الفهرس 
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Abstract
Hepatocellular carcinoma (HCC) is the happening of primary liver cancer in liver
parenchyma cells or on the other hand hepatocytes. The dominating risk factors and
etiological agents answerable for hepatocellular carcinoma in humans have been
distinguished as chronic infection with hepatitis B infection or hepatitis C infection,
exposure to aflatoxin B1 or other chemical carcinogens, alcoholic cirrhosis and cirrhosis
related with genetic liver diseases.
HCC is a forceful disease with a poor outcome. The treatment of numerous HCC
patients was belated because of the slur of early symptoms. At present, the treatment of
liver cancer primarily incorporates surgery and chemotherapy. However, the therapeutic
impacts of the current chemotherapeutic drugs are sufficiently bad and they have various
symptoms. As of late, the naturally occurring substances have been accepting expanded
consideration by researchers and have been the subject of numerous strict scientific
studies.
Due to the importance for seeking novel compounds with higher activity and fewer
side effects for chemoprevention and treatment of HCC, the present study was designed to
evaluate the anti-proliferative effects of metformin and pomegranate against
N-nitrosodiethylamine induced hepatocellular carcinoma in male rats using histological,
immunohistochemical and biochemical studies
The present study was carried out on 40 male Sprague Dawley rats 5 - 6 weeks old
weighing 100 – 120 g obtained from the animal house of Medical Research Institute
(Appendix 1, Guiding Principles for Biomedical Research Involving Animals, 2011).
Rats were selected randomly and divided into 2 main groups:
group 1: Eight normal rats (negative control).
group 2: Thirty-two rats were intraperitoneally injected with a single dose of DEN,
200 mg/kg body weight freshly dissolved in sterile 0.9% saline followed
by subcutaneous injections of 1 ml/kg body weight 10% CCl4 dissolved in
sunflower oil, 3 alternative day/week for 6 weeks.
After HCC establishing; the rats were divided into three subgroups:
group 2a: Eight HCC rats received no treatment (untreated HCC).
group 2b: Eight HCC rats were treated with 150 mg/kg body weight metformin daily
for 5 weeks (metformin treated). The dose of metformin was selected by
performing an effective dose fixation study.
group 2c: Eight rats with HCC were treated with 250 mg/kg body weight
pomegranate daily for 5 weeks (pomegranate treated).
group 2d: Eight HCC rats were treated with 150 mg/kg body weight metformin and 250
mg/kg body weight pomegranate, all at once, daily for 5 weeks (combined
treated).
Summary, Conclusion & Recommendations
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Metformin and pomegranate were intragastrically given by gavage using a cannula
fitted to a feeding needle. Rats of all groups were sacrificed by decapitation at the end of
experiment. Liver tissue was removed from each animal, divided into two parts, one part
was embedded into 10% neutral buffered formalin and prepared for the histochemical
stains (hematoxylin and eosin, toluidine blue and reticulin stains) and determination of
fibroblast growth factor 2 (FGF 2) by immunohistochemical technique, while the other part
was homogenized (1:5) PBS and stored at -20°C for estimation of Hepatic Nuclear factor
erythroid 2-related factor 2 (Nrf 2) content, Hepatic 8-Hydroxydesoxyguanosine (8-OHdG)
content and Hepatic Survivin content. Blood samples were collected to separate serum and
stored at -20°C for measuring of liver function tests.
Results of the present work revealed that untreated HCC rat liver had widely
distributed malignant cells in trabecular pattern and multiple nodular round masses of
different sizes of localized lesions separated by bands of fibrous tissue. Treatment with
metformin decreased neoplastic foci and hepatocytes proliferation and improved
vascularization. However, pomegranate showed preserved hepatic lobular architecture with
normal looking hepatocytes and few dilated sinusoids.
Furthermore, results of the present study demonstrated that combined therapy
produced the most favorable response on inhibition of angiogenesis, reduction of oxidative
stress and increasing apoptosis in treated groups. Its effect was elicited through reducing
FGF 2 expression, hepatic Nrf 2 content, hepatic 8-OHdG content and hepatic survivin
content.
Pomegranate was in the second rank following combined therapy in decreasing HCC
damage and inhibits tumor propagation. Although metformin had the least effect on
decreasing liver enzymes activities and also hepatic Nrf 2, 8-OHdG and survivin contents,
it had notable effect on liver architecture improvement.
Taken together, this work provided evidence that combined metformin and
pomegranate therapy, pomegranate and lesser extent metformin alone exerted antitumor
effect on hepatocellular carcinoma induced in rats via numerous mechanisms of actions
including reducing oxidative stress and DNA oxidative damage and finally induce
apoptosis of HCC cancerous cells.