curves of H3-strychnine to the right. Moreover, intrathecal injection of strychnine also totally blocked koumine and glycine mechanical antiallodynia in neuropathic pain. Since gelsemine and koumine are principal active ingredients of Gelsemium sempervirens Ait. and Gelsemium elegans Benth. [1], [2], [3], [4], it would be safe to conclude that activation of α3 glycine receptors contributes to Gelsemium-induced antinociception.