RESUMO
ETHNOPHARMACOLOGICAL RELEVANCE: Tabebuia aurea (Silva Manso) Benth. & Hook. f. ex S. Moore is used as anti-inflammatory, analgesic and antiophidic in traditional medicine, though its pharmacological proprieties are still underexplored. In the bothropic envenoming, pain is a key symptom drove by an intense local inflammatory and neurotoxic event. The antivenom serum therapy is still the main treatment despite its poor local effects against pain and tissue injury. Furthermore, it is limited to ambulatorial niches, giving space for the search of new and more inclusive pharmacological approaches. AIM OF THE STUDY: evaluation of Tabebuia aurea hydroethanolic extract (HEETa) in hyperalgesia and neuronal injury induced by Bothrops mattogrossensis venom (VBm). MATERIALS AND METHODS: Stem barks from Tabebuia aurea were extracted with ethanol and water (7:3, v/v) to yield the extract HEETa. Then, HEETa was analyzed by LC-DAD-MS and its constituents were identified. Snake venoms were extracted from adult specimens of Bothrops mattogrossensis, lyophilized and kept at -20⯰C until use. Male Swiss mice, weighting 20-25â¯g, were used to hyperalgesia (electronic von Frey), motor impairment (Rotarod test) and tissue injury evaluation (histopatology and ATF-3 immunohistochemistry). Therefore, three experimental groups were formed: VBm (1â¯pg, 1â¯ng, 0.3⯵g, 1⯵g, 3 and 6⯵g/paw), HEETa orally (180, 540, 720, 810 or 1080â¯mg/kg; 10â¯mL/kg, 30â¯min prior VBm inoculation) and VBm neutralized (VBm: HEETa, 1:100 parts, respectively). In all set of experiments a control (saline group) was used. First, we made a dose-time-response course curve of VBm's induced hyperalgesia. Next, VBm maximum hyperalgesic dose was employed to perform HEETa orally dose-time-response course curve and analyses of VBm neutralized. Paw tissues for histopathology and DRGs were collected from animals inoculated with VBm maximum dose and treated with HEETa antihyperalgesic effective dose or neutralized VBm. Paws were extract two or 72â¯h after VBm inoculation and DRGs, in the maximum expected time expression of ATF-3 (72â¯h). RESULTS: From HEETa extract, glycosylated iridoids were identified, such as catalpol, minecoside, verminoside and specioside. VBm induced a time and dose dependent hyperalgesia with its highest effect seen with 3⯵g/paw, 2â¯h after venom inoculation. HEETa effective dose (720â¯mg/kg) decreased significantly VBm induced hyperalgesia (3⯵g/paw) with no motor impairment and signs of acute toxicity. HEETa antihyperalgesic action starts 1.5â¯h after VBm inoculation and lasted up until 2â¯h after VBm. Hyperalgesia wasn't reduced by VBm: HEETa neutralization. Histopathology revealed a large hemorragic field 2â¯h after VBm inoculation and an intense inflammatory infiltrate of polymorphonuclear cells at 72â¯h. Both HEETa orally and VBm: HEETa groups had a reduced inflammation at 72â¯h after VBm. Also, the venom significantly induced ATF-3 expression (35.37⯱â¯3.25%) compared with saline group (4.18⯱â¯0.68%) which was reduced in HEETa orally (25.87⯱â¯2.57%) and VBm: HEETa (19.84⯱â¯2.15%) groups. CONCLUSION: HEETa reduced the hyperalgesia and neuronal injury induced by VBm. These effects could be related to iridoid glycosides detected in HEETa and their intrinsic reported mechanism.
Assuntos
Analgésicos/uso terapêutico , Anti-Inflamatórios/uso terapêutico , Bothrops , Hiperalgesia/tratamento farmacológico , Extratos Vegetais/uso terapêutico , Venenos de Serpentes/toxicidade , Tabebuia , Fator 3 Ativador da Transcrição/metabolismo , Analgésicos/farmacologia , Animais , Anti-Inflamatórios/farmacologia , Gânglios Espinais/lesões , Hiperalgesia/metabolismo , Masculino , Camundongos , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Fitoterapia , Extratos Vegetais/farmacologia , Caules de PlantaRESUMO
Transcription factors are proteins that modulate the transcriptional rate of target genes in the nucleus in response to extracellular or cytoplasmic signals. Activating transcription factors 2 (ATF2) and 3 (ATF3) respond to environmental signals and maintain cellular homeostasis. There is evidence that inflammation and nerve injury modulate ATF2 and ATF3 expression. However, the function of these transcription factors in pain is unknown. The purpose of this study was to investigate the contribution of ATF2 and ATF3 to nerve injury-induced neuropathic pain. L5/6 spinal nerve ligation induced tactile allodynia and thermal hyperalgesia. Moreover, nerve damage enhanced ATF2 and ATF3 protein expression in injured L5/6 dorsal root ganglia and spinal cord but not in uninjured L4 dorsal root ganglia. Nerve damage also enhanced ATF2 immunoreactivity in dorsal root ganglia and spinal cord 7 to 21 days post-injury. Repeated intrathecal post-treatment with a small-interfering RNA targeted against ATF2 (ATF2 siRNA) or anti-ATF2 antibody partially reversed tactile allodynia and thermal hyperalgesia. In contrast, ATF3 siRNA or anti-ATF3 antibody did not modify nociceptive behaviors. ATF2 immunoreactivity was found in dorsal root ganglia and spinal cord co-labeling with NeuN mainly in non-peptidergic (IB4+) but also in peptidergic (CGRP+) neurons. ATF2 was found mainly in small- and medium-sized neurons. These results suggest that ATF2, but not ATF3, is found in strategic sites related to spinal nociceptive processing and participates in the maintenance of neuropathic pain in rats.
Assuntos
Fator 2 Ativador da Transcrição/metabolismo , Fator 3 Ativador da Transcrição/metabolismo , Hiperalgesia/etiologia , Hiperalgesia/metabolismo , Fator 2 Ativador da Transcrição/genética , Fator 3 Ativador da Transcrição/genética , Animais , Peptídeo Relacionado com Gene de Calcitonina/metabolismo , Modelos Animais de Doenças , Feminino , Gânglios Espinais/metabolismo , Gânglios Espinais/patologia , Regulação da Expressão Gênica , Lectinas/metabolismo , Masculino , Microscopia Confocal , Medição da Dor , Traumatismos dos Nervos Periféricos/complicações , Traumatismos dos Nervos Periféricos/patologia , Fosfopiruvato Hidratase/metabolismo , RNA Interferente Pequeno/administração & dosagem , Ratos , Ratos Wistar , Nervos Espinhais/metabolismo , Nervos Espinhais/patologia , Tato/fisiologiaRESUMO
The purpose of this study was to investigate whether 1%, 2% or 5% formalin injection produce hypersensitivity with characteristics of the neuropathic pain induced by spinal nerve injury. Formalin injection (1%, 2% and 5%) produced concentration-dependent long-lasting (at least 14 days) mechanical allodynia and hyperalgesia in both paws. Likewise, L5/L6 spinal nerve ligation induced allodynia and hyperalgesia in both paws. The intensity of hypersensitivity was greater in the ipsilateral than in the contralateral paw in all models. Systemic gabapentin or morphine completely reduced 1% formalin-induced hypersensitivity. In contrast, both drugs were not able to fully diminish 2-5% formalin- and nerve injury-induced hypersensitivity. Indomethacin produced a significant effect in the chronic 1% formalin test. Conversely, this drug did not modify 2 or 5% formalin- and nerve injury-induced hypersensitivity. Spinal nerve injury and 2-5%, but not 1%, formalin injection enhanced ATF3 protein expression and immunofluorescence in dorsal root ganglia (DRG) in a time-dependent manner. Furthermore, 2-5%, but not 1%, formalin injection or spinal nerve injury also enhanced α2δ-1 subunit protein levels in DRG. Our results suggest that 5% and, at lesser extent, 2% formalin injection produces long-lasting hypersensitivity with a pharmacological and molecular pattern that resembles neuropathic pain induced by spinal nerve ligation.