HSOR belonging to the aldoketo reductase superfamily

3α-HSOR, belonging to the aldoketo reductase superfamily, is a rate-limiting enzyme for the synthesis of 3α-reduced steroids, such as allopregnanolone and tetrahydrodeoxycorticosterone. 3α-HSOR was extensively expressed in the spinal dorsal horn and was upregulated following peripheral nerve injury, and has been considered as a potential target molecule for the management of neuropathic pain [12], [42], [47]. Previous experiments showed that treatment with gelsemine and koumine stimulated the allopregnanolone biosynthesis via activation of 3α-HSOR in the spinal cord and hippocampus [12], [22], [23]. Our study further demonstrated that intrathecal injection of gelsemine, koumine and glycine markedly increased 3α-HSOR gene Solithromycin in the spinal cords of neuropathic rats. Although it is well believed that glycine receptors are located on the inhibitory transduction pathway in primary afferent neurons [48]. The double immunofluorescence staining studies exhibited that 3α-HSOR was also expressed in neurons, microglia and astrocytes in the spinal cord [12], [49]. It is thus possible that glycine receptor activation leads to 3α-HSOR biosynthesis in glial cells. To identify the cell type specificity of glycine, gelsemine and koumine we used primary culture of spinal neurons, microglia and astrocytes originated from the spinal cord. Likewise glycine, treatment with gelsemine and koumine in spinal neurons, but not in microglia or astrocytes, significantly enhanced 3α-HSOR mRNA expression, which was completely blocked by pretreatment with strychnine, indicating a neuronal rather than glial role of 3α-HSOR activity following glycine receptor activation. The notion is further supported by the following findings that 1) functional glycine receptors were not present in microglia [50], and that 2) the microglial inhibitor minocycline failed to significantly affect gelsemine-, koumine- and glycine-stimulated 3α-HSOR mRNA expression in spinal neurons and their mechanical antiallodynia in neuropathic rats. Minocycline has been extensively demonstrated to have inhibitory effects on activation of astrocytes and particularly microglia, but have no direct effects on neurons [34], [35], [36], [37], [38], [39]. The biochemical pathways responsible for the cell specificity have not been fully illustrated. However, minocycline indeed failed to alter morphine- or smaller dose of dynorphin A-induced mechanical antiallodynic effects which are known to be mediated by neurons [29], [51], whereas it completely blocked the antihypersensitivity effects of aconitines, cynandione A, interleukin-10 and the GLP-1 receptor agonists exenatide and shanzhiside methylester, which are known to be mediated by microglia [26], [28], [29], [30], [31], [51], [52]. We further demonstrated that intrathecal injection of the 3α-HSOR inhibitor MPA in neuropathic rats completely blocked spinal gelsemine, koumine and glycine mechanical antiallodynia. In addition, the antinociceptive effects of subcutaneous koumine (approximately 40-fold less potent than gelsemine) was dose-dependently eliminated by MPA in neuropathic pain, diabetes pain and inflammatory pain [8], [12], [16]. However, the potency of MPA was very low and high doses had to be used in these studies. To rule out the possible nonspecificity of MPA on the 3α-HSOR activity due to high doses, spinal gene ablation technique using multiple daily injections of siRNA/3α-HSOR was employed. Multiple daily intrathecal injection of siRNA/3α-HSOR significantly reduced spinal 3α-HSOR expression by more than 50%, and blocked glycine-, gelsemine- and koumine-induced mechanical antiallodynia. All these results proved the causal role of 3α-HSOR in gelsemine- and koumine-induced mechanical antiallodynia. Superficial layers of the dorsal horn have dense population of Glycinergic/GABAergic inhibitory interneurons where neurosteroids are prominently expressed [53]. Allopregnanolone is a positive allosteric modulator of GABAA receptors and binds to the neurosteroid binding site of GABAA receptors. It opens the associated chloride ion channels, inhibits the nerve impulse transmission to higher brain centers [54], [55], [56], and produces antinociception in many animal models of chronic pain [57], [58]. Thus glycine receptor activation-induced allopregnanolone biosynthesis via 3α-HSOR, activates GABAA receptors, probably through both autocrine and paracrine mechanisms. Indeed, intrathecal injection of the selective GABAA receptor antagonist isoallopregnanolone, specifically at the neurosteroid binding site [44], [45], completely inhibited allopregnanolone-, and gelsemine-, koumine- as well as glycine-induced mechanical antiallodynia. Moreover, bicuculline, an antagonist specifically acting at the GABA binding site, significantly blocked koumine-induced antinociception in postoperative pain [14]. In contrast, strychnine failed to block allopregnanolone mechanical antiallodynia. Taken together, all these results suggest that activation of the spinal neuronal α3 glycine receptor/3α-HSOR/allopregnanolone/GABAA receptor pathway in painful hypersensitivity states induces antihypersensitivity effects, which mediates gelsemine- and koumine-induced mechanical antiallodynia. The schematic illustration of the postulated mechanical antiallodynic pathway is presented in Fig. 9.