Healing effect of heparin in the course of acute cerulein-induced pancreatitis

Introduction


There is evidence that initiation and progression of acute pancreatitis are associated with disturbance in the pancreatic microcirculation, leading to formation of thrombi in capillaries, activation of leukocytes, release of proteolytic enzymes, and formation of oxygen-derived free radicals and pro-inflammatory cytokines. Coagulative disorders are related to the severity of acute pancreatitis [1, 2]. In this disease, activation of the haemostatic system may range from scattered intravascular thrombosis to severe disseminated intravascular coagulation (DIC) [3]. Inflammation and coagulation are closely linked processes [4] and inflammatory cytokines activate coagulation by increasing expression of tissue factor on monocytes and endothelium, leading to thrombin formation [5].
Heparin, a heteroglycan containing alternating sulphated units of glucuronic acid and glucosamine, exhibits numerous biological activities [6]. Heparin, in complex with antithrombin III, prevents coagulation and, in large doses, may inhibit platelet aggregation [6]. Beside anticoagulative properties related to direct or indirect inhibition of protease involved in the coagulation cascade, heparin also inhibits other proteases present in plasma and tissues, including pancreatic enzymes. Heparin directly or indirectly reduces activity of trypsin [7, 8] and chymotrypsin [9], and inhibits conversion of trypsinogen to trypsin [10, 11].
Previous experimental and some clinical studies have shown that pretreatment or early treatment with heparin during induction of acute pancreatitis exhibits a protective effect on the pancreas, inhibiting the development of acute pancreatitis evoked by cerulein [12], bile [13], taurocholate [14], pancreatic ischaemia followed by reperfusion [15] or endoscopic retrograde cholangiopancreatography (ERCP) [16]. This effect of heparin may be useful in the prevention of acute pancreatitis, but its clinical value is considerably limited. Clinically patients are usually seen several hours or days after the onset of acute pancreatitis and therapy is started after admission to hospital. For this reason, it is more important to answer the question whether treatment with heparin after development of acute pancreatitis can affect the course of acute pancreatitis. Our previous study [15] has shown that administration of heparin after development of ischaemia/reperfusion-induced pancreatitis exhibits a therapeutic effect in this disease. However, this experimental model of acute pancreatitis depends on a primary vascular mechanism related to severe pancreatic ischaemia followed by reperfusion. The effect of heparin administration on the course of acute pancreatitis evoked by a primary non-vascular mechanism is unknown. For this reason, the present study was designed to determine the influence of treatment with unfractionated heparin on the course of acute cerulein-induced pancreatitis.

Material and methods


Studies were performed on male Wistar rats weighing 160-180 g. The experimental protocol is in agreement with the “European Convention for the Protection of Vertebrate Animals used for Experimental and other Scientific Purpose” and was approved by the Local Commission of Ethics for the Care and Use of Laboratory Animals. Animals were housed in cages with wire mesh bottoms, with normal room temperature and a 12-h light-dark cycle.
Acute pancreatitis was induced by cerulein (Sigma-Aldrich, GmbH, Steinheim, Germany) admini-stered intraperitoneally (i.p.) 5 times with 1 h intervals at a dose of 50 mg/kg/dose.
Unfractionated heparin (Heparinum, Polfa, Warszawa, Poland) was administered subcutaneously at the dose of 150 U/kg, twice a day, starting 24 h after the last injection of cerulein. The dose of heparin, 150 U/kg, was chosen because this dose caused a two-fold increase in activated partial thromboplastin time (aPTT) in a preliminary study (data not shown).
Pancreatic blood flow was measured using a laser Doppler flowmeter (PeriFlux 4001 Master monitor, Perimed AB, J rf lla, Sweden).
Plasma lipase activity was determined with a Kodak Ectachem DT II System analyzer (Eastman Kodak Company, Rochester, NY, USA) using Lipa DT Slides (Vitros DT Chemistry System, Johnson & Johnson Clinical Diagnostic, Inc., Rochester, NY, USA). Plasma concentration of interleukin-1b (IL-1b) was measured using the BioSource Cytoscreen rat IL-1b kit (BioSource International, Camarillo, California, USA) based on ELISA. Activated partial thromboplastin time (aPTT) was determined in fresh plasma, using Plastelin LS (Organon Teknika Corporation, Dirham, NC, USA). Plasma D-dimer concentration was determined using a latex-enhanced immunoturbidimetric assay (D-dimer test, Roche Diagnostics).
Pancreatic DNA synthesis was measured by incubation of minced pancreatic tissue at 37°C for 45 min in 2 ml of medium containing 8 mCi /ml of [3H] thymidine ([6-3H]-thymidine, 20-30 Ci/mmol, Institute for Research, Production and Application of Radio-isotopes, Prague, Czech Republic), as described previously [17]. DNA synthesis was expressed as [3H] thymidine disintegrations per minute per microgram DNA (dpm/mg DNA).
Morphological examination of pancreatic tissue was performed in haematoxylin and eosin stained slides as described previously in detail [17]. The histological grading of oedema, leukocytic inflammatory infiltration, vacuolization of acinar cells, haemorrhages and necrosis was made using a scale raging from 0 (absent) to 3 for maximal alteration. Results of histological examination were expressed as a predominant histological grading in each experimental group of animals.
Statistical analysis, except histological data, was made by analysis of variance followed by Tukey’s multiple comparison test. A difference with a p value of less than 0.05 was considered significant.


Results


Treatment with heparin after induction of acute pancreatitis reduced the severity of this disease and accelerated pancreatic regeneration. In histological examination, a reduction in pancreatic oedema, inflammatory infiltration, vacuolization of acinar cells, and haemorrhages was observed (Table I). Pancreases of animals treated with heparin after induction of acute pancreatitis recovered during 7 days from induction of acute pancreatitis, whereas pancreases of animals without treatment with heparin reached normal morphology after 10 days (Table I). Also, treatment with heparin reduced biochemical indices of the severity of acute pancreatitis. Treatment with heparin significantly reduced the pancreatitis-evoked increase in plasma activity of lipase (Fig. 1) and plasma concentration of pro-inflammatory IL-1b (Fig. 2). Pancreatic DNA synthesis (Fig. 3) and pancreatic blood flow (Fig. 4) were increased in animals with acute pancreatitis and treated with heparin. Treatment with heparin after induction of acute pancreatitis prolonged aPTT (Fig. 5) and this effect was associated with a significant reduction in plasma concentration of D-dimer (Fig. 6).

Discussion


Previous studies have shown that pretreatment with heparin inhibits the development of acute pancreatitis in different experimental models of this disease [12-15], as well as protecting the pancreas against acute pancreatitis evoked by ERCP in a clinical study in humans [16]. Our present study confirms and extends these observations. In a previously used model of acute pancreatitis, acute pancreatitis was evoked by severe pancreatic ischaemia followed by reperfusion, where a vascular mechanism with extensive intravascular coagulation was the primary cause of this disease. In our present study we induced acute pancreatitis using an experimental model of this disease with a primary non-vascular aetiology. We have found that administration of heparin demonstrates a healing effect in the course of cerulein-induced acute pancreatitis. This observation suggests that heparin exhibits a therapeutic effect in acute pancreatitis independently of the primary aetiology of this disease. The therapeutic effect of heparin in the course of cerulein-induced pancreatitis was manifested as a reduction in the severity of acute pancreatitis and a faster pancreatic recovery. Morphological features of pancreatic tissue have shown that treatment with heparin reduces pancreatic oedema, necrosis, haemorrhages and leukocyte infiltration. Reduction in pancreatic leukocyte infiltration was in harmony with the reduction in plasma interleukin-1b observed by us. Activation of leukocytes and release of pro-inflammatory cytokines are responsible for local pancreatic damage and development of systemic inflammatory response syndrome (SIRS) and multiple organ failure (MOF) in the course of acute pancreatitis [18]. Pro-inflammatory cytokines, such as IL-1b, IL-6 and tumour necrosis factor-a (TNF-α), are produced within the pancreas and subsequently within distant organs, leading to the development of MOF in severe acute pancreatitis [19]. IL-1b plays the most important role in the induction of the systemic acute phase response and in the release of other members of the pro-inflammatory cytokine cascade [20]. Pro-inflammatory cytokine production is well correlated with severity of acute pancreatitis [21].
This anti-inflammatory effect of heparin in the course of cerulein-induced acute pancreatitis seems to be dependent on direct and indirect mechanisms. The concept of direct anti-inflammatory action of heparin is supported by numerous studies. Already in 1984, Laghi Pasini et al. [22] showed in an in vitro study that heparin inhibits granulocyte aggregation and degranulation stimulated by chemotactic factor FMLP (N-formyl- methionyl-leucyl-phenylalanine) or by zymosan activated serum, and reduces the FMLP-dependent superoxide anion generation. In agreement with these data is the observation that heparin inhibits neutrophil aggregation stimulated by FMLP or PAF (platelet- activating factor), as well as reducing the release of elastase by these cells [23].
Our present study has shown that the anti- inflammatory effect of heparin in the course of acute cerulein-induced pancreatitis is also related to its influence on the plasma activity of pancreatic digestive enzymes. The increase in plasma activity of lipase and amylase is a well established index of acute pancreatitis severity with high sensitivity and specificity [24]. In our present study, pretreatment with heparin reduced the pancreatitis-evoked increase in serum activity of lipase. This observation is further evidence of the therapeutic effect of heparin in acute pancreatitis. A reduction in plasma activity of pancreatic enzymes may be a result of direct action of heparin on these enzymes or a result of improvement of pancreatic condition, or both these effects. The concept of direct inhibitory action of heparin on pancreatic enzymes is supported by the observation that heparin directly reduces activity of pancreatic enzymes [7-11]. On the other hand, there is a study showing interaction between pancreatic enzymes and intravascular activation of leukocytes. Keck et al. [25] showed that presence of active trypsin and elastase in the circulation up-regulates the expression of adhesion molecules on leukocytes and endothelial cells. This effect leads to an increase in leukocyte-endothelial interaction, promoting pancreatic microcirculatory failure. For this reason, the heparin- evoked reduction in plasma activity of pancreatic enzymes can be a result and/or cause of its therapeutic effect in acute pancreatitis.
Another important mechanism of the therapeutic effect of heparin in acute cerulein-induced pancreatitis seems to be dependent on its influence on pancreatic blood flow. Pancreatic ischaemia plays an important role in the development of acute pancreatitis [26] and the severity of acute pancreatitis is closely correlated with the disturbance of pancreatic circulation. Our present study has shown that treatment with heparin improves pancreatic blood flow and reduces the severity of acute pancreatitis. This effect seems be a result and cause of the therapeutic effect of heparin because the improvement of pancreatic blood flow, during induction of acute pancreatitis, reduces the severity of pancreatic damage [27], but simultaneously a reduction in pancreatic damage improves pancreatic blood flow.
It is most likely that the circulatory effect of heparin is, at least in part, related to its well-known anticoagulant activity. In our present study, induction of acute pancreatitis by cerulein led to a reduction in pancreatic blood flow and this effect was associated with an almost two-fold increase in aPTT and a twelve-fold increase in D-dimer concentration. These data indicate that development of acute pancreatitis, also with primary non-vascular aetiology, is associated with formation of thrombi within the pancreatic and systemic circulation. Changes in aPTT are a result of consumption of factors involved in coagulation, whereas the increase in D-dimer concentration indicates subsequent activation of coagulation. Our present study has shown that treatment with heparin accelerated reduction in plasma D-dimer concentration in animals with acute pancreatitis. This observation indicates that treatment with heparin inhibits coagulation and for this reason reduces consumption of coagulation factors and creation of products of fibrinolysis.
Pancreatic DNA synthesis is an index of cell proliferation in the pancreas and the reduction in pancreatic DNA synthesis is well correlated with pancreatic damage in acute pancreatitis [27-28]. Our present study has shown that administration of heparin increases pancreatic DNA synthesis in the early stage of acute pancreatitis. This observation is additional evidence of the beneficial effect of heparin in acute pancreatitis and may explain our observation that heparin accelerates pancreatic regeneration in this disease.

Conclusions


Heparin, a safe and well-known medicine, exhibits a strong therapeutic effect in the course of acute pancreatitis independently of the primary cause of this disease. This observation suggests that heparin may be useful in routine clinical management of acute pancreatitis.


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Address for correspondence: Prof. Artur Dembiński
MD, PhD, Department of Physiology
Jagiellonian University Medical College
16 Grzegórzecka Street
31-531 Kraków
phone +48 12 421 10 06
fax +48 12 422 54 78
e-mail: mpdembin@cyf-kr.edu.pl