RESUMO
BACKGROUND: Cinobufagin is the major bufadienolide of Bufonis venenum (Chansu), which has been traditionally used for the treatment of chronic pain especially cancer pain. The current study aimed to evaluate its antinociceptive effects in bone cancer pain and explore the underlying mechanisms. METHODS: Rat bone cancer model was used in this study. The withdrawal threshold evoked by stimulation of the hindpaw was determined using a 2290 CE electrical von Frey hair. The ß-endorphin and IL-10 levels were measured in the spinal cord and cultured primary microglia, astrocytes, and neurons. RESULTS: Cinobufagin, given intrathecally, dose-dependently attenuated mechanical allodynia in bone cancer pain rats, with the projected Emax of 90% MPE and ED50 of 6.4 µg. Intrathecal cinobufagin also stimulated the gene and protein expression of IL-10 and ß-endorphin (but not dynorphin A) in the spinal cords of bone cancer pain rats. In addition, treatment with cinobufagin in cultured primary spinal microglia but not astrocytes or neurons stimulated the mRNA and protein expression of IL-10 and ß-endorphin, which was prevented by the pretreatment with the IL-10 antibody but not ß-endorphin antiserum. Furthermore, spinal cinobufagin-induced mechanical antiallodynia was inhibited by the pretreatment with intrathecal injection of the microglial inhibitor minocycline, IL-10 antibody, ß-endorphin antiserum and specific µ-opioid receptor antagonist CTAP. Lastly, cinobufagin- and the specific α-7 nicotinic acetylcholine receptor (α7-nAChR) agonist PHA-543613-induced microglial gene expression of IL-10/ß-endorphin and mechanical antiallodynia in bone cancer pain were blocked by the pretreatment with the specific α7-nAChR antagonist methyllycaconitine. CONCLUSIONS: Our results illustrate that cinobufagin produces mechanical antiallodynia in bone cancer pain through spinal microglial expression of IL-10 and subsequent ß-endorphin following activation of α7-nAChRs. Our results also highlight the broad significance of the recently uncovered spinal microglial IL-10/ß-endorphin pathway in antinociception.
Assuntos
Bufanolídeos/farmacologia , Dor do Câncer/metabolismo , Hiperalgesia/metabolismo , Microglia/efeitos dos fármacos , Animais , Neoplasias Ósseas/complicações , Feminino , Interleucina-10/metabolismo , Masculino , Microglia/metabolismo , Ratos , Ratos Wistar , Medula Espinal/efeitos dos fármacos , Medula Espinal/metabolismo , Receptor Nicotínico de Acetilcolina alfa7/metabolismo , beta-Endorfina/metabolismoRESUMO
BACKGROUND: The G protein-coupled receptor 40 (GPR40), broadly expressed in various tissues such as the spinal cord, exerts multiple physiological functions including pain regulation. This study aimed to elucidate the mechanisms underlying GPR40 activation-induced antinociception in neuropathic pain, particularly related to the spinal glial expression of IL-10 and subsequent ß-endorphin. METHODS: Spinal nerve ligation-induced neuropathic pain model was used in this study. ß-Endorphin and IL-10 levels were measured in the spinal cord and cultured primary microglia, astrocytes, and neurons. Double immunofluorescence staining of ß-endorphin with glial and neuronal cellular biomarkers was also detected in the spinal cord and cultured primary microglia, astrocytes, and neurons. RESULTS: GPR40 was expressed on microglia, astrocytes, and neurons in the spinal cords and upregulated by spinal nerve ligation. Intrathecal injection of the GPR40 agonist GW9508 dose-dependently attenuated mechanical allodynia and thermal hyperalgesia in neuropathic rats, with Emax values of 80% and 100% MPE and ED50 values of 6.7 and 5.4 µg, respectively. Its mechanical antiallodynia was blocked by the selective GPR40 antagonist GW1100 but not GPR120 antagonist AH7614. Intrathecal GW9508 significantly enhanced IL-10 and ß-endorphin immunostaining in spinal microglia and astrocytes but not in neurons. GW9508 also markedly stimulated gene and protein expression of IL-10 and ß-endorphin in cultured primary spinal microglia and astrocytes but not in neurons, originated from 1-day-old neonatal rats. The IL-10 antibody inhibited GW9508-stimulated gene expression of the ß-endorphin precursor proopiomelanocortin (POMC) but not IL-10, whereas the ß-endorphin antibody did not affect GW9508-stimulated IL-10 or POMC gene expression. GW9508 increased phosphorylation of mitogen-activated protein kinases (MAPKs) including p38, extracellular signal-regulated kinase (ERK), and c-Jun N-terminal kinase (JNK), and its stimulatory effects on IL-10 and POMC expression were blocked by each MAPK isoform inhibitor. Spinal GW9508-induced mechanical antiallodynia was completely blocked by intrathecal minocycline, IL-10 neutralizing antibody, ß-endorphin antiserum, and µ-opioid receptor-preferred antagonist naloxone. CONCLUSIONS: Our results illustrate that GPR40 activation produces antinociception via the spinal glial IL-10/ß-endorphin antinociceptive pathway.
Assuntos
Hiperalgesia/etiologia , Hiperalgesia/metabolismo , Interleucina-10/metabolismo , Neuralgia , Neuroglia/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Transdução de Sinais/fisiologia , beta-Endorfina/metabolismo , Animais , Animais Recém-Nascidos , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Hiperalgesia/tratamento farmacológico , Interleucina-10/genética , Masculino , Metilaminas/uso terapêutico , Proteínas do Tecido Nervoso/metabolismo , Neuralgia/complicações , Neuralgia/metabolismo , Neuralgia/patologia , Medição da Dor , Propionatos/uso terapêutico , RNA Mensageiro/metabolismo , Ratos , Ratos Wistar , Receptores Acoplados a Proteínas G/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacosRESUMO
The glucagon-like peptide-1 (GLP-1) receptor agonist exenatide stimulates microglial ß-endorphin expression and subsequently produces neuroprotection and antinociception. This study illustrated an unrecognized autocrine role of IL-10 in mediation of exenatide-induced ß-endorphin expression. Treatment with exenatide in cultured primary spinal microglia concentration dependently stimulated the expression of the M2 microglial markers IL-10, IL-4, Arg 1, and CD206, but not the M1 microglial markers TNF-α, IL-1ß, IL-6, or CD68. Intrathecal exenatide injection also significantly upregulated spinal microglial expression of IL-10, IL-4, Arg 1, and CD206, but not TNF-α, IL-1ß, IL-6, or CD68. Intrathecal injection of exenatide stimulated spinal microglial expression of IL-10 and ß-endorphin in neuropathic rats. Furthermore, treatment with IL-10 (but not IL-4) stimulated ß-endorphin expression in cultured primary microglia, whereas treatment with ß-endorphin failed to increase IL-10 expression. The IL-10-neutralizing antibody entirely blocked exenatide-induced spinal microglial expression of ß-endorphin in vitro and in vivo and fully blocked exenatide mechanical antiallodynia in neuropathic rats. Moreover, specific cAMP/PKA/p38 signal inhibitors and siRNA/p38ß, but not siRNA/p38α, completely blocked exenatide-induced IL-10 expression in cultured primary microglia. Knock-down of IL-10 receptor-α mRNA using siRNA fully inhibited exenatide-induced spinal microglial ß-endorphin expression and mechanical antiallodynia in neuropathy. Exenatide also markedly stimulated phosphorylation of the transcription factor STAT3 in cultured primary microglia and ß-endorphin stimulation was completely inhibited by the specific STAT3 activation inhibitor. These results revealed that IL-10 in microglia mediated ß-endorphin expression after GLP-1 receptor activation through the autocrine cAMP/PKA/p38ß/CREB and subsequent IL-10 receptor/STAT3 signal pathways.SIGNIFICANCE STATEMENT Activation of GLP-1 receptors specifically and simultaneously stimulates the expression of anti-inflammatory cytokines IL-10 and IL-4, as well as the neuroprotective factor ß-endorphin from microglia. GLP-1 receptor agonism induces ß-endorphin expression and antinociception through autocrine release of IL-10. Activation of GLP-1 receptors stimulates IL-10 and ß-endorphin expression subsequently through the Gs-cAMP/PKA/p38ß/CREB and IL-10/IL-10 receptor-α/STAT3 signal transduction pathways.
Assuntos
Comunicação Autócrina/fisiologia , Receptor do Peptídeo Semelhante ao Glucagon 1/biossíntese , Interleucina-10/biossíntese , Microglia/metabolismo , Medula Espinal/metabolismo , beta-Endorfina/biossíntese , Animais , Animais Recém-Nascidos , Comunicação Autócrina/efeitos dos fármacos , Células Cultivadas , Relação Dose-Resposta a Droga , Exenatida , Expressão Gênica , Receptor do Peptídeo Semelhante ao Glucagon 1/agonistas , Receptor do Peptídeo Semelhante ao Glucagon 1/genética , Interleucina-10/genética , Interleucina-10/farmacologia , Masculino , Microglia/efeitos dos fármacos , Peptídeos/farmacologia , Ratos , Ratos Wistar , Medula Espinal/citologia , Medula Espinal/efeitos dos fármacos , Peçonhas/farmacologia , beta-Endorfina/genéticaRESUMO
Liposome-encapsulated clodronate (LEC) is a specific depletor of macrophages. Our study characterized the LEC depletory effects, given intrathecally, on spinal microglia and assessed its effects on initiation and maintenance of neuropathic pain. Measured by using the MTT assay, LEC treatment specifically inhibited cell viability of cultured primary microglia, but not astrocytes or neurons, from neonatal rats, with an IC50 of 43⯵g/mL. In spinal nerve ligation-induced neuropathic rats, pretreatment (1 day but not 5 days earlier) with intrathecal LEC specifically depleted microglia (but not astrocytes or neurons) in both contralateral and ipsilateral dorsal horns by the same degree (63% vs. 71%). Intrathecal injection of LEC reversibly blocked the antinociceptive effects of the GLP-1 receptor agonist exenatide and dynorphin A stimulator bulleyaconitine, which have been claimed to be mediated by spinal microglia, whereas it failed to alter morphine- or the glycine receptor agonist gelsemine-induced mechanical antiallodynia which was mediated via the neuronal mechanisms. Furthermore, intrathecal LEC injection significantly attenuated initial (one day after nerve injury) but not existing (2 weeks after nerve injury) mechanical allodynia. Our study demonstrated that LEC, given intrathecally, is a specific spinal microglial inhibitor and significantly reduces initiation but not maintenance of neuropathic pain, highlighting an opposite role of spinal microglia in different stages of neuropathic pain.
Assuntos
Ácido Clodrônico/uso terapêutico , Microglia/patologia , Neuralgia/tratamento farmacológico , Medula Espinal/patologia , Aconitina/análogos & derivados , Alcaloides , Animais , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Ácido Clodrônico/farmacologia , Exenatida , Feminino , Hiperalgesia/complicações , Hiperalgesia/tratamento farmacológico , Hiperalgesia/patologia , Injeções Espinhais , Lipossomos , Masculino , Microglia/efeitos dos fármacos , Microglia/metabolismo , Neuralgia/complicações , Neuralgia/patologia , Peptídeos , Ratos Wistar , PeçonhasRESUMO
The transcription factor Gli2 plays crucial roles in the transduction of Hedgehog (Hh) signals, yet the mechanisms that control Gli2 degradation remain unclear. Here we have identified the eubiquitinating enzyme otubain2 (OTUB2) as a regulator of Gli2 protein degradation. We found that OTUB2 was coimmunoprecipitated with Gli2. Knockdown of OTUB2 decreased Gli2 protein level while the proteasome inhibitor MG-132 treatment restored Gli2 expression. Additionally, OTUB2 overexpression stabilized Gli2 protein in U2OS cells and extended the half-life of Gli2. We also found that knockdown of OTUB2 reduced deubiquitination of Gli2 in vivo. In vitro deubiquitination assay showed that ubiquitinated Gli2 was decreased by wild-type OTUB2 but not OTUB2 mutations. We also found that OTUB2 knockdown suppressed the ALP activity and the expression of the common markers BMP2 and RUNX2 during osteogenesis of MSCs in response to Shh and Smo agonists, which indicated OTUB2 may have effect on osteogenic differentiation by regulating Hh signaling.
Assuntos
Enzimas Desubiquitinantes/metabolismo , Tioléster Hidrolases/metabolismo , Ubiquitinação , Proteína Gli2 com Dedos de Zinco/metabolismo , Animais , Diferenciação Celular/genética , Linhagem Celular , Linhagem Celular Tumoral , Enzimas Desubiquitinantes/genética , Células HEK293 , Humanos , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Mutação , Osteogênese/genética , Ligação Proteica , Estabilidade Proteica , Interferência de RNA , Tioléster Hidrolases/genética , Proteína Gli2 com Dedos de Zinco/genéticaRESUMO
Interleukin 10 (IL-10) is antinociceptive in various animal models of pain without induction of tolerance, and its mechanism of action was generally believed to be mediated by inhibition of neuroinflammation. Here we reported that intrathecal IL-10 injection dose dependently attenuated mechanical allodynia and thermal hyperalgesiain male and female neuropathic rats, with ED50 values of 40.8â¯ng and 24â¯ng, and Emax values of 61.5% MPE and 100% MPE in male rats. Treatment with IL-10 specifically increased expression of the ß-endorphin (but not prodynorphin) gene and protein in primary cultures of spinal microglia but not in astrocytes or neurons. Intrathecal injection of IL-10 stimulated ß-endorphin expression from microglia but not neurons or astrocytes in both contralateral and ipsilateral spinal cords of neuropathic rats. However, intrathecal injection of the ß-endorphin neutralizing antibody, opioid receptor antagonist naloxone, or µ-opioid receptor antagonist CTAP completely blocked spinal IL-10-induced mechanical antiallodynia, while the microglial inhibitor minocycline and specific microglia depletor reversed spinal IL-10-induced ß-endorphin overexpression and mechanical antiallodynia. IL-10 treatment increased spinal microglial STAT3 phosphorylation, and the STAT3 inhibitor NSC74859 completely reversed IL-10-increased spinal expression of ß-endorphin and neuroinflammatory cytokines and mechanical antiallodynia. Silence of the Bcl3 and Socs3 genes nearly fully reversed IL-10-induced suppression of neuroinflammatory cytokines (but not expression of ß-endorphin), although it had no effect on mechanical allodynia. In contrast, disruption of the POMC gene completely blocked IL-10-stimulated ß-endorphin expression and mechanical antiallodynia, but had no effect on IL-10 inhibited expression of neuroinflammatory cytokines. Thus this study revealed that IL-10 produced antinociception through spinal microglial ß-endorphin expression, but not inhibition of neuroinflammation.
Assuntos
Hiperalgesia/tratamento farmacológico , Interleucina-10/farmacologia , beta-Endorfina/metabolismo , Analgésicos/farmacologia , Animais , Astrócitos , Citocinas/metabolismo , Feminino , Hiperalgesia/metabolismo , Injeções Espinhais , Interleucina-10/metabolismo , Masculino , Microglia/efeitos dos fármacos , Microglia/metabolismo , Microglia/fisiologia , Minociclina/farmacologia , Naloxona/farmacologia , Neuralgia/metabolismo , Neurônios , Cultura Primária de Células , Ratos , Ratos Wistar , Medula Espinal/efeitos dos fármacos , Medula Espinal/metabolismo , Coluna Vertebral/efeitos dos fármacos , Coluna Vertebral/metabolismo , beta-Endorfina/efeitos dos fármacosRESUMO
Recent discoveries established that activation of glucagon-like peptide-1 receptors (GLP-1Rs) mediates neuroprotection and antinociception through microglial ß-endorphin expression. This study aimed to explore the underlying signaling mechanisms of microglial ß-endorphin. GLP-1Rs and ß-endorphin were coexpressed in primary cultures of microglia. Treatment with the GLP-1R agonist exenatide concentration-dependently stimulated microglial expression of the ß-endorphin precursor gene proopiomelanocortin (POMC) and peptides, with EC50 values of 4.1 and 7.5 nM, respectively. Exenatide also significantly increased intracellular cAMP levels and expression of p-protein kinase A (PKA), p-p38, and p-cAMP response element binding protein (CREB) in cultured primary microglia. Furthermore, exenatide-induced microglial expression of POMC was completely blocked by reagents that specifically inhibit adenylyl cyclase and activation of PKA, p38, and CREB. In addition, knockdown of p38ß (but not p38α) using short interfering RNA (siRNA) eliminated exenatide-induced microglial p38 phosphorylation and POMC expression. In contrast, lipopolysaccharide increased microglial activation of p38, and knockdown of p38α (but not p38ß) partially suppressed expression of proinflammatory factors (including tumor necrosis factor-α, interleukin-1ß, and interleukin-6). Exenatide-induced phosphorylation of p38 and CREB was also totally blocked by the PKA inhibitor and siRNA/p38ß, but not by siRNA/p38α Seven-day intrathecal injections of siRNA/p38ß (but not siRNA/p38α) completely blocked exenatide-induced spinal p38 activation, ß-endorphin expression, and mechanical antiallodynia in rats with established neuropathy, although siRNA/p38ß and siRNA/p38α were not antiallodynic. To our knowledge, our results are the first to show a causal relationship between the PKA-dependent p38ß mitogen-activated protein kinase/CREB signal cascade and GLP-1R agonism-mediated microglial ß-endorphin expression. The differential role of p38α and p38ß activation in inflammation and nociception was also highlighted.
Assuntos
Sistema de Sinalização das MAP Quinases , Microglia/metabolismo , Peptídeos/farmacologia , Peçonhas/farmacologia , beta-Endorfina/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Animais , Células Cultivadas , AMP Cíclico/metabolismo , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Citocinas/metabolismo , Exenatida , Receptor do Peptídeo Semelhante ao Glucagon 1/metabolismo , Hiperalgesia/metabolismo , Hiperalgesia/patologia , Mediadores da Inflamação/metabolismo , Injeções Espinhais , Lipopolissacarídeos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Masculino , Microglia/efeitos dos fármacos , Modelos Biológicos , Fosforilação/efeitos dos fármacos , Pró-Opiomelanocortina/metabolismo , RNA Interferente Pequeno/metabolismo , Ratos WistarRESUMO
Increased ubiquitin-specific protease 5 (USP5) has been associated with tumorigenesis of malignancy including glioblastoma, melanoma and hepatocellular carcinoma. However, the role of USP5 in tumorigenesis of pancreatic ductal adenocarcinoma (PDAC) has not been studied yet. In this study, we demonstrated that USP5 was significantly upregulated in a panel of PDAC cell lines and correlated with FoxM1 protein expression. USP5 knockdown inhibited proliferation of PANC-1 and SW1990, two PDAC cell lines. In the mouse xenografted pancreatic tumor model, suppression of USP5 significantly decreased tumor growth, correlated with down regulation of FoxM1. Additionally, we found that overexpression of USP5 stabilized the FoxM1 protein in PDAC cells. Overexpression of USP5 extended the half-life of FoxM1. Knockdown of USP5 in PANC-1 cells decreased FoxM1 protein level while the proteasome inhibitor MG-132 treatment restored FoxM1 expression. We also found that endogenous USP5 was coimmunoprecipitated with an endogenous FoxM1 from PANC-1 cells while FoxM1 was also coimmunoprecipitated with USP5. Furthermore, we also confirmed that USP5 regulated proliferation of PDAC via FoxM1 by rescuing the inhibitory effect of USP5 knockdown with ectopic expression of FoxM1 in USP5-depleted cells. Taken together, our study demonstrates that USP5 plays a critical role in tumorigenesis and progression of pancreatic cancer by stabilizing FoxM1 protein, and provides a rationale for USP5 being a potential therapeutic approach against PDAC.
Assuntos
Carcinogênese , Progressão da Doença , Endopeptidases/metabolismo , Proteína Forkhead Box M1/metabolismo , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , Animais , Proliferação de Células , Sobrevivência Celular , Células Cultivadas , Endopeptidases/genética , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Neoplasias Experimentais/metabolismo , Neoplasias Experimentais/patologia , Estabilidade ProteicaRESUMO
Cynanchi Wilfordii Radix (baishouwu), a medicinal herb, has been widely used in Asia to treat a variety of diseases or illnesses. Cynandione A isolated from C. Wilfordii is the principle acetophenone and exhibits neuroprotective and anti-inflammatory activities. This study aims to evaluate the antihypersensitivity activities of cynandione A in neuropathy and explored its mechanisms of action. Intrathecal injection of cynandione A dose-dependently attenuated spinal nerve ligation-induced mechanical allodynia and thermal hyperalgesia, with maximal possible effects of 57% and 59%, ED50s of 14.9µg and 6.5µg, respectively. Intrathecal injection of cynandione A significantly increased ß-endorphin levels in spinal cords of neuropathic rats and its treatment concentration-dependently induced ß-endorphin expression in cultured primary microglia (but not in neurons or astrocytes), with EC50s of 38.8 and 20.0µM, respectively. Cynandione A also non-selectively upregulated phosphorylation of mitogen-activated protein kinases (MAPKs), including p38, extracellular signal regulated kinase (ERK1/2), and extracellular signal regulated kinase (JNK) in primary microglial culture; however, cynandione A-stimulated ß-endorphin expression was completely inhibited by the specific p38 activation inhibitor SB203580, but not by the ERK1/2 or JNK activation inhibitors. Knockdown of spinal p38ß but not p38α using siRNA also completely blocked cynandione A-induced ß-endorphin expression in cultured microglial cells. Furthermore, cynandione A-induced antiallodynia in neuropathy was totally inhibited by the microglial inhibitor minocycline, SB203580, anti-ß-endorphin antibody, and µ-opioid receptor antagonist CTAP (but not the κ- or δ-opioid receptor antagonist). These results suggest that cynandione A attenuates neuropathic pain through upregulation of spinal microglial expression ß-endorphin via p38ß MAPK activation.
Assuntos
Analgésicos/uso terapêutico , Compostos de Bifenilo/uso terapêutico , Microglia/efeitos dos fármacos , Neuralgia/tratamento farmacológico , Transdução de Sinais/efeitos dos fármacos , Medula Espinal/efeitos dos fármacos , beta-Endorfina/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Analgésicos/farmacologia , Animais , Comportamento Animal/efeitos dos fármacos , Compostos de Bifenilo/farmacologia , Células Cultivadas , Masculino , Microglia/metabolismo , Neuralgia/metabolismo , Medição da Dor , Ratos , Ratos Wistar , Teste de Desempenho do Rota-Rod , Medula Espinal/metabolismoRESUMO
BACKGROUND: Aconiti brachypodi Radix (Xue-shang-yi-zhi-hao) has been prescribed to manage chronic pain, arthritis, and traumatic injuries. Bullatine A, a C20-diterpenoid alkaloid, is one of its principle effective compounds. This study aimed to investigate the anti-hypersensitivity of bullatine A in a variety of rat pain models and explore its mechanisms of action. METHODS: Rat neuropathic pain, inflammatory pain, diabetic neuropathic pain, and bone cancer pain models were used. Dynorphin A and pro-inflammatory cytokines were measured in the spinal cord and cultured primary microglia. Double immunofluorescence staining of dynorphin A and glial and neuronal cellular markers was also measured in the spinal cord. RESULTS: Subcutaneous and intrathecal injection of bullatine A dose-dependently attenuated spinal nerve ligation-, complete Freud's adjuvant-, diabetes-, and bone cancer-induced mechanical allodynia and thermal hyperalgesia, with the efficacies of 45-70 % inhibition, and half-effective doses of 0.9-1.9 mg/kg for subcutaneous injection. However, bullatine A was not effective in blocking acute nociceptive response in the normal condition. Bullatine A specifically stimulated dynorphin A expression in microglia in the spinal cord in vivo and cultured primary microglia in vitro; the stimulatory effects were completely inhibited by the microglial inhibitor minocycline. In contrast, bullatine A did not have an inhibitory effect on peripheral nerve injury- or lipopolysaccharide-induced pro-inflammatory cytokine expression. The spinal anti-allodynic effects of bullatine A were entirely blocked by intrathecal injection of minocycline, the specific dynorphin A antiserum, and the selective k-opioid receptor antagonist. CONCLUSIONS: We, for the first time, demonstrate that bullatine A specifically attenuates pain hypersensitivity, regardless of the pain models employed. The results also suggest that stimulation of spinal microglial dynorphin A expression mediates bullatine A anti-nociception in pain hypersensitivity conditions.
Assuntos
Alcaloides/uso terapêutico , Modelos Animais de Doenças , Diterpenos/uso terapêutico , Dinorfinas/biossíntese , Hiperalgesia/tratamento farmacológico , Hiperalgesia/metabolismo , Medula Espinal/metabolismo , Alcaloides/farmacologia , Analgésicos/farmacologia , Analgésicos/uso terapêutico , Animais , Animais Recém-Nascidos , Células Cultivadas , Diterpenos/farmacologia , Relação Dose-Resposta a Droga , Dinorfinas/genética , Feminino , Expressão Gênica , Injeções Subcutâneas , Masculino , Neuralgia/tratamento farmacológico , Neuralgia/metabolismo , Ratos , Ratos Sprague-Dawley , Ratos Wistar , Medula Espinal/efeitos dos fármacosRESUMO
Dezocine is an opioid analgesic widely used in China, occupying over 45% of the domestic market of opioid analgesics. We have recently demonstrated that dezocine produced mechanical antiallodynia and thermal antihyperalgesia through spinal µ-opioid receptor activation and norepinephrine reuptake inhibition in neuropathic pain. This study further explored the dual µ-opioid receptor and norepinephrine reuptake mechanisms underlying dezocine-induced mechanical antiallodynia in bone cancer pain, compared with tapentadol, the first recognized analgesic in this class. Dezocine and tapentadol, given subcutaneously, exerted profound mechanical antiallodynia in bone cancer pain rats in a dose-dependent manner, yielding similar maximal effects but different potencies: ED50s of 0.6 mg/kg for dezocine and 7.5 mg/kg for tapentadol, respectively. Furthermore, their mechanical antiallodynia was partially blocked by intrathecal injection of the specific µ-opioid receptor antagonist CTAP, but not κ-opioid receptor antagonists GNTI and nor-BNI or δ-opioid receptor antagonist naltrindole. Intrathecal administrations of the specific norepinephrine depletor 6-OHDA (but not the serotonin depletor PCPA) for three consecutive days and single injection of the α-adrenoceptor antagonist phentolamine/α2-adrenoceptor antagonist yohimbine partially blocked dezocine- and tapentadol-induced mechanical antiallodynia. Strikingly, the combination of CTAP and yohimbine nearly completely blocked dezocine- and tapentadol-induced mechanical antiallodynia. Our results illustrate that both dezocine and tapentadol exert mechanical antiallodynia in bone cancer pain through dual mechanisms of µ-opioid receptor activation and norepinephrine reuptake inhibition, and suggest that the µ-opioid receptor and norepinephrine reuptake dual-targeting opioids are effective analgesics in cancer pain.
Assuntos
Analgésicos Opioides/farmacologia , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Dor do Câncer/tratamento farmacológico , Hiperalgesia/prevenção & controle , Receptores Opioides mu/metabolismo , Inibidores da Recaptação de Serotonina e Norepinefrina/farmacologia , Tapentadol/farmacologia , Tetra-Hidronaftalenos/farmacologia , Animais , Comportamento Animal/efeitos dos fármacos , Neoplasias Ósseas , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Feminino , Injeções Espinhais , Ratos , Ratos Sprague-Dawley , Ratos WistarRESUMO
Electroacupuncture produces analgesia in chronic pain patients and animal models of pain hypersensitivity. The current study aims to illustrate the mechanisms underlying electroacupuncture-attenuated neuropathic pain. Neuropathic rats, induced by tight ligation of L5/L6 spinal nerves, markedly reduced mechanical thresholds in the ipsilateral hindpaws relative to the contralateral hindpaws. Low frequency (2 Hz) electroacupuncture stimulation for a period of 20 min alleviated neuropathic pain in the ipsilateral hindpaws of neuropathic rats in a time-dependent manner. The same electroacupuncture treatment also stimulated spinal gene and protein expression of IL-10 and ß-endorphin but not dynorphin A, measured by real-time quantitative PCR and ELISA kits. Intrathecal injection of the specific IL-10 antibody in neuropathic rats completely blocked electroacupuncture-increased spinal expression of ß-endorphin, but the ß-endorphin antibody failed to alter electroacupuncture-stimulated spinal IL-10 expression. Using a double fluorescence immunostaining technique, we observed that electroacupuncture stimulated spinal IL-10 and ß-endorphin expression in microglia but not in neurons or astrocytes in the spinal dorsal horn of neuropathic rats. Pretreatment with intrathecal injection of the microglial inhibitor minocycline, specific IL-10 antibody and ß-endorphin antiserum (but not the dynorphin A antibody), or selective µ-opioid receptor antagonist CTAP (but not κ- or δ-opioid receptor antagonist) completely blocked electroacupuncture-induced attenuation of neuropathic pain. These results suggest that low frequency electroacupuncture alleviates neuropathic pain through stimulation of the spinal microglial expression of IL-10 and subsequent expression of ß-endorphin.
Assuntos
Eletroacupuntura/métodos , Interleucina-10/metabolismo , Microglia/metabolismo , Neuralgia/metabolismo , Neuralgia/terapia , beta-Endorfina/metabolismo , Animais , Feminino , Masculino , Ratos , Ratos Wistar , Transdução de Sinais/fisiologia , Medula Espinal/metabolismoRESUMO
Gelsemine, the principal active alkaloid from Gelsemium sempervirens Ait., and koumine, the most dominant alkaloids from Gelsemium elegans Benth., produced antinociception in a variety of rodent models of painful hypersensitivity. The present study explored the molecular mechanisms underlying gelsemine- and koumine-induced mechanical antiallodynia in neuropathic pain. The radioligand binding and displacement assays indicated that gelsemine and koumine, like glycine, were reversible and orthosteric agonists of glycine receptors with full efficacy and probably acted on same binding site as the glycine receptor antagonist strychnine. Treatment with gelsemine, koumine and glycine in primary cultures of spinal neurons (but not microglia or astrocytes) concentration dependently increased 3α-hydroxysteroid oxidoreductase (3α-HSOR) mRNA expression, which was inhibited by pretreatment with strychnine but not the glial inhibitor minocycline. Intrathecal injection of gelsemine, koumine and glycine stimulated 3α-HSOR mRNA expression in the spinal cords of neuropathic rats and produced mechanical antiallodynia. Their spinal mechanical antiallodynia was completely blocked by strychnine, the selective 3α-HSOR inhibitor medroxyprogesterone acetate (MPA), 3α-HSOR gene silencer siRNA/3α-HSOR and specific GABAA receptor antagonist isoallopregnanolone, but not minocycline. All the results taken together uncovered that gelsemine and koumine are orthosteric agonists of glycine receptors, and produce mechanical antiallodynia through neuronal glycine receptor/3α-HSOR/allopregnanolone/GABAA receptor pathway.
Assuntos
Alcaloides/metabolismo , Gelsemium/metabolismo , Hiperalgesia/metabolismo , Alcaloides Indólicos/metabolismo , Pregnanolona/biossíntese , Receptores de Glicina/metabolismo , Medula Espinal/metabolismo , Alcaloides/uso terapêutico , Animais , Animais Recém-Nascidos , Células Cultivadas , Relação Dose-Resposta a Droga , Feminino , Hiperalgesia/induzido quimicamente , Hiperalgesia/tratamento farmacológico , Alcaloides Indólicos/uso terapêutico , Masculino , Ratos , Ratos Wistar , Medula Espinal/efeitos dos fármacosRESUMO
BACKGROUND AND PURPOSE: d-Amino acid oxidase (DAAO) is a flavine adenine dinucleotide-containing flavoenzyme and specifically catalyses oxidative deamination of d-amino acids. This study aimed to explore the association between increased cerebral DAAO expression or enzymic activity and the development of cerebral ischaemia. EXPERIMENTAL APPROACH: A mouse model of transient (90 min) middle cerebral artery occlusion (MCAO) was established, and western blotting, enzymic activity assay, and fluorescent immunostaining techniques were used. KEY RESULTS: The expression and enzymic activity of DAAO increased over time in the cortical peri-infarct area of the mice subjected to transient MCAO. The DAAO was specifically expressed in astrocytes, and its double immunostaining with the astrocytic intracellular marker, glial fibrillary acidic protein, in the cortical peri-infarct area was up-regulated following ischaemic insult, with peak increase on Day 5 after MCAO. Single intravenous injection of the specific and potent DAAO inhibitor Compound SUN reduced the cerebral DAAO enzymic activity and attenuated neuronal infarction and neurobehavioural deficits with optimal improvement apparent immediately after the MCAO procedure. The neuroprotective effect was dose dependent, with ED50 values of 3.9-4.5 mg·kg-1 . Intracerebroventricular injection of the DAAO gene silencer siRNA/DAAO significantly reduced cerebral DAAO expression and attenuated MCAO-induced neuronal infarction and behavioural deficits. CONCLUSIONS AND IMPLICATIONS: Our results, for the first time, demonstrated that increased cerebral astrocytic DAAO expression and enzymic activity were causally associated with the development of neuronal destruction following ischaemic insults, suggesting that targeting cerebral DAAO could be a potential approach for treatment of neurological conditions following cerebral ischaemia.
Assuntos
Isquemia Encefálica/metabolismo , D-Aminoácido Oxidase/metabolismo , Infarto da Artéria Cerebral Média/metabolismo , Animais , Isquemia Encefálica/induzido quimicamente , Modelos Animais de Doenças , Infarto da Artéria Cerebral Média/induzido quimicamente , Injeções Intraventriculares , Masculino , Camundongos , Pentobarbital/administração & dosagemRESUMO
GLP-1 receptor agonists, exenatide and GLP-1, promoted M2 type polarization in monocytes/macrophages and microglial cells. This study explored the signal basis underlying exenatide-stimulated expression of M2 microglia-specific genes, including the cytoplasmic marker Arg 1, surface marker CD206, and secretion protein marker IL-4. Treatment with exenatide in cultured primary microglial cells concentration dependently stimulated the expression of Arg 1, CD206 and IL-4, but did not significantly alter LPS-stimulated expression of TNF-α, IL-1ß and IL-6. The stimulatory effects of exenatide were completely prevented by the GLP-1 receptor antagonist exendin(9-39), but not altered by application of LPS. Furthermore, the adenylyl cyclase inhibitor DDA, PKA inhibitor H89 and CREB inhibitor KG501 completely blocked exenatide-induced overexpression of Arg 1, CD206 and IL-4. In addition, exenatide-stimulated expression of Arg 1 and CD206 was totally blocked by the p38 MAPK inhibitor SB203580 and gene silencer siRNA/p38ß (but not siRNA/p38α), whereas the expressed IL-4 was not significantly altered by the p38 inhibitor or other MAPK subtype inhibitors. These findings revealed that both classic Gs-cAMP/PKA/CREB and alternative Gs-cAMP/PKA/p38ß/CREB mediated GLP-1 receptor agonism-induced overexpression of M2 microglial biomarkers.
Assuntos
Diferenciação Celular/fisiologia , Exenatida/farmacologia , Microglia/efeitos dos fármacos , Microglia/metabolismo , Transdução de Sinais/fisiologia , Animais , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , AMP Cíclico/metabolismo , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Feminino , Receptor do Peptídeo Semelhante ao Glucagon 1/agonistas , Incretinas/farmacologia , Masculino , Proteína Quinase 11 Ativada por Mitógeno/metabolismo , Ratos , Ratos Wistar , Transdução de Sinais/efeitos dos fármacosRESUMO
Dezocine is the number one opioid painkiller prescribed and sold in China, occupying 44% of the nation's opioid analgesics market today and far ahead of the gold-standard morphine. We discovered the mechanisms underlying dezocine antihypersensitivity activity and assessed their implications to antihypersensitivity tolerance. Dezocine, given subcutaneously in spinal nerve-ligated neuropathic rats, time- and dose-dependently produced mechanical antiallodynia and thermal antihyperalgesia, significantly increased ipsilateral spinal norepinephrine and serotonin levels, and induced less antiallodynic tolerance than morphine. Its mechanical antiallodynia was partially (40% or 60%) and completely (100%) attenuated by spinal µ-opioid receptor (MOR) antagonism or norepinephrine depletion/α2-adrenoceptor antagonism and combined antagonism of MORs and α2-adenoceptors, respectively. In contrast, antagonism of spinal κ-opioid receptors (KORs) and δ-opioid receptors (DORs) or depletion of spinal serotonin did not significantly alter dezocine antiallodynia. In addition, dezocine-delayed antiallodynic tolerance was accelerated by spinal norepinephrine depletion/α2-adenoceptor antagonism. Thus dezocine produces antihypersensitivity activity through spinal MOR activation and norepinephrine reuptake inhibition (NRI), but apparently not through spinal KOR and DOR activation, serotonin reuptake inhibition or other mechanisms. Our findings reclassify dezocine as the first analgesic of the recently proposed MOR-NRI, and reveal its potential as an alternative to as well as concurrent use with morphine in treating pain.
Assuntos
Analgésicos/farmacologia , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Norepinefrina/antagonistas & inibidores , Receptores Opioides mu/agonistas , Tetra-Hidronaftalenos/farmacologia , Animais , Relação Dose-Resposta a Droga , Hiperalgesia , RatosRESUMO
Spinal transient receptor potential ankyrin 1 (TRPA1) channel is associated with various pain hypersensitivity conditions. Spinally, TRPA1 is expressed by central terminals of nociceptive nerve fibers and astrocytes. Among potential endogenous agonists of TRPA1 is H2O2 generated by d-amino acid oxidase (DAAO) in astrocytes. Here we studied whether prolonged block of the spinal TRPA1 or astrocytes starting at time of injury attenuates development and/or maintenance of neuropathic hypersensitivity. Additionally, TRPA1 and DAAO mRNA were determined in the dorsal root ganglion (DRG) and spinal dorsal horn (SDH). Experiments were performed in rats with spared nerve injury (SNI) and chronic intrathecal catheter. Drugs were administered twice daily for the first seven injury days or only once seven days after injury. Mechanical hypersensitivity was assessed with monofilaments. Acute and prolonged treatment with Chembridge-5861528 (a TRPA1 antagonist), carbenoxolone (an inhibitor of activated astrocytes), or gabapentin (a comparison drug) attenuated tactile allodynia-like responses evoked by low (2g) stimulus. However, antihypersensitivity effect of these compounds was short of significance at a high (15g) stimulus intensity. No preemptive effects were observed. In healthy controls, carbenoxolone failed to prevent hypersensitivity induced by spinal cinnamaldehyde, a TRPA1 agonist. TRPA1 and DAAO mRNA in the DRG but not SDH were slightly increased in SNI, independent of drug treatment. The results indicate that prolonged peri-injury block of spinal TRPA1 or inhibition of spinal astrocyte activation attenuates maintenance but not development of mechanical (tactile allodynia-like) hypersensitivity after nerve injury.
Assuntos
Junções Comunicantes/efeitos dos fármacos , Doenças do Sistema Nervoso Periférico/tratamento farmacológico , Canais de Cátion TRPC/antagonistas & inibidores , Aminas/farmacologia , Animais , Carbenoxolona/farmacologia , Ácidos Cicloexanocarboxílicos/farmacologia , D-Aminoácido Oxidase/genética , D-Aminoácido Oxidase/fisiologia , Gabapentina , Injeções Espinhais , Masculino , Doenças do Sistema Nervoso Periférico/fisiopatologia , Ratos , Canal de Cátion TRPA1 , Canais de Cátion TRPC/genética , Ácido gama-Aminobutírico/farmacologiaRESUMO
The study with gnotobiotic microcosm showed within the range of test soil moisture contents, the feeding activity of Caenorhabditis elegans on Bacillus subtilis promoted the latter' s proliferation, and enhanced soil respiration significantly. The increment of B. subtilis number varied with soil moisture content, and decreased in the order of 23% > 17% > 28% . The interaction of C. elegans and B. subtilis increased the contents of soil mineral N and NH4 + -N significantly, indicating that soil nitrogen mineralization was markedly promoted. A higher nitrogen mineralization rate was observed under 23% soil moisture content.