FORSKOLIN PREVENTS ISCHEMIA/REPERFUSION INJURY AFTER SMALL INTESTINAL TRANSPLANTATION IN RATS

Abstract

O257 Aims: Forskolin (FS), a diterpene isolated from the roots of Coleus forskohlii, directly activates adenylyl cyclase in a wide variety of cells and increases intracellular 3’5’-cyclic monophosphate (cAMP), which is a crucial second messenger mediating important physiological responses. Ischemia/reperfusion (I/R) injury associating with small intestinal transplantation (SITx) remains a major problem. Since cold ischemia is known to deplete cAMP from preserved grafts, exogenous FS and increase of graft tissue cAMP levels would prevent I/R injury associating with transplantation. Based on this hypothesis, the study examines the efficacy of donor pretreatment with FS on intestinal cold I/R injury using rejection-free rat syngenic SITx model. Methods: Syngenic heterotopic SITx was performed in Lewis rats after 6 hrs graft preservation in UW solution at 4°C. Forskolin (FS: 1mg/kg) was infused into donor intestine lumen 10 min before harvesting. Blood and graft samples were obtained at 1, 3, and 24 hrs after SITx. Graft cAMP levels (cAMP immunoassay), circulating proinflammatory cyokines (ELISA), and graft mRNA levels for ICAM-1 and cytokines (real time PCR) were measured. The severity of I/R injury was evaluated in routine histopathology and polymorphonuclear neutrophiric infiltration (MPO stain). Phospho-PKA and F-actin were also determined in immunofluorescent stain. Results: Infusion of FS into normal intestinal lumen effectively increased cAMP levels to 34.9±16.2 from 24.8±12.4 nmol/mg. In untreated intestinal grafts, cAMP levels strikingly decreased to 10-15 nmol/mg during cold preservation and for 24 hours after SITx. Severe I/R injury in untreated grafts associated with widespread erosion and MPO+ infiltrates in the lamina propria. With FS treatment, tissue cAMP levels significantly increased and histopathological severity of I/R injury were significantly improved. I/R-induced upregulation of ICAM-1 and proinflammatory cytokines (IL6, TNFα) in untreated controls was significantly reduced with FS treatment. Furthermore, FS treatment increased PKA phosphorylation in the intestinal epithelium and maintained intestinal epithelial microvilli (F-actin positive). In addition, the elevation of cAMP with FS efficiently prevented an increase of graft permeability associating with intestinal I/R injury. Conclusions: FS administration into the intestinal graft lumen reduces cold I/R injury associating with prolonged preservation and SITx. FS mediates the efficacy by increasing intracellular cAMP levels, activating PKA, protecting intestinal barrier function, inhibiting neutrophil recruitments, and downregulating inflammatory cytokine responses during intestinal I/R injury.