Acute pancreatitis is an inflammatory disorder of exocrine pancreas, which carries considerable morbidity and mortality, and its pathophysiology remains obscure . Pancreatic injury is mild in 80–90% of patients who recover without complications. The remaining patients may develop severe disease with local complications such as acinar cell necrosis, abscess and remote organ injury, including lung injury .
Gallstones and ethanol account for over 80% of causes of acute pancreatitis. Drugs are implicated in only 2% of cases, whereas hypertriglyceridaemia, hypercalcaemia and infection are rare causes. The identification of a causative agent is important, as removal of it will facilitate recovery and reduce its recurrence .
Orlistat is a powerful inhibitor of gastrointestinal lipase, and as such reduces fat absorption . It acts by binding covalently to the serine residue of the active site of gastric and pancreatic lipases. When administered with fat-containing foods, orlistat partially inhibits the hydrolysis of triglycerides, thus reducing the subsequent absorption of monoglycerides and free fatty acids .
The use of orlistat has been associated with several mild-to-moderate gastrointestinal adverse effects, such as oily stools, diarrhoea, abdominal pain and faecal spotting. A few cases of serious hepatic adverse effects (cholelithiasis, cholostatic hepatitis and subacute liver failure) have been reported. There have been several reports of pancreatitis associated with the use of this drug [6,7].
Nitric oxide (NO) is a gaseous neurotransmitter, a vasodilator and paracrine regulator. In the pancreas, it regulates the normal pancreatic exocrine and endocrine secretions in addition to regulation of pancreatic microvascular blood flow. NO has multiple species, and is produced de novo by three NO synthase enzymes; endothelial NO synthase (eNOS), neuronal NO synthase and inducible NO synthase (iNOS). The eNOS reduces the severity of the initial phase of experimental acute pancreatitis .
In the pancreas, eNOS and neuronal NO synthase are constitutively expressed in the vessels and in the neurons, respectively. However, eNOS was weakly expressed in the acini . Both types are calcium-dependent enzymes with a low-level of NO output, which is known to be a key mediator in normal and inflamed pancreata .
Overproduction of NO by induction of the iNOS is an important factor in the haemodynamic disturbances of several inflammatory states . NO is synthesized from L-arginine by constitutive enzymes. It is important for nonspecific host defense, helping to kill tumours and intracellular pathogens. However, cytotoxicity can result due to its massive formation . Its physiological actions are mainly mediated by cyclic guanosine monophosphate . In the pancreas, intracellular cyclic guanosine monophosphate concentration is increased in response to certain secretagogues, such as cholecystokinin. NO thereby plays an active role in pancreatic secretion. Outside the pancreatic cells, NO plays a part in the control of systemic blood pressure and active dilation of blood vessels, and is additionally involved in platelet aggregation, leukocyte activation and adhesion [10,14,15].
The prevalence of obesity has markedly increased worldwide, and many people are interested to reduce their weight by the nonprescription administration of orlistat. As many cases of pancreatitis are clinically found in patients treated with orlistat, this study aimed to study the histological changes that may occur in the pancreas of adult female albino rats after treatment with orlistat, and evaluate the possibility of recovery after its withdrawal. Also, the distribution of iNOS was studied immunohistochemically in an attempt to verify the mechanism of pancreatic affection.
Materials and methods
This study was carried out on 48 adult female albino rats, weighing 180–200 g each. Animals were housed in clean, properly ventilated cages under the same environmental conditions, and were fed on a standard laboratory diet. The female rats were used in this research because the prevalence of obesity is more in female than in male rats .
All female rats were kept in separate cages for 1 month to be sure that all rats were not pregnant. The experiment was approved by the Local Ethics Committee of the Faculty of Medicine, Tanta University. The animals were divided into three equal groups:
Group I (control group)
Group I was subdivided into two equal subgroups; animals of the first subgroup were kept without any treatment throughout the whole period of the experiment, and the other subgroup received 1 ml of fish oil (the vehicle for orlistat) orally by a gastric tube once daily for 8 weeks.
Group II (treated group)
Group II received orlistat dissolved in fish oil  orally by a gastric tube at a dose of 32 mg/kg/day for 8 weeks. The dose was calculated after applying the interspecies dosage conversion scheme by Paget and Branes  on a daily human dose. Orlistat was purchased as a capsule with a trade name (Xenical) manufactured by Hoffmann-La Roche, Germany.
Group III received the same dose of orlistat by the same route and duration as in group II, and was left for another 8 weeks without treatment.
Two rats of group II and one rat of group III died through the period of this study. At the appropriate time, the rats were killed, and the pancreas was dissected out and divided into two halves by longitudinal section. One half was processed for light microscopic examination, whereas the other was processed for electron microscopic examination.
For light microscopy, the specimens were fixed in a 10% formalin solution for 24 h, and were processed and embedded in paraffin wax by routine protocol. Five micrometre-thick sections were obtained for
- (1) Haematoxylin and eosin  staining.
- (2) Immunohistochemistry in which the paraffin sections were processed for detection of iNOS. After deparaffinization in xylene, the sections were rehydrated in descending grades of alcohol. Then, the endogenous peroxidase activity was blocked with 3% H2O2 in methanol and the nonspecific binding sites with a protein blocker.
The sections were incubated for 32 min with a 1 : 100 dilution of iNOS primary antibody (Rabbit antirat iNOS polyclonal antibody, Thermo Fisher Scientific, Massachusetts, USA). Then, biotinylated secondary antibody was added at a concentration of 2% for 30 min (37°C), followed by addition of the avidin–biotin–peroxidase complex. Visualization of the reaction was performed using 3,3-diaminobenzidine. The sections were then counterstained with haematoxylin. The iNOS cytoplasmic sites of reaction were stained brown, and nuclei were stained blue. The negative control included sections incubated in the absence of the primary antibody , whereas the lung was the positive control.
Quantitative measurements were carried out using an image analysis system (Leica Qwin 500 C Imaging System Ltd., Cambridge, England) at National Research Centre to measure the mean of optical density of iNOS reaction at magnification of ×400 in five nonoverlapping fields from each animal in all groups. Values were represented as mean±standard deviation. The data were analyzed by unpaired Student's t-test using Minitab software, version 16 for Windows, State College, Pennsylvania. Differences were regarded as significant if probability value, P less than 0.05.
For electron microscopic examination, parallel tissue specimens were fixed at 4C in phosphate buffered 2.5% glutaraldehyde (pH 7.3) for 2 h, rinsed in 0.1 mol/l phosphate buffer and post fixed in phosphate buffered 1% osmium tetroxide for 1 h, then dehydrated in ascending grades of ethanol. After immersion in propylene oxide, the specimens were embedded in an epoxy resin mixture. Semithin sections (1 μm thick) were stained with 1% toluidine blue, and were examined by a light microscope for proper orientation. Ultrathin sections (80–90 nm) were stained with uranyl acetate and lead citrate to be examined by a JEOL electron microscope at 80 kV in Faculty of Medicine, Tanta University, Egypt .
Light microscopic results
Histological examination of all specimens from the control animals (group 1) was similar, and showed the normal histological structure of the exocrine pancreas. It consisted of closely packed pancreatic acini with narrow lumen, and was separated from each other by very little connective tissue septa. Ducts were present in between the acini. Each acinar cell had basal basophilia and apical acidophilia. The nuclei appeared rounded and basal. The centres of the acini were occupied by centroacinar cells that appeared small with oval euchromatic nuclei and a pale staining cytoplasm (Figs 1 and 2).
Compared with control specimens, examination of orlistat-treated animals (group II) showed that 11 rats from the 14 rats that completed the experiment had variable histological changes. Most of the specimens were affected, and revealed focal lesions in the form of variable degrees of cellular degeneration. These lesions lead to disturbance of the normal architecture of the pancreas.
There was an increase of interlobular connective tissue with dilated congested blood vessels, which was observed to contain many leukocytes (Fig. 3). Areas of highly vacuolated acini were also noticed. The acini were separated by wide spaces that may be due to interstitial oedema (Figs 4 and 5). Focal areas of acinar degeneration that were completely destroyed leaving empty spaces were detected (Fig. 6). In some areas, there was decrease in the basal basophilia and apical acidophilia with cellular infiltrate around the blood vessels (Fig. 7).
As regards animals of group III, most specimens showed preserved normal pancreatic architecture, and most acinar cells and their nuclei appeared more or less similar as in control group (Fig. 8).
The iNOS immunohistochemical-stained sections of the control group showed that the immunoreactions were abolished in the negative control specimens when the antibody was replaced with normal rabbit serum. By the use of affinity-purified anti-iNOS antibody in control pancreas sections, iNOS was not demonstrated in the serous acinar cells (Fig. 9).
The iNOS immunohistochemical-stained sections of the orlistat-treated group showed that most of the acini revealed marked cytoplasmic reaction in the form of darkly brown granules compared with those of the control group (Fig. 10). The immunohistochemical-stained sections of the recovery group showed that most of the acini revealed reduced iNOS expression compared with those of the orlistat-treated group, but was more when compared with the controls (Fig. 11).
Using an Image analyzer, quantitative assessment revealed a significant increase (P<0.05) in the mean optical density of iNOS in group II in which the animals were treated with orlistat compared with control animals. However, there was a nonsignificant difference (P>0.05) in the mean optical density of iNOS in group III compared with controls (Table 1).
Electron microscopic results
Sections from the control group revealed that the exocrine pancreatic acinar cells contained rounded basal nuclei with dispersed chromatin and prominent nucleolus surrounded by numerous parallel-arranged cisternae of rough endoplasmic reticulum (rER). RER is characteristic of these cells. The apical part occupied by many variable-sized zymogene granules that appeared as spherical and homogenously and densely stained. Mitochondria were present in the basal part of the cell and in between the RER. The luminal surface of the acinar cells was provided with numerous microvilli (Fig. 12).
With regard to the animals of group II that received orlistat, most of the acini showed affection of cytoplasmic organelles in the form of dilated RER and destroyed mitochondria. With regard to nuclear changes, they were in the form of irregularly shaped nucleus with dark clumped chromatin (Figs 13 and 14).
Other acinar cells showed an apparent decrease in the number of zymogene granules with variation in their size, shape and density. Some of these granules revealed peripheral dissolution, which appeared in the form of empty vacuoles with central electron-dense content, whereas others were completely empty. Secondary lysosomes of variable size and shape were found. Some inflammatory cells were observed between the acinar cells. These cells were macrophages and lymphocytes (Figs 15 and 16).
Examination of pancreatic specimens from group III showed that most of the acinar cells and their organelles and nuclei appeared more or less similar to those in the control group excluding some mitochondria that were still degenerated (Fig. 17).
This study revealed that orlistat induced histological and ultrastructural changes in the exocrine part of the pancreas. In accordance with these results, it has been reported that the use of orlistat was associated with pancreatitis in some cases clinically. It was reported that the most obvious links between orlistat and pancreatitis seem to be due to cholelithiasis and increased alcohol consumption by obese participant. However, orlistat was associated with acute pancreatitis with no evidence of biliary disease in many cases [6,7].
In this study, light microscopic examination of the pancreas of orlistat-treated rats showed focal lesions in the pancreatic acini. Disturbance of normal architecture of the pancreas, variable degrees of acinar destruction, vacuolization of the acinar cells and inflammatory cellular infiltrate were found. The same results were observed in a previous study and were described as pancreatitis .
Dilation and congestion of the blood vessels, which were detected in this study, were explained by excess production of NO arising from inducible NO synthase, which may be an important factor in the systemic and local homodynamic disturbances [11,23].
With regard to the result of immunostaining, this study revealed strong positive reaction of iNOS in pancreatitis in orlistat-treated rats. This finding was reported by many other investigators who detected that NO and other free radicals take part in the aggravation of oxidative stress and play important roles in the pathogenesis of acute pancreatitis [24,25]. In addition, other researchers documented that iNOS is expressed at an increasing rate in inflammatory processes [26,27].
Inducible NOS is not a normal cellular component, but it can be expressed in a wide variety of cells, and generate a large amount of NO. Excessive production of NO causes vasodilation and hypotension that is refractory to vasoconstriction. These changes initiate anaerobic metabolism. In addition, reaction of NO with superoxide causes formation of peroxynitrite, which is a powerful cytotoxic agent that may play an important role in cellular damage [11,28].
Inducible NOS activation may induce apoptosis mediators, such as interferon-γ, by a reactive oxygen species-mediated mechanism. The mechanism of NO-mediated apoptosis involves accumulation of the tumour suppressor protein p53, damage of different mitochondrial functions, alterations in the expression of members of the Bcl-2 family, activation of the caspase cascade, and DNA fragmentation. Depending on the amount, duration and the site of NO production, this molecule may mediate apoptosis in the target cells [29,30]. These effects may explain the nuclear and cytoplasmic structural alterations that were detected in this study.
Electron microscopic results in this study confirmed the light microscopic results. These results were in the form of dilated RER and destroyed mitochondria. Similar findings were reported by other investigators in cases of acute pancreatitis [31,32]. The peripheral dissolution of zymogene granules was explained by a defect in the synthesis of the submembranous matrix of zymogene granules, which is involved in granule formation in pancreatic acinar cells. It acts through adhesion of the granular content to its surrounding membrane . Generally, it was documented that the early changes in pancreatitis occur in the acinar cells. These events include dysregulation of the digestive enzyme secretion, premature intrapancreatic trypsinogen activation and activation of necrosis factor leading to upregulation of proinflammatory cytokines. These resulted in inflammatory cellular infiltrate and parenchymal cell death [34,35]. Zymogene granules contain trypsinogen that is appropriately targeted to lysosomes within acinar cells. Thus, trypsinogen will be activated inside the cells rather than the lumen of the duodenum leading to cellular destruction . The acinar cell damage was followed by production of free radicals that may exacerbate cell damage causing lesions of the cell membrane and cytoskeleton, impairing function of intercellular proteins damaging DNA and decreasing the level of antioxidant .
However, the exact mechanism by which the orlistat causes pancreatitis is not clarified. It may be through an immunoallergic reaction or through disturbed gall bladder emptying and cholecystokinin release .
With regard to the recovery group, improvement in the pancreatic tissue after stoppage of the orlistat was noticed. These data are in accordance with the findings detected in case reports of many patients who improved and were discharged from the hospitals [3,39].
It was suggested that orlistat can induce pancreatitis, and hence it must be used under medical observation. Thus, it is necessary to pay more attention to the side effects of orlistat on pancreatic function particularly in patients at risk of pancreatic injury.
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