PANCREATIC PROTEASES ARE NECESSARY FOR INCREASED GUT EPITHELIAL PERMEABILITY DURING TRAUMA/HEMORRHAGIC SHOCK

Abstract

Shock-induced distant organ injury is associated with increased gut permeability which contributes to gut-origin multiple organ dysfunction syndrome (MODS). Thus, understanding the pathogenesis of trauma- hemorrhagic shock (T/HS)-induced gut injury is important. In this context, we propose the hypothesis that pancreatic proteases play a key role in loss of gut barrier function after T/HS. To test this hypothesis, male Sprague- Dawley rats were subjected to T/HS (MAP 30-35 mmHg × 90 min) with and without pancreatic duct ligation (PDL). After 3 hours of reperfusion, ileal permeability was assessed using FITC-labeled dextran (in-vivo closed loop technique). The magnitude of gut permeability was approximately 3-fold greater in the T/HS rats as compared to T/HS rats with PDL (18.3 ± 4 vs 6.5 ± 5.1 μg/ml P<0.05). Next, we directly tested whether pancreatic proteases increase enterocyte permeability using an in-vitro enterocyte monolayer model. Caco-2 monolayers were incubated for 3 hr or 18 hr with either fetal bovine serum (FBS) or supernatants from homogenized rat pancreas (1:6 w/v PBS). Permeability was assessed using a 10,000 MW rhodamine probe applied to a 3 μm-pore sized transwell cell culture system. We demonstrated a dose-dependent response at both 3hr (r2 = 0.997) and 18 hr (r2 = 0.998) incubation using 5%, 10%, 25% dilutions of initial homogenized pancreas supernatants. Furthermore, we demonstrated that at 18 hr monolayer permeability was approximately 5-fold greater in the cells treated with 25% pancreatic supernatants as opposed to 25 % FBS (16 ± 5 vs 3.3 ± 0.3 % permeability p<0.05). Conclusion: These results support out hypothesis that pancreatic proteases are involved in the pathogenesis of T/HS-induced gut permeability and are able to directly increase enterocyte monolayer permeability.