AAV-glycine receptor α3 alleviates CFA-induced inflammatory pain by downregulating ERK phosphorylation and proinflammatory cytokine expression in SD rats

BackgroundGlycine receptors (GlyRs) play key roles in the processing of inflammatory pain. The use of adeno-associated virus (AAV) vectors for gene therapy in human clinical trials has shown promise, as AAV generally causes a very mild immune response and long-term gene transfer, and there have been no reports of disease. Therefore, we used AAV for GlyR alpha 1/3 gene transfer in F11 neuron cells and into Sprague-Dawley (SD) rats to investigate the effects and roles of AAV-GlyR alpha 1/3 on cell cytotoxicity and inflammatory response.MethodsIn vitro experiments were performed using plasmid adeno-associated virus (pAAV)-GlyR alpha 1/3-transfected F11 neurons to investigate the effects of pAAV-GlyR alpha 1/3 on cell cytotoxicity and the prostaglandin E2 (PGE2)-mediated inflammatory response. In vivo experiment, the association between GlyR alpha 3 and inflammatory pain was analyzed in normal rats after AAV-GlyR alpha 3 intrathecal injection and after complete Freund's adjuvant (CFA) intraplantar administration. Intrathecal AAV-GlyR alpha 3 delivery into SD rats was evaluated in terms of its potential for alleviating CFA-induced inflammatory pain.ResultsThe activation of mitogen-activated protein kinase (MAPK) inflammatory signaling and neuronal injury marker activating transcription factor 3 (ATF-3) were evaluated by western blotting and immunofluorescence; the level of cytokine expression was measured by ELISA. The results showed that pAAV/pAAV-GlyR alpha 1/3 transfection into F11 cells did not significantly reduce cell viability or induce extracellular signal-regulated kinase (ERK) phosphorylation or ATF-3 activation. PGE2-induced ERK phosphorylation in F11 cells was repressed by the expression of pAAV-GlyR alpha 3 and administration of an EP2 inhibitor, GlyR alpha s antagonist (strychnine), and a protein kinase C inhibitor. Additionally, intrathecal AAV-GlyR alpha 3 administration to SD rats significantly decreased CFA-induced inflammatory pain and suppressed CFA-induced ERK phosphorylation, did not induce obvious histopathological injury but increased ATF-3 activation in dorsal root ganglion (DRGs).ConclusionsAntagonists of the prostaglandin EP2 receptor, PKC, and glycine receptor can inhibit PGE2-induced ERK phosphorylation. Intrathecal AAV-GlyR alpha 3 administration to SD rats significantly decreased CFA-induced inflammatory pain and suppressed CFA-induced ERK phosphorylation, did not significantly induce gross histopathological injury but elicited ATF-3 activation. We suggest that PGE2-induced ERK phosphorylation can be modulated by GlyR alpha 3, and AAV-GlyR alpha 3 significantly downregulated CFA-induced cytokine activation.