Abstract
Aim To investigate the potential influence of nuclear factor κB (NF-κB) activation on the inflammatory mediators secreted by alveolar macrophages (AMs) in rats with acute necrotizing pancreatitis (ANP) and to evaluate the effect of an inhibitor of NF-κB-N-acetylcysteine (NAC).
Methods Ninety male Sprague-Dawley rats were randomly divided into 3 groups, 30 of each: control, ANP, and ANP plus NAC groups. The ANP rat models were established by a retrograde injection of 5% sodium taurocholate into the pancreatic duct. In addition to sodium taurocholate, the ANP plus NAC group received intravenous infusion of NAC (25 mg/100 g). At the sixth hour after modeling, the protein content of the bronchoalveolar lavage fluid, the myeloperoxidase in the lung tissue, and the transforming growth factor α and the nitric oxide (NO) secreted by AMs were determined. The histopathologic changes of the pancreas and the lung were observed under light microscope, and NF-κB activation of AMs was detected.
Results The protein content of the bronchoalveolar lavage fluid and the myeloperoxidase level of the lung tissue showed a significant increase in the ANP group as compared with the NAC-administered group. The levels of transforming growth factor α and NO secreted by AMs in the ANP and the ANP plus NAC group rose significantly over that in the control group, and there was a significant difference between them. Although they were still higher than those in the control group, the pancreas destruction and the lung injury were slighter in the ANP plus NAC group and the activation of NF-κB was lower in the ANP plus NAC group as compared with that in the ANP group.
Conclusions The correlation between the NF-κB activation, the up-regulation of the inflammatory mediators secreted by AMs, and the tissue damage suggests a key influence of NF-κB in the pathogenesis of ANP. Inhibition of NF-κB activation may reverse the lung injury of ANP.
Acute necrotizing pancreatitis (ANP) is a life-threatening necroinflammatory disease with high morbidity and mortality, and it is often complicated by systemic inflammatory response syndrome and multiple organ failure.1,2Within the first few days after the onset of ANP, acute lung injury (ALI) occurs as a frequent consequence of ANP.3Sepsis is a dominant cause for ALI and mortality in the later phase of the disease process.4However, the exact mechanisms of ALI are indefinite, but once the disease process is initiated, monocyte macrophages are stimulated excessively. Simultaneously, common inflammatory and repair pathways are invoked.5By far, in-depth understanding of the pathogenesis of ALI in ANP has not turned up trumps, and none of the clinical trials using novel therapeutic agents has indicated an improvement in patient outcome. Consequently, effective therapeutic interventions are thus called for.
Nuclear factor κB (NF-κB) is one of the most important transcription factors that control proinflammatory gene expression during ANP and known to be involved in inflammatory and immune responses.6It plays an important role in physiologic and pathologic conditions as an inducible NF.7In most cells, NF-κB is normally sequestered in the cytoplasm in an inactive form associated with a class of inhibitory proteins called IκBs. During ANP, NF-κB is rapidly activated and translocated to the nucleus, binds to specific κB sequences in the promoter regions, and transactivates the downstream genes, including interleukins, chemokines, adhesion molecules, receptors, and specific inducible isoform of nitric oxide synthase enzymes.8Recent experimental studies appeared to have shed some light on the intracellular signaling pathway in the inflammatory cascade in ANP.9The role of NF-κB in ANP has attracted more and more attention. For example, it has been shown to play a critical role in the pathogenesis of ANP by regulating the expressions of many proinflammatory genes in the pancreas.10
Hence, on the basis aforementioned, this study was conducted to evaluate the potential influence of NF-κB activation on the inflammatory mediators secreted by alveolar macrophages (AMs) in rats with ANP and to evaluate the effect of an inhibitor of NF-κB-N-acetylcysteine (NAC).
MATERIALS AND METHODS
Animal Model and Groups
Ninety adult male Sprague-Dawley (SD) rats weighing between 280 and 300 g were provided by the Experimental Animal Center, Xiangya Medical School, Changsha, China, and were housed under standardized conditions with free access to food and water. The animals were randomly divided into a control, an ANP, and an ANP plus NAC group. The rat ANP model was established by the retrograde injection of 5% sodium taurocholate (Sigma, St. Louis, MO) at 0.1 mg/100 g into the pancreatic duct as described previously.11In the control group, rats were given a saline injection. In addition to sodium taurocholate, rats in the ANP plus NAC group received intravenous infusion of NAC (Sigma) at 25 mg/100 g, whereas the control and the ANP groups received the same amount of saline instead. All studies were performed with the approval of the experimental animal committee at our university.
Histopathologic Examination
Six hours after the ANP modeling, rats were killed by dislocation of the cervical vertebrae. The pancreas and the lung tissues were sectioned and then fixed in 10% formalin and embedded in paraffin, followed by hematoxylin-eosin staining. Pulmonary lesion was scored after the criteria of Lei et al.11
Protein Content of the Bronchoalveolar Lavage Fluid
After killing the rats, a thoracotomy was performed. Then, the whole lungs together with the trachea were taken out, the left bronchus was ligated, and the left lung was harvested for the measurements of the activity of myeloperoxidase (MPO); the right lung together with the trachea was used for lavage purpose. The surface blood was washed away with isotonic sodium chloride. A total of 5 mL of isotonic sodium chloride was repeatedly lavaged 5 times. The lavage fluid was collected and centrifuged, and the supernatant was obtained for protein content measurement.
Myeloperoxidase Activity in the Lung Tissues
Myeloperoxidase activity in the lung tissues of the 3 groups was assessed by the modification of the method described previously.12Frozen tissues were thawed and washed twice with 50-mmol/L potassium-phosphate buffer (pH 6.0), then homogenized in the hexadecyltrimethyl ammonium bromide suspension buffer (50-mmol/L potassium phosphate and 0.5% hexadecyltrimethyl ammonium bromide at pH 6.0; 50 mg of tissue/1 mL of buffer). It was homogenized at 13,500 rpm on ice for 1 minute. The homogenate was frozen and thawed on 2 consecutive occasions before a final 30-second homogenization and then centrifuged at 20,000g at 4°C for 15 minutes. The upper layer was incubated at 60°C for 2 hours and centrifuged at 2000g at 4°C for 15 minutes. The clear supernatants were collected for the assay of MPO. To measure MPO activity, 100 μL of supernatants was incubated at 37°C with 100 μL of 0.1% tetradimethylbenzidine dissolved in dimetylsulfoxide and 750 μL of 0.5-mmol/L hydrogen peroxide dissolved in 50-mmol/L potassium phosphate buffer (pH = 5.4). The enzyme activity was assessed photometrically at 655 nm by using an MPO calibration curve (Cat No. 475911; Calbiochem, La Jolla, CA). The within-run coefficient of variations was 5.8 for the lung tissues. The protein concentrations of the supernatants were measured by the Lowry method. The MPO activity was expressed as units per gram of protein.
Levels of Transforming Growth Factor α and NO Secreted by AMs
The whole bronchoalveolar lavage fluid (BALF) was centrifuged at 280 rpm for 10 minutes at 4°C. Cell pellets were resuspended (1 × 105 cells/mL) in the Royal Park Memorial Institute 1640 medium (Nikken, Osaka, Japan). Cell suspension was then placed in plastic Petri dishes (Nunc, Roskilde, Denmark) and incubated at 37°C for 1 hour in a CO2 incubator (50-mL/L CO2 + 95% air). The levels of transforming growth factor α (TNF-α) and NO secreted by AMs were measured by the enzyme-linked immunosorbent assay (ELISA) method according to the instruction of the kits (TPI Ltd, St. Louis, MO). Nonadherent cells were removed from adherent macrophages by washing with the Royal Park Memorial Institute 1640 medium. Purified AMs were recovered by gently rubbing the dishes with a rubber policeman. Nuclear protein was extracted from purified AMs as previously described.13Protein was stored at −70°C for subsequent examination of NF-κB activity.
Nuclear Factor κB Activation of AMs
Nuclear factor κB activation of AMs was determined using TransAM NF-κB p65 Chemi ELISA kit (Active Motif, Carlsbad, CA) by Central LB 960 microplate luminometer (Berthold, Bad Wildbad, Germany) and expressed as relative light units.
Statistics
The statistical package SPSS 10.0 (SPSS Inc, Chicago, IL) was used for all analyses. All values were expressed as mean (SD). Statistical differences among different groups were compared using analysis of variance with the Bonferroni/Dunn post hoc tests. The correlation of the NF-κB activation of AMs with the histopathologic score of the lung injury, the protein content of BALF, the MPO activity in the lung tissues, and the levels of TNF-α and NO secreted by AMs was analyzed using the Spearman rank correlation test. P < 0.05 was considered to be statistically significant.
RESULTS
Histopathologic Analysis
The histopathologic study of the pancreas revealed an extensive necrosis of the pancreatic tissue, an intense edema, and an inflammatory infiltrate (Fig. 1A). The necrosis of the pancreatic tissue and the edema in the ANP plus NAC group were similar to those in the ANP group (Fig. 1B). The control group was normal. In addition, the representative light microscopic view of the lung injury, including the alveolar septal thickening, the interstitial edema, the diffuse alveolar blood stasis, the infiltration of the inflammatory cells, mostly neutrophils, and the destruction of the alveolar wall in the ANP group, was shown in Figure 1C. The lung histopathologic scores of the ANP group were significantly higher than those in the control group (P < 0.01, Table 1). In the ANP plus NAC group, the major histopathologic findings were mild edema of the alveolar walls and mild alveolar blood stasis with slight infiltration of neutrophils (Fig. 1D). The lung histopathologic scores of the ANP plus NAC group decreased significantly compared with those of the ANP group (P < 0.05).
Protein Content of BALF
After modeling, the protein content in BALF of the ANP group rose rapidly. Although it was significantly higher than that in the control group (P < 0.05), the protein content of the ANP plus NAC group was still significantly lower than that in the ANP group (P < 0.05; Table 2).
Myeloperoxidase Activity in Lung Tissues
Table 3 shows the MPO activity of the lung tissues in the 3 groups. The MPO levels in the ANP group were significantly higher than those in the control group (P < 0.01). In the ANP plus NAC group, the lung MPO levels were significantly decreased (P < 0.01).
Levels of TNF-α and NO Secreted by AMs
Levels of TNF-α and NO secreted by AMs in both the ANP (both P < 0.01) and the ANP plus NAC (both P < 0.05) group rose significantly over those in the control group, whereas the former was also significantly higher than the latter (P < 0.01; Table 4).
Nuclear Factor κB Activation of AMs
Figure 2 shows that the NF-κB activation of AMs in the ANP group was significantly higher than that in the control group (P < 0.01) and was significantly decreased in the ANP plus NAC group (P < 0.01).
Correlation Analysis
The NF-κB activation of AMs was positively correlated with the histopathologic score of the lung injury (r = 0.66 and 0.69, respectively; both P < 0.01), the protein content of BALF (r = 0.72 and 0.75, respectively; both P < 0.01), the MPO activity in the lung tissues (r = 0.62 and 0.65, respectively; both P < 0.01), and the levels of TNF-α and NO secreted by AMs (r = 0.83 and 0.80 and r = 0.85 and 0.83, respectively; both P < 0.01).
DISCUSSION
Acute necrotizing pancreatitis as an inflammatory disorder may cause various systemic complications. Among them, ALI is the most dreadful and impending catastrophe that leads to a mortality rate of 25% and is difficult to deal with clinically.14It has been indicated that oxygen-free radicals, platelet-activating factor, cyclooxygenase-2, cytokines, and arachidonic acid metabolites play an important role in the progression of ALI in ANP.15In addition, pancreatic proteolytic enzymes or activated phospholipase A2 released into the circulatory system determines the development of lung injury.16Furthermore, other mediators in the lung tissue such as the platelet-activating factor and the arachidonic acid metabolites can stimulate inflammatory cell activation. In addition, the interaction of polymorphonuclear granulocytes, endothelium, and endothelium-derived mediators seems to be important to amplify lung damage.17However, the mechanisms of the development of this disease and the effective therapies for preventing or reversing it remain obscure. Randomized studies of ANP in the clinical setting do have limitations. In this regard, reliable ANP animal models are of paramount importance. Therefore, this study was conducted to investigate a mechanism of the progression of ALI-the potential influence of NF-κB activation on the inflammatory mediators secreted by AMs and the histopathologic changes in rat models with ANP-which has not been reported previously.
Nuclear factor κB is a kind of pleiotropic regulative protein of transcription, which regulates the expressions of a large number of genes that are critical for the regulation of apoptosis, viral replication, tumorigenesis, inflammation, and various autoimmune diseases. The activation of NF-κB is thought to be part of a stress response because it is activated by many divergent stimuli, including proinflammatory cytokines such as TNF-α, interleukin 1β, epidermal growth factor, T- and B-cell mitogens, bacteria and lipopolysaccharides, viruses, viral proteins, double-stranded RNA, and physical and chemical stresses. Its activation also takes part in the pathogenesis of ANP, and the inhibition of the action can ameliorate the rat with ANP.18Hypoxemia and ischemia can activate NF-κB and subsequently act on genes for proinflammatory cytokines, chemokines, immune receptors, and adhesion molecules. All the previously mentioned cytokines and inflammatory mediators can further aggravate the injury to multiple organs in severe acute pancreatitis, particularly the lung tissue, and thus accelerate acute respiratory distress syndrome or damage to other organs.6,19Moreover, in recent years, some researchers also reported that the activation of AMs may play an important role in lung injury associated with ANP and that TNF-α and NO secreted by AMs are increased significantly in rats with ANP.20,21Transforming growth factor α has been considered as a prognostic factor of ANP. Most authors believed that the increase in TNF-α activity was due to the excessive stimulation of the mononuclear macrophage by endotoxin.22Excessive production of NO causes vasodilatation and hypotension leading to organ hypoperfusion, edema, and organ dysfunction. The reaction of NO with superoxide causes the formation of peroxynitrite, which is a powerful oxidant and cytotoxic agent and may play an important role in the cellular damage associated with the overproduction of NO.23,24
Our study provides evidence that the injection of sodium taurocholate can cause ANP, with manifestation of the damage to the pancreas, and inflammatory infiltration. The rise of protein content in BALF, the increase of MPO activity, and the TNF-α and the NO levels secreted by AMs were also the indicators of lung damage caused by ANP. Accompanied with these, ELISA results also suggested that NF-κB was activated at the onset of ANP. The lower expression of the NF-κB p65 subunit in the ANP plus NAC group indicated that the treatment of NAC inhibited the NF-κB activation. Nuclear factor κB-N-acetylcysteine also led to a slighter histological damage to the pancreas and lung and a lower increase in the protein content in BALF, in MPO activity, and in TNF-α and NO levels secreted by AMs. All these results are consistent with previous studies, substantiating that NF-κB activation is involved in the pathogenesis of ANP and inhibition of the activation may reverse rat ANP.25,26Correlation between NF-κB activation, tissue damage, protein content in BALF, MPO activity, and TNF-α and NO levels secreted by AMs is a direct evidence that NF-κB activation plays a key role in the pathogenesis of lung injury in the ANP model.
In summary, we may draw a conclusion that the correlation between NF-κB activation, up-regulation of inflammatory mediators secreted by AMs, and tissue damage suggests a key influence of NF-κB in the pathogenesis of ANP. The use of NF-κB inhibitors can block the activation of the signal transduction pathway to block the synthesis of inflammatory cytokines, which may be the future direction of the treatment of lung injury occurring in ANP.