887 Analgesic Action of Resolvin D1 to Combat Visceral Pain in a Rat Model of Visceral Hypersensitivity

Abstract

G A A b st ra ct s root ganglia (DRG) neurons. The aim of this study was to examine the downstream signaling pathways mediating these effects, with the focus on Smad 3 and TRPV1. Methods: In vitro patch clamp studies were performed on dissociated DRG neurons. For in vivo studies, CP was induced by intraductal injection of trinitrobenzene sulfonic acid (TNBS) in adult male rats, with controls receiving the same volume of vehicle, followed by intrapancreatic injection of DiI, a retrograde neuronal tracer, to label the DRG neurons that project to pancreas. Three weeks later, SIS3 (2.5mg/kg i.p. daily) was administered for 1 week. Subsequently, thoracic DRGs were harvested and stained for pSmad3 using immunohistochemistry. Phosphorylated Smad3 (pSmad3), Smad3 and TRPV1 protein levels in the thoracic DRGs (T8T12) were measured using immunoblot assay. Results: DRG cells were cultured with vehicle (1μg/ml DMSO-solvent for SIS3), TGF β1(10ng/ml), SIS3-TGF β1(10ng/ml-0.3μM), respectively for 48 hours and then TRPV1 currents were determined by the response to 1μM of capsaicin. TGFβ1 significantly enhanced TRPV1 currents in DRG neurons in culture (99.06±24 pA/pF; n=6) compared to controls (44.77±14 pA/pF; n=8), an effect that was blocked by SIS3 (26.23±4.4 pA/pF; n=6) (p<0.01 by ANOVA). However, sis3 was unable to block TRPA1 currents induced by mustard oil, attesting to the relative specificity of this effect. In vivo, as shown previously, SIS3 significantly attenuates both somatic referred hypersensitivity (measured by VFF) and pancreatic hyperalgesia (measured by behavioral response to pancreatic electrical stimulation) in rats with CP. pSmad3 positive neurons in the DiI-labeled thoracic neurons were significantly increased in CP rats (79.06±4.72% compare with control 67.15±1.53%, p<0.05), which can be reduced by treatment with SIS3 (63.64±2.21%, P<0.05). Immunoblot confirmed that pSmad3 levels, but not total Smad3, were significantly reduced in the DRGs (T8-T12) of rats treated with SIS3 relative to vehicle treated rats (P<0.05). Finally, compared with vehicle treatment we found that SIS3 significantly reduced TRPV1 protein levels in rats with CP (106.11±9.44 vs 70.32±6.17, P<0.05). Conclusions: The present results demonstrate a novel link between TGFβ1 signaling and TRPV1 function and expression, an effect that appears to be mediated by Smad3. These results provide the fundamental basis for developing novel therapeutic approaches for CPinduced pain.