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Abstract
Orlistat is a specific long-acting reversible lipase inhibitor. It is a member of new class of drugs proposed to be used for long-term treatment of obesity. Many workers had reported the harmful consequences of increased fecal fat excretion on colonic mucosa. So, the current study was designed to evaluate the effect of orlistat on colonic mucosa in presence of other risk factors (directly related to colon carcinogenesis) as high fat diet and colon carcinogen di-methyl hydrazine (DMH).
The experiment was conducted on 60 adult male albino rats with average weight 180 - 200 gm. They were divided into six groups:
Group I : served as the control group. All rats were fed balanced diet. They were divided into two subgroups: Subgroup (Ia): formed of five rats that received no intervention. Subgroup (Ib): formed of five rats that were injected subcutaneously by two equivalent doses of saline, at the beginning of the study and after one week. Thereafter, they were allowed to live for further four weeks.
Group II: included 10 rats which were fed high fat diet for five weeks.
Group III: included 10 rats which were fed high fat diet and received orlistat orally by gastric tube in a dose of 32 mg/kg (therapeutic dose) daily for five weeks.
Group IV: included 10 rats which were fed high fat diet and received orlistat orally by gastric tube at a dose of 96 mg/kg (three times the therapeutic dose) daily for five weeks.
Group V: included 10 rats which were fed high fat diet and were injected subcutaneously by DMH at a dose of 25 mg/kg, at the beginning of the study and after one week, then were allowed to live for further four weeks. The dose of DMH was chosen just to initiate colon carcinogenesis in a way to mimic genetic predisposition.
Group VI: included 10 rats which were fed high fat diet and received DMH (similar to group V) together with therapeutic dose of orlistat (32 mg/kg) daily and orally by gastric tube for five weeks.
After the end of the experiment, animals were anesthetized and distal colons were dissected out.
Paraffin sections were prepared and stained with:
1- Hematoxylin and eosin.
2- Combined Alcain blue – PAS technique.
3- Modified Avidin – biotin immunoperoxidase technique for PCNA.
Statistical study was done to compare the difference in the means of the incidence of aberrant crypts and the PCNA index between different groups.
The present study revealed that rats which were fed high fat diet alone (group II) showed statistically significant increase in the incidence of aberrant crypts. However, the only type of aberrant crypts detected was typical aberrant crypts which appeared having dilated lumen.
By combined Alcian blue-PAS reaction, most crypts showed distended goblet cells with predominance of blue acidic mucin.
Regarding PCNA index, insignificant increase was detected in group II compared to control group. Positively stained nuclei for PCNA remained at the lower third of the crypts.
Administration of therapeutic dose of orlistat together with HFD was not associated with statistically significant increase in the incidence of aberrant crypts and in PCNA index compared to high fat diet alone. Typical aberrant crypts were also the only type of aberrant crypts observed in this group.
A relative increase in the intensity of reaction using combined Alcian blue-PAS technique was noticed.
Interestingly, tripling the dose of orlistat (group IV) resulted in insignificant increase in aberrant crypts and PCNA index as well (compared to group III). However, an additional type of aberrant crypts (hyperplastic type) started to be recognized in group IV but was hardly detected.
By combined Alcian blue-PAS reaction, goblet cells that contained acidic mucin were the most numerous type. In most crypts, goblet cells appeared distended with mucin secretion particularly in the upper part of the crypts. While, few crypts showed a relative decrease in goblet cells number or appeared containing few mucin granules.
Positively stained nuclei of PCNA appeared upwardly elevated reaching up to the half of the crypts.
However, on comparing the increase in PCNA index between group IV and group III, it was statistically insignificant.
DMH together with HFD (group V) was found to cause severe injury to colonic mucosa. In addition, three types of aberrant crypts were detected; typical, hyperplastic and dysplastic. Features of inflammation were more obvious as severe mononuclear cell infiltration, apparent reduction in mucosal thickness with subsequent crypt loss. Branched crypts were frequently detected as well.
Combined Alcian blue – PAS reaction showed scarcity of goblet cells in some crypts with predominance of acidic mucin.
Significant increase in PCNA index was noticed in group V compared to control group. PCNA labeled nuclei reached the upper third in most of crypts.
Moreover, therapeutic dose of orlistat (group VI) was found to have a potentiating effect on DMH and HFD (group V). This was proved by significant increase in colonic cell proliferation and in aberrant crypts incidence (especially of dysplastic type) in group VI compared to group V. Areas of distorted crypt architecture, reduced mucosal thickness, loss of surface epithelium and villiform appearance of mucosal surface were observed frequently.
By combined Alcian blue – PAS reaction, most of crypts showed mucus depletion and acidic mucin predominance.
Since, aberrant crypts formation and increased colonic cell proliferation had been generally accepted as preneoplastic markers for colon cancer, therefore the current study revealed that orlistat, in presence of risk factors as HFD and genetic predisposition, might represent a new risk factor for cancer colon.