RESUMEN
Increased invasion of synovial fibroblasts and their involvement in cartilage damage are characteristic phenotypes of rheumatoid arthritis (RA). To identify low molecular weight compounds that suppress synovial fibroblast invasion, a panel of inhibitors (n = 330) was initially screened using a real-time cell analysis system for human synovial fibroblasts that were enzymatically isolated from surgical samples of RA patients. To evaluate the effects of the inhibitors identified in the screen, synovial fibroblast migration was measured using a wound-healing assay, and phosphorylation of intracellular signaling molecules was determined by immunoblots. Several candidate inhibitors were identified in the screen, including inhibitors against platelet-derived growth factor receptor (PDGFR), Akt, PI3K, and glycogen kinase synthetase 3 (GSK-3). These inhibitors strongly suppressed synovial fibroblast migration after 72 h and downregulated phosphorylation of Akt (Ser473) at 48 h. When the inhibitors were removed from the culture conditions, both migration and phosphorylated Akt (Ser473) levels were restored. Furthermore, all the categories of inhibitors except for PDGFR inhibitor IV decreased cell proliferation as well as IL-6 production in synovial fibroblasts. Interestingly, GSK-3 inhibitors increased anti-inflammatory cytokine IL-10 production but suppressed IL-23 production from LPS-primed macrophages obtained from healthy donors. In conclusion, blocking PDGFR, PI3K, or GSK-3 could have therapeutic value as an RA treatment that targets the invasion/migration of synovial fibroblasts.
Asunto(s)
Antiinflamatorios , Artritis Reumatoide/tratamiento farmacológico , Movimiento Celular/efectos de los fármacos , Fibroblastos/inmunología , Membrana Sinovial/inmunología , Anciano , Antiinflamatorios/química , Antiinflamatorios/farmacología , Artritis Reumatoide/inmunología , Artritis Reumatoide/patología , Movimiento Celular/inmunología , Evaluación Preclínica de Medicamentos , Femenino , Fibroblastos/patología , Humanos , Masculino , Persona de Mediana Edad , Membrana Sinovial/patologíaRESUMEN
Peripheral tissue inflammation or injury causes glutamate release from nociceptive axons, keratinocytes, and Schwann cells, resulting in thermal hypersensitivity. However, the detailed molecular mechanisms underlying glutamate-induced thermal hypersensitivity are unknown. The aim of this study was to clarify the involvement of peripheral transient receptor potential (TRP) TRP vanilloid 1 (TRPV1), TRP ankyrin 1 (TRPA1), and protein kinase C epsilon (PKCε) in glutamate-induced pain hypersensitivity. The amount of glutamate in the facial tissue was significantly increased 3 days after facial Complete Freund's adjuvant injection. The head-withdrawal reflex threshold to heat, cold, or mechanical stimulation was significantly decreased on day 7 after continuous glutamate or metabotropic glutamate receptor 5 (mGluR5) agonist (CHPG) injection into the facial skin compared with vehicle-injected rats, and glutamate-induced hypersensitivity was significantly recovered by mGluR5 antagonist MTEP, TRPA1 antagonist HC-030031, TRPV1 antagonist SB366791, or PKCε translocation inhibitor administration into the facial skin. TRPV1 and TRPA1 were expressed in mGluR5-immunoreactive (IR) trigeminal ganglion (TG) neurons innervating the facial skin, and mGluR5-IR TG neurons expressed PKCε. There was no significant difference in the number of GluR5-IR TG neurons among glutamate-injected, saline-injected, and naive rats, whereas that of TRPV1- or TRPA1-IR TG neurons was significantly increased 7 days after continuous glutamate injection into the facial skin compared with vehicle injection. PKCε phosphorylation in TG was significantly enhanced following glutamate injection into the facial skin. Moreover, neuronal activity of TG neurons was significantly increased following facial glutamate treatment. The present findings suggest that sensitization of TRPA1 and/or TRPV1 through mGluR5 signaling via PKCε is involved in facial thermal and mechanical hypersensitivity.
Asunto(s)
Hiperalgesia/metabolismo , Umbral del Dolor/fisiología , Receptor del Glutamato Metabotropico 5/metabolismo , Transducción de Señal/fisiología , Canal Catiónico TRPA1/metabolismo , Canales Catiónicos TRPV/metabolismo , Acetanilidas/farmacología , Animales , Modelos Animales de Enfermedad , Inhibidores Enzimáticos/farmacología , Adyuvante de Freund/toxicidad , Ácido Glutámico/metabolismo , Ácido Glutámico/toxicidad , Glicina/análogos & derivados , Glicina/farmacología , Hiperalgesia/etiología , Masculino , Neuronas/efectos de los fármacos , Neuronas/fisiología , Dolor/inducido químicamente , Dolor/complicaciones , Umbral del Dolor/efectos de los fármacos , Fenilacetatos/farmacología , Estimulación Física/efectos adversos , Purinas/farmacología , Piridinas/farmacología , Ratas , Ratas Sprague-Dawley , Piel/inervación , Canal Catiónico TRPA1/antagonistas & inhibidores , Canales Catiónicos TRPV/antagonistas & inhibidores , Tiazoles/farmacología , Ganglio del Trigémino/citologíaRESUMEN
BACKGROUND: In order to evaluate mechanisms that may underlie the sensitization of trigeminal spinal subnucleus caudalis (Vc; the medullary dorsal horn) and upper cervical spinal cord (C1-C2) nociceptive neurons to heat, cold and mechanical stimuli following topical capsaicin treatment of the facial skin, nocifensive behaviors as well as phosphorylation of extracellular regulated-kinase (pERK) in Vc and C1-C2 neurons were studied in rats. RESULTS: Compared to vehicle application, capsaicin application to the lateral facial skin produced 1 hour later a flare in the skin, and also induced significantly greater nocifensive behaviors to heat, cold or mechanical stimulus of the lateral facial skin. The intrathecal (i.t.) injection of the MEK inhibitor PD98059 markedly attenuated the nocifensive behaviors to these stimuli in capsaicin-treated rats. Moreover, the number of pERK-like immunoreactive (pERK-LI) cells in Vc and C1-C2 was significantly larger following the heat, cold and mechanical stimuli in capsaicin-treated rats compared with vehicle-treated rats. The number of pERK-LI cells gradually increased following progressive increases in the heat or mechanical stimulus intensity and following progressive decrease in the cold stimulus. The ERK phosphorylation in Vc and C1-C2 neurons was strongly inhibited after subcutaneous injection of the capsaicin antagonist capsazepine in capsaicin-treated rats. CONCLUSION: The present findings revealed that capsaicin treatment of the lateral facial skin causes an enhancement of ERK phosphorylation in Vc and C1-C2 neurons as well as induces nocifensive behavior to heat, cold and mechanical simulation of the capsaicin-treated skin. The findings suggest that TRPV1 receptor mechanisms in rat facial skin influence nociceptive responses to noxious cutaneous thermal and mechanical stimuli by inducing neuroplastic changes in Vc and C1-C2 neurons that involve in the MAP kinase cascade.