RESUMO
Platelet-rich plasma (PRP) and growth factors have been increasing in popularity for the orthopedic treatment of degenerative and traumatic diseases. The treatment concept is based on the substitution of growth-inducing substances in tissues with low or absent regeneration capacity (cartilage, tendons) as well as for the induction or further acceleration of growth and regeneration (bone, muscle). This review article provides an overview on the clinical feasibility of usage and a summary of the current study situation.
Assuntos
Sistema Musculoesquelético , Plasma Rico em Plaquetas , Cartilagem , Peptídeos e Proteínas de Sinalização Intercelular , TendõesRESUMO
BACKGROUND: Extracellular lysophosphatidic acid (LPA) species transmit signals via six different G protein-coupled receptors (LPAR1-6) and are indispensible for brain development and function of the nervous system. However, under neuroinflammatory conditions or brain damage, LPA levels increase, thereby inducing signaling cascades that counteract brain function. We describe a critical role for 1-oleyl-2-hydroxy-sn-glycero-3-phosphate (termed "LPA" throughout our study) in mediating a motile and pro-inflammatory microglial phenotype via LPAR5 that couples to protein kinase D (PKD)-mediated pathways. METHODS: Using the xCELLigence system and time-lapse microscopy, we investigated the migrational response of microglial cells. Different M1 and M2 markers were analyzed by confocal microscopy, flow cytometry, and immunoblotting. Using qPCR and ELISA, we studied the expression of migratory genes and quantitated the secretion of pro-inflammatory cytokines and chemokines, respectively. Different transcription factors that promote the regulation of pro-inflammatory genes were analyzed by western blot. Reactive oxygen species (ROS) and nitric oxide (NO) production, phagocytosis, and microglial cytotoxicity were determined using commercially available assay kits. RESULTS: LPA induces MAPK family and AKT activation and pro-inflammatory transcription factors' phosphorylation (NF-κB, c-Jun, STAT1, and STAT3) that were inhibited by both LPAR5 and PKD family antagonists. LPA increases migratory capacity, induces secretion of pro-inflammatory cytokines and chemokines and expression of M1 markers, enhances production of ROS and NO by microglia, and augments cytotoxicity of microglial cell-conditioned medium towards neurons. The PKD family inhibitor blunted all of these effects. We propose that interference with this signaling axis could aid in the development of new therapeutic approaches to control neuroinflammation under conditions of overshooting LPA production. CONCLUSIONS: In the present study, we show that inflammatory LPA levels increased the migratory response of microglia and promoted a pro-inflammatory phenotype via the LPAR5/PKD axis. Interference with this signaling axis reduced microglial migration, blunted microglial cytotoxicity, and abrogated the expression and secretion of pro-inflammatory mediators.
Assuntos
Movimento Celular/efeitos dos fármacos , Lisofosfolipídeos/farmacologia , Microglia/efeitos dos fármacos , Proteína Quinase C/metabolismo , Receptores de Ácidos Lisofosfatídicos/metabolismo , Transdução de Sinais/efeitos dos fármacos , Animais , Animais Recém-Nascidos , Ácidos Carboxílicos/farmacologia , Células Cultivadas , Córtex Cerebral/citologia , Citocinas/genética , Citocinas/metabolismo , Inibidores Enzimáticos/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Neurônios/efeitos dos fármacos , Fosforilação , Proteína Quinase C/genética , Pirimidinas/farmacologia , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Receptores de Ácidos Lisofosfatídicos/antagonistas & inibidoresRESUMO
OBJECTIVE: To investigate the combination of mild mechanical stimuli and a disease modifying osteoarthritis drug (DMOAD) in inflammatory activated chondrocytes and to study the combination of drug and mechanical tension on the cellular level as a model for an integrated biophysical approach for osteoarthritis (OA) treatments. METHODS: Interleukin-1beta (IL-1ß) stimulated C28/I2 cells underwent mild mechanically treatment while cultured in the presence of the DMOAD diacerein. The pharmacological input of diacerein was evaluated by cell viability and cell proliferation measurements. Inflammation and treatment induced changes in key regulatory proteins and components of the extracellular matrix (ECM) were characterized by quantitative real-time PCR (qPCR). The effects on metalloproteinase-1 (MMP-1) activity and glycosaminoglycan (GAG) concentration in cell supernatants of treated cells were investigated. RESULTS: C28/I2 cells demonstrated significant changes in expression of inflammatory and cartilage destructive proteins in response to IL-1ß stimulation. The chondroprotective action of diacerein in mechanically stimulated cells was mediated by a decrease in interleukin-8 (IL-8), fibronectin-1 (FN-1), collagen type I (Col 1) and MMP-1 expression levels, respectively. Augmented expression of interleukin-6 receptor (IL-6R) and the fibroblast growth factor receptors (FGFRs) by diacerein was not abolished by mechanical treatment. The observed effects were accompanied by a reduced cell proliferation rate, attenuated cell viability and extenuated MMP-1 activity. CONCLUSION: Diacerein diversely regulates the expression of main regulatory proteins as well as components important to regenerate and set up ECM. Mechanical stimulation does not negatively influence the chondroprotective effect induced by diacerein treatment in immortalized human C28/I2 chondrocytes.
Assuntos
Antraquinonas/farmacologia , Antirreumáticos/farmacologia , Condrócitos/efeitos dos fármacos , Condrócitos/patologia , Inflamação/prevenção & controle , Estimulação Física , Estresse Mecânico , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Condrócitos/metabolismo , Colágeno Tipo I/metabolismo , Fibronectinas/metabolismo , Glicosaminoglicanos/metabolismo , Humanos , Inflamação/metabolismo , Inflamação/patologia , Interleucina-1beta/farmacologia , Interleucina-8/metabolismo , Metaloproteinase 1 da Matriz/metabolismo , Osteoartrite/metabolismo , Osteoartrite/patologia , Osteoartrite/prevenção & controleRESUMO
The molecular target of arachidonic-acid-derived metabolites, serving as second messengers that activate atrial acetylcholine-activated potassium current (IK.ACh) in addition to G-protein beta/gamma subunits (Gbeta/gamma), is unknown. Co-expression of two isoforms of G-protein-activated, inwardly rectifying potassium channels (GIRKs) in oocytes of Xenopus laevis revealed that these heterologous co-expressed GIRKs, which are responsible for the formation of IK.ACh in the atrium, are activated by arachidonic acid metabolites, like their counterparts in atrial cells. The expression of homooligomeric GIRK1(F137S) and GIRK4wt channels revealed that this activatory mechanism is specific to the GIRKI subunit. Sequestrating available Gbeta/gamma by overexpression of C-betaARK (a Gbeta/gamma binding protein) failed to abolish the activation of GIRK currents by arachidonic acid. From our experiments we conclude that the GIRKI subunit itself is the molecular target for regulation of GIRK channels by arachidonic acid metabolites.