Acute pancreatitis is an inflammatory intra-abdominal process, which in approximately 15–20% of patients presents in a severe form, with a gradual establishment of multiple organ dysfunction or local complications, including necrosis, pseudocyst, and abscess [
The deficit in our understanding of the mechanism driving the inflammatory process in acute pancreatitis is a reason why our therapeutic strategy has failed to reduce mortality, despite ongoing research. The aetiology of early mortality after acute pancreatitis is multiple organ failure. When acute pancreatitis leads to the establishment of acute kidney injury, there is a 5- to 10-fold rise in mortality, which can reach 70% [
Eugenol (1-allyl-4-hydroxy-3-methoxybenzene) is a naturally occurring substance, found in the essential oil of commonly consumed spices such as clove oil as well as cinnamon, basil, and nutmeg oils [
106 male Wistar rats, aged 3-4 months and weighing 220–350 gr, were used in this study. They were housed in cages under standard laboratory conditions (12 hr light-dark cycles, 22–25°C room temperature, and 55–58% humidity), with free access to food and water. The animals were procured from the Hellenic Pasteur Institute (Athens, Greece). The experiment took place at the ELPEN Experimental Research Center (Pikermi, Greece), while the histological analysis was carried out at the Lab of Histology, Embryology, Medical School, Democritus University of Thrace. The experimental surgical procedures and the general handling of the animals conformed to the international guidelines of Directive 86/609/EEC on the protection of animals used for experimental and other scientific purposes. The animals were randomly assigned in 3 groups: Sham (
The animals were anaesthetized initially by being placed in a glass box containing isoflurane and then through administration of 0.25 mL of butorphanol (Dolorex; Intervet/Schering/Plough Animal Health, Boxmeer, Holland) by subcutaneous injection. The animals were intubated with a 16 G venous catheter, which was then connected to a ventilator set at 70 breaths/min and a tidal volume of 3 mL. After confirmation of the success of intubation, anaesthesia was maintained by a mixture of 93% O2, 5% CO2, and 2% isoflurane. Acute pancreatitis was induced according to a previously described model [
Postoperatively, analgesia was maintained through subcutaneous administration of 2 mL/Kg butorphanol (Dolorex; Intervet/Schering/Plough Animal Health, Boxmeer, Holland). Euthanasia was performed at a predetermined time for each animal with the use of ketamine (Narcetan; Vetoquinol, Buckingham, UK) 0.3–0.6 mL and xylazine (Rompun; Bayer, Uxbridge, UK) 0.1–0.3 mL, followed by a midline laparotomy and exsanguination of the abdominal aorta. Time points for analysis were 6, 12, 24, 48, and 72 hours postoperatively. Serum samples for measurement of urea and creatinine as well as specimens from both kidneys for histopathological examination were acquired.
Pure eugenol (eugenol 99%, Aldrich Chemistry, St. Louis, MO, USA) was purchased and prepared in an oily solution in the chemical laboratory of Elpen Pharmaceutical Co. Inc. (ELPEN Pharmaceutical Co. Inc., Pikermi Attica, Greece). This was achieved with the admixture of pure eugenol in a corn oil solution in a concentration of 1.5 mg eugenol/mL.
Samples were placed in 10% buffered formalin solution, and 4
Semiquantitative scoring system.
Variable | Result | Score |
---|---|---|
Histopathological variables | ||
Hyperemia and dilation of renal parenchyma capillaries | None |
|
Hyperemia and dilation of renal corpuscles capillaries | Mild |
|
Inflammatory infiltration of renal parenchyma | ||
Edema | Moderate |
|
Acute tubular necrosis | Severe |
|
|
||
Immunohistochemical variables | ||
IL-6 | None |
|
TNF- |
Mild |
|
Moderate |
| |
MPO | Severe |
|
Immunohistochemical staining was applied to detect the possible expression of inflammatory cytokines like IL-6, TNF-
The buffers, blocking solutions, secondary antibodies, avidin-biotin complex reagents, and chromogen were supplied in a detection kit (EnVision HRP, Mouse/Rabbit detection system (K 5007), DAKO). To inhibit endogenous peroxidase, the specimens were incubated with 3% H2O2 (200 mL H2O and 6 mL H2O2) for 15 min in a dark room. Before the primary antibody was applied, the sections were immersed in 10 mM citrate buffer (pH 6.0), rinsed in tris-buffered saline, and subsequently heated in a microwave oven (650–800 W) for three cycles of 5 min. The slides were washed with tris-buffered saline before application of the primary antibody in order to reduce nonspecific binding of antisera. Control slides were used as common negative controls for all antibody staining. Sections were then briefly counterstained with Mayer’s hematoxylin, mounted, and examined under a Nikon Eclipse 50i microscope (Nikon Instruments Inc, NY, USA).
Scoring was assigned according to the proportion of cells with cytoplasmic staining. The positivity of the expression was determined by counting the number of stained cells. The average labeling index was assessed according to the proportion of positive cells, after scanning the entire section of the specimen. Sections with greater than 10% stained cells were considered as being positive. The results were graded as negative (0) for <10% of stained cells, low (1) for >10% and <30% of cells stained, moderate (2) for >30% and <70% cells stained, and high expression (3) for >70% cells stained (Table
The statistical analysis of the results was completed with the use of the 20th version of SPSS (Statistical Package for the Social Sciences, SPSS Inc., Chicago, IL, USA). We performed an analysis in which the data were treated as qualitative using Fisher’s exact test (this test was preferable to
The operation was concluded successfully on all animals and all animals survived the operation. The animals resumed normal diet and activity with normal bowel function. Six animals died before the predetermined time point for their euthanasia. These animals were all part of the Control group. They were substituted and were not included in the statistical analysis.
The statistical analysis showed that none of the variables followed a normal distribution. Thus, nonparametric tests were used to analyze the results.
Eugenol administration resulted in a lower histological score in rats with acute pancreatitis. The difference between the Eugenol and Control groups is apparent at 48 and 72 hours after induction of pancreatitis (Figures
Different levels of inflammation and the corresponding histopathological score (H&E, ×200). (a) Sham group: score 0. (b) Control group: score 3. (c) Eugenol group: score 5.5. (d) Control group: score 9.
Comparative results of histopathological analysis (statistically significant differences are marked by arrows). (a) Histological score. (b) Hyperemia and dilatation of renal parenchymal capillaries. (c) Hyperemia and dilatation of renal corpuscles capillaries. (d) Edema. (e) Acute tubular necrosis.
Eugenol administration lowers hyperemia and dilation of renal parenchyma capillaries and the difference was statistically significant for the 48 and 72 hour time points and for the whole sample. The Eugenol group exhibited lower values than the Control group and both exhibited higher values than the Sham group. The same was true for hyperemia and dilation of renal corpuscles capillaries for the 48- and 72-hour time points, but not for the whole sample.
There were no inflammatory infiltrations in any of the animals in our experimental model and measurement of this factor did not produce any results.
Edema was reduced through the administration of eugenol and, again, the difference to the Control group was significant for the 48- and 72-hour time points and the whole sample. The Control group had higher values than the Sham group at 48 hours and also higher values than both the Sham and Eugenol groups at 72 hours. When values of the whole sample were considered, the Control group had higher values than the Eugenol group, which in turn had higher values than the Sham group.
Finally, eugenol did not reduce acute tubular necrosis in our experimental model. There was no statistically significant difference at any of the time points studied. Analysis of the whole sample showed only higher values for the Eugenol and Control groups when compared to the Sham group.
There was no clear difference regarding IL-6 expression between the different groups (Figures
Different levels of immunohistochemical staining expression (×200). (a) Control group: mild expression of IL-6 after 72 hours. (b) Eugenol group: moderate expression of IL-6 after 72 hours. (c) Sham group: no expression of TNF-
Serum urea and creatinine and immunohistochemical staining results (statistically significant differences are marked by arrows). (a) IL-6. (b) TNF-
Eugenol administration resulted in lower serum levels of urea and creatinine especially at the 48- and 72-hour time points, compared to the Control group. Urea and creatinine levels were higher for both the Eugenol and Control groups, when they were compared to the Sham group (Figure
The results of this study suggest that eugenol attenuates the intensity of the histopathological changes and the expression of TNF-
To evaluate the extent of kidney injury, we decided to evaluate serum urea and creatinine levels and the histopathological changes in the kidney, as well as the expression of TNF-
The histopathological evaluation showed that the histologic score was lower for the Eugenol group in comparison to the Control group at 48 and 72 hours from the initiation of the inflammatory process (means: 3.75/6.5 and 4.12/7.62, resp.) and this difference was statistically significant. This difference between the two groups was also present for individual histological changes such as hyperemia and dilation of renal parenchyma and renal corpuscles capillaries and edema. The difference observed in the degree of acute tubular necrosis and inflammatory infiltration was not statistically significant.
Regarding the expression of inflammatory mediators, TNF-
We chose the bile-pancreatic duct ligation model as it is a well-characterized model of acute pancreatitis, which mimics acute pancreatitis caused by biliary obstruction, which is a frequent clinical scenario and results in multiorgan failure similar to that observed in humans [
Eugenol has been shown to possess a multitude of pharmacological effects [
A number of authors have proposed strategies to reduce kidney injury caused by acute pancreatitis. Zhang et al. have tried dexamethasone administration in an experimental model of retrograde injection of sodium taurocholate in the pancreatic duct [
There are some limitations to our experimental protocol. The half life of eugenol in the rat has been determined to be 18,3 hours [
In conclusion, the administration of eugenol in a rat model of acute pancreatitis was protective for the kidneys in our experimental model. Further research is necessary to determine the possible role of eugenol in the management of acute pancreatitis.
The authors alone are responsible for the content and writing of the paper.
The authors report no conflict of interests.
This study was funded with a scholarship by the Experimental Research Center ELPEN Pharmaceuticals (ERCE), Athens, Greece, which also provided the research facilities for this project. The authors would like to thank A. Zacharioudaki DVM, MLAS, E. Karampela, MSc, K. Tsarea, M. Karamperi, N. Psychalakis, A. Karaiskos, S. Gerakis, and E. Gerakis, staff members of the ERCE, for their important assistance during the experiments, and Myrto Kogevina for the editing of the paper.