Mechanism About Ischemia-Reperfusion Of The Pancreas Induced Lung Injury In Rats

IntroductionIschemia-Reperfusion (I/R) injury to the pancreas remains an important clinical problem during shock, pancreatic surgery, and pancreas transplantation. Oxygen free radicals are involved in I/R-related pancreatic injury. Many studies have demonstrated that I/R of the pancreas induce systemic inflammatory responses by increasing the blood white blood cell count, oxygen radical production, and cytokine release. Acute pancreatitis can lead to inappropriate activation of pancreatic enzymes through a mechanism involving proteolytically derived activators by which inflammatory cells are activated, interleukin released, oxidative and nitrosative stress occur, changing airway reactivity. In this study, we characterized whether I/R of the pancreas induced inflammation reactivity changes in airways upon challenge with a cholinergic agonist.Some reports have indicated that intercellular adhesion molecule-1 (ICAM-1) plays an important role in the development and progression of acute pancreatitis complicated by acute lung injury, and the severity of the lung injury correlates well with the expression levels of ICAM-1 protein. ICAM-1, a single-chain transmembrane glycoprotein with a molecular weight of 80-110 KDa, consists of five Ig-like domains, a hydrophobic transmembrane domain and a short cytoplasmic C-terminal domain. Its ligand includes lymphocyte function-associated antigen-1 (LFA-1) and macrophage antigen-1 (Mac-1). Under physiological conditions, ICAM-1 is expressed at a low level in endothelial cells and epithelial cells or constitutively on the surface of alveolar cells, providing the underlying molecular basis for cell recognition, activation, proliferation, differentiation and motility, and thereby helping to stabilize the internal environment of the body. Moreover, ICAM-1 also plays a key role during pathological conditions, such as inflammatory reaction etc. For these reasons, a comprehensive and objective understanding of ICAM-1 is needed. It is obvious that NF-kappa B plays a critical role in the expression of ICAM-1. Therefore, research on the use of NF-kappa B inhibitor to alleviate inflammation response has become a hotspot. Calpain I inhibitor and pyrrolidine dithiocarbamate (PDTC) are antioxidants which are potent inhibitors of NF-kappa B. Calpain I inhibitor and pyrrolidine dithiocarbamate (PDTC) can lessen lung injury in rats with acute pancreatitis, decrease the activation of NF-kappa B as well as the expression of ICAM-1 protein, and can retain the soakage of inflammatory cells and mitigate the microvascular impairment of the lungs, which reduces the incidence rate of pneumonedema. After Hietaranta et al. first reported that MG132, a prosome inhibitor, could depress the activation of NF-kappa B in acute pancreatitis, some researchers demonstrated that MG132 also had the effect of protecting lung tissue in rats with acute pancreatitis which may be associated with the function of inhibiting NF-kappa B activation. The use of the NF-kappa B inhibitor may be considered as another effective path in the treatment of acute pancreatitis complicated by acute lung injury, and associated clinical research is required.MIF was originally identified as a cytokine derived from activated T cells. However, MIF is now considered to exert various biologic functions in macrophage activation. Moreover, MIF is thought to play a central role in exacerbation of inflammation and sepsis. Importantly, a recent report has suggested that gene expression of TLR-4 in macrophages can be upregulated by MIF. Thus, hyporesponsiveness of MIF-deficient macrophages to LPS has been demonstrated by a marked reduction in the activity of NF-κB and the production of TNF-a, which is strictly associated with downregulation of TLR-4. During AP, MIP-2 is involved in neutrophil activation and sequestration in the pancreas and lungs. MIP-2 is a potent rodent chemokine, homologous to GRO-b, which binds to the C-X-C chemokine receptor-2. We found that cerulein induced AP was associated with a significant increase in serum MIP-2 concentrations, and that leptin treatment substantially decreased the MIP-2 concentration. The effect of leptin administration on serum MIP-2 concentration is not clearly established. To the best of our knowledge, this is the first study demonstrating the effect of leptin on serum MIP-2 concentration. A decrease in the concentration of MIP-2 may therefore play an important role in reducing neutrophil adhesion and sequestration. Moreover, Ob-R is present in the pancreas and lungs.Material and Methods1. Animal samples (1) Experimental DesignAnimals were randomly divided into two groups:(1) The I/R group (n=8) underwent 2 hours of gastroduodenal and splenic artery occlusions followed by 6 hours of reperfusion. The rats were not given treatment except saline prior to clamping the arteries. (2) The sham group hosts (n=7) were prepared in the same manner as in the I/R group, but the vessels were not clamped.(2) Preparation of AnimalsMale Sprague-Dawley rats (300-350 g) were anesthetized with pentobarbitals the right femoral vein was cannulated for blood sampling. The gastroduodenal artery and the splenic artery were exposed and ischemia induced by clamping for 2 hours followed by 6-hour reperfusion.2. Experimental Methods(1) Quantification of Pancreatitis by Measuring Plasma Activities of AmylaseBlood samples were collected for WBC measurement. After centrifugation, plasma was isolated for amylase measurement using a Kodak Ektachem DT60 analyzer (Rochester, NY) and expressed in IU/L.(2) Measurement of Nitric Oxide by High-Performance Liquid ChromatographyHigh-performance liquid chromatography was used to measure blood levels of nitrite and nitrate anions derived from nitric oxide (NO).(3) Methylguanidine Measured by SpectrofluorometerWe measured the levels of methylguanidine in blood as a reflection of I/R-induced hydroxyl radical production.(4) WBC Counts in Lung LavagesLung lavages were performed with 5 mL saline at the end of the experiment. WBC in lavage samples were measured using a cell counter.(5) Quantitation of Tumor Necrosis Factor-a by Enzyme-Linked lmmunosorbent AssayThe tumor necrosis factor-a (TNFa) concentrations in blood samples were measured separately with an enzyme-linked immunosorbent assay kit according to the manufacturer's instructions (Endogen, Woburn, MA).(6) Measurement of Bronchial Responsiveness to MethacholineAirway responses to methacholine challenge were measured by unrestrained. whole-body plethysmography (Buxco Co). Rats placed inside Plexiglas chambers underwent measurements of their respiratory rate and breathing volume using pressuresensitive transducers that had been calibrated for the experimental conditions. After an acclimation period of about 10 minutes, the baseline enhanced pause (Penh), a measure of airway resistance, was determined by exposing the animals to a saline aerosol and calculating Penh according to an algorithm developed by Buxco. Methacholine at specifically metered dose rates was then fed into the chambers and Penh measured again.(7) RNA Isolation and Real-Time Polymerase Chain ReactionIsolation of mRNA from lung tissues was performed using an mRNA Isolation Kit (QIAGEN RNeasy kits, QIAGEN Inc, Valencia, CA). The mRNA isolated from each lung tissue sample was reversely transcribed to cDNA following the manufacturer's recommendation. Polymerase chain reaction (PCR) primers and TaqMan-MGB probes were designed using Primer Express V.2.0 software (Applied Biosystems Inc, Foster City, CA) based on sequences from GenBank. TaqMan-MGB probes were labeled with 6-carboxy-fluorescein as the reporter dye. PCR reactions were monitored in real time using an ABI PRISM 7000 Sequence Detector (Applied Biosystems Inc).(8) ICAM-1 expression in lung tissue by Western blotWestern blot analysis was performed as described previously. Briefly, proteins were separated by SDS-polyacrylamide gel electrophoresis (PAGE; 12% separating, 4% stacking) and transferred to NC membranes (Amersham Pharmacia Biotech, Inc., Piscataway, NJ). After the membranes were blocked in 5% nonfat dry milk in PBS buffer containing 0.1%, the protein signal was amplified and visualized via chemiluminescence using the ECL Western blotting detection system and Hyperfilm ECL autoradiograhpy film (Amersham Pharmacia Biotech, Inc.). Images were quantified using the Labworks v3.0.2 image scanning and analysis software.(8) Total MIF mRNA isolation and real time RT-PCRTotal RNA was isolated using the acid guanidinium thiocyanate-phenol-chloroform method. The quality and quantity of the isolated RNA was determined before using the RNA. One microgram of total RNA was reverse transcribed using Advantage RT-for-PCR kit. Real-time RT-PCR was done using Smart Cycler (Cepheid, Sunnyvale, CA) in which 2μl cDNA,10μl Sybergreen Master mix, and 0.5μlof 20 μM gene-specific primers were used. The specificity and size of the PCR products were tested by adding a melt curve at the end of the amplifications and running it on a 2% agarose gel. All values were normalized to 18S expression.(9) Level of MP-2 in Lung tissueConcentrations of MIP-2 in culture supernatants were determined by enzyme-linked immunosorbent assay (ELISA) using commercially available kits.3. Data AnalysisData were expressed as mean values±standard errors of the means. Comparisons within each group for a given parameter used paired Student t tests. Values of P<0.05 were considered statistically significant.Results1.Ischemia-Reperfusion of the Pancreas Induced Hyperresponsiveness of the Airways in RatsThis protocol resulted in significant elevations of the blood concentrations of nitric oxide, hydroxyl radical, amylase, TNFa, and white cells among the I/R group. The mRNA expressions of iNOS and of TNFαin the lung tissues were significantly increased after I/R. Pulmonary function data showed that I/R of the pancreas induced significant increases in the responses to methacholine challenge:Penh was significantly increased in the I/R group compared with the sham group. Lavage white cells were significantly increased in the I/R group.2. Efect of NF-κB and Intercellular Adhesion Molecule-1 on Ischemia-Reperfusion of the Pancreas Associated Lung Injury in RatsThe histologic scores of pancreas and lung in I/R group were 5.94±0.72 and 6.42±0.65 respectively, which was significantly higher than that in sham group (0.20±0.14 and 0.27±0.31, respectively)(p<0.05). The levels of amylase and myeloperoxidase activity were increase significantly in I/R group (3719.6±523.8, 0.74±0.06, respectively) than in sham group (1198.4±121.7). The overexpression of ICAM-1 protein and mRNA level was related with lung injury in I/R rats (0.47±0.03 and 1.12±0.07), comparing with the control group. The level of NF-κB activity also significantly increased. 3. Role of Macrophage Inflammatory protein 2 and Macrophage Migration Inhibitory Factor in Ischemia-Reperfusion of the Pancreas-associated Lung InjuryThe histologic scores of pancreas and lung in I/R group were 8.52±1.17 and 4.71±0.30 respectively, which was significantly higher than that in sham group (2.14±0.06 and 0.37±0.14, respectively) (p< 0.05).The levels of amylase and myeloperoxidase activity were increase significantly in I/R group. The overexpression of MIF protein and mRNA level was related with lung injury in I/R rats comparing with the control group. The level of MIP-2 in I/R group (91.5±12.1) was significantly hisher than that in sham group (23.9±5.8) (p< 0.05).ConclusionI/R of the pancreas induced systemic inflammatory responses and increased white cell sequestration in the lung. Hyperresponsive responses in the airways of the reperfusion group may be due to airways inflammation, which increased white cell sequesteration in the lung and the expressions iNOS and TNF-a inflammatory mediators in lung tissues.