Inflammation, Vol. 30, No. 6, December 2007 (# 2007) DOI: 10.1007/s10753-007-9038-y

Effects of Enalaprilat on Acute Necrotizing Pancreatitis in Rats Serdar Turkyilmaz,1,4 Etem Alhan,1 Cengiz Ercin,2 and Birgul Kural Vanizor3

Abstract—The aim of this study was to investigate the influence of enalaprilat on acute necrotizing pancreatitis (ANP) induced by glycodeoxycholic acid in rats. The induction of ANP resulted in a significant increase in the mortality rate, pancreatic necrosis, serum activity of amylase, alanine aminotransferase (ALT), and interleukin-6 (IL-6), lactate dehydrogenase (LDH) in bronchoalveolar lavage (BAL) fluid, serum concentration of urea, and tissue activity of myeloperoxidase (MPO) and maondialdehyde (MDA) in the pancreas and lung, and a significant decrease in concentrations of calcium, blood pressure, urine output and p02. The use of enalaprilat inhibited the changes in urine output, blood pressure, serum concentration of urea, p02, and tissue activity of MPO and MDA in the pancreas and lungs. It reduced the mortality and pancreatic damage. Enalaprilat demonstrated a beneficial effect on the course of ANP in rats; therefore, it may be used in the treatment of acute pancreatitis. KEY WORDS: acute necrotizing pancreatitis; reninYangiotensin system inhibitor; enalaprilat; experimental.

20.8% in spite of the improved fluid management, respiratory care and nutritional support [1, 2]. Auto digestion of the pancreas and impairment of pancreatic microcirculation are two important parts in the pathophysiology of acute pancreatitis [2Y4]. The severity of the local inflammation and the systemic complications are related to excessive up-regulation of cytokines, secondary mediators including histamine, prostaglandins, thromboxanes, leukotrienes, nitric oxide, and platelet activating factor, and polymorphonuclear leukocyte functions, the amount of pancreatic acinar necrosis, and the development of bacterial contamination [1, 2, 5, 6]. In the early stage of the disease, hypovolemia, resulting from fluid sequestration into peripancreatic areas and the abdominal cavity, and in the late stage, a septic complication, caused by bacteria translocated from the gut resulting in systemic inflammatory response, may cause increased metabolic demands progressing to clinical multi-organ failure [1, 2, 7].

INTRODUCTION In most cases, acute pancreatitis is a mild and selflimiting disease, but severe necrotizing forms associated with a significant mortality rate are not infrequent. In recent years, the mortality seen in acute necrotizing pancreatitis (ANP) has been reported to vary from 6.2 to Part of this study was presented at the 40th Congress of the European Surgical Research Society on 25Y29 May 2006, in Rostock, Germany. 1

Department of Surgery, Farabi Hospital, School of Medicine, Karadeniz Technical University, 61080 Trabzon, Turkey. 2 Department of Pathology, School of Medicine, Kocaeli University, Kocaeli, Turkey. 3 Department of Biochemistry, School of Medicine, Karadeniz Technical University, 61080 Trabzon, Turkey. 4 To whom correspondence should be addressed at Department of Surgery, Farabi Hospital, School of Medicine, Karadeniz Technical University, 61080 Trabzon, Turkey. E-mail: serdarturkyilmaz@yahoo. com

205 0360-3997/07/0600-0205/0 # 2007 Springer Science + Business Media, LLC

206 The hormonal reninYangiotensin system (RAS) has important physiological functions. These are regulation of blood pressure and fluid and electrolyte metabolism [8]. Renin in the circulating blood produces angiotensin I from hepatic angiotensinogen. The product, angiotensin I, is hydrolyzed to angiotensin II by the pulmonary angiotensin converting enzyme (ACE). Angiotensin II is potent vasoconstrictor agent in the organism. However, alternate enzymes to renin and ACE can generate different RAS productsVangiotensins III, IV, VI and VII [9]. It has been demonstrated that many tissues/organs exhibit their own RAS products and activities, which have autocrine and paracrine action [9, 10]. These actions are cell proliferation, anti-proliferation, apoptosis, superoxide generation and vasoconstriction as well as vasodilatation [9, 10]. Recently, some new studies have indicated that the pancreas has a local RAS [10]. Local pancreatic RAS is responsive to a number of physiological stimuli and clinical conditions [9, 10]. The available data indicate that a pancreatic RAS would be important for regulating the exocrine and endocrine functions such as acinar digestive enzyme secretion, islet hormonal secretion and ductal anion secretion [11]. It has previously been shown that acute pancreatitis could markedly upregulate the expression of RAS components, thus implicating its clinical relevance to pancreatitis [12]. We, therefore, have examined the effect of the reninYangiotensin inhibitor (RAI) enalaprilat on the extent of acinar cell injury, mortality, systemic cardiorespiratory variables, renal and hepatic function, and some enzyme markers for pancreatic and lung tissues during ANP in rats.

MATERIALS AND METHODS Seventy-two male Sprague Dawley rats weighing 300Y350 g were used. They were housed in rooms maintained at 21 T 1-C and a 12-h light/dark cycle. Animals were fasted overnight before the experiment, but had free access to water. The care was provided in accordance with the Ethics Committee of Karadeniz Technical University, Trabzon, Turkey. Anesthesia was induced with vaporized ether and maintained by an intraperitoneal injection of ketamine 50 mg/kg (Ketalar, EczacŒbasŒ, Istanbul, Turkey). The right internal jugular vein and carotid artery were cannulated (Luer Lock, ID 0.5 mm, Braun AG, and

Turkyilmaz, Alhan, Ercin, and Vanizor Melsungen, Germany). The catheters were tunnelled subcutaneously to the suprascapular area. During the experiment, the animals were housed in metabolic cages, which enabled quantitative assessment of urine production. The acute pancreatitis was induced by an intravenous infusion of cerulein (Sigma and Aldrich Chemie GmbH, Steinheim, Germany) at a dose of 5 mg/kg/h over 6 h superimposed on a standard infusion of 1.2 ml/kg glycodeoxycholic acid (10 mmol/l, Sigma, St. Louis, USA) into the biliary-pancreatic duct for 10 min at 30 mmHg as described by Schmidt et al. [13]. A special infusion pump for pressure and volume control (Hampshire, United Kingdom, Alaris Medical Systems, RG22, 4BS, IVAC P 7000) was used. Cerulein was reconstituted in physiological saline and infused at 8 ml kgj1 hj1 as the baseline hydration. The animals of the sham group were given intraductal saline followed by a 6-h intravenous infusion of saline. The rats were divided into four experimental groups (Fig. 1). Those in the first group (sham + saline, n = 6) had arterial and venous lines placed and were given intraductal saline followed by a 6-h intravenous infusion of saline. Following the 6-h period, 1 ml saline was given intravenously, and saline was infused intravenously at 6 ml/kg for the last 18 h. Eighteen hours later, mortality was recorded and the cardiorespiratory function was assessed by monitoring the arterial blood gases, mean arterial pressure (MAP), heart rate (HR), and renal function by the collection of urine using metabolic cages. Thereafter, the blood samples were taken from the carotid artery for the measurements of serum concentrations of electrolytes, calcium, urea, creatinine and glucose and activities of amylase, alanine aminotransferase (ALT), and interleukin-6 (IL-6). A midline sternotomy was performed and the left main bronchus was clamped. The bronchoalveolar lavage (BAL) of the right lung was performed with 2 ml phosphate-buffered saline containing 0.07 M ethylene diamine tetra-acetic acid and this procedure was repeated twice. The combined lavage of approximately 6 ml was centrifuged at 1,500 rpm for 20 min at 4-C, frozen at j20-C, and assessed subsequently for lactate dehydrogenase (LDH) measurement [14]. The left lung was harvested for the measurements of myeloperoxidase (MPO) and malondialdehyde (MDA) levels. The excised lung tissues were rinsed in saline, blotted dry, frozen in liquid nitrogen, and stored frozen at j80-C until thawing for measurement of MPO and MDA activity. At the end of the experiment, laparotomy was

207

Effects of Enalaprilat on Pancreatitis in Rats

saline Sham groups

The beginning of sham operation and the induction of ANP

End of the induction of ANP

End of the experiment and collection of data

enalaprilat

Rats ANP groups

saline

0

6

enalaprilat

24

time/hours

Receipt of saline or enalaprilat

Fig. 1. Experimental design and time schedule of the study. ANP Acute necrotizing pancreatitis.

performed and the entire pancreas was removed. The pancreas was divided into two, one section for the histological examination and the other for the measurements of MPO and MDA activity. The second group (sham + enalaprilat, n =6) was treated in the same way as group 1, but 0.03 mg/kg of enalaprilat (Hexal AG, 83607 Holzkirchen, Germany), was given intravenously at 6 h. The dose of enalaprilat was obtained in a pilot study prior to the experiment. The third group (ANP + saline, n=6) was treated after the induction of ANP in the same manner as group 1. In the

fourth group (ANP + enalaprilat, n = 6), ANP was induced and enalaprilat was given as in group 2. Saline or enalaprilat in pancreatitis groups was given six hours after the induction of pancreatitis, since ANP in small animals is 4Y6 times faster than in humans and most patients with acute pancreatitis are admitted 24Y36 h after the onset of pancreatitis. Hence, the period of 6 h is closer to the clinical situation [15]. In the second part of the study, 12 rats were studied in similar groups. Survival of all rats was monitored for 24 h.

Table 1. Histopathologic Scoring Criteria Score Edema 0 0.5 1 1.5 2 2.5 3 3.5 4 Acinar necrosis 0 0.5 1 1.5 2 2.5 3 3.5 4> Inflammation and perivascular infiltrate 0, 0-1 0.5, 2Y5 1, 6Y10 1.5, 11Y15 2, 16Y20 2.5, 21Y25 3, 26Y30 3.5, >30 4, >35

Description Absent Focal expansion of interlobal septae Diffuse expansion of interlobar septae Same as 1 + focal expansion of interlobal septae Same as 1 + diffuse expansion of interlobar septae Same as 2 + focal expansion of interacinar septae Same as 2 + diffuse expansion of interacinar septae Same as 3 + focal expansion + intercellular spaces Same as 3 + diffuse expansion + intercellular spaces Absent Focal occurrence of 1Y4 necrotic cells/high power field Diffuse occurrence of 1Y4 necrotic cells/high power field Same as 1 + focal occurrence of 5Y10 necrotic cells/high power field Diffuse occurrence of 11Y16 necrotic cells/high power field Same as 2 + focal occurrence of 11Y16 necrotic cells/high power- field Diffuse occurrence of 11Y16 necrotic cells/high power field Same as 3 + focal occurrence of >16 cells/high power- field Necrotic cells/high power field (Extensive confluent necrosis) Intralobular or perivascular leukocytes /high power field Intralobular or perivascular leukocytes /high power field Intralobular or perivascular leukocytes /high power field Intralobular or perivascular leukocytes /high power field Intralobular or perivascular leukocytes/high power field Intralobular or perivascular leukocytes /high power field Intralobular or perivascular leukocytes /high power field Leukocytes/high power field or focal microabscesses Leukocytes/high power field or confluent

208

Turkyilmaz, Alhan, Ercin, and Vanizor Table 2. Systemic Hemodynamic Variables, Blood Gas Analysis at 24 h and Mortality at 24 h (Data are Given as Mean T SEM)

MAP (mmHg) HR (beats/min) PH PO2 (mmHg) PCO2 (mmHg) Urine (ml/h) Death

Sham + saline (n = 6)

Sham + enalaprilat (n = 6)

ANP + saline (n = 6)

ANP + enalaprilat (n = 6)

134 T 8 320 T 23 7.36 T 0.04 110 T 6 34 T 3 0.7 T 0.1 0/12

125 T 7 327 T 23 7.35 T 0.05 104 T 5 35 T 4 0.8 T 0.1 0/12

83 T 4* 390 T 18* 7.3 T 0.04 81 T 5* 37 T 5 0.2 T 0.1** 5/12*

104 T 6 385 T 22* 7.31 T 0.04 95 T 4 36 T 5 0.5 T 0.1 1/12

*P < 0.05 **P < 0.01 versus sham groups

Blood pressure and HR were measured with a pressure monitor (Petas KLM 200, Istanbul, Turkey) by connecting the arterial line to a pressure transducer. The blood gases were analyzed using a Ciba Corning 865 analyzer (Chiro Diagnostica Co, East Walpole, USA). The serum activities of amylase and ALT and the concentrations of glucose, creatinine, urea, calcium, LDH in BAL and the electrolytes were measured by an auto analyzer (Vitros 750 auto analyzer, Johnson & Johnson, Rochester, USA). Serum IL-6 concentration was measured with commercial ELISA kit (IL-6, Biosource Cat no. BMS 625) and an ELISA reader (Sanofi Diagnostic Pasteur LP 35, Marnes-LA-Coquette, France). The tissue-associated MPO activity was assessed by a modification of the method described by Schierwagen et al. [16]. Protein concentrations of supernatants were measured by the method of Lowry et al. [17]. MPO activity was expressed as U/mg protein.

The lipid peroxidation in tissues was assessed by measuring the concentration of MDA using a colorimetric reaction with thiobarbituric acid by modification of the method described by Buege and Aust [18]. The protein concentrations of supernatants were measured by Lowry_s method [6]. MDA concentrations were expressed as nmol/mg protein. Finally, half of the pancreas was fixed in 10% buffered formalin and after sectioning, it was stained with hematoxylin and eosin. Two pathologists with expertise in pancreatic pathology made the histopathological evaluation. They were unaware of the induction technique and the additional drugs given. Edema, acinar necrosis, and inflammation were assessed using a scoring system from 0 (no pathologic changes) to 3 (maximum inflammatory infiltration, total necrosis of the pancreas), as previously described [13]. The detailed description of histological scoring criteria was given in Table 1.

Table 3. Serum IL-6, Tissue Concentrations of Myeloperoxidase (MPO) and Malondialdehyde (MDA), LDH Levels in Bronchoalveolar Lavage (BAL), Serum Levels of Amylase and the Other Biochemical Parameters at 24 h in Animals without and with ANP Treated with Saline or Enalaprilat (Data are Given Mean T SEM)

IL-6 (pg/ml) Amylase (U/l) Glucose (mg %) Urea (mg %) Creatinine (mg %) ALT (U/dl) Calcium (mg %) BAL LDH (U/dl) Lung MPO (U/mg protein) Lung MDA (nmol/mg protein) Pancreatic MPO (U/mg protein) Pancreatic MDA (nmol/mg protein)

Sham + saline (n = 6)

Sham + enalaprilat (n = 6)

ANP + saline (n = 6)

ANP + enalaprilat (n = 6)

48 T 6 1,177 T 16 66 T 5 14 T 2 0.47 T 0.1 62 T 2 8.8 T 0.2 336 T 28 6.2 T 1.2 0.24 T 0.02 0.34 T 0.08 0.36 T 0.03

52 T 6 1,327 T 33 73 T 5 16 T 3 0.43 T 0.1 59 T 3 9.2 T 0.2 313 T 22 4.9 T 0.7 0.27 T 0.03 0.36 T 0.07 0.34 T 0.03

98 T 7* 10,469 T 371** 75 T 5 49 T 12*,*** 0.52 T 0.1* 200 T 14* 8.2 T 0.2* 701 T 92* 11.3 T 2.3*,*** 0.44 T 0.03*,*** 0.92 T 0.13*,*** 0.66 T 0.15*,***

85 T 4* 8,385 T 312** 88 T 10 20 T 3 0.45 T 0.1 146 T 13* 8.5 T 0.2 774 T 69* 6.86 T 1.2 0.22 T 0.03 0.39 T 0.07 038 T 0.03

*P < 0.05 **P < 0.01 versus sham groups ***P < 0.05 compared with ANP + enalaprilat group

209

Effects of Enalaprilat on Pancreatitis in Rats Table 4. Histological Assessment of Edema, Necrosis and Inflammation (Values are Given Mean T SEM)

Edema Necrosis Inflammation

Sham + saline (n = 6)

Sham + enalaprilat (n = 6)

ANP + saline (n = 6)

ANP + enalaprilat (n = 6)

0.6 T 0.1 0.2 T 0.2 0.2 T 0.2

0.6 T 0.2 0.2 T 0.2 0.2 T 0.2

1.1 T 0.1* 1.5 T 0.2** 1.2 T 0.3**

0.3 T 0.1 1 T 0.2*,*** 0.8 T 0.2*

*P < 0.05 **P < 0.01 compared with sham groups ***P < 0.05 compared with ANP + saline group

Results are presented as the mean T (SEM). The significance of the differences in survival rates were assessed by Fisher_s exact test, and histopathological results and enzyme activities by the KruskalYWallis and MannYWhitney U tests, and the differences were considered significant at P <0.05.

RESULTS The mortality rate was found as 0% in the sham + saline group, 0% in the sham + enalaprilat group, 41%

in the ANP + saline group, and 8% in the ANP + enalaprilat group. There was a significant difference between the ANP + saline and the other groups (P< 0.05, Table 2). The induction of ANP leads to a significant decrease in MAP, an increase in HR and a reduction in urine volume. The use of enalaprilat reversed these alterations except HR (Table 2). The induction of pancreatitis resulted in a significant increase in serum amylase, ALT and IL-6 activity in pancreatitis groups (Table 3). Serum urea and creatinine increased only in the ANP + saline group

Fig. 2. a Dual composition of normal pancreas in sham + saline group (H&E 100); AG lobular units of acini of the exocrine component, L Langerhans_ islets of the endocrine component. b A similar pattern in sham + enalaprilat group (H&E 100); AG lobular units of acini, L Langerhans islets. c Severe acinar necrosis, leukocyte infiltration and edema in interlobar space in ANP+ saline group (H&E 100); SN severe necrosis, SLO severe leukocyte infiltration and edema. d Moderate acinar necrosis, leukocyte infiltration and edema in interlobar space in ANP+ enalaprilat group (H&E 100); MN moderate acinar necrosis; MLO moderate leukocyte infiltration and edema.

210 (P < 0.05, Table 3). A significant decrease in the serum calcium value was observed in the ANP + saline group (Table 3). Serum glucose value showed no changes in all groups (Table 3). A significant increase in LDH in BAL in pancreatitis groups (P <0.05, Table 3) and a decrease in p02 value only in the ANP + saline group (P< 0.05, Table 2) were observed. Considerable increases in MPO and MDA activities in the lung and pancreatic tissues occurred after the induction of acute pancreatitis (Table 3). The use of enalaprilat in the ANP + enalaprilat group decreased these changes to the levels of the sham groups. According to the histological examination, the ANP groups had greater edema, necrosis, and leukocyte infiltration than the sham groups (Table 4). The use of enalaprilat decreased edema and necrosis in the ANP + enalaprilat group compared to the ANP + saline group (Table 4, Fig. 2).

DISCUSSION In this study, the ANP model described by Schmidt et al. [13] was used. This model provides a superior opportunity to study an innovative treatment by standard processes. ANP in small animals is 4Y6 times faster than in humans; most patients with acute pancreatitis are admitted 24Y36 h after the onset of pancreatitis [15]. Therefore, we administered enalaprilat 6 h after the induction of pancreatitis. We chose the severe form of acute pancreatitis as an induction method because mild or moderate forms of acute pancreatitis can be treated with minimal morbidity and mortality [2]. The first step in the treatment of acute pancreatitis is appropriate fluid replacement, so we used the aggressive fluid replacement to maximize the organ function (6 ml kgj1 hj1) after the induction of ANP. Pancreatic necrosis is an indicator of severe pancreatitis [19]. Pancreatic necrosis occurs by the autodigestion due to activated digestive enzymes and increased free oxygen radicals with lipid peroxidation during acute pancreatitis [2, 4, 20]. It has been shown that the local RAS plays a very important role in the physiopathology of acute pancreatitis. Pancreatic ACE activity increases by sixfold in pancreatitis induced by cerulein [12, 21]. Enalaprilat applied after the induction of ANP decreased the levels of MPO and MDA in the pancreatic tissue, the mortality rate, pancreatic necrosis and edema. This was consistent with the information presented by Tsang et al. [22], who reported that

Turkyilmaz, Alhan, Ercin, and Vanizor pancreatic RAS activation played an important role in the severity of the pancreatitis and pancreatic injury. Acute respiratory distress syndrome (ARDS) may occur often in the early term of severe pancreatitis and may be related with sudden death [23]. Activation of leukocytes and increase in capillary permeability play an important role in the pathogenesis of ARDS. Activated leukocytes damage the pulmonary basal membrane by production of free oxygen radicals and increase capillary permeability. In order to evaluate capillary permeability, we measured the levels of LDH in BAL fluid. ANP induction increased the level of LDH in BAL fluid by increasing MPO and MDA activities in the pulmonary tissue while decreasing pO2. After the application of enalaprilat, pO2 levels increased by decreasing MPO and MDA activities in the pulmonary tissue. RAS was reported to induce the synthesis and secretion of vascular permeability factors [24, 25]. We thus expected that enalaprilat application would decrease the level of LDH in BAL fluid, but in fact it had the opposite effect and the level increased, though not statistically significant. This condition led us to think that the positive effect on the lung is related to blockade of free oxygen radical production by the local RAS, with the help of enalaprilat. There are articles in the literature reporting that ACE inhibitors or angiotensin II receptor antagonists may be useful in the treatment of diffuse parenchymal pulmonary diseases and that they may be assessed as a new potential treatment of fibrotic pulmonary diseases [26]. Biochemical abnormalities occur during the course of acute pancreatitis, and altered metabolic pathways inside the affected cells and reflected changes in some organs may be seen. Severe parenchymal injury in the liver, lung, kidneys and heart has been reported in bileinduced pancreatitis [2, 6]. Patients and animals with pancreatitis die of multi-organ failure and systematic inflammatory response to pancreatic injury by vasoactive substances, cytokines and activation of some enzymes [27]. In our study, hypotension, increased serum urea concentration and ALT activity, and decreased serum calcium concentration and urine volume were observed. Enalaprilat application was determined to preserve the renal function of the subjects by decreasing serum urea concentration and increasing urine volume. There are other studies presenting the positive effects of RAS blockade on the kidneys. Mezzano et al. [28] reported that angiotensin II took place at the renal inflammatory process and RAS blockade may have useful effects. Brewster and

Effects of Enalaprilat on Pancreatitis in Rats Perazella [29] deduced that RAS inhibitors should be used in order to prevent renal injury, decrease proteinuria and reach the blood pressure goals. In addition, it was indicated that ACE inhibition, independent from its systemic reninYangiotensin effect, prevented interstitial fibrosis caused by cyclosporin A in the kidneys but did not affect the cytotoxicity in the proximal tubules [30]. Though it was reported that RAS played an important role in hepatic fibrosis induced by carbon tetrachloride or following bile duct obligation and that ACE inhibitors decreased hepatic fibrosis progression, in our study no improvement was observed in the level of liver enzymes [31, 32]. On the other hand, there are no studies available in the literature with which we can compare our results. It was stated that addition of exogenous angiotensin II induced digestion enzyme secretion (amylase and lipase) by isolated pancreatic acinar cells and that digestion enzyme secretion was inhibited considerably by losartan application, but specific AT2 receptor blocker PD123319 had no suppressive effect [33]. However, we observed that enalaprilat had no important effect on the level of amylase. A possible explanation may be the severe pancreatitis induced by our induction method. Some markers, such as trypsinogen activating peptide, C-reactive protein, tumor necrosis factor (TNF)-a, IL-6, and IL-10, may be used as indicators for the severity and consequences of the disease [34]. Of these markers, only serum IL-6 was measured in this study, and enalaprilat was observed not to alter the level of IL-6. This result is not correlated with the pancreatic necrosis and mortality rate. This may be related not with the systemic but rather with the local autocrine and paracrine effects of enalaprilat [9, 10]. We determined that multi-organ failure induced by free oxygen radicals produced by active leukocytes had an important role in the progression of acute pancreatitis and that enalaprilat, as an ACE inhibitor, preserves renal and pulmonary functions during the course of ANP and decreases the mortality rate due to pancreatic injury. This result indicates that the local pancreatic RAS plays an important role in the pathogenesis of ANP. Enalaprilat application may be useful in the treatment of ANP.

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