Attenuation of graft ischemia-reperfusion injury by urinary trypsin inhibitor in mouse intestinal transplantation

Abstract

Aim: Ischemia/reperfusion (I/R) injury is one of the major obstacles for intestinal transplantation (ITx). Urinary trypsin inhibitor (Ulinastatin, UTI) suppresses proteases and stabilizes lysosomal membranes. We supposed that Ulinastatin would diminish I/R injury of intestinal graft.

Methods: UTI- treated group and untreated control group were investigated by histological assessment at 1.5, 4, 24, and 72 h after ITx. Myeloperoxidase (MPO) activity was used as the activity of neutrophils, and malondialdehyde (MDA) was used as an index of lipid peroxidation. TNFalpha and i-NOS mRNA expression in graft tissue were measured by semi-quantitative RT-PCR. CD11b+Gr1+ cells in graft lamina propria were analyzed by flow cytometry.

Results: Histological scores of the graft showed that the tissue injury was markedly attenuated by UTI treatment at different time points after ITx, with reduced MPO and MDA value in the grafts. The expression of TNFalpha and i-NOS mRNA was profoundly inhibited, while the infiltration of CD11b+ Gr1+ cells into the intestinal graft was decreased in UTI group.

Conclusion: Urinary trypsin inhibitor attenuates I/R injury in mouse intestinal transplantation by reducing monocytes infiltration and down-regulation of TNFalpha and i-NOS mRNA expression.

Figure 1

At 1.5, 4, 24, and 72 h after intestinal transplantation, the histological scores of tissue injury in the UTI-treated group were apparently less than the untreated group.

Figure 2

MPO activity in the intestinal grafts in the UTI-treated group remained at a lower level when compared with the control group in the earlier time points after IR (1.5, 4, and 24 h after transplantation respectively, ^aP<0.05 vs control group).

Figure 3

The MDA level at 1.5 and 4 h after transplantation was significantly suppressed in UTI-treated group, ^aP<0.05 vs the untreated control group.

Figure 4

The sub-population of CD11b⁺ Gr1⁺ cells in the monocyte gate of graft lamina propria was found reduced in the UTI-treated group on the third post-transplant day.

Figure 5

In UTI group, the expression of i-NOS mRNA was apparently inhibited at 1.5, 4, 24, and 72 h after intestinal transplantation. The upper bands show the representative images of the PT-PCR results for i-NOS. M: band for molecular marker; S: Sham operation group; C1-C4: bands for untreated group at 1.5, 4, 24 and 72 h after transplantation respectively; T1-T4: bands for UTI-treated group at 1.5, 4, 24 and 72 h after transplantation, respectively.

Figure 6

The expression of TNFα mRNA was inhibited at 1.5 and 4 h after intestinal transplantation in UTI-treated group, while no difference was found at 24 and 72 h after transplantation. The upper bands show the representative images of the PT-PCR results for TNFα. M: band for molecular marker; S: Sham operation group; C1-C4: bands for untreated group at 1.5, 4, 24 and 72 h after transplantation respectively; T1-T4: bands for UTI-treated group at 1.5, 4, 24 and 72 h after transplantation, respectively.