Baicalin relieves neuropathic pain by regulating α2-adrenoceptor levels in rats following spinal nerve injury.

In the present study, the ability of baicalin to relieve neuropathic pain due to spinal nerve ligation in rats was explored, and the relationship between baicalin and α2-adrenoceptors (α2-AR) was determined. The neuropathic pain model was established by ligating the L5-L6 spinal nerves in Sprague-Dawley rats. Several α2-AR antagonists were injected into the intramedullary sheath to evaluate the role of baicalin in neuropathic pain. The antagonists included nonselective α2-AR antagonist idazoxan, α2a-AR antagonist BRL 44408, α2b-AR antagonist ARC 239 and α2c-AR antagonist JP 1302. The rats were divided into an untreated control group, saline group, baicalin group and baicalin + α2-AR antagonist groups. Paw withdrawal threshold (PWT) was tested to assess the level of pain felt by the rats. The levels of α2-AR mRNA were tested by reverse transcription-quantitative PCR. Inflammatory factors, including tumor necrosis factor (TNF)-α, interleukin (IL)-6, IL-17 and IL-1ß, were analyzed by ELISA. The histopathological changes were assessed by hematoxylin and eosin staining. Flow cytometry was used to examine the percentage of CD4+ peripheral blood mononuclear cells (PBMCs). Compared with the saline group, the PWT value increased after treating with baicalin. However, intrathecal injection of α2-AR antagonist reversed the antinociceptive effects of baicalin. Compared with the saline group, the expression of α2a-AR and α2c-AR mRNA was upregulated significantly in the baicalin group (P<0.05). Levels of α2-AR mRNA were also decreased in the baicalin + idazoxan group compared with the baicalin group (P<0.05). The levels of TNF-α, IL-6, IL-17 and IL-1ß were raised after treatment with baicalin. In addition, baicalin treatment ameliorated the histological damage in the spinal cord. The percentage of CD4+ PBMCs was increased in the saline group compared with the control group (P<0.05). Compared with the baicalin group, the percentage of CD4+ PBMCs was raised after treatment with the α2-AR antagonists. In conclusion, intrathecal injection of baicalin produced an antiallodynic effect in a spinal nerve ligation-induced neuropathic pain model. The mechanism may be related to the regulation of a2-AR expression.

Molecular cloning and characterization of clyA genes in various serotypes of Salmonella enterica.

Cytolysin A (ClyA) is a pore-forming hemolytic protein encoded by the clyA gene. It has been identified in Salmonella enterica serovars Typhi and Paratyphi A. To identify and characterize the clyA genes in various Salmonella enterica strains, 21 different serotypes of strains isolated from clinical specimens were presently examined. Full-length clyA genes were found in S. enterica serovar Brandenburg, Indiana, Panama, and Schwarzengrund strains by polymerase chain reaction amplification. The ClyA proteins from these four strains showed >97% amino acid identity to that of S. enterica serovar Typhi. Although all four serovars expressed detectable levels of ClyA as determined by Western blot analysis, they did not show a strong hemolytic effect on blood agar, indicating that ClyA may not be efficiently expressed or secreted. Escherichia coli transformed with clyA genes from the four serovars enhanced production of ClyA proteins and hemolytic activities to a level similar to S. enterica serovar Typhi ClyA. The present results suggest that ClyA may play a role in the pathogenesis of S. enterica serovar Brandenburg, Indiana, Panama and Schwarzengrund.

Effect of sildenafil on neuropathic pain and hemodynamics in rats.

PURPOSE: The inhibition of phosphodiesterase 5 produces an antinociception through the increase of cyclic guanosine monophosphate (cGMP), and increasing cGMP levels enhance the release of gamma-aminobutyric acid (GABA). Furthermore, this phosphodiesterase 5 plays a pivotal role in the regulation of the vasodilatation associated to cGMP. In this work, we examined the contribution of GABA receptors to the effect of sildenafil, a phosphodiesterase 5 inhibitor, in a neuropathic pain rat, and assessed the hemodynamic effect of sildenafil in normal rats. MATERIALS AND METHODS: Neuropathic pain was induced by ligation of L5/6 spinal nerves in Sprague-Dawley male rats. After observing the effect of intravenous sildenafil on neuropathic pain, GABAA receptor antagonist (bicuculline) and GABAB receptor antagonist (saclofen) were administered prior to delivery of sildenafil to determine the role of GABA receptors in the activity of sildenafil. For hemodynamic measurements, catheters were inserted into the tail artery. Mean arterial pressure (MAP) and heart rate (HR) were measured over 60 min following administration of sildenafil. RESULTS: Intravenous sildenafil dose-dependently increased the withdrawal threshold to the von Frey filament application in the ligated paw. Intravenous bicuculline and saclofen reversed the antinociception of sildenafil. Intravenous sildenafil increased the magnitude of MAP reduction at the maximal dosage, but it did not affect HR response. CONCLUSION: These results suggest that sildenafil is active in causing neuropathic pain. Both GABAA and GABAB receptors are involved in the antinociceptive effect of sildenafil. Additionally, intravenous sildenafil reduces MAP without affecting HR.

Synergistic antinociception of intrathecal sildenafil with clonidine in the rat formalin test.

Spinal sildenafil (phosphodiesterase 5 inhibitor) and clonidine (alpha-2 adrenoceptor agonist) have shown antinociception. The author examined the properties of drug interaction after concurrent administration of intrathecal sildenafil-clonidine, and further clarified the reciprocity of sildenafil and clonidine. Catheters were inserted into the intrathecal space of male Sprague-Dawley rats. The formalin test was used as a nociceptive test, which was induced by subcutaneous injection of 50 microl of 5% formalin solution into the hindpaw. The pharmacological interaction was characterized using an isobolographic analysis. Intrathecal sildenafil and clonidine dose-dependently suppressed the flinching response observed during phase 1 and phase 2 in the formalin test. Isobolographic analysis revealed a synergistic interaction after intrathecal delivery of sildenafil-clonidine in both phases. Intrathecal yohimbine antagonized the antinociceptive action of intrathecal sildenafil during both phases in the formalin test. However, intrathecal ODQ failed to antagonize the antinociceptive action of intrathecal clonidine. These results suggest that sildenafil and clonidine, and the mixture of the two are effective against acute pain and facilitated pain state at the spinal level. Furthermore, synergism was noted after delivery of sildenafil-clonidine mixture. The antinociception of sildenafil can be modulated by spinal alpha-2 adrenoceptor, while the effect of clonidine is independent on the guanyly cyclase.

Evaluation for the interaction between intrathecal melatonin and clonidine or neostigmine on formalin-induced nociception.

AIMS: We examined the nature of pharmacological interaction after coadministration of melatonin with clonidine or neostigmine on formalin-induced nociception at the spinal level. Further, the role of melatonin receptor subtypes in melatonin-induced antinociception was clarified. MAIN METHODS: Catheters were inserted into the intrathecal space of male Sprague-Dawley rats. Pain was assessed using the formalin test (induced by a subcutaneous injection of 50 microl of a 5% formalin solution to the hindpaw). Isobolographic analysis was used for the evaluation of drug interaction between melatonin and clonidine or neostigmine. Non-selective MT1/MT2 receptors antagonist (luzindole), MT2 receptor antagonist (4-P-PDOT), and MT3 receptor/alpha-1 adrenoceptor antagonist (prazosin) were intrathecally given to verify the involvement of the melatonin receptor subtypes in the antinociception of melatonin. Furthermore, the effect of intrathecal MT3 receptor ligand (GR 135531) was observed. KEY FINDINGS: Intrathecal melatonin, clonidine, and neostigmine dose-dependently suppressed the flinching response during phase 1 and phase 2 in the formalin test. Isobolographic analysis showed additivity between melatonin and clonidine or neostigmine in both phases. The antinociceptive effect of melatonin was antagonized by luzindole, 4-P-PDOT, and prazosin in the spinal cord. Intrathecal GR 135531 was ineffective against the formalin-induced flinching response. SIGNIFICANCE: These results suggest that melatonin interacts additively with clonidine and neostigmine in the formalin-induced nociception at the spinal level. Furthermore, the antinociception of melatonin is mediated through the MT2 receptor, but not the MT3 receptor. However, it seems that alpha-1 adrenoceptor plays in the effect of melatonin.

Roles of opioid receptor subtypes on the antinociceptive effect of intrathecal sildenafil in the formalin test of rats.

Recently, it has been known that the antinociception of sildenafil, a phosphodiesterase 5 inhibitor, is mediated through the opioid receptors. There are common three types of opioid receptors mu, delta, and kappa. We characterized the role of subtypes of opioid receptor for the antinociception of sildenafil at the spinal level. Intrathecal catheters were placed for drug delivery and formalin solution (5%, 50 microl) was injected for induction of nociception within male SD rats. The effect of mu opioid receptor antagonist (CTOP), delta opioid receptor antagonist (naltrindole), and kappa opioid receptor antagonist (GNTI) on the activity of sildenafil was examined. Intrathecal sildenafil decreased the flinching responses during phases 1 and 2 in the formalin test. Intrathecal CTOP and naltrindole reversed the antinociception of sildenafil during both phases in the formalin test. Intrathecal GNTI reversed the effect of sildenafil during phase 2, but not phase 1. These results suggest that sildenafil is effective to acute pain and the facilitated pain state at the spinal level. Both mu and delta opioid receptors are involved. However, it seems that kappa opioid receptors play in the effect of sildenafil.

Additive antinociception between intrathecal sildenafil and morphine in the rat formalin test.

The possible characteristics of spinal interaction between sildenafil (phosphodiesterase 5 inhibitor) and morphine on formalin-induced nociception in rats was examined. Then the role of the opioid receptor in the effect of sildenafil was further investigated. Catheters were inserted into the intrathecal space of male Sprague-Dawley rats. For induction of pain, 50 microL of 5% formalin solution was applied to the hind-paw. Isobolographic analysis was used for the evaluation of drug interaction between sildenafil and morphine. Furthermore, naloxone was intrathecally given to verify the involvement of the opioid receptor in the antinociception of sildenafil. Both sildenafil and morphine produced an antinociceptive effect during phase 1 and phase 2 in the formalin test. The isobolographic analysis revealed an additive interaction after intrathecal delivery of the sildenafil-morphine mixture in both phases. Intrathecal naloxone reversed the antinociception of sildenafil in both phases. These results suggest that sildenafil, morphine, and the mixture of the two drugs are effective against acute pain and facilitated pain state at the spinal level. Thus, the spinal combination of sildenafil with morphine may be useful in the management of the same state. Furthermore, the opioid receptor is contributable to the antinocieptive mechanism of sildenafil at the spinal level.