Therapeutic effects of ellagic acid on L-arginin ınduced acute pancreatitis 1

PURPOSE: To investigate the therapeutic effects of ellagic acid on L-arginin ınduced acute pancreatitis in rats. METHODS: Thirty-two were split into four groups. Group 1 (control) rats were performed only laparotomy, no drugs were administered. Group 2 (control+EA) rats were administered 85mg/kg EA orally. Rats were sacrificed by cardiac puncture 24 hours after the administration. Group3 (AP) 24 hours after intraperitoneal L-arginine administration, rats were sacrificed by cardiac puncture. Group 4 (EA)-(AP): 85mg/kg EA was administered orally after the L-arginine administration. 24 hours later, rats were sacrificed by cardiac puncture. Serum TNF-α, IL-1β, IL-6, total oxidative status (TOS), total antioxidant capacity (TAC), amylase levels were determined in all groups. RESULTS: Group 3 (AP) rats showed significantly raised TOS level as compared to Group1 (control) rats (p<0.001). Following the EA therapy, a decrease in TOS was observed in Group 4 (AP+EA). TAC levels were significantly raised in the Group 4 (AP+EA) compared to the Group 3 (AP) (p=0.003). Group 3 (AP) showed significantly increased TNF-α, IL-1β and IL-6 serum levels as compared to Group 4 (AP+EA). Histopathological changes were supported our result. CONCLUSION: The healing effects of ellagic acid on inflammatory and oxidative stress were confirmed by histopathological and biochemical evaluations of the pancreatic tissue.


Introduction
Acute pancreatitis (AP) is an inflammatory disease caused by stimulation of inflammatory macrophages, neutrophil penetration, together with development of necrosis in the pancreatic tissue 1 .The mean incidence around the world is 40/100 2,3 .Many studies have shown that cytokines such as IL-1β, IL-6, and TNF-α 4,5 , which are secreted due to ductal obstruction and ductal injury as well as oxidative stress, are involved in the pathogenesis of AP 6,8 .Oxidative stress, through reactive oxygen species, can damage the membrane resulting in lipid peroxidation, and alter the cytosol dynamics leading to early activation of pancreatic digestive enzymes that initiate pancreatic damage.The inflammatory cytokines along with oxidative stress are important in the development of AP and cause local damage in the pancreas and systemic diseases such as ARDS, shock and multi-organ failure 8,9 .
Many preventative agents have been researched; however, an ideal treatment has yet to be found.
Many studies on rats have proven that high dose essential amino acids can damage the pancreas and cause acute pancreatitis.
A semi-essential amino acid, L-arginine, was proven to induce the development of acute pancreatitis and cause IL-6 and TNF-α levels to significantly increase.The increase in these cytokines was hypothesized to occur due to the stimulation of peritoneal macrophages following an intraperitoneal injection of L-arginine or the activation of monocytes/macrophages in severe pancreatic damage 10 .Mizunuma et al. 11 have used this property of L-arginine and administered high dose L-arginine intraperitoneally to develop an experimental model for acute pancreatitis.This model is ideal due to its non-invasive and non-invasive nature and the fact that it causes dose-dependent acinar cell necrosis, allowing studies on the pathogenesis of acute pancreatitis.Szaboles et al. 12 have also used L-arginine to develop pancreatitis from which they have earned good results.EA (ellagic acid) is a known antioxidant that is derivatized from phenolic acid.The chemical components of EA result in its antioxidant activity.The four hydroxyl groups are known to enhance oxidant protection and reduce lipid peroxidation as well as protect cells from acute and chronic oxidative damage.
EA functions acts directly on oxidative stress through the induction of cellular antioxidant signaling systems 15 .Despite its proven antiinflammatory and antioxidant effects, it wasn't shown to have any effect on acute pancreatitis.In this study, we aimed to evaluate the effects of EA that has an antioxidant and antiinflammatory effect on acute pancreatitis.

Methods
This study was conducted with the permission of Dicle Thirty-two adult male Sprague-Dawley rats (250-350g) had free access to water and standart food, were keeped to standard animal care conditions (22±2°C) with a 12:12-h light: dark cycle.They were randomly allocated to four groups as follows: sham, EA, AP and EA-AP groups.Each group consists of 8 rats.Acute pancreatitis was induced by 2 doses of 250 mg/100 g of L-arginine (Sigma Chemical) prepared with 20% 0.15 M NaCl and administered intraperitoneally at a 1 hour interval.
Group 1: Sham Group: Intraperitoneal saline was injected at a dose of 1ml per rat.24 hours after saline administration, rats were sacrificed by cardiac puncture.No drugs were administered.All surgical procedures were performed using 10 mg/ mL xylazine (Rompun; Bayer) and 50 mg/mL ketamine (Ketalar; JHP), given intramuscularly in the right rear leg of the rats at a dose of 0.25 mL/100 g of body weight before the procedures.24 hours later, experiment was terminated.Rats were sacrificed by cardiac puncture.Biochemical and histopathological tissue samples were obtained from all the groups of rats by laparotomy.Blood samples were separated immediately by centrifugation at 4000 rpm for 5 minutes and stored at -20°C for biochemical analysis.Specimens from the pancreas were fixed in 10% formalin, embedded in paraffin and stained with hematoxylin-eosin.

Biochemical assays
In all groups, amylase levels, TNF-α, IL-1β, IL-6, serum total antioxidant capacity (TAC), total oxidative status (TOS) were determined.Total antioxidant capacity (TAC) was performed for each blood sample.Total oxidant status (TOS) and TAC analyses were performed for each tissue sample.TOS Analysis is a fully automatic colorimetric method developed by Erel.In this analysis, color intensity was measured spectrophotometrically.The TOS values of the tissues were calculated as nmol H 2 O 2 equiv./mg protein.TAC analysis method is a fully automatic method developed by Erel 16 and is capable of measuring TAC of the body against strong free radicals 17 .The TAC values of the blood were calculated as μmol Trolox equiv./L,and the TAC values of the tissues were calculated as nmol Trolox equiv./mgprotein.
The serum levels of amylase were determined using the Olympus Autoanalyser (Olympus Instruments, Tokyo, Japan).
The IL-β, IL-6 and TNF-a levels of blood samples were using enzyme-linked immunosorbent assay (ELISA) kit specific for the previously mentioned rat cytokines (Biosource International, Belgium).

Histopathological evaluation
Pathological findings were assessed by one of the author blinded to group allocations.Acute pancreatitis was evaluated and documented in each tissue sections.We histopathologically evaluated edema, acinar cell necrosis, hemorrhage and the degree of inflammation in the pancreas.The scoring system used by Schmidt et al. 18 was used for histopathological evaluation.

Statistical analysis
SPSS 16 package program (SPSS Inc., Chicago, IL, USA) was used for statistical analysis.All data were presented as mean ± SD.Comparisons were made using Mann-Whitney U test and one way ANOVA test.p<0.05 values were considered significant.

Results
Biochemical parameters are compared in Table 1.AP and AP+EA groups were high amylase level.Serum levels of amylase, TNF-α, IL-1β and IL-6 were meaningful lower in the AP-EA group than the AP group (p=0.009,p=0.002, p=0.004, respectively) (Table 1).Serum TAC level increased significantly in AP-EA group than AP group.AP group was the lowest level of serum TAC in all groups (Table 2).Following the EA therapy, a decrease in TOS was observed in AP+EA group (Table 2).AP group was the highest level of serum TOS in all groups (Table 2).
The histopathological evaluation of the AP-EA group showed that edema, necrosis, hemorrhage and inflammation was significantly lower than AP group (Table 3 and Figure 1).inflammatory cytokines in this pathway, especially the secretion of TNF-α, IL-1β, and IL-6 20 .With the mediators it induces (proteolytic enzymes, Phospholipase A2, free oxygen radicals), TNF-α is responsible for the systemic effects.When TNF-α is neutralized, these mediators and amylase levels are observed to decrease and acinar cells begin the healing process 2,3,19 .When studies on TNF-α are examined, it is observed that anti-TNF-α treatment is effective on pancreatic cell healing 19 .In addition, many conducted studies have shown that an increase in IL-1β and IL-6 levels may be precursor markers of pancreatic damage and these levels were seen to decrease after treatment 5 .In this study, EA was seen to correct increased IL-1β, IL-6, and TNF-α levels seen during AP in the AP-EA group, and these results were confirmed by histopathological evaluations as well.Many studies have shown that free radicals were important in the pathogenesis of AP 21 .Due to the high chemical reactivity of the radicals, they interact with many important molecules like lipids, proteins, deoxyribonucleic acid (DNA) and carbohydrates, causing lipid and protein oxidation, DNA breakage, and a dysfunction in the immune system by killing immunity cells and loss of cell function 22 .Free radicals are usually either neutralized or prevented from forming by antioxidants.Depending on the increase of oxidant levels and/ or the decrease of antioxidants, the oxidative/antioxidative balance

Discussion
In this study, we determined that ellagic acid is an effective biochemical and histological therapeutic agent for the treatment of inflammation and oxidative stress in AP.
Despite current therapies, AP still causes serious mortality and morbidity.Approaches that aim to decrease the systemic inflammatory process and oxidative stress have gained momentum, but a consensus has not yet been formed for a drug that administers effective treatment 6,7,18 .
Pancreatitis starts by the cessation of secretion from acinar cells, which causes zymogene granules to accumulate intracellularly.This results in cell damage due to the combination of intracellular lyzozomal enzymes and zymogens.Increased amylase is indicative of acinar cell damage 19 and an important parameter for diagnosis.In our study, amylase was increased in all rats with induced AP (Table 1).
Cathepsin B, a lyzozomal enzyme, activates the precursor, trypsinogen, in the zymogene granules into trypsin, which is followed by the proteolysis of other digestive enzyme precursors (chymotrypsinogen, proleastase, prophospholipase, procolipase, callicreinogen) into active enzymes, resulting in pancreatic tissue damage.Many studies have shown the importance of various pro-shifts in favor of the oxidative system, causing oxidative stress, which is linked to the pathogenesis of many diseases 23 .From aging to cancer, anti-oxidants have drawn attention in the treatment of many diseases 22,23 .The measurement of total antioxidant capacity (TAC) or total oxidant stress (TOS) is much easier, practical, and economically convenient than many other analytical methods 16,17 .
In this study, we observed that TOS levels, an indicator of lipid peroxidation, were increased and TAC levels were decreased in the AP group when compared to the other groups (Table 2).In addition, we determined a marked decrease in TOS levels and a marked increase in TAC levels in the AP-EA group, which was related to the anti-oxidant and free-radical-eliminating effects of EA.Being a strong antioxidant, EA was observed to have a marked beneficial effect on TAC and TOS levels.In many studies, despite proving the antioxidant effects of EA.To our knowledge, this is the first study to shown that EA can reduce lipid peroxidation and increase protection against AP 15 .
In this study, our results show that edema, hemorrhage, severe acinar necrosis, and fibrosis were augmented in the pancreatic tissues of experimental models of rat AP.We also were able to find inflammatory cell infiltration in the inflammed pancreatic sections.
Interestingly, EA treatment resulted in a reduction of inflamed pancreas and lower levels of infiltrating neutrophils in the AP-EA rats pancreas sections.This data represents an important additional mechanism whereby EA protects the pancreas from oxidative and inflammatory damage.
The lack of treatment of pancreatitis leads to mortality and morbidity in acute pancreatitis.Along with many inflammatory cytokines, IL-1β, IL-6, TNF-α, and free oxygen radicals cause increased permeability in vessels and disruption of microcirculation.These functional disorders, unless corrected appropriately, damage intestine barriers which allow the transition of endotoxins and intestinal bacteria into the blood stream, resulting in endotoxemia and multi organ disorder and failure caused by the infection 24 .IL-6 is an early biomarker, which has been determined to cause serious organ failure and mortality in acute pancreatitis.While many cytokines were reported to play a role in ARDS, IL-6 and TNF-α were demonstrated to increase phospholipase A2, cause lung damage by affecting phospholipids and surfactant in the lung, increase vessel permeability in the lung, and result in acute lung damage and ARDS 25,26 .In our study, we determined that EA was significantly beneficial in the treatment of AP through the reduction of serum levels of IL6, IL-1β, and TNF-α in the AP-EA group compared to the AP group.

Conclusions
Ellagic acid was shown to reduce inflammatory and oxidative stress in pancreatitis and cause marked improvements in the fatal damage by these two important mechanisms.The healing effects of EA on inflammatory and oxidative stress were confirmed by histopathological and biochemical evaluations of the pancreatic tissue.Ellagic acid is a drug that decreases the damage following acute pancreatitis in both the systemic response and the pancreatic tissue.

Group 2 :
Ellagic acid (EA) group: 85mg/kg Ellagic acid was administered orally.Rats were sacrificed by cardiac puncture 24 hours after the administration.Group 3: Acute Pancreatitis (AP) group: 24 hours after intraperitoneal L-arginine administration, rats were sacrificed by cardiac puncture.Group 4: Ellagic acid (EA)-Acute Pancreatitis (AP) group: 85mg/kg Ellagic acid was administered orally after the L-arginine administration.24 hours later, rats were sacrificed by cardiac puncture.

TABLE 1 -
Serum biochemical parametres in all the experimental groups.
*Compare groups III to the others goups AP: Acute pancreatitis; EA: Ellagic acid; TNF: tumor necrosis factor-alpha; IL: interleukin All data were expressed as median(min-max)

TABLE 2 -
The Parametres of serum oxidative status in all the experimental groups.
AP: acute pancreatitis; EA: ellagic acid; TAC: total antioxidant capacity; TOS: total oxidant status All data were expressed as median(min-max)

TABLE 3 -
Histological injury criteria in pancreatic tissues in all experimental groups.
AP: acute pancreatitis; EA: ellagic acid All data were expressed as median(min-max).a =X 2 test