Effects Of MMP-9 And Inhibitors On Endotoxin Induced Acute Lung Injury In Rabbits

ObjectiveThe pathological change of acute lung injury is the damage of alveolar -capillary basement membrane caused by uncontrolled inflammatory responses mediated by multiple inflammatory cells in lung. It is characterized by high alveolar - capillary leakage, hemorrhage and edema in alveolar, hyaline membranes formation, low lung compliance and the dysfunction of gas changing. It presented in clinic as respiratory distress and refractory hypoxemia. The alveolar — capillary basement membrane is extracellular matrix in itself. The main enzyme to degrade the extracellular matrix in lung tissue is matrix metalloproteinase - 9 (MMP - 9). We research the course of endotoxin induced acute lung injury in rabbits by studying the pulmonary oxygenation, histomorphology and biochemistry changing and the activity of MMP - 9 in lung tissues. We also investigated the effects of tetracycline, the inhibitor of MMP - 9, to the acute lung injury and tried to afford the evidence for prevention the endotoxin induced acute lung injury in clinical practice.MethodsPART 1: Ten healthy male rabbits were randomly divided into two groups. (1) ET group(n = 5) : All rabbits received continuous infusion of lipopolysac-charide ( E. Coli O₁11:B₄ LPS, 100μg/kg) within 4 hours. The changes of Cdyn, PaO₂/FiO₂, MAP, and artery blood pH were studied. We had observed the rabbits for 14 hours. The plasma level of Nitric Oxide (NO) , Xanthine Oxi-dase ( XOD) , SOD and TNF - α, IL- 10 were measured at the time before LPSinfusion (T0) , after 2 hours (T6) and after 10 hours (T,4). The pathological histology examination of the lung was carried out after rabbits sacrificed by 10% KC1 5ml intravenous injection. Another 5 rabbits were Sham group(n =5) , u-sing 0.9% saline instead of LPS.PART 2: There are 20 healthy male rabbits in this part. Sham group ( n = 5) was according to part one. Another fifteen rabbits were received lipopolysac-charide (E. Coli O₁11;B4 LPS, 100μg/kg) intravenous infusion within 4 hours and then randomly divided into ET + NS group ( n = 5 ) , ET + T group ( n = 5 ) and ET + D group ( n = 5 ). The rabbits received saline ( NS) , tetracycline (25mg/kg) and dexamethasone ( 10mg/kg) respectively. The changes of PaO2/FiO2 and Cdyn were recorded before LPS infusion ( TQ) , the end of infusion (T4) , 2 hours (T6) and 10 hours (T14) after infusion. After 14 hours studying, all rabbits were sacrificed by 10% KC1 5ml intravenous injection. The ratio of lung wet/dry (W/D) and the blood cell counts, the contents of albumin , the activity of NO, SOD in bronchoalveolar lavage fluid ( BALF) were measured. The activity of myeloperoxidase (MPO) , sodium potassium ATP enzyme (Na+ ,K+ - ATPase) in lung tissue was calculated. We used enzyme — linked immunosorbent assay (ELISA) to assess the MMP - 9 concentrations in the plasma and the lung tissues. The histomorphology of lung tissues were examined.ResultsPART 1: (1) There were no change of artery pH, MAP, Cdyn and PaO2/ FiO2 in Sham group. All parameters had no significantly change besides SOD elevating in T6( P <0.01) , IL - 10 elevating in T14 ( P < 0. 01) in Sham group. (2) In ET group, PaO2/FiO2, Cdyn began to decline 2 hours after LPS infusion (P <0.01 or P < 0. 05 ). The plasma level of XOD, NO and TNF - a in ET group was higher than those in Sham group (P<0.01 or P <0. 05). The lung in ET group consolidated and hemorrhaged obviously.PART 2: ( 1) The PaO2/FiO2 and Cdyn in ET + NS group, ET + T group and ET + D group were aggressively decreasing after T6, especially the decreaseof PaO/FiO2 ( P < 0. 01) . At the end of observation, the PaO2/FiO2 in ET + T group and the Cdyn in ET + D group were much higher than those in ET + NS group (P <0. 05). (2) The lung W/D, the activity of MPO, and the counts of WBC, RBC and albumin, the level of NO in BALF in ET + T group and ET + D group were higher than those in Sham group (P <0. 01 or P <0. 05) , but less than those in ET + NS group (P<0.01). (3) The activity of Na+ , K+ - AT-Pase in ET + NS group was lowest among three groups (P <0.01). The lung W/ D, the activity of MPO, and the counts of WBC, RBC and albumin, the level of NO in BALF in ET + D group were higher than those in ET + T group ( P > 0. 05). (4)The plasma level of MMP -9 in ET + NS group, ET + T group and ET+ D group were higher than those in Sham group (P <0. 01). The content of MMP - 9 in lung tissues in ET + NS group, ET + T group, ET + D group was higher than that in Sham group ( P <0.01). The lung tissue MMP -9 in ET + T group, ET + D group was much less than that in ET + NS group (P<0.01), e-ven less in ET + T group. (5) The pathological change of alveolar in ET + NS group were much severe, neutrophil infiltration, alveolar wall thickening and in-traalveolar hemorrhage and edema fluid, the normal anatomical shape of alveolar>vas disappeared. But the injury in ET + T group and ET + D group was less. E-ven less lung lessen rabbits were seen in ET + T group.Discussion1. In our experiment, we found the plasma level of inflammatory mediators such as TNF-a, IL- 10, XOD, NO were aggressively elevating 2 hours after the rabbits received LPS infusion. On the other hand, the artery blood pressure, the pH of artery was decreasing followed by the reduction of PaO2/FiO2 and Cdyn. At T14 in ET group, the above parameters were significantly changed. The mechanism of systemic inflammatory reaction caused by LPS may be as follows : (1) the activation of XOD which stimulated to produce a large amount of oxygen free radicals; (2) inducing the excessive production of NO; (3) the increase of the plasma level of inflammatory mediators, such as TNF - a, IL - 10.LPS infusion resulted in systemic inflammatory reaction followed by the a-cute lung injury. It demonstrated that endotoxin induced acute lung injury is the lung reaction during systemic inflammatory reaction. How to regulate the systemic inflammatory reaction is much important to prevent the endotoxin induced a- , cute lung injury.2. We have found that lung water content was increased but the PaO2/FiO2 and Cdyn were aggressively decreased in endotoxin induced acute lung injury. The pathological change was the alveolar - capillary basement membranes thicken , lung consolidated and hemorrhaged. The increased lung W/D and the alveolar wall thickening in ET + NS group, ET + T group and ET + D group showed pulmonary edema. LPS induced systemic inflammatory reaction and inflammatory mediators released by inflammatory cells recruitment in lung, which resulted in the damaging to alveolar capillary endothelia cells and the alveolar - capillary basement membranes. The high activity of MPO in lung tissue illuminated the recruitment of neutrophil. The recruited neutrophil took part in the lung injury.MMPs can degrade the extracellular matrix. It was activated neutrophil, which can produce a large numbers of MMPs. The MMP - 9 can degrade the extracellular matrix that compounded the alveolar - capillary basement membranes. We found in our experiment that the higher content of MMP -9 in lung tissues was coincided with the increased MPO, pulmonary function impairment and the histomorphology change. It is evidence that MMP - 9 is the fundament for LPS induced lung injury.The mechanism of LPS induced lung injury included: ( 1) the recruitment of inflammatory cell in lung tissues, and releasing inflammatory mediators; (2 ) the activity of MMP - 9 upregulation in lung tissues to degrade the alveolar -capillary basement membranes and increase alveolar - capillary leakage; (3 ) the inhibition of Na+ ,K+ - ATPase and the SOD activity.3. The traditional antibiotic tetracycline had both antibiotic and anti - inflammatory effects, which could inhibit the MMP - 9 activity. We found both tetracycline and dexamethasone could reduce the contents of MMP - 9 in lung tissues, and attenuates the lung injury caused by endotoxin.