Inflammation ( # 2013) DOI: 10.1007/s10753-013-9702-3

Effects of Clotrimazol on the Acute Necrotizing Pancreatitis in Rats Arif Burak Cekic,1 Etem Alhan,2,6 Arif Usta,3 Serdar Türkyılmaz,2 Birgül Vanizor Kural,4 and Cengiz Erçin5

Abstract—This study aims to investigate the influence of clotrimazol (CLTZ) on acute necrotizing pancreatitis (ANP) induced by glycodeoxycholic acid in rats. Rats were divided into five groups as sham+saline, sham+CLTZ, sham+polyethylene glycol, ANP+saline, and ANP+CLTZ. ANP in rats was induced by glycodeoxycholic acid. The extent of acinar cell injury, mortality, systemic cardiorespiratory variables, functional capillary density (FCD), renal/hepatic functions, and changes in some enzyme markers for pancreatic and lung tissue were investigated during ANP in rats. The use of CLTZ after the induction of ANP resulted in a significant decrease in the mortality rate, pancreatic necrosis, and serum activity of amylase, alanine aminotransferase, interleukin-6, lactate dehydrogenase in bronchoalveolar lavage fluid, serum concentration of urea, and tissue activity of myeloperoxidase, and malondialdehyde in the pancreas and lung and a significant increase in concentrations of calcium, blood pressure, urine output, pO2, and FCD. This study showed that CLTZ demonstrated beneficial effect on the course of ANP in rats. Therefore, it may be used in the treatment of acute pancreatitis. KEY WORDS: acute pancreatitis; clotrimazol.

INTRODUCTION In most cases, acute pancreatitis is a mild and selflimiting disease, but severe necrotizing forms associated with a significant mortality rate are infrequent. However, in recent years, the mortality of acute necrotizing pancreatitis (ANP) has been reported to vary from 6.2 to 20.8 % despite improved fluid management, respiratory care, and nutritional support [1, 2]. Autodigestion of the pancreas and impairment in pancreatic microcirculation are two important parts in the pathophysiology of acute pancreatitis [2–4]. Excessive

1

Department of Surgery, State Hospital, Trabzon, Turkey Department of Surgery, Karadeniz Technical University, Trabzon, Turkey 3 Department of Surgery, State Hospital, Karabük, Turkey 4 Department of Biochemistry, Karadeniz Technical University, Trabzon, Turkey 5 Department of Pathology, Kocaeli University, Kocaeli, Turkey 6 To whom correspondence should be addressed at Department of Surgery, Karadeniz Technical University, Trabzon, Turkey. E-mail: [email protected] 2

upregulation of cytokines and secondary mediators such as histamines prostaglandins, thromboxanes, leukotrienes, nitric oxide, and platelet-activating factor play an important role during the course of acute pancreatitis [1, 2, 5, 6]. In the early stage of the disease, hypovolemia, resulting from fluid sequestration into the abdominal cavity, and in the late stage, a sepsis caused by bacteria translocated from the gut resulting in systemic inflammation may cause clinical multiorgan failure [1, 2, 7]. Reactive oxygen species (ROS) play an important mediator function in the early and late course of acute pancreatitis [8]. In the initial stages of this disease, the exact site and source of ROS is not clear. In later stages, the disease attracts and activates leukocytes in the pancreas producing enhanced ROS [9]. ROS have a direct influence on the lipids and proteins in the cell membrane and disrupt their functions. Indirectly, they act on the arachidonic metabolism in two ways [10, 11]. First, they increase the secretion of thromboxane, which decreases tissue circulation because of its platelet aggregating and vasoconstricting effects. Second, it increases the production of leukotriene B4, which enhances the

0360-3997/13/0000-0001/0 # 2013 Springer Science+Business Media New York

Cekic, Alhan, Usta, Türkyılmaz, Kural, and Erçin activation of leukocytes and the release of lysosomal enzymes [10]. These effects lead to a further cell damage of the pancreas. In experimental and clinical studies performed on the role of ROS during acute pancreatitis [8, 12–14], ROS scavengers were observed to decrease the characteristic changes of the disease significantly but did not completely inhibit them [8]. Clotrimazol (CLTZ) is an antifungal drug which has long been used in the treatment of fungal infections in clinical praxis. CLTZ triggers the inhibition of sterol 14 ademethylase, a microsomal cytochrome P45o-dependent enzyme of the endoplasmic reticulum [15–17]. Additionally, CLTZ has been shown to be potent inhibitors of many mammalian cytochrome P450 oxidases and has several effects on cellular metabolism and signaling especially Ca2-dependent processes [15–18]. These studies have also reported that CLTZ prevent excessive calcium influx into the cells during hypoxia/ischemia [15–18]. Calcium overload activates the calcium-dependent proteases, nitric oxide synthase, the generation of free oxygen species, and results in the destruction of the mitochondria [18]. CLTZ, as a potent inhibitor of the plasma membrane calcium channels and free radical scavenger, has been shown in several cells [18]. This study examined the effect of CLTZ on the extent of acinar cell injury, mortality, systemic cardiorespiratory variables, functional capillary density (FCD), renal/hepatic functions, and changes in some enzyme markers for pancreatic and lung tissue during ANP in rats.

MATERIALS AND METHODS Seventy-eight male Sprague–Dawley rats weighing 300–350 g were used. They were housed in rooms maintained at 21±1 °C and a 12:12-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 (number 35, 18.2.2010). Anesthesia was induced with vaporized ether and maintained by an intraperitoneal injection of ketamine at 50 mg/kg (Ketalar, Eczacıbası, Istanbul, Turkey). The right internal jugular vein and carotid artery were cannulated (ID 0.5 mm, Luer Lock, Braun AG, Melsungen, Germany). The catheters were tunneled subcutaneously to the suprascapular area. During the experiment, the animals were housed in metabolic cages, which enabled quantitative assessment of urine production.

Acute pancreatitis was induced by an intravenous infusion of cerulein (Sigma & Aldrich Chemie GmbH, Steinheim, Germany) at a dose of 5 μg kg−1 h−1 over 6 h superimposed on a standard infusion of 1.2 mL/kg glycodeoxycholic acid (10 mmol/L; Sigma, St. Louis, MO, USA) into the biliary-pancreatic duct for 10 min at 30 mmHg as described by Schmidt et al. [19]. A special infusion pump for pressure and volume control (IVAC P 7000, Alaris Medical Systems, Hampshire, UK) was used. Cerulein was reconstituted in physiological saline and infused at 8 mL kg−1 h−1 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 randomized into five experimental groups (Fig. 1). Those in the first group (sham+saline, n= 10) had arterial and venous lines placed and were given intraductal saline followed by a 6-h intravenous infusion of saline at 8 mL kg−1 h−1. 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. At the 24 h from the beginning of the experiment, the cardiorespiratory function was assessed by monitoring the arterial blood gases, mean arterial pressure (MAP), renal function by the collection of urine using metabolic cages, and survival. As microcirculation of the pancreas cannot be measured under unstable conditions, the rats with MAP of <80 mmHg, p02 of <80 mmHg, pC02 of>50 mmHg, and pH of <7.3 and the dead rats were excluded from the study. At 24 h, the rats were again anesthetized by ketamine and laparotomy was performed. The pancreas and spleen were exposed on an adjustable stage. The orthogonal polarization imaging video microscope (Cytoscan A/R, Cytometrics, Philadelphia, PA, USA) was attached to the moveable shaft and the microcirculation was recorded in six different capillary regions of the exocrine pancreas at least 20 s [20]. The images were stored in AVI format on a computer (Sony VGN-FW 230J/H). 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 (IL)-6. At 24 h after the blood was withdrawn, a midline sternotomy was performed and the left main bronchus was clamped. Bronchoalveolar lavage (BAL) of the right lung was performed with 2 mL phosphate-buffered saline containing 0.07 M ethylenediamine tetraacetic 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 −20 °C, and assessed

Effects of Clotrimazol on the ANP in Rats subsequently for lactate dehydrogenase (LDH) measurement [21]. The left lung was harvested for the measurements of the activity of the myeloperoxidase (MPO) and malondialdehyde (MDA) levels. The excised lung tissues were rinsed in saline, blotted dry, frozen in liquid nitrogen, and stored frozen at −80 °C until thawing for measurement of MPO and MDA activities. At the end of the experiment, the entire pancreas was removed. The pancreas was divided into two parts, one for the histological examination and the other for the measurements of MPO and MDA activity. For the second group (sham+CLTZ, n=10), 1 mL CLTZ at 30 mg/kg body weight dissolved in 3 % polyethylene glycol (C6019, Sigma-Aldrich, Italy) was given intraperitonaly at 6 h [15]. The other procedures were the same as described for the first group. The third group (ANP+saline, n=16) was treated according to the protocol of group 1 after the induction of ANP. In the fourth group (ANP+CLTZ, n=16 group), ANP was induced and CLTZ was given, as in group 2. Saline or CLTZ in pancreatitis groups were given 6 h after the induction of pancreatitis. As ANP in small animals occurs four to six times faster in humans and most patients with acute pancreatitis are admitted 24–36 h after the onset of pancreatitis; this period is closer to the clinical situation [22]. Blood pressure was 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, MA, 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 autoanalyzer (Vitros 750 auto analyzer, Johnson & Johnson, Rochester, NY, USA). Orthogonal polarization imaging has been suggested for recording and quantifying changes in the microcirculation [20]. The technique uses optical filtration of polarized light that is absorbed by hemoglobin so that red blood cells appear dark. The recorded images were analyzed by software using MAS image analysis system. MAS was developed at the Academic Medical Center, University of Amsterdam, the Netherlands by Dr. Iwan Dobbe, Professor Can Ince and Dr. K.R Mathura. FCD, identified as the best parameter for the measurement of the microcirculation, was defined by Messmer [20]. FCD was defined as the length of red blood cell-perfused capillaries (in centimeters) per observation area (in square centimeters). We thus selected FCD as the parameter for measurement of the microcirculation.

Serum IL-6 concentrations were measured with commercial ELISA kit (IL-6, Bio source 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. [23]. MPO activity was expressed as units per milligram of 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 [24]. MDA concentrations were expressed as nanomoles per milligram of protein. Protein concentrations of supernatants (for MDA and MPO levels) were measured by Lowry's method [25]. Finally, half of the pancreas was fixed in 10 % buffered formalin, and after sectioning, it was stained with hematoxylin and eosin. Two pathologists expert in pancreatic pathology conducted the histopathological evaluation. They were blinded to both 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 [19]. Results are presented as the mean± SEM. The significance of the differences in survival rates was assessed by Fisher's exact test, and histopathological results and enzyme activities by the Kruskal–Wallis and Mann–Whitney U test, 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+CLTZ group, 37.5 % in the ANP+saline group, and 18.75 % in the ANP+CLTZ group. There was a significant difference between the pancreatitis and sham groups (P<0.05; Table 1). The use of CLTZ in the ANP + CLTZ group decreased the mortality (P<0.05). The induction of ANP led to a significant decrease in MAP, and a reduction of urine volume, p02 and FCD. The use of CLTZ restored these alterations (Table 1). The induction of pancreatitis resulted in a significant increase in serum activity of amylase, ALT, IL-6, and serum urea (Table 2) and a decrease in serum calcium concentration in the pancreatitis groups (P<0.05; Table 2). The use of CLTZ corrected these values. Serum glucose

Cekic, Alhan, Usta, Türkyılmaz, Kural, and Erçin

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

CLTZ

Rats ANP groups

saline

0

CLTZ

6

24

time/hours

receipt of saline or CLTZ

Fig. 1. Experimental design and time schedule of the study. CLTZ clortrimazol, ANP acute necrotizing pancreatitis.

and creatinine values showed no changes in any group (Table 2). A significant increase in LDH in BAL and in MPO and MDA activities in the lung and pancreatic tissues occurred in the ANP+saline group (P<0.05; Table 2). CLTZ decreased this increase in ANP+CLTZ group (Table 2). According to the histological examination, the ANP groups had greater edema, necrosis, and leukocyte infiltration than the sham groups (Table 3). CLTZ in the ANP+CLTZ group corrected pancreatic necrosis compared with the ANP+saline group (Table 3; Fig. 2).

DISCUSSION Our results indicate that the induction of ANP resulted in a significant increase in the mortality rate; pancreatic necrosis; serum activity of amylase, ALT, and IL-6; serum concentration of urea, LDH in BAL fluid; tissue activity of MPO; and MDA in the pancreas and lung and a significant decrease in serum concentrations of calcium, blood pressure, urine output, pO2, and FCD. The

administration of CLTZ restored these changes significantly. In this study, the ANP model described by Schmidt et al. [19] was used. This model provides a superior opportunity to study an innovative treatment by standard processes. We chose severe forms of acute pancreatitis as an induction method for pancreatitis because mild or moderate forms of acute pancreatitis can be treated with minimal morbidity and mortality [2]. ANP in small animals is four to six times faster than in humans, and most patients with acute pancreatitis are admitted 24–36 h after its onset [22]. Therefore, we administered CLTZ 6 h after the induction of pancreatitis. We gave 30 mg/kg body weight CLTZ intraperitonaly at 6 h for single dose [15]. The first step in the treatment of acute pancreatitis is appropriate fluid replacement, so we used appropriate fluid replacement to restore the organ function (6 mL kg−1 h−1) after the induction of ANP. Since we used severe form of pancreatitis model and this aggressive model does not allow long-term survival, we completed our experiment at the 24-h post-pancreatitis endpoint. CLTZ, as potent antimycotic drug acting via the inhibition of sterol-14 demethylase, is a cytochrome P450-dependent enzyme [16]. CLTZ has also antioxidant,

Table 1. Systemic Hemodynamic Variables, Blood Gas Analysis, Mortality and Functional Capillary Density (FCD) at 24 h (Data Are Given as Mean±SEM)

MAP (mmHg) HR (beat/min) pH pO2 (mmHg) pCO2 (mmHg) Urine (mL/h) Death (%) FCD (cm/cm2)

Sham+saline (n=10)

Sham+CLTZ (n=10)

ANP+saline (n=10)

ANP+CLTZ (n=13)

131±4 137±22 7.36±0.02 97.9±1.4 37±3 1.01±0.12 0 32±3.2

99±4 153±26 7.32±0.06 95.6±7.8 38±4 0.75±0.12 0 31.9±2.6

84±4* 104±18 7.32±0.03 80±4* 39±3 0.24±0.09* 37.5* 23±2.6*

96±8 102±20 7.36±0.03 93±2 36±4 0.44±0.09 18.7*, α 34±2.8α

MAP mean arterial pressure *P<0.05, versus sham groups; αP<0.05 versus ANP+saline group

Effects of Clotrimazol on the ANP in Rats Table 2. Serum IL-6, Tissue Concentrations of MPO and MDA, LDH levels in BAL, Serum Levels of Amylase, and the Other Biochemical Parameters at 24 h in Animals with or without ANP Treated with Saline or Clortrimazol (Data Are Given as Mean±SEM) Sham+saline (n=10)

Sham+CLTZ (n=10)

ANP+saline (n=9)

ANP+CLTZ (n=13)

122±25 869±101 137±22 17±3.3 0.38±0.06 59±21 9.6±0.4 334±38 5.36±0.3 0.24±0.02 0.34±0.08 0.36±0.02

208±32 1,852±89 153±26 12.6±3 0.4±0.04 61±23 9.1±0.4 343±40 4.3±0.2 0.27±0.03 0.36±0.07 0.34±0.03

723±112* 4,518±608* 104±18 45±8* 0.5±0. 288±28* 7.2±0.7* 1,002±188* 11.3±2.3* 0.44±0.03* 1.13±0.07* 0.66±0.15*

346±88 3,092±630* 102±20 27±5 0.53±.22 132±35 8.3±0.7 472±88 6.86±1.2*, α 0.22±0.03 0.7±0.08*α 0.38±0.03

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

*P<0.05 versus other groups; **P<0.01 versus other groups; αP<0.05 versus ANP+saline group MPO myeloperoxidase, MDA malondialdehyde, LDH lactate dehydrogenase, BAL bronchoalveolar lavage, ANP acute necrotizing pancreatitis, IL interleukin, ALT alanine aminotransferase

immune-stimulating, antineoplastic, anti-inflammatory, and apoptotic effects. In addition, CLTZ has inhibitory effects on NF-KB by inhibition of intermediate-conductance of Ca2+-activated K+ channel 3.1 that was first described by Gardos, and partially inhibition of the Nmethyl-D-aspartate receptors, which in turn cause both reduced Ca2+ overload and a reduced probability of mitochondrial potential collapse [15,26–35]. Pancreatic necrosis is the hallmark of severe pancreatitis and shows strong relation with the mortality [36]. Autodigestion of the pancreas by activated digestive enzymes, ischemia of the pancreas, apoptosis, and ischemia-related ROS-associated lipid peroxidation cause pancreatic damage during acute pancreatitis [2–4, 36, 37]. Microcirculation disturbance is an important pathogenetic factor during acute pancreatitis [3–5]. The microcirculation of the pancreas can be measured by diffuse reflectance spectroscopy, intravital microscopy, multiple indicator dilution technique, and orthogonal polarization imaging [3, 4, 20, 38]. We measured pancreatic microcirculation by orthogonal polarization imaging in this study. In our study, the induction of pancreatitis resulted in a decrease in FCD and increase in ischemic mediators such

as pancreatic tissue MPO and MDA. Many authors have reported similar results using intravital microscopy, diffuse reflectance spectroscopy, or orthogonal polarization imaging during acute pancreatitis [3, 4, 20, 38]. In this study, the use of CLTZ restored the decreased FCD value, and increased pancreatic tissue ischemia mediator MPO and MDA activities which reflect oxidative stress [6, 8]. The free radical scavenger effects of CLTZ has been demonstrated in an experimental spinal cord ischemia–reperfusion injury in the rat by Usul et al., in a normothermic ischemia–reperfusion injury animal model in the liver by Iannelli et al., and on ovarian ischemia–reperfusion injury in a rat ovarian-torsion model by Osmanagaoglu et al. [15, 29, 34]. These studies support our study that CLTZ has a protective effect as an antioxidant during ANP. Recently, it has been realized that the release of intracellular product such as bioactive peptides, heparin sulphate, high mobility group bax-1, heat shock proteins, hyaluronic acid, and RNA from damaged and injured cells can have paracrine- and endocrine-like effects on distant tissue to activate the inflammatory response [39]. These molecules that are released from cells are known as

Table 3. Histological Assessment of Edema, Necrosis, and Inflammation (Values Are Given as Mean±SEM)

Edema Necrosis Inflammation

Sham+saline (n=10)

Sham+CLTZ2 (n=10)

ANP+saline (n=10)

ANP+CLTZ (n=13)

0.4±0.3 0.25±0.2 0.2±0.2

0.432±0.35 0.1±0.2 0.12±0.2

1.15±0.3* 2.3±0.2** 2.1±0.3**

0.76±0.2*, α 0.7±0.2*, α 1.16±0.3*, α

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

Cekic, Alhan, Usta, Türkyılmaz, Kural, and Erçin

Fig. 2. a Dual composition of normal pancreas in sham+saline group (hematoxylin–eosin (H&E), ×100 magnification); PA lobular units of acini of the exocrine pancreas, L Langerhans islets of the endocrine component. b A similar pattern in the sham+CLTZ group (H&E, ×100). c Severe acinar necrosis and leukocyte infiltration in the ANP+saline group (H&E, ×40); SI severe leukocyte infiltration, SN severe necrosis. d Minimal necrosis and leukocyte infiltration in the ANP+CLTZ group (H&E, ×40).

damage-associated molecular patterns (DAMPs) [39]. DAMPs are recognized by cell surface receptors to effect intracellular signaling that primes and amplifies the immune response. These receptors are known as pattern recognition receptors (PRRs) and include the Toll-like receptors (TLRs) [40]. The bacterial products including lipopolysaccharide and DAMPs through TLRs activated intracellular signal pathways such as NF-KB, p38 mitogen kinase and JNK in leukocytes [40]. NF-KB is a family of transcription factors. NF-KB activation has been demonstrated in experimental pancreatitis in pancreatic tissue, lung, liver, peripheral blood mononuclear cells, peritoneal and alveolar macrophages, and endothelial cells [41]. All these pathways included NF-KB result in cytokine and mediator production in the leukocyte and leukocyte endothelia interaction. Activated leukocyte is known to mediate micro vascular and parenchymal injury by the release of various mediators such as ROS, proteases, leukotrienes, eicosanoids, platelet activating factor, and

nitric oxide resulting in vasomotor changes, endothelial injury, and loss of the microvascular integrity, which finally lead to cell death and organ failure [42]. Weidenhach et al. reported that animals with pancreatitis die due to multi-organ failures through the development of systemic inflammatory response to pancreatic injury by the activation of various enzymes, cytokines, and vasoactive substance [43]. Acute lung injury and acute respiratory distress syndrome (ARDS) occurs often in the early stage of severe pancreatitis and may be related to early death [41, 44]. The production of ROS by the activated leukocytes resulting increased capillary permeability play a major role in the pathogenesis of ARDS [21, 44]. We used LDH level in BAL fluid for the assessment of capillary permeability [21, 44]. The induction of ANP resulted in increased LDH in BAL fluid, decreased pO2, and increased activities of MPO and MDA in lung tissue. Administration of CLT normalizes the lung injury after

Effects of Clotrimazol on the ANP in Rats the induction of acute pancreatitis in this study. Ivey et al. have showed the similar results on canine lung permeability after heart failure [45]. After the induction of ANP in our study, increased concentration of serum urea and ALT activity and decreased concentration of serum calcium and urine volume occurred. The use of CLTZ improves these changes. Iannelli et al. reported that CLTZ protects the liver against normothermic ischemia–reperfusion injury. Conversely, NF-KB has an important role during the progression from the localized disease to multiorgan failure in acute pancreatitis and CLTZ inhibited the activation of NF-KB [31, 34, 46]. This effect may explain the protective effects of CLTZ on the other organs. Some markers, such as trypsinogen activated peptide, C-reactive protein, TNF-α, IL-6, and IL-10 can be used as an index for the severity and outcome of the disease in experimental studies [47]. We measured serum IL-6 as a marker. The use of CLTZ improves serum IL-6 level in our study. This finding correlated with the pancreatic damage and mortality rate. CLTZ inhibited cytokine release by inhibition of NF-KB after ischemia–reperfusion injury [31, 46]. This result supports our findings during acute pancreatitis. In addition, CLTZ has a stimulant effect on the immune cell function. This effect conducts low mortality in the ANP+CLTZ group in our study [35].

CONCLUSIONS We concluded that ischemia of the pancreas is a major cause of pancreatic necrosis. Multiorgan failure induced through ischemia-related ROS, which was produced by activated leukocytes, has an important role in the progression of acute pancreatitis, and the administration of CLTZ did improve cardiovascular, hepatic, lung, renal functions, serum IL-6, and pancreatic microcirculation. CLTZ reduces pancreatic mortality and pancreatic damage during acute pancreatitis. As CLTZ has positive effects on the course of ANP in rats, it may be used in the treatment of acute severe experimental pancreatitis in rats.

REFERENCES 1. Schwarz, M., J. Thomson, H. Meyer, M.W. Büchler, and H.G. Beger. 2000. Frequency and time course of pancreatic and extra

pancreatic bacterial infection in experimental pancreatitis in rats. Surgery 127: 427–432. 2. Saluja, A.K., and M.L. Steer. 1999. Pathophysiology of pancreatitis. Role of cytokines and inflammatory mediators. Digestion 60: 27–33. 3. Klar, E., K. Messmer, A.L. Warshaw, and C. Herfarth. 1990. Pancreatic ischemia in experimental acute pancreatitis: mechanism, significance and therapy. British Journal of Surgery 77: 1205–1210. 4. Alhan, E., U. Kücüktülü, C. Ercin, O. Deger, and R. Cicek. 2001. The effects of dopexamine on acute necrotizing pancreatitis in rats. The European Journal of Surgery 167: 761–766. 5. Norman, J. 1998. The role of cytokines in the pathogenesis of acute pancreatitis. American Journal of Surgery 1175: 76–83. 6. Alhan, E., N.I. Kalyoncu, B. Vanizor, and C. Erçin. 2004. Effects of melatonin on acute pancreatitis in rats. Zeitschrift für Gastroenterologie 42: 967–972. 7. Hg, B., R. Bittner, and M. Büchler. 1986. Hemodynamic data pattern in patients with acute pancreatitis. Gastroenterology 90: 74–79. 8. Rau, B., B. Poch, F. Gansauge, A. Bauer, K.A. Nüssler, T. Nevalainen, M.H. Schoenberg, and H.G. Beger. 2000. Pathophysiologic role of oxygen free radicals in acute pancreatitis. Annals of Surgery 31: 352–360. 9. Yamamoto, Y. 1985. Oxidation of biological membranes and its inhibition. Free radical chain oxidation of erythrocyte ghost membranes by oxygen. Biochimica et Biophysica Acta 819: 29–36. 10. Faiber, J.L., M.E. Kyle, and J.B. Coleman. 1990. Mechanisms of cell injury by activated oxygen species. Laboratory Investigation 62: 670–679. 11. Hotter, G., D. Closa, N. Prats, F. Pi, E. Gelpi, and J. Rosello-Catafau. 1997. Free radical enhancement promotes leukocytes recruitment through a PAF and LTB4 dependent mechanism. Free Radical Biology & Medicine 22: 947–954. 12. Stanfey, H., G.B. Bulkley, and J.L. Cameron. 1984. The role of oxygen-derived free radicals in the pathogenesis of acute pancreatitis. Annals of Surgery 200: 405–413. 13. Schulz, H.U., and C. Niederau. 1994. Oxidative stress-induced changes in pancreatic acinar cells: Insight from in vitro studies. Hepato-Gastroenterology 41: 309–312. 14. Schoenberg, M.H., D. Birk, and H.G. Beger. 1995. Oxidative stress in acute and chronic pancreatitis. The American Journal of Clinical Nutrition 62: 1306S–1314S. 15. Osmanağaoğlu, M.A., M. Kesim, E. Yulug, A. Menteşe, and C. Karahan. 2012. Ovarian-protective effects of clotrimazole on ovarian ischemia/reperfusion Injury in a rat ovarian-torsion model. Gynecologic and Obstetric Investigation 74: 125–130. 16. Vanden Bossche, H., P. Marichal, J. Gorrens, M.C. Coene, G. Willemsens, D. Bellens, I. Roels, H. Moereels, and P.A. Janssen. 1989. Biochemical approaches to selective antifungal activity. Focus on azole antifungals. Mycoses 32: 35–52. 17. Alvarez, J., M. Montero, and J. Garcia-Sancho. 1992. High affinity inhibition of Ca2+-dependent K+ channels by cytochrome P450 inhibitors. The Journal of Biological Chemistry 267: 11789–11793. 18. Isaev, N.K., E.V. Stelmashook, U. Dirnagl, N.A. Andreeva, L. Manuhova, V.S. Vorobjev, I.N. Sharonova, V.G. Skrebitsky, I.V. Victorov, J. Katchanov, M. Weih, and D.B. Zorov. 2002. Neuroprotective effects of the antifungal drug clotrimazole. Neuroscience 113: 47–53. 19. Schmidt, J., M.D. Rattner, and K. Lewandrowski. 1992. A better model of acute pancreatitis for evaluating therapy. Annals of Surgery 215: 44–56. 20. Dobschuetz, E., P. Biberthaler, T. Mussack, S. Langer, K. Messmer, and T. Hoffmann. 2003. Noninvasive in vivo assessment of the pancreatic microcirculation: Orthogonal polarization spectral imaging. Pancreas 26: 139–143. 21. Sookhal, S., J.J. Wang, M. McCourt, W. Kwan, D.H. Bouchier, and P. Redmond. 2002. A novel therapeutic strategy for attenuating neutrophil-mediated lung injury in vivo. Annals of Surgery 235: 283–291.

Cekic, Alhan, Usta, Türkyılmaz, Kural, and Erçin 22. Lankisch, P.G., U. Pohl, J. Otto, and G. Rahlf. 1988. When should treatment of acute experimental pancreatitis be started? The early phase of bile-induced acute pancreatitis. Research in Experimental Medicine 188: 123–129. 23. Schierwagen, C., A.C. Bylund-Fellenius, and C. Lundberg. 1990. Improved method for quantification of tissue PMN accumulation measured by myeloperoxidase activity. Journal of Pharmacological Methods 23: 179–186. 24. Buege, J.A., and S.D. Aust. 1978. Microsomal lipid peroxidation. Methods in Enzymology 52: 302–310. 25. Lowry, O.H., N.J. Rosebrough, A.L. Farr, and R.J. Randall. 1951. Protein measurement with the Folin phenol reagent. The Journal of Biological Chemistry 193: 265–275. 26. Jensen, B.S., M. Hertz, P. Christophersen, and L.S. Madsen. 2002. The Ca2+-activated K+ channel of intermediate conductance: A possible target for immune suppression. Expert Opinion on Therapeutic Targets 6: 623–636. 27. Lang, F., C. Birka, S. Myssina, K.S. Lang, P.A. Lang, V. Tanneur, C. Duranton, T. Wieder, and S.M. Huber. 2004. Erythrocyte ion channels in regulation of apoptosis. Advances in Experimental Medicine and Biology 559: 211–217. 28. Miller, B.A. 2006. The role of TRP channels in oxidative stressinduced cell death. The Journal of Membrane Biology 209: 31–41. 29. Usul, H., E. Arslan, T. Cansever, et al. 2008. Effects of clotrimazole on experimental spinal cord ischemia/reperfusion injury in rats. Spine 33: 2863. 30. Chou, C.C., C.A. Lunn, and N.J. Murgolo. 2008. KCa3.1: target and marker for cancer, autoimmune disorder and vascular inflammation? Expert Review of Molecular Diagnostics 8: 179–187. 31. Thapa, D., J.S. Lee, S.Y. Park, et al. 2008. Clotrimazole ameliorates intestinal inflammation and abnormal angiogenesis by inhibiting interleukin-8 expression through a nuclear factor-Kβ-dependent manner. The Journal of Pharmacology and Experimental Therapeutics 327: 353–364. 32. Bowles, D.K. 2009. The intermediate-conductance Ca2+-activated K+ channel (KCa3.1) in vascular disease. Cardiovascular & Hematological Agents in Medicinal Chemistry 7: 1–11. 33. Marcondes, M.C., M. Sola-Penna, and P. Zancan. 2010. Clotrimazole potentiates the inhibitory effects of ATP on the key glycolytic enzyme 6-phosphofructo-1-kinase. Archives of Biochemistry and Biophysics 497: 62–67. 34. Iannelli, A., G. de Sousa, N. Zucchini, M.C. Saint-Paul, J. Gugenheim, and R. Rahmani. 2011. Anti-apoptotic pro-survival effect of clotrimazole in a normothermic ischemia reperfusion injury animal model. The Journal of Surgical Research 171: 101–107. 35. Horng, C.T., N.N. Chiang, I.L. Chen, W.Z. Liang, I.S. Chen, D.H. Kuo, P.C. Shieh, and C.R. Jan. 2013. Effect of clotrimazole on

36. 37. 38.

39.

40.

41.

42. 43. 44.

45.

46.

47.

cytosolic Ca(2+) rise and viability in HA59T human hepatoma cells. Journal of Receptor and Signal Transduction Research 33: 89–95. Banks, P.A., S. Tenner, E.C. Noordhoek, G. Sica, S. Feng, and M. Zinner. 1996. Does pancreatic necrosis predict severity in patients with acute necrotizing pancreatitis? Digestion 5: 218–223. Bhatia, M., F.L. Wong, Y. Cao, H.Y. Lau, J. Huang, P. Puneet, and L. Chevali. 2005. Pathophysiology of acute pancreatitis. Pancreatology 5: 132–134. Alhan, E., A. Cinel, C. Ercin, S. Türkyılmaz, B.V. Kural, M. Alper, and D. Flinte. 2011. The effects of p53 inhibition using pifithrin-α on acute necrotizing pancreatitis in rats. Turkish Journal of Medical Sciences 41: 673–683. Lotze, M.I., H.J. Zeh, A. Rubartelli, L.J. Sparvero, A.A. Amoscato, N.R. Washburn, M.E. Devera, X. Liang, M. Tör, and T. Billiar. 2007. The grateful dead: Damage-associated molecular pattern molecules and reduction/oxidation regulate immunity. Immunological Reviews 220: 60–81. Sharif, R., R. Dawra, K. Wasiluk, P. Phillips, V. Dudeja, E. KurtJones, R. Finberg, and A. Saluja. 2009. Impact of toll-like receptor 4 on the severity of acute pancreatitis and pancreatitis-associated lung injury in mice. Gut 58: 813–819. Alhan, E., C. Türkyılmaz, C. Ercin, B.V. Kural, and D. Flinte. 2012. Does nuclear factor-Kappa B in peripheralmononuclear cells have a prognostic role during acute necrotizing pancreatitis in rats? European Surgical Research 48: 34–39. Zhang, X., D. Wu, and X. Jiang. 2009. ICAM-1 and acute pancreatitis complicated by acute lung injury. Journal of the Pancreas 10: 8–14. Weidenhach, H., M.M. Lerch, T.M. Gress, D. Pfaff, S. Turi, and G. Adler. 1995. Vasoactive mediators and the progression from edematous to necrotizing experimental acute pancreatitis. Gut 37: 434–440. Murakami, H., A. Nakao, W. Kishimoto, M. Nakano, and H. Takagel. 1995. Detection of O2− generation and neutrophil accumulation in rat lungs after acute necrotizing pancreatitis. Surgery 118: 547–554. Ivey, C.L., A.L. Stephenson, and A.M. Townsley. 1998. Involvement of cytochrome P-450 enzyme activity in the control of microvascular permeability in canine lung. American Journal of Physiology. Lung Cellular and Molecular Physiology 275: 756–776. Thapa, D., J.S. Lee, M.A. Park, M.Y. Cho, Y.J. Park, H.G. Choi, T.C. Jeong, and J.A. Kim. 2009. Inhibitory effects of clotrimazole on TNF-alpha-induced adhesion molecule expression and angiogenesis. Archives of Pharmacal Research 32: 593–603. Borgström, A., S. Appelros, C.A. Müller, W. Uhl, and M.W. Büchler. 2002. Role of activation peptides from pancreatic proenzymes in the diagnosis and prognosis of acute pancreatitis. Surgery 131: 125–128.