الفهرس 
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Abstract
The pancreas is a multifunctional organ (a mixed exocrine-endocrine gland that produces both digestive enzymes and hormones) with a firm, lobulated smooth surface, measuring 10–20 cm in length and 75–125 g in weight in an average adult human.
The pancreas is located retroperitoneal at the level of L1 and L2 vertebrae. It can be anatomically divided into four anatomical parts: head, neck, body, and tail, along with one accessory lobe or uncinate process, with the head lying within the curve of the second part of the duodenum.
Roche Pharmaceutical Company has introduced orlistat at the end of 1998 in UK. This drug received wide media coverage and represented as a magic medicine for obesity without pain of dieting. It has been approved by the Food and Drug Administration (FAD) in 1999 for the weight-reduction in critical obesity conditions in conjunction with lifestyle modifications such as sport and low food intake.
Orlistat is a specific long acting reversible lipase inhibitor that acts locally in the lumen of alimentary tract by binding covalently to the serine residue of the active site of gastric and pancreatic lipase enzymes. Thus, digestion and absorption of fat are inhibited leading to oily stool, oily spotting and flatulence.
Beta-carotene belongs to the group of carotenoids consisting of isoprene units. It is the most abundant form of carotenoid and it is a precursor of the vitamin A. It is an antioxidant that can be found in yellow, orange and green leafy vegetables and fruits.
Beta-carotene is FDA approved to be used as a nutrient supplement and to be even added in infant formula as a source of vitamin A. It is also approved to be used as a color additive for food products, drugs and cosmetics.
Materials and methods:
Drugs
- Orlistat: It is available in the form of capsules with a trade name (Xenical 120 mg) manufactured by Hoffmann La Roche, Germany. The content of each capsule was evacuated and then the human therapeutic dose of orlistat (360 mg/day) was converted to animal dose.
- B-Carotene: It was purchased as a capsule with a trade name (Beta carotene forte) produced by Arab company for gelatin and pharmaceutical products for (MEPACO-MEDIFOOD). Each capsule contains natural B- carotene 15 mg (equivalent to natural vit. A 25000 IU).
Animals
Fifty adult female albino rats, weighing 150-200 gm were employed in the present study. They were housed in four stainless steel cages in clean well-ventilated room. They were allowed free access to laboratory rat chow diet and water ad-libitum. Strict care and hygiene were taken to maintain healthy environment for all rats all the time. The general conditions and behavior of the animals were noticed. All animals’ protocols were approved and observed via the Animal Care Committee of the Research Laboratory of Experimental Animals at the College of Medicine, Menoufia University, Egypt.
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Experimental protocol
The animals were divided into four main groups.
group I (control group): included 10 rats, they received 1ml distilled water daily by oral gavage for 5 weeks.
group II (B- carotene treated group): included 10 rats, they received B- carotene at a dose of 0.52 mg/kg/ d by oral gavage for 5 weeks
group III (Orlistat treated group): included 20 rats, they received a therapeutic dose of Orlistat 32 mg/kg/d dissolved in 1 ml distilled water, orally for 5 weeksthen 10 rats were scarified (subgroup IIIa) and the others 10 rats were left without treatment for another 5 weeks, withdrawal group (subgroup IIIb).
group IV (Orlistat and B- carotene treated group): included 10 rats, they received B-carotene as group II one hour before the administration of orlistat as group III a for 5 weeks.
All over the experiment, the animals were noticed for food habits and physical activities. At the end of each detected period, the animals were weighted and sacrificed by i.p. injection of 50 mg / kg pentobarbital sodium. Blood samples were collected into heparin coated tubes and centrifuged for 1 min. plasma samples were stored at -20 for biochemical study. The pancreas of each rat was dissected out and divided into 2 parts. One part was fixed in 10% buffered formalin overnight for histological and immunohistochemical study. The other part was cut into small pieces and rapidly fixed in 1% phosphate buffered glutaraldehyde, then processed for electron Microscopic study.
I- Biochemical study:
Blood glucose level
Plasma glucose levels were estimated by utilizing a commercial glucose Colorimetric Assay Kit. It was performed in the central laboratory, faculty of medicine, Menoufia University.
II- Histological study
Tissue samples fixed in 10 % buffered formalin were processed to get the ordinary paraffin blocks. Sections of 5-6 μm thick were cut and stained with Hematoxylin & Eosin stain (Hx&E) to show the histological structure and Mallory trichrome stain to detect collagen fibers.
III- Immunohistochemical study
Inducible nitric oxide synthase immunostaining (iNOS):
Paraffin sections were incubated with inducible Nitric oxide synthase (iNOS) rabbit polyclonal antibody. Negative control sections were processed by replacing the primary antibody with buffer alone. Human liver tissue was used as a positive control.
Insulin immunostaining:
Paraffin sections were incubated with anti-insulin antibody; insulin Ab-6 (INS04 + INS05) Mouse Monoclonal Antibody. Negative control sections were processed by
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replacing the primary antibody with buffer alone. Pancreas was used as a positive control.
IV- Electron microscopic study:
Parallel small (1×1 mm) sized pieces of pancreatic tissue were fixed at 4°C phosphate buffered gluteraldehyde solution (pH 7.4) for 4 h. rinsed three times with phosphate buffer (two changes) and post fixed in 1% aqueous buffered osmium tetroxide at room temperature for 2h. After that, the specimens were dehydrated in ascending grades of alcohol, and embedded at the apex of inverted polythene beam capsule filled with liquid resin. The sections were cut using ultramicrotome into semithin (0.5 μm thickness) and ultrathin sections. The semithin sections were stained with toluidine blue for detection of secretory granules. Ultrathin sections (80–90 nm) were stained with uranyl acetate and lead citrate to be examined by Transmission Electron Microscope at faculty of medicine, Tanta University.
V- Morphometric and Statistical analysis
By using image analyzer system (Image J 1.47v national institute of health, USA) we measured the intensity of the brown color of anti-insulin immune expression at 400×magnification of 5 non-overlapping fields from randomly selected light microscopic slides stained with anti-insulin monoclonal antibody per group. The number of zymogen granules was counted at five fields of randomly selected electron microscopic slides per group.
The collected data were analyzed and compared by student’s t-test. The p-value was utilized to test the significant change in the experimental groups in each parameter in comparison with the control group. The data were tabulated as mean ± SD and analyzed utilizing statistical package for the Social Science Software (SPSS). P value was set at 0.05, P>0.05 non-significant, P value<0.05 significant and P value <0.001 highly significant.
Results
I- General appearance of animals
group I (control) and group II (B- carotene treated):
All animals of these groups were in a good general condition and showed normal behavior, activity and appetite.
Orlistat treated subgroup (Subgroup IIIa):
Throughout the experiment, the animals treated with Orlistat became less active. Diarrhea accompanied by a decrease in body weight was noticed.
Withdrawal subgroup (Subgroup IIIb):
Animals of this group showed improvement in their activity. Also they showed apparent decrease in diarrhea.
Orlistat and B-carotene treated group (group IV):
The animals of this group showed good general condition and normal activity. Diarrhea accompanied by a decrease in body weight was also noticed.
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II- Light microscopic results:
Hematoxylin & Eosin stained sections of pancreas of group I (control) and group II (B- carotene treated): were the same and revealed different sized lobules of closely packed serous acini which lined by pyramidal cells. The cells had apical cytoplasmic acidophilia and basal basophilia with rounded vesicular nuclei. Intra lobular duct lined by cubical epithelium with rounded nuclei was observed. Islet of Langerhans showed central cells with acidophilic cytoplasm and rounded vesicular nuclei.
Sections of pancreas of subgroup III a (orlistat treated subgroup) exhibited disturbance in the normal pancreatic architecture. The pancreatic lobules were separated by wide spaces. Some acini were distorted and acinar cells displayed faint basal basophilia. And intracytoplasmic vacuoles, also, some nuclei were deeply stained and pyknotic. Dilated ducts with retained secretion were observed. Many dilated congested blood vessels with inflammatory cellular infilterate were seen. The islet cells appeared with deeply acidophilic cytoplasm and dark pyknotic nuclei.
Sections of pancreas of subgroup III b (withdrawal subgroup) displayed some improvement in the form of restoration of normal pancreatic architecture and most of the acinar cells and their nuclei appeared more or less similar to that of control group. But some acini still revealing disappearance of basal basophilia and others still revealing vacuolated cytoplasm and pyknotic nuclei. There were wide spaces inbetween acini.
group IV (Orlistat and B-carotene treated group) showed good improvement in structure of pancreatic tissue as revealed by restoration of the normal pancreatic architecture with nearly normal acinar cells.The ducts and their linning epithelium appeared nearly normal .The islets of langerhans appeared normal but some cells with small pyknotic nuclei in the center of the islet were observed.
Semi thin sections of groups I& II (control &B- carotene treated groups) revealed abundant apical zymogene granules in acinar cytoplasm. While subgroup III a (orlistat treated subgroup) exhibited apparent decrease in zymogene granulesin some acini and abscense of these granules in others. Subgroup III b (withdrawal subgroup) revealed restoration of zymogene granules in some acinar cells.moreover, group IV (orlistat and B-carotene treated group) revealed abundant zymogene granules in acinar cytoplasm.
Mallory trichrome stainedSections of pancreas of groups I& II (control and B- carotene treated) revealed minimal amount of collagen fibers in the connective tissue septae and around pancreatic acini and duct. While subgroupIII a (Orlistat treated subgroup) exhibited large amount of collagen fibers between pancreatic lobules, around congested blood vessels and duct. Subgroup III b (withdrawal subgroup) revealed decrease in the amount of collagen fibers inbetween acini and surrounding ducts and blood vessels. group IV (orlistat and B-carotene treated group) displayed minimal amount of collagen fibers in between pancreatic acini and around duct.
Regarding iNOS marker expression, pancreatic tissues obtained from groups I&II (contral and B- carotene treated groups) were similar and displayed negative cytoplasmic immune reactivity for iNOS. SubgroupIII a (Orlistat treated subgroup)
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revealed strong positive immune reactivity in the acinar cytoplasm and cytoplasm of B- cells of islet of langerhans. While subgroupIII b (withdrawal subgroup) displayed weak positive cytoplasmic immune reactivity in acinar cells and negative reaction in cytoplasm of islets cells. group IV (orlistat and B-carotene treated group) showed negative cytoplasmic immune reaction.
Regarding anti-insulin marker expression pancreatic tissues obtained from groups I&II (contral and B- carotene treated groups) were similar and revealed strong positive cytoplasmic immune reactivity in ᵦ -cells of islets of Langerhans. SubgroupIII a (Orlistat treated subgroup) revealed apparent decrease in the immunostaining of in ᵦ-cells when compared with control group. Moreover, subgroupIII b (withdrawal subgroup) displayed strong positive cytoplasmic immune reaction for insulin in ᵦ -cells of islets of Langerhans. group IV (orlistat and B-carotene treated group) showed moderate positive cytoplasmic immune reaction for insulin in ᵦ -cells of islets of Langerhans.
III- Transmission electron microscopic results:
Ultra-thin pancreatic sections of group I &II (control & B-carotene treated groups) revealed the pancreatic acinar cells with rounded basal euchromatic nuclei and prominent nucleolus, numerous parallel cisternae of rough endoplasmic reticulum (rER) occupying the base of the acinar cells. The apical part occupied by many spherical homogenous electron dense zymogene granules. The luminal surface was supplied by microvilli. The centroacinar cells had rounded euchromatic nuclei, lysosomes, mitochondria and junctional complexes between adjacent cells.
An electron microscopic examination of subgroupIII a (orlistat treated subgroup) revealed that most of the acini exhibited irregular shaped nucleus with dilated perinuclear space and dilated rER and an apparent decrease in the number of zymogen granules with variable size and density. Dilated duct with retained secretion and irregularly shaped nuclei of the linning epithelium were noticed.
Examination of pancreatic specimens from sub group III b (withdrawal subgroup) displayed irregular shaped nuclei in some acinar cells, packed cisternae of rER, and apparent decrease in the number of zymogene granules.
While group IV (Orlistat and B-carotene treated group) revealed that most of the acinar cells and their nuclei and organelles were more or less similar to those in the control group.
IV- Statistical results:
1- Body weight:
There was non-significant increase in the mean body weight of B- carotene treated group (group II) as compared to control group (p>0.05), highly significant decrease in the mean body weight of Orlistat treated and protected group (subgroup III a and group IV respectively) as compared to control group (p<0.001) and significant decrease in the mean body weight of the withdrawal group (subgroup III b) as compared to control group (p<0.05).
There was highly significant increase in the mean body weight of the withdrawal subgroup (subgroup III b) as compared to Orlistat treated subgroup (subgroup III a) (p ˂0.001) and non-significant change in the mean body weight of protected group (group
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IV) when compared with Orlistat treated group (p>0.05) and highly significant decrease when compared with withdrawal group (p ˂0.001)
2- Blood glucose level:
There was highly significant increase in mean blood glucose level in treated group (III a) and protected group (IV) compared to control group (p ˂ 0.001). While there was no significant increase in mean blood glucose level of B- carotene treated group and the withdrawal group (III b) compared to control group (p ˃ 0.05)
Moreover, withdrawal group (subgroup IIIb) and protected group (group IV) exhibited a highly significant decrease in mean blood glucose level in comparison with Orlistat treated (subgroup IIIa)(p ˂ 0.001) while protected group (group IV) exhibited a highly significant increase in mean blood glucose level in comparison with withdrawal subgroup (subgroup IIIb) (p ˂ 0.001)
3- Mean intensity of insulin immune expression:
There was non-significant decrease in the mean intensity of the brown color of anti-insulin immune expression of ᵦ- cells of islets of Langerhans in B- carotene treated group (group II) (p ˃0.05) as compared to control group. There was highly significant decrease in the mean intensity of the brown color of anti-insulin immune expression in Orlistat treated subgroup (subgroup III a) (p˂0.001) as compared to control group. While, there was non-significant decrease in the mean intensity of the brown color of anti-insulin immune expression in the withdrawal subgroup (subgroup III b) (p˃ 0.05) as compared to control group. Also, there was highly significant increase as compared to the treated group (p˂0.001). The protected group showed highly significant decrease in the mean intensity of the brown color of anti-insulin immune expression as compared to control group (p˂0.001). While, there was highly significant increase in withdrawal subgroup and protected group as compared to the treated group (p ˂0.001).The protected group showed significant decrease of the brown color of anti-insulin immune expression as compared to the withdrawal group (p ˂0.05)
4- Mean number of secretory granules:
There was non-significant increase in the mean number of secretory granules in B- carotene treated group (group II) (p ˃0.05) as compared to control group. There was highly significant decrease in the mean number of secretory granules in Orlistat treated subgroup (subgroup III a) (p˂0.001) as compared to control group. There was significant decrease in the mean number of secretory granules in the withdrawal subgroup (subgroup III b) (p˂0.05) as compared to control group. Also, there was highly significant increase as compared to the treated group (p˂0.001). The protected group (group IV) showed non- significant decrease in the mean number of secretory granules as compared to control group (p˃0.05).
The protected group showed highly significant increase as compared to the treated group (p˂0.001) and significant increase as compared to the withdrawal group(p˂0.05)