Protective Effects Of Urinary Trypsin Inhibitor Combined With LPD Solution On Lung Graft Ischemia-reperfusion Injury By Using An In-situ Cold Ischemic Rabbit Model

Background:Lung transplantation (LTx) has gained widespread acceptance as a therapeutic option in a selected number of patients with end-stage lung disease refractory to other forms of medical treatment. However, overall mortality following LTx is still notable. Primary graft dysfunction secondary to ischemia-reperfusion injury is the major complication following LTx and is the main cause of death in the early postoperative period, also works on long-term prognosis. Thus, reducing lung ischemia-reperfusion injury is a key pathway to improve the outcomes. Over the past decade, better understanding of the mechanism of ischemia-reperfusion injury and development of lung preservation technique have been associated with a reduction in the incidence of severe primary graft failure. The mechanism of ischemia-reperfusion injury is still not clearly demonstrated, potentially related to sodium pump inactivation, intracellular calcium overload, oxidative stress, upregulation of cell surface adhesion molecules, release of inflammatory mediators(TNF- α , cytokines, PAF, complement, etc. ), leukocyte activation, NO and endothelin, microcirculation disorder, edema and apoptosis. Trying to attenuate this injury is always under studying, such as refinement of preservation solution, precondition and drug inhibition.At present, urinary trypsin inhibitor(UTI) is used to treat the pancreatitis or protect giant organs at perioperation. Recent studies have indicated its protective function on reperfusion injury in liver, kidney, and small intestine transplantation. UTI has binding sites of various enzymes including trypsin, alpha-chymotrypsin, alidase, elastase, fibrinolysin and so on, it can inhibit the function of these enzymes simultaneously. It was also reported that UTI had effect on stabilizing lysosome member, inhibiting proinflammatory mediators releasing, inhibiting neutrophils activation. So we investigate whether UTI has protective role in donor lung ischemia-reperfusion injury and its possible mechanism.Materials and Methods:Twelve New Zealand white rabbits weighting 2. 0². 5kg were randomly divided into 2 groups: control group and UTI group. According to our rabbit model, a catheter was inserted through the main pulmonary artery trunk to left pulmonary artery, lung protection solution was supplied to the left lung via the catheter. In control group(L group), single LPD solution was employed and in UTI group(L+U group), 50,000U/Kg UTI was added to LPD solution. After perfusion, the left artery, vein, bronchus were clamped, ice-cold gauze was covered on left lung and cold ischemia was maintained for 120min. At the end of this period, the left vein, artery, bronchus were declamped, reperfusion and reventilation were carried out for 90min. Mean pulmonary artery pressure(MPAP) was continually recorded before ischemia and after reperfusion. Blood gas analysis(PaO₂ ) and serum TNF- α level were measured before ischemia and at the 15min, 30min, 60min, 90min after reperfusion.The lung tissue samples were obtained at the end of reperfusion. The pulmonary water index(D/W), tissure myeloperoxidase (MPO) activity, tissue malondialdehyde (MDA) content were measured, and microscopic examination of left lung was conducted.Results:All items measured before ischemia between two groups have no statistical difference. At 15min after reperfusion, PaC>2 in both groups decreased significantly(L, 312.33±25.18mmHg vs 225.67 + 35.38, P<0.01;L+U, 321.67+19. 17mmHg vs 250.83 + 47. 21mmHg, P<0.01), but in L+U group, PaO2was increased gradually and returned to normal level at the end of reperfusion, while it did not in L group. At the 90min after reperfusion, PaO2in L+U group(304.00+20. 90 mmHg) was significantly higher when compared to L group(240.67 + 41. 87mmHg, P<0.01). MPAP after reperfusion are significantly higher than it before ischemia in both groups, at the 60min, 90min after reperfusion, MPAP in L+U group(14.667±0.817mmHg^ 15.333 + 0.516 mmHg) were significantly lower than in L group(15.833+0.753 mmHg, P<0.05> 17.333+0.817 mmHg, P<0.01). Serum TNF- a level significantly increased at 30min after reperfusion than before ischemia in both L group(45.07+3.98 pg/ml vs 60.62+ 7.82 pg/ml, P<0.01)andL+Ugroup(45.77+4. 13 pg/ml vs 55.35 + 6.77 pg/ml, P<0.05). After reperfusion 60min, 90min, Serum TNF- a was significantly lower in L+U group(71.62±8. 04 pg/mK 73.47+5. 88 pg/ml) than in L group(86.90+3. 45 pg/ml, P<0.01;90.17±2. 37 pg/ml, P<0.01). The MPO activity was significantly lower in L+U group(9.8367 + 0.6949U/g) compared to L group(12.4279 + 1. 1302U/g, P<0.01), MDA leverls were significantly lower in L+U group(4.1187± 1. 9752nmol/mg) when compared to L group(7.2088+1.0497 nmol/mg, P<0.01). The ratio of DAV was significantly higher in L+U group(0.1886+0. 0085) compared to L group(0.1650±0. 0096, P<0.01). In addition, the microscopic examination also showed less severe alveolar edema formation and less red blood cell effusion in L+U group compared to L group.Conclusions:This study showed that the addition of UTI to LPD solution as pulmonary flush solution in this in situ cold ischemic rabbit lung model attenuated ischemia-reperfusion injury by improving oxygenation, decreasing pulmonary pressure and lung edema, further increased the lung protective effect of LPD solution.