Effect of pancreatin on acute pancreatitis resulting from L-arginine administration in mice, a morpho-histopathological and biochemical study

Abstract Acute pancreatitis (AP) is a life-unpleasant situation with contradictory and inadequate treatments. In this regard, the present study evaluated the effect of the possible pretreatment of lipase-pancreatin on L-arginine-induced AP. Forty adult mice were selected and divided into five groups: I) control group, II and III) AP groups (i.p.) receiving L-arginine of 2×300 and 2×400 mg/100 g body weight (b.w.), IV) AP (2×300 L-arginine) group + pancreatin (mice were i.p. injected by 350 U-lipase), and V) AP (2×400 L-arginine) group + pancreatin (mice were i.p. injected by 350 U-lipase). All AP groups displayed a significant increase in serum levels of ALT, AST, TBARS, and TNF-alpha compared to the control group. Moreover, pancreatic tissue edema, inflammation, and vacuolization of acinar cells were significantly higher in the untreated L-arginine group compared to the control and pancreatin groups. Conversely, the diameter of pancreatic islets significantly declined after induction of pancreatitis compared with control and pancreatin groups. Pancreatin treatment can be used in pancreatic dysfunction, however, this medicine showed no protective effect against L-arginine-induced AP in the mouse model.


INTRODUCTION
AP is a serious health-threatening disease whose incidence rate has gradually increased worldwide (Abdelzaher et al., 2020;Mederos, Reber, Girgis, 2021;Mirmalek et al., 2016).Systemic inflammatory response and limb dysfunction syndrome resulting from AP eventually lead to fibrosis and subsequent organ failure (Abu-Hilal et al., 2006;Melo et al., 2010).Complexity in the pathophysiology of AP shows the involvement of numerous inflammatory pathways in the occurrence of this disease.Many reasons suggest the fundamental role of oxygen radicals in the pathophysiology of AP; however, some active sites of reactive oxygen species (ROS) are related to the pathogenesis of AP.ROS are overproduced in AP and inflammatory reactions through tissue necrosis pathways which may eventually lead to increased amylase and lipase activity and zymogen degranulation (Abu-Hilal et al., 2006).Some studies have evaluated the

Effect of pancreatin on acute pancreatitis resulting from L-arginine administration in mice, a morphohistopathological and biochemical study
Page 2/11 Braz.J. Pharm.Sci.2023;59: e21494 Rahmat Allah Fatahian Dehkordi, Iraj Karimi, Behnaz Karimi, Reyhaneh Ghasemi Eshkaftaki, Reza Abtahi, Hanieh Mohammadi relation between pancreatic inflammation and oxidative alterations.These findings show the incidence of pancreatic oxidative stress during the early stages of AP induction.Oxidative stress is severely elicited following the increase in the production of ROS and irregularity created in the antioxidant capacity balance (Abdelzaher et al., 2020;Sindhu et al., 2005;Vaziri et al., 2007).
It has been assumed that free-radical oxygen scavengers have a clinically protective effect and could be efficient in patients with AP (Fusco et al., 2020;Swentek, Chung, Ichii, 2021;Zhang et al., 2012).Oxidative stress caused by acute pancreatitis accelerates the inflammatory process and disturbs cellular metabolism regulation by activating more complex inflammatory processes, leading to cell death (Gukovsky et al., 2013;Pérez et al., 2015).Experimental investigations have shown the extensive production of pro-inflammatory mediators such as tumor necrosis factor (TNF) -α, nitric oxide (NO), adhesion molecules, and interleukin (IL) -1β, as inflammation precursors, during AP in addition to ROS (Abdel-Gawad, 2015).Inflammatory mediators amplify the inflammatory cytokine cascade and expand AP from localized disease to a problematic systemic disease (Bhatia et al., 2000;Makhija, Kingsnorth, 2002).
The possible benefits of the pancreatic enzyme supplementation (pancreatin) in managing exocrine pancreatic insufficiency, acute and chronic pancreatitis, pancreatectomy, pancreatic duct obstruction, and pancreatic cancer have been indicated (Ianiro et al., 2016;Johnson, Hillier, 2011).
The first L-arginine induction model in mice (2×4 g/1000 g, i.p.) was experimentally proposed by Dawra et al., (2007) for the development of AP.L-arginine (a type of amino acid) has been increasingly used as a model to induce AP due to its unique properties such as cost-effectiveness and non-invasiveness as well as a feasible injection for AP induction (Dawra, Saluja, 2012;Kui et al., 2014).L-arginine can stimulate the production of oxygen and nitrogen free radicals and eliminate the cell membrane of zymogen granules, thus increasing the secretion of digestive enzymes and the rate of inflammatory mediators (El-Ashmawy et al., 2018).
Stereology is the first selective method capable of predicting three-dimensional information of structural values (volume, surface, diameter, length, and number) in different tissue components (Fatahian Dehkordi, Hamid, 2015;Mayhew, 1991;Mayhew, 1992).Qualitative variables such as hypoplasia or hypertrophy and atrophy can be studied through stereological techniques and expressed as quantitative data.Therefore, the values obtained from the stereological evaluation can be explored to provide information.
Regarding the recently documented effect of pancreatic enzyme supplements on the outcome of acute pancreatitis (Kahl et al., 2014), the present study is aimed to investigate the effect of pancreatin on L-arginine-induced AP by determining the levels of oxidative stress parameters (TBARS, AST, and ALT) along with the assessment of pancreatic morpho-histopathological changes.

Animals
Forty adult male BALB/C mice weighing 28-32 g was used in this study.The animals were fed by commercial mouse food and tap water, and in a standard temperature (around 21 °C) and humidity conditions (21-33%) with a 12-12 h light-dark-cycle were maintained.To create a suitable environment during the test period, the cage f loors were covered with fresh and soft sawdust and the cages were cleaned every three days to maintain hygiene.Mice were randomly divided into the experimental groups described as follows.

Material
L-arginine hydrochloride powder (Sigma, UT, USA) was dissolved in 0.9% saline and set at a pH=7.4 with 0.1 M sodium hydroxide (NaOH) solution.Before each injection, the fresh solution was prepared from L-arginine.The mouse model of acute pancreatitis was developed by intraperitoneal injections (i.p.) of L-arginine hydrochloride.Two different doses of 2×300 and 2×400 mg/100 g of L-arginine were established for administration to mice via i.p. injection (at an interval of 2 h).Control animals were injected i.p. as a placebo with normal saline.Several samples were selected and after tissue slide preparation, pancreatitis was confirmed by a pathologist microscopically.
At the end of the experiment and 6 hours after the last treatment, blood samples were collected from the heart under general anesthesia.Blood sampling was performed to determine levels of TNF-alpha, ALT, AST, and TBARS.The samples were centrifugated for 10 minutes at 3000 rpm at 4 °C using a Universal centrifuge (Hettich, Tuttlingen, Germany) set.The obtained clear sera were stored at -70 °C until use.In addition, pancreatic tissue was removed by laparotomy after dissection of surrounding tissues.Specimens trimmed and fixed in 10% neutral buffered formalin solution for histopathological examinations and the microscopic serially slides were stained with H&E.Finally, the pathological analysis was performed under light microscopy by an expert pathologist.

TNF-α assay
The serum TNF-alpha concentration was determined by enzyme-linked immunosorbent assay using Mouse/ Rat TNF-α ELISA Kit (Diaclone SAS, mAbexperts group Company -France).The kit includes an assay range of 31.25 pg/ml-1000 pg/ml and a highly sensitive 25 pg/ml.

Diameter of pancreatic islets histologically
Preparation of tissue sections was performed using morphological techniques.Thus, the quantitative technique was based on the serial sections and according to our previous study (Amiri et al., 2018) on the random selection protocol from sections to measure the diameter of pancreatic islets.The mean diameter of the islets was measured by tracing the cross-sectional boundary of the pancreatic islets with the pancreatic exocrine glands.Since the islands lacked a regular geometric shape, the mean of the smallest and largest diameter was considered as average diameter, and then measured diameter was introduced as the final diameter.

Pathology examinations
Pancreatic histology slides were prepared according to the process of procurement the tissue sections and stained with hematoxylin and eosin.Pancreatic sections were evaluated by an intervening pathologist according to the experiment protocol.The extent of the injuries was interpreted based on edema, inflammation and histopathological changes.

ALT activity assay:
ALT activity was measured by using Pars Azmoon kit (Tehran, Iran).The reaction is evaluated based on the transferring of an amino group from alanine to α-ketoglutarate resulting in the production of glutamate and pyruvate.Then, in parallel reaction, the measurement of the absorbance change of NADH concentration at 340 nm is assayed based on the pyruvate reaction with lactate dehydrogenase (LDH).ALT activity in serum samples was measured as U/L.

AST activity assay:
AST activity was measured by using Pars Azmoon kit (Tehran, Iran).The reaction is based on the reversible transamination between aspartate and α-ketoglutarate to form glutamate and oxaloacetate.AST activity is assayed by monitoring the rate of NADH oxidation at 340 nm in the presence of oxaloacetate and malate dehydrogenase (MDH).AST activity was measured as U/L.

Thiobarbituric Acid Reactive Substances Assay
The measurement of Thiobarbituric Acid Reactive Substances (TBARS) was performed by the method of Olatosin et al., (2014).Briefly, a stock solution of trichloroacetic acid (TCA), thiobarbituric acid (TBA) and Hydrochloric acid (HCl) containing 15g TCA, 0.375g TBA and 0.25N HCl was prepared.2ml of TCA-TBA-HCl mixture was added to the 1 ml of serum.The mixture was placed in boiling water for 50 min, cooled to room temperature and centrifuged at 1000 rpm for 10 min.Thereafter, the absorbance of the supernatant was read at the wavelength of 535 nm against blank reference.TBARS levels were expressed as nmol/ml.

Statistical analysis
To compare data between treatment and control groups, SPSS software version 23 was used.Data analysis was performed using One-Way Analysis of Variance (ANOVA), followed by LSD test as a post-hoc test and were calculated as means ± SD.Significant differences were observed between the groups at a 95% probability level with a significant level of P<0.05.

Diameter of Pancreatic islets
In this investigation, the mean diameter of islets (μm) showed a statistically significant difference among the groups as shown in Table I.L-arginine groups (2×300 and 400) significantly reduced the mean diameter of islets by 103.97±24.8 and 121.89±23.6 respectively compared to the control group 149.24±23.5 (P<0.05).Whereas, experimental groups treated with pancreatin (350 units, groups IV and V) exhibited higher diameter of islets compared with untreated pancreatitis groups (groups II and III); however, there was no statistically significant difference between groups IV and V compared to groups II and III (p>0.05)(Figure 1).

Histopathological changes
As shown in figures 2 and 3, histopathological appraisal of the pancreatic tissue from treated and untreated L-arginine groups (groups II to V) showed fluid accumulation, disturbance of tissue architecture, vacuolization of acinar cells, edema and infiltration of inflammatory cells (neutrophils).The observations also showed that the pancreatic islets appeared to be smaller (shrinkaged) compared to the control group.Besides, there were mild histopathology changes in the pancreas in L-arginine plus pancreatin-treated groups.

TNF-alpha level
The progression of primary injury to the exocrine pancreas occurs in the local and systemic inflammatory response, resulting in AP (Hegyi et al., 2011).Hence, the serum level of pancreatic TNF-α was determined using ELISA in all experimental groups.It was shown that the blood serum TNF-α level of mice (Figure 4) with AP (groups II and III) were significantly increased in comparison with the mice in the control group (p<0.05).Pancreatin in treated groups (the last two groups) decreased the serum TNF-α level of mice with AP, although, these differences were not significant (P>0.05).In addition, it was found that there was a significant difference between the pancreatitis groups treated with pancreatin compared to the control group (P<0.05).

ALT and AST
According to Table I, comparison of the mean serum levels of ALT and AST between the different groups showed that parameters mentioned were significantly increased in the L-arginine groups 2×300 and 400 (group II and III) compared to the other groups (P<0.05).Nevertheless, the levels of ALT and AST parameters when were compared among the groups, showed a decrease nonsignificant in treated pancreatitis groups by pancreatin than to untreated L-arginine groups (P>0.05).Although the pancreatin-treated groups showed a decrease compared to the untreated pancreatitis groups, however, increasing significance in the pancreatin-treated group compared with the control group was quite evident (P<0.05), in fact, pancreatin could not show a healing effect.AP is an invasive inflammation of the pancreas following pancreatic dysfunction.Regarding its increasing incidence, AP is considered common acute damage (El Morsy, Ahmed, 2020;Mirmalek et al., 2016).In this regard, remarkable attention has been paid to cell biology, pathophysiology, and cell damage mechanisms in AP.The present experimental study histopathologically and biochemically evaluated the effect of lipase-pancreatin to show the possible treatment of pancreatin on L-arginineinduced pancreatitis.
The generation of Reactive Oxygen Species (ROS) and as a result, oxidative stress is one of the most important challenges in AP (Pasari et al., 2019).The present study showed a significant difference in serum TBARS, ALT, AST, and TNF-alpha levels in the L-arginine-induced pancreatitis groups as compared with the controls and a non-significant difference in pancreatin treatment groups.As observed in numerous studies, excessive concentrations of amino acids (often L-arginine) can seriously damage the structure of the pancreas through an unknown mechanism (Biczó et al., 2011;Kui et al., 2014).Nevertheless, L-arginineinduced pancreatitis is considered a well-defined pattern in the development of pancreatitis that resembles those of AP in humans (Zhang et al., 2019).L-arginine induces AP as a nitric oxide donor through activation of nitric oxide synthetase, leading to endoplasmic reticulum stress and free radical-induced oxidative stress (Abdelzaher et al., 2020;Aziz, Kamel, Rifaai, 2017).These mechanisms are in line with the findings of other researchers, who introduced pancreatitis-induced oxidative stress as the basis of AP disease (Aziz, Kamel, Rifaai, 2017;El Morsy, Ahmed, 2020;Mirmalek et al., 2016).The pathophysiology of AP is highly complex involving damage to the intracellular and extracellular structure; the generation of free radicals results in the development of AP (Affourtit, Jastroch, Brand, 2011;Hernández et al., 2011).The pancreatic tissue is vulnerable to oxidative stress due to the poor expression of antioxidant enzymes in pancreatic islet cells, and hence its poor antioxidant capacity (Miki et al., 2018).Following AP, the oxygen-free radicals disrupt pancreatitis acinar cell damage and play a key role in pancreatic dysfunction (Abdelzaher et al., 2020).
The intraperitoneal injection of L-arginine solution to intact mice showed an increase in serum levels of TBARS, ALT, AST, and TNF-alpha; however, the intraperitoneal administration of pancreatin led to a partial reduction in

TBARS
Table II, displays that in serum isolated from the untreated pancreatitis mice (groups II and III), TBARS values were significantly higher than the control group and those from pancreatin-treated mice (groups IV and V).Whereas, pancreatin treatment decreased TBARS values non-significantly in serum from the treated pancreatitis mice compared with the untreated pancreatitis mice (P>0.05).Despite the decreasing effects of pancreatin treatment groups in TBARS values compared to L-arginine-induced pancreatitis groups, however, there was still a significant increase in TBARS values in pancreatin treatment group compared to the control group (P<0.05).
the mentioned parameters (P>0.05).Collectively, it does not seem that pancreatin can even partially control the destructive effects of pancreatitis; thus, it does not play a significant role in reducing the effects of antioxidant disruptors in this study.Following the induction of AP, microvascular failure in the pancreas leads to ischemia and inflammation (Yenicerioglu et al., 2013).With the increase of the inflammation severity, the accumulation of toxic mediators becomes noticeable in the pancreatic tissue.Monocytes and macrophages begin to secrete cytokines with the spread of inflammation.Reactive oxygen intermediates (ROI) generated by macrophages also contribute to the increased oxidative stress, an additional destructive intermediary in the increasing aggressiveness of pancreatitis (Shrivastava, Bhatia, 2010).Therefore, plasma levels of some cytokines are elevated in patients suffering from severe AP (Hu et al., 2020).
Given this anatomical relationship and the proximity of the bloodstream, the distribution of oxygen free radicals in pancreatitis enters the liver, triggering the process of liver damage (Abdelzaher et al., 2020).According to these findings, it can be inferred that the development of inflammation leads to an increase in the level of AST, and ALT, as well as, an increment in proinflammatory cytokines (TNF-α).TIBARS, a marker of lipid peroxidation, was also enhanced in L-argininetreated mice.Lipid peroxidation produced by steps of the free-radical mediated chain reaction impairs the functional and structural integrity of membranes (Biradar, Veeresh, 2012).This could be due to the accumulation of L-arginine-produced free radicals.Hernández et al., (2011) in their study showed that in severe AP there are high degrees of lipid peroxidation.
The other parameter measured was the elliptical and Feret's diameters for each of the pancreatic islets.The elliptical diameter was a representation of an elliptical model from the diameter of the islands.The Feret's diameter in a structure was the longest distance (largest distance) from a given origin (Dražić, Sladoje, Lindblad, 2016).In the current work, morphometrical assessment of the diameter of pancreatic islets showed a significant reduction in L-arginine-induced pancreatitis groups compared to the control group.L-arginine induced a dramatic decrease in islet diameter in a dose-dependent mode.The pancreatic islets appear to be shrunken and this may be the main reason for the decrease in the diameter of the islets in L-arginineinduced pancreatitis groups.Therefore, L-arginine negatively affects the structure of the pancreas.These findings were accordant with the results obtained from Wahba et al., (2016) investigations, which showed that the diameter of pancreatic islets after L-arginineinduced pancreatitis had reduced.Abdelzaher et al., (2021) also found a significant decrease in the mean diameter of pancreatic islets and concluded that the degeneration of the pancreatic islets is one of the reasons for the decrease in the diameter of the islands.

CONCLUSION
This study was carried out on mice suffering from AP induced by L-arginine and pancreatin administration.The results indicated that pancreatin did not play an impressive role in protecting the pancreas of mice with AP.However, pancreatin showed some remarkably subtractive effects on the evaluated parameters in contrast to the L-arginineinduced pancreatitis group.Administration of pancreatin as an enzymatic-gastrointestinal treatment of pancreatitis requires several clinical trials at different doses and for various durations.In any case, L-arginine administration significantly changed the histopathology evaluation and biochemistry parameters of the pancreas.

FIGURE 2 -
FIGURE 2 -Pancreas photomicrograph in the control group.It shows a normal slide of pancreatic islands and acini without pathologic alterations; (H&E "hematoxylin and eosin", magnification was 20×).

FIGURE 3 -
FIGURE 3 -Pancreas photomicrograph in L-arginine-induced pancreatitis group.A slide showing pancreatitis in the form of a disorder in the architecture of the pancreas, meaningful interstitial edema marked with a black asterisk, necrosis of the acinar cell marked with the black arrow, extensive infiltration of inflammatory cells marked with red arrows, vacuolation of acinar cells marked by bold arrows, small areas of bleeding marked with a square; (H&E "hematoxylin and eosin", magnification was 20×).

TABLE I -
Effect of pancreatin on ALT and AST level (IU/L) in L-arginine-induced pancreatitis mice

TABLE II -
Effect of pancreatin on TBARS level (µm) in L-arginine-induced pancreatitis mice Values with different superscripts differed significant at p<0.05.