W. Yang1, S. Matsuo2, A. J. Chaung1, P. Wang1 1Hofstra North Shore-LIJ School Of Medicine,Surgery,Mnahasset, NY, USA 2Tokyo Women’s Medical University,Surgery,Tokyo, , Japan
Introduction: Intestinal ischemia-reperfusion (I/R) occurs in various clinical settings, such as transplantation, acute mesenteric arterial occlusion, trauma and shock. I/R injury causes severe systemic inflammation, leading to multiple organ dysfunction associated with high rates of morbidity and mortality. Ubiquitin proteasome pathway has been indicated in the regulation of inflammation, particularly through NF-κB signaling pathway. PYR-41 is a small molecule compound that selectively inhibits ubiquitin-activating enzyme E1. We hypothesized that administration of PYR-41 attenuated organ injury after intestinal I/R.
Methods: Male C57BL/6 mice (20-25 g) were subjected to intestinal ischemia by clamping the superior mesenteric artery for 45 min, followed by reperfusion. At the beginning of reperfusion, PYR-41 (5 mg/kg BW) or 20% DMSO (vehicle) in normal saline was administered intravenously (n=5/group). After 4 h, blood and tissues were collected for various measurements. Apoptotic cells in tissue sections were detected by TUNEL assay. Cytokines were measured by ELISA. Ubiquitination and protein levels were determined by Western blotting. Gene expression was assessed by qPCR.
Results:PYR-41 significantly reduced LDH, AST and IL-6 serum levels from 752.8 to 155.3 IU/L, 76.7 to 47.0 IU/L and 10.4 to 5.1 ng/ml, respectively, compared to the vehicle group (P < 0.05). The integrity of microscopic structures of the small intestine and lungs was much more preserved in the PYR-41-treated group than in the vehicle group (Figure). PYR-41 decreased the number of TUNEL-positive cells in the small intestine and lungs from 144 to 32 and 120 to 38 cells/field, respectively, which were associated with a reduction of cleaved caspase-3 levels in both organs. The IL-6 and IL-1β levels were decreased from 166.8 to 64.8 and 65.8 to 37.7 pg/mg protein in the intestine as well as from 53.2 to 38.3 and 132.6 to 70.7 pg/mg protein in the lungs, respectively, with PYR-41 treatment. Myeloperoxidase activity in the intestine and lungs is reduced by 69% and 38%, respectively, with PYR-41 treatment. PYR-41 also inhibited the expression of KC and MIP-2 mRNA in the intestine as well as lungs. In addition, PYR-41 inhibited the degradation of IκB in the intestine and the ubiquitination in the lungs after intestinal I/R.
Conclusion: Treatment with PYR-41 effectively attenuates intestinal and pulmonary injuries through inhibiting NF-κB activation to reduce local and systemic inflammation after intestinal I/R. Thus, ubiquitination may be a potential therapeutic target for treating patients suffering from intestinal I/R.
