Clinical management of severe acute pancreatitis (AP) remains a difficult problem mainly in cases with intense systemic inflammatory response syndrome usually associated with a high mortality rate (1, 2).
Local alterations in AP are characterized mainly by increased capillary permeability, tissue infiltration of inflammatory cells, and release of cytokines that increase tissue damage (3, 4). Systemic alterations involve severe hemodynamic alterations, acute lung injury secondary to AP, increased plasma levels of inflammatory cytokines, bowel damage, and increased bacterial translocation.
It has been previously demonstrated that administration of hypertonic saline (HS) during hemorrhagic shock corrects the hemodynamic alterations and decreases capillary and sinusoidal luminal narrowing in resuscitation of hemorrhagic shock (5, 6). Hypertonic saline resuscitation of hemorrhagic shock also reduces neutrophil rolling and adherence into endothelial cells, therefore diminishing vascular lesions (7, 8).
Treatment with HS was shown to be also beneficial in experimental AP (9-11). Studies from our group have shown that administration of HS to rats with AP induced by taurocholic acid avoided mortality, reduced hemodynamic alterations, and was associated with a 10-fold decrease of IL-6 and IL-10 serum levels. Pancreatic infection and pulmonary and liver alterations were also reduced (12). We hypothesized that these effects are related not only to systemic inflammatory reduction, but also to a reduction of the pancreatic lesion.
In the present study, we investigated the effects of HS administration in the same experimental model of AP focusing on the pancreatic inflammation. We found that the severity of pancreatic lesions was not affected by HS treatment, whereas inflammation markers (iNOS, cyclooxygenase 2 [COX-2], TNF-α, and IL-6) in pancreatic tissue were all reduced by this treatment. The volume of ascitic fluid and the level of TNF-α in ascitic fluid were also reduced by HS.
MATERIALS AND METHODS
Adult male Wistar rats weighing 230 to 270 g were housed in individual cages and kept under standard conditions (12 h of light/dark cycle and temperatures between 22°C and 28°C) with free access to a standard rat chow and water. The experimental protocol was approved by the Ethics Committee for Animal Research from the Medical School of São Paulo University. All Animals received care in accordance with the Guide for the Care and Use of Laboratory Animals.
Ketamine chloride (Ketalar; Parke Davis, São Paulo, Brazil); TNF-α, IL-6, and IL-10 assay kits (Biosource International, Camarillo, Calif); TAP radioimmunoassay kits; Bicinchoninic Acid Protein Assay kit (Pierce, Rockford, Ill); ECL chemiluminescence kit (Amersham Biosciences Corp, Piscataway, NY); Mini-Gel System and Trans-Blot SD-Semidry Transfer Cells (Bio-Rad Laboratories, Hercules, Calif); rabbit polyclonal antibodies to COX-2 or rabbit antiserum to iNOS (1:500; Cayman Chemical, Ann Arbor, Mich); Rainbow protein molecular weight markers (Amresco Inc, Solon, Ohio); immunocomplexed peroxidase-labeled antibodies. The other chemical reagents were obtained from Sigma Chemical Co (St Louis, Mo).
Induction of AP
Surgical anesthesia was induced with ketamine chloride 50 mg/mL (0.2 mL/g body weight). Acute pancreatitis was induced in anesthetized animals by retrograde injection of 0.5 mL of 2.5% sodium taurocholate in saline into the main pancreatic duct during 1 min at a constant rate by using an infusion pump (0.5 mL/min). A clamp was applied to the proximal part of the hepatic duct during the injection (13, 14).
Animals were randomized to four groups:
- Control group (C): 10 rats without induction of AP
- NT group: 16 animals submitted to AP and not treated
- NS group: 16 rats received 34 mL/kg of normal saline solution (NaCl 0.9%) injected via the dorsal penial vein for 5 min 1 h after induction of AP
- HS group: 16 rats received 4 mL/kg of HS solution (NaCl 7.5%) injected via the dorsal penial vein for 5 min 1 h after induction of AP (12, 15, 16)
Distinct volumes of saline were used in the NS and HS groups to guarantee that both received the same amount of sodium (5.13 mEq/kg).
At 2 or 24 h after the induction of AP, animals were killed and serum and/or ascitic fluid samples were assayed for amylase activity (17), trypsinogen activation peptides (TAPs), and TNF-α, IL-6, and IL-10. The volume of the ascitic fluid was also evaluated. Pancreatic tissue was collected for evaluation of protein expression of COX-2 and iNOS, evaluation of malondialdehyde (MDA) contents, myeloperoxidase (MPO) activity, and levels of TNF-α, IL-6, and IL-10. A portion of the pancreas was fixed in 10% buffered formalin for histological analysis.
TAP determination in serum and ascitic fluid
The formation of anodal TAP (TAP-A) and cathodal TAP (TAP-C) derived from trypsinogen activation was measured by a radioimmunoassay, and results were expressed as the ratio TAP-A/TAP-C, as previously described (18).
Expression of COX-2 and iNOS
The protein content was determined in the supernatant of pancreatic homogenates using the Bicinchoninic Acid Protein Assay kit, according to the protocol provided by the manufacturer. Samples containing 20 µg protein were separated on 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels and transferred to nitrocellulose membrane using the Bio-Rad Mini-Gel System and Trans-Blot SD-Semidry Transfer Cells. For immunoblotting, the nitrocellulose membranes were incubated in Tris-Buffered Saline Tween-20 (TBS-T) buffer (150 mM NaCl, 20 mM Tris, 1% Tween 20, pH 7.4) containing 5% nonfat milk dried milk for 1 h. The blot was treated with 1:1,000 dilutions of rabbit polyclonal antibodies to COX-2 or rabbit antiserum iNOS for 2 h at room temperature, then were washed three times with TBS-T, and incubated with 1:2,000 dilutions of peroxidase-conjugated monoclonal antirabbit IgG for 1 h at room temperature. Protein bands at 72 kd (COX-2) or at 130 kd (iNOS) were identified by comparison with Rainbow protein molecular weight markers from Amresco Inc. The immunocomplexed peroxidase-labeled antibodies were visualized by an ECL chemiluminescence kit following manufacturer's instructions from Amersham Biosciences Corp and exposed to photographic film. Finally, blots were stripped with 200 mM glycine, pH 3.0, for 10 min, washed with TBS-T three times for 30 min each, and reprobed with β-actin (1:10,000), followed by antimouse secondary antibody (1:2,000). The band densities were determined by densitometric analysis using the AlphaEase FC program (Alpha Innotech, San Leandro, Calif). Density values of bands were normalized to the total β-actin present in each lane and expressed in percentage of control.
Levels of TNF-α, IL-6, and IL-10 were measured in homogenates of pancreas and in the ascitic fluid by a solid-phase sandwich enzyme-linked immune absorbent assay according to manufacturer's instructions.
Malondialdehyde formation was used as indicative of tissue lipid peroxidation occurrence and was estimated as thiobarbituric acid-reactive substances. Pancreatic tissue (100 mg/mL) was homogenized in 1.15% KCl buffer and centrifuged at 14,000g for 20 min. An aliquot of the supernatant was then added to a reaction mixture consisting of 1.5 mL 0.8% thiobarbituric acid, 200 μL 8.1% (vol/vol) sodium dodecyl sulfate, 1.5 mL 20% acetic acid (pH 3.5), and 600 μL distilled water. The mixture was then heated at 90°C for 45 min. After cooling to room temperature, the samples were cleared by centrifugation (10,000g for 10 min), and the absorbance was measured at 532 nm using malondialdehyde bis-(dimethyl acetal) as an external standard. The content of lipid peroxides was expressed as nanomole MDA per milligram protein (19).
Pancreatic MPO activity
Myeloperoxidase activity was used as an indicator of neutrophil tissue infiltration. Samples of pancreatic tissue were homogenized with a Polytron homogenizer using a homogenization buffer that contains 0.5% hexadecyltrimethyl ammonium bromide, 5 mmol/L EDTA, and 50 mmol/L phosphate at pH 6.0. Homogenized samples were sonicated and centrifuged (3,000g, 30 min) at 4°C. Myeloperoxidase activity in the supernatant was assayed by measuring the change in A460 resulting from the decomposition of H2O2 in the presence of O-dianisidine. Results were expressed as optical density at 460 mm (20, 21).
Histological analysis of the pancreas
Pancreatic tissue samples were fixed in 10% buffered formalin for standard hematoxylin and eosin staining. Histological evaluation of the pancreatic sections was performed by the same pathologist in a blinded way. The histological lesion severity was analyzed in accordance with Schmidt et al. (22). This score includes the graded assessment of pancreatic edema, inflammatory infiltration, pancreatic necrosis, pancreatic hemorrhage, and extrapancreatic fat necrosis, and a scale of 0 to 4 was used.
Results are presented as mean ± SEM. Continuous variables (ascitic volume, amylase activity, TAP, MDA, TNF-α, IL-6, IL-10, COX-2, and iNOS expression, and MPO activity) were compared using ANOVA. Results from the histological analysis were compared using the Kruskal-Wallis test. The level of P < 0.05 was considered statistically significant. The GraphPad Prism software (GraphPad Software, San Diego, Calif) was used for statistical analysis.
Effect of HS on selected features of AP
Serum amylase activity was significantly elevated in all groups with AP (NT, NS, and HS) compared with the control group (values in parentheses). Higher levels were detected after 24 h of AP induction. Trypsinogen activation peptides, expressed as the ratio TAP-A/TAP-C, were only detected in the serum of animals with AP after 24 h of induction and were not detected in the control group. Administration of HS had no significant effect on the serum levels of amylase and TAP. In the ascitic fluid, characteristic of the AP groups, amylase activity and TAP were measured only after 2 h of AP induction because after 24 h, the ascitic fluid has been already absorbed. Again, the administration of HS had no effect on the levels of amylase and TAP in ascitic fluid. Malondialdehyde content in the pancreas was used as measure of lipid peroxidation in the organ, and it was significantly increased. Higher levels were found 2 h after the induction of AP compared with 24 h, and there were no significant differences among the AP groups (NT, NS, and HS). These results are presented in Table 1.
Effect of HS on pancreatic inflammation
Pancreatic tissue sections were examined for edema, inflammatory cell infiltration, necrosis, and hemorrhage, and results were expressed as histological scores. These scores include a graded assessment of the pathological parameters, and a scale of 0 to 4 was used. The scores were significantly increased both at 2 and 24 h after AP induction. We found no differences in histological scores among groups with AP (NT, NS, and HS) at 2 and 24 h after AP induction (Fig. 1A).
Neutrophil infiltration in tissues can be estimated by the levels of MPO. Figure 1B shows that pancreatic MPO activity increased at 2 and 24 h after induction of the AP; however, there were no differences among the groups with AP (NT, NS, and HS).
A marked increase of pancreatic tissue levels of TNF-α (Fig. 2A) and IL-6 (Fig. 2B) and IL-10 (Fig. 2C) was observed at 2 and 24 h after AP induction. Treatment with HS significantly reduced the levels of TNF-α and IL-6 but did not affect the levels of IL-10. However, a marked increase of IL-10/TNF-α and IL-10/IL-6 ratios were observed in animals of the HS group when compared with animals of NT and NS groups. This suggests that upon HS treatment, there is a predominance of anti-inflammatory (IL-10) over proinflammatory (TNF-α and IL-6) cytokines.
The inducible enzymes COX-2 and iNOS, responsible for the synthesis of prostaglandins and nitric oxide, respectively, can be used as markers of inflammation. We found that their expression in the pancreatic tissue was significantly elevated 2 h after AP induction compared with the control group. Treatment with HS significantly reduced the expression of both enzymes (Fig. 3).
Effect of HS on peritoneal inflammation
During AP, there is an accumulation of ascitic fluid in the peritoneal cavity. It was observed a significant decrease of ascitic fluid volume in the animals of HS group when compared with NT and NS groups (Fig. 4A). Levels of TNF-α and IL-10 in ascitic fluid after 2 h of AP induction were significantly lower in animals of the HS group when compared with animals of the NT and NS groups. No differences were observed in IL-6 levels in ascitic fluid among animals with AP (NT, NS, and HS) (Fig. 4, B-D). The ratio IL-10/TNF-α is increased in the HS group compared with the NS group, whereas the ratio IL-10/IL-6 is similar in these groups.
Hypertonic saline solution has been proposed for treatment of severe hemorrhagic shock not only by its beneficial effects on disrupted hemodynamic conditions (15) but also because of its effects on modulation of the postinjury inflammatory response (23-25).
Hypertonic saline solution restores mean arterial pressure through a transcapillary osmotic gradient that increases water transposition from the interstitium, endothelial cells, and red blood cells. It thus increases peripheral tissue perfusion, cardiac contractility, and oxygen consumption by vasodilatation of precapillary resistance vessels and cardiac preload enhancement (15, 26-28). Besides, the beneficial effect on hemodynamic parameters in hemorrhagic shock, HS has been shown to have potent anti-inflammatory effects, diminishing the systemic inflammatory response syndrome (16).
In vitro studies have demonstrated that exposure to hyperosmolarity changes the expression of adhesion molecules (l-selectin, intercellular adhesion molecule I, and CD11b) on neutrophils and endothelial cells (29). It also decreases cytokines and elastase release by neutrophils in response to LPS stimulation and interferes in the cytoskeleton reorganization signal transduction (29-31). Hypertonic saline solution also has an anti-inflammatory effect on macrophages as has been demonstrated in in vitro studies (32).
Inadequate upregulation of cytokine-producing cells is believed to be an important step in the host's systemic inflammation and multiple organ dysfunction in AP (4). Therefore, besides its beneficial effect on hemodynamic parameters, the anti-inflammatory effects of HS could be useful in the treatment of AP.
In fact, administration of HS has been shown to be beneficial in AP (9-11). Previous studies from our laboratory (12) also demonstrated that administration of HS avoids mortality, reduces hemodynamic alterations, and is associated with a 10-fold decrease of IL-6 and IL-10 serum levels. Pancreatic infection and pulmonary and liver alterations were also reduced in experimental AP.
In the present study, we investigated whether the protective effects of HS in AP depend upon a reduction of the pancreatic inflammation and lesions. We found that the histopathologic findings (edema, acinar necrosis, hemorrhage, fat necrosis, and inflammatory cell infiltration) and MDA content were not significantly different among the groups treated or not with HS. Based on these observations, we can conclude that HS does not interfere with the pancreatic lesions in AP. Moreover, HS has no effect on serum amylase activity and TAP levels that are elevated during the AP. Hypertonic saline treatment did not affect these parameters also in ascitic fluid, although it caused a significant reduction in the ascitic fluid volume and in the levels of TNF-α.
Ascitic fluid volume and TNF-α levels are related to activated pancreatic enzyme leakage into the peritoneal cavity (33). Because the pancreatic enzyme activation in the HS group was not reduced, we may conclude that ascitic fluid volume and TNF-α level reductions are caused by a dampening of the proinflammatory response of peritoneal macrophages, as been demonstrated in in vitro studies (32-34).
In the present study, higher levels of TNF-α, IL-6, and IL-10 were observed in the pancreatic tissue of animals with AP when compared with the control group. However, in animals treated with HS, there was a significant reduction of TNF-α and IL-6 levels in the pancreatic tissue when compared with untreated (NT) and treated with normal saline (NS) groups. We also observed increases of the IL-10/TNF and IL-10/IL-6 ratios in HS-treated animals, which suggests that this treatment has an anti-inflammatory effect in the pancreatic tissue. However, this was not sufficient to change the pancreatic lesion scores.
Inflammation involves activation of intracellular signaling pathways, which leads to increased expression of specific genes such as those for the inducible forms of NOS and COX (35, 36). Thus, these enzymes can be used as markers of inflammation. In the present study, we observed a decreased expression of these enzymes in the pancreatic tissue in the HS group when compared with the NT and NS groups.
The reduction of local inflammatory mediators in the HS group without improvement of pancreatic histology suggests that the mediators measured in this study are not the main factors responsible for the pancreatic tissue damage.
Some data suggest that activated polymorphonuclear cells sequestered in organs unleash a cytotoxic arsenal of protease and oxygen radicals that may cause injury in endothelium and vascular leakage (37-39). The mechanisms underlying neutrophil sequestration into the pancreatic tissue are probably related to TNF-α production by pancreatic acinar cells (40). In the present study, there was no difference in pancreatic MPO activity among groups with AP (NT, NS, and HS), indicating that neutrophil infiltration was not affected by HS treatment.
A previous study from our group showed that treatment of rats with AP by administration of HS reduced the mortality, and in parallel, it reduced the systemic effects of AP, namely the lung inflammation and pancreatic infection. In the present study, the decreased expression of COX-2 and iNOS in the pancreas of the HS group could also be attributed to the decreased systemic inflammation. We also showed here that HS had no beneficial effect on pancreatic tissue damage, but it modulated the cytokines in pancreas toward an anti-inflammatory profile. Thus, the beneficial effect of HS in this model of AP seems to be related to improvement of the systemic inflammation rather than reduction of the pancreatic lesions, which are possibly related mainly to enzymatic activation (41). These results strengthen the therapeutic use of HS in AP.
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