International Journal of
Molecular Sciences Article
Protective Effect of Pretreatment with Acenocoumarol in Cerulein-Induced Acute Pancreatitis 3, Zygmunt Warzecha 1, *, Paweł Sendur 1,2 , Piotr Ceranowicz 1 , Marcin Dembinski ´ 1 4 5 Jakub Cieszkowski , Beata Ku´snierz-Cabala , Rafał Olszanecki , Romana Tomaszewska 6 , 1 Tadeusz Ambro˙zy 7 and Artur Dembinski ´ 1
2 3 4 5 6 7
*
Department of Physiology, Faculty of Medicine, Jagiellonian University Medical College, 16 Grzegórzecka St., 31-531 Cracow, Poland;
[email protected] (P.S.);
[email protected] (P.C.);
[email protected] (J.C.);
[email protected] (A.D.) Department of Anesthesiology and Intensive Therapy, Faculty of Medicine, Jagiellonian University Medical College, 31-501 Cracow, Poland The Second Department of General Surgery, Faculty of Medicine, Jagiellonian University Medical College, 31-501 Cracow, Poland;
[email protected] Department of Clinical Biochemistry, Faculty of Medicine, Jagiellonian University Medical College, 31-501 Cracow, Poland;
[email protected] Department of Pharmacology, Faculty of Medicine, Jagiellonian University Medical College, 31-531 Cracow, Poland;
[email protected] Department of Pathology, Faculty of Medicine, Jagiellonian University Medical College, 31-531 Cracow, Poland;
[email protected] Faculty of Physical Education and Sport, University of Physical Education, 31-571 Cracow, Poland;
[email protected] Correspondence:
[email protected]; Tel.: +48-12-421-10-06; Fax: +48-12-422-54-78
Academic Editor: Srikumar Chellappan Received: 14 August 2016; Accepted: 30 September 2016; Published: 12 October 2016
Abstract: Coagulation is recognized as a key player in inflammatory and autoimmune diseases. The aim of the current research was to examine the effect of pretreatment with acenocoumarol on the development of acute pancreatitis (AP) evoked by cerulein. Methods: AP was induced in rats by cerulein administered intraperitoneally. Acenocoumarol (50, 100 or 150 µg/kg/dose/day) or saline were given once daily for seven days before AP induction. Results: In rats with AP, pretreatment with acenocoumarol administered at the dose of 50 or 100 µg/kg/dose/day improved pancreatic histology, reducing the degree of edema and inflammatory infiltration, and vacuolization of acinar cells. Moreover, pretreatment with acenocoumarol given at the dose of 50 or 100 µg/kg/dose/day reduced the AP-evoked increase in pancreatic weight, serum activity of amylase and lipase, and serum concentration of pro-inflammatory interleukin-1β, as well as ameliorated pancreatic DNA synthesis and pancreatic blood flow. In contrast, acenocoumarol given at the dose of 150 µg/kg/dose did not exhibit any protective effect against cerulein-induced pancreatitis. Conclusion: Low doses of acenocoumarol, given before induction of AP by cerulein, inhibit the development of that inflammation. Keywords: acute pancreatitis; coagulation; inflammation; interleukin-1β; lipase
1. Introduction There is close bidirectional relationship between coagulation and inflammation [1,2]. Inflammation can be both a cause and a result of induction of coagulation [1–4]. Similarly, coagulation results not only in thrombosis but also in activation of inflammatory process [1,2,5]. Acute pancreatitis (AP) is associated with coagulative disorders [6–9]. The stage of these disorders depends on the severity of AP. Mild AP is associated with scattered intravascular thrombosis, Int. J. Mol. Sci. 2016, 17, 1709; doi:10.3390/ijms17101709
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whereas severe AP may lead to the development of disseminated intravascular coagulation [6–9]. Measurement of disseminated intravascular coagulation parameters is useful in the assessment of AP severity and prediction for poor outcome of this disease at admission [7,8]. There are animal and clinical studies showing protective and therapeutic effect of a well-known anticoagulant, heparin, in AP. Animals studies have indicated that pretreatment with heparin inhibits the development of AP evoked by bile [9], taurocholate [10], cerulein [11] and pancreatic ischemia followed by reperfusion [12]. Administration of heparin after induction of AP has been shown to accelerate the recovery in acute pancreatitis evoked by cerulein [13] and pancreatic ischemia followed by reperfusion [12]. Clinical reports suggests that pretreatment with heparin reduces frequency of post-ERCP (endoscopic retrograde cholangiopancreatography) pancreatitis [14,15]. Moreover, treatment with heparin given together with insulin is recommended as a gold standard in the management of hyperlipidemia-induced AP [16–18]. Heparin has also been found to be effective in the prevention of encephalopathy in the course of severe AP [19]. Mechanism of anticoagulant activity of heparin requires the presence of antithrombin III. Antithrombin III is a protease inhibitor and complex heparin–antithrombin III inactivates thrombin and active forms of clotting factors, IX, X, XI and XII, as well as inhibits the formation of thrombin and active forms of clotting factors [20,21]. Observations concerning anti-inflammatory effects of heparin in AP led to the question whether other anticoagulants such as coumarins also exhibit anti-inflammatory activity in this disease. Coumarins are vitamin K antagonists. Reduced vitamin K is a necessary cofactor for the hepatic γ-glutamyl carboxylase activity. That enzyme adds a carboxyl group to glutamic acid residues in immature clotting factors II, VII, IX, and X as well as proteins S, C and Z. During activity of γ-glutamyl carboxylase, vitamin K is oxidized. Coumarins inhibit the vitamin K epoxide reductase, an enzyme that reduces vitamin K back to its active form. Coumarins reduce plasma levels of vitamin K-dependent clotting factors causing a reduction in blood coagulability [21,22]. Our recent study has shown that inhibition of coagulation by pretreatment with low doses of acenocoumarol, a drug from the group of coumarins, exhibits protective effect in the pancreas leading to reduction of the severity of ischemia/reperfusion-induced AP [20]. On the other hand, there are no data on whether protective effect of acenocoumarol in the pancreas depends on the cause of AP or exhibits universal nature and occurs regardless of the primary agent causing this inflammation. The effect of pretreatment with acenocoumarol on the development of AP evoked by a primary non-vascular mechanism is unknown. Therefore, the aim of our present study was to investigate whether the pretreatment with low doses of acenocoumarol affect the development of cerulein-induced AP. 2. Results In control saline-treated rats without induction of AP, international normalized ratio (INR) reached a value of 1.08 ± 0.10 (Figure 1) while the average weight of the pancreas was 618 ± 22 g (Figure 2). Morphological features showed that the pancreas in this group of animals exhibits regular histology without damage of the gland (Figure 3 and Table 1). In rats without induction of AP, administration of acenocoumarol given for seven days caused a dose-dependent increase in INR (Figure 1). Acenocoumarol given at the dose of 50, 100 or 150 µg/kg/dose caused around three-, four- and six-fold increase in INR, respectively. In these rats, acenocoumarol given at the doses used did not cause a statistically significant effect on the weight of the pancreas (Figure 2), pancreatic blood flow (Figure 4) and pancreatic DNA synthesis (Figure 5). In addition, serum activity of lipase (Figure 6) and amylase (Figure 7), serum concentrations of interleukin-1β (IL-1β) (Figure 8), and plasma D-dimer concentration (Figure 9) were not affected by acenocoumarol given in rats without causing pancreatitis. Morphological features showed that acenocoumarol given alone caused in half of cases slight interlobular edema of the pancreas (Table 1). Moreover, in contrast to lower doses,
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contrast doses, given of µg/kg/dose led appear single acenocoumarol given at theacenocoumarol dose of 150 µg/kg/dose leddose to appear hemorrhagic in pancreatic contrast to to lower lower doses, acenocoumarol given at at the the dose of 150 150single µg/kg/dose led to to foci appear single Int. J. Mol. Sci. 2016, 17, 1709 3 of 14 hemorrhagic in in animals of hemorrhagic foci in pancreatic pancreatic tissue in half halfofof ofpancreatitis animals without without induction of pancreatitis pancreatitis (Table (Table 1). 1). tissue in half offoci animals withouttissue induction (Tableinduction 1). contrast to lower doses, acenocoumarol given at the dose of 150 µg/kg/dose led to appear single 7 7 aa aa hemorrhagic foci in pancreatic tissue in half of animals without induction of pancreatitis (Table 1). 6 6
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Figure on the prothrombin time measured as Figure Impact of of pretreatment pretreatmentwith withacenocoumarol acenocoumarol prothrombin measured Figure1.1. 1. Impact Impact of pretreatment with acenocoumarol on on theAPthe prothrombin time time measured as non-AP ratio (INR) in or cerulein-induced pancreatitis. Key: asinternational internationalnormalized normalized ratio (INR) in with rats or without cerulein-induced pancreatitis. international normalized ratio (INR) in rats rats with with or without without cerulein-induced pancreatitis. Key: 1. AP Impact pretreatmentacute with acenocoumarol on the prothrombin time measured asacute C == cerulein-induced pancreatitis; non-AP == groups without induction of Key: Ccontrol; = control; AP = of cerulein-induced acute pancreatitis; non-AP = groups without induction of C ==Figure control; AP cerulein-induced acute pancreatitis; non-AP groups without induction of acute acute international normalized ratio (INR) in rats with or without cerulein-induced pancreatitis. Key: pancreatitis; AC 50 µg/kg/day; == = 150 µg/kg/day. pancreatitis; = =acenocoumarol; 100 === 100 100 µg/kg/day; µg/kg/day; 150 150 µg/kg/day. pancreatitis;AC AC = acenocoumarol; acenocoumarol;50 50===5050 50µg/kg/day; µg/kg/day; 100 100 100 µg/kg/day; 150 150 150 µg/kg/day. C =±control; AP = in cerulein-induced acuteaa apancreatitis; non-APto=control. groups without induction of acute p < 0.05 compared Mean SEM. n = 10 each group of rats. Mean ± SEM. n = 10 in each group of rats. p < 0.05 compared to control. Mean ± SEM. n = 10 in each group of rats. p < 0.05 compared to control. aa
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Figure of pretreatment with acenocoumarol on weight of in or Figure2.2. 2. Impact Impact with acenocoumarol on the theon weight of the the pancreas pancreas in rats rats with with Figure Impactofofpretreatment pretreatment with acenocoumarol the weight of the pancreas in orrats without cerulein-induced pancreatitis. Key: C = control; AP = cerulein-induced acute pancreatitis; Figure 2. Impact of pretreatment with acenocoumarol on the weight of the pancreas in rats with or without cerulein-induced pancreatitis. Key: C = control; cerulein-induced acute pancreatitis; with or without cerulein-induced pancreatitis. Key: CAP = =control; AP = cerulein-induced acute without cerulein-induced pancreatitis. Key: C = control; AP acute pancreatitis; non-AP == groups without induction of pancreatitis; AC acenocoumarol; 50 == 50 µg/kg/day; non-AP groups without induction of acute acute pancreatitis; AC===cerulein-induced acenocoumarol; 50 = 50 µg/kg/day; pancreatitis; non-AP = groups without induction of acute pancreatitis; AC acenocoumarol; a p < 0.05 = groups without of acuteMean pancreatitis; acenocoumarol; 50 of = 50 µg/kg/day; 100non-AP == 100 150 == induction 150 µg/kg/day. ±± SEM. nn === 10 in group rats. a p < 0.05 100 µg/kg/day; µg/kg/day; 150 150 µg/kg/day. Mean SEM.AC 10 in each each group of rats. 50100 = 50 µg/kg/day; 100 = 100 µg/kg/day; 150 = 150 µg/kg/day. Mean ± SEM. n = 10 in each group a b p < 100 = 100 µg/kg/day; 150 = 150 µg/kg/day. Mean ± SEM. n = 10 in each group of rats. p < 0.05 0.05 compared to AP alone. compared to control; b p < 0.05 compared AP alone. control; b p to of compared rats. a p < to 0.05 compared to control; < 0.05 compared to AP alone. compared to control; b p < 0.05 compared to AP alone.
Figure 3. Representative observedinin controlsaline saline treated Figure 3. Representativemorphological morphologicalimages images of of the the pancreas pancreas observed control treated Figure Representative morphological images saline treated Figure 3. 3.Representative morphological images of of the the pancreas pancreasobserved observedinincontrol control saline treated rats (A); rats pretreated of 50 µg/kg/day without induction rats (A); rats pretreatedwith withacenocoumarol acenocoumarol given given the the dose of 50 µg/kg/day without induction of of rats (A); rats pretreated with acenocoumarol given the dose of 50 µg/kg/day without induction of rats (A); rats pretreated with acenocoumarol given the dose of 50 µg/kg/day without induction acute pancreatitis (B);rats ratswith withcerulein-induced cerulein-induced acute acute pancreatitis pancreatitis with acute pancreatitis (B); (C); and rats pretreated with acute pancreatitis (B); rats with cerulein-induced acute pancreatitis (C); (C);and andrats ratspretreated pretreated with of acute pancreatitis(given (B); rats withofcerulein-induced acute pancreatitis (C); and rats pretreated with acenocoumarol thedose dose 50µg/kg/day) µg/kg/day) before before induction cerulein acenocoumarol (given of induction of acute pancreatitis cerulein acenocoumarol (given the the dose of 50 50 µg/kg/day) before induction of ofacute acutepancreatitis pancreatitisbyby by cerulein acenocoumarol (given the dose of 50 µg/kg/day) before induction (D). Hematoxylin–eosin counterstain, original magnification 200×. of acute pancreatitis by cerulein (D). (D). (D). Hematoxylin–eosin Hematoxylin–eosin counterstain, counterstain, original original magnification magnification 200×. 200×. Hematoxylin–eosin counterstain, original magnification 200×.
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