الفهرس 
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Abstract
Hepatic fibrosis is a critical clinical disease that has significant impacts on patient morbidity and mortality. Hepatic fibrosis is the common scarring reaction associated with chronic liver injury that results from prolonged parenchymal cell inflammation.This disease is characterized by progressive accumulation of extracellular matrix which destroys the physiological architecture of the liver. Obesity is associated with an increased risk of nonalcoholic fatty liver disease which is associated with the development of hepatic fibrosis. Hepatic fibrosis can occure as a result of a number of factors including hepatotropic virus infections, metabolic syndrome, toxins, drugs, hereditary factors, cholestasis and parasites.
Aim of the work
Up till now, there is no approved therapy exists for hepatic fibrosis, thus there is an urgent need to develop mechanistic-based therapies for treating fibrosis. Hence, more supplementary researches in order to achieve promising drugs for treatment of hepatic fibrosis of trustable efficacy and safety should be conducted. Therefore, the purposes of this study were to explore the possible protective / therapeutic influence of lycopene and resveratrol against hepatic fibrosis induced in obese rats and to shed light on the precise mode of action and the related signal pathway underlying the possible effects of these remedies to combat hepatic fibrosis.
Materials and Methods
This study was conducted on eighty adult female albino Wistar rats with weight range from 120-130g and age of 3 months. Hepatic fibrosis model was achieved by thioacetamide (TAA) administration (200 mg/kg b.wt, i.p) twice a week for 6 weeks. Obese model was generated by feeding the rats with high fat diet (HFD) for 24 weeks. After this period, the success of obesity model was confirmed by measuring the anthropometric parameters including the abdominal circumference (AC) (immediately anterior to the forefoot), thoracic circumference (TC) (immediately behind the foreleg), body length (nose-to-anus or nose-anus length) of the anaesthetized rats. The body weight and body length were used to determine the body mass index (BMI). Then, these animals were fasted overnight, anaesthetized by sodium pentobarbital and 1ml blood was withdrawn from each rat in the anti-coagulant coated tubes to obtain plasma. Plasma glucose level was measured colorimetrically and plasma insulin level was estimated by enzyme linked immunosorbent assay (ELISA). The homeostasis model assessment of basal insulin resistance (HOMA-IR) was used to calculate the index from the product of the fasting concentration of plasma glucose (mmol/L) and plasma insulin (mU/ml) divided by 22.5.
After that, the rats were assigned into 8 groups (10 rats/group) as follow:
(1) Lean control group was administered saline intraperitoneally twice a week for 6 week after feeding a standard chow diet for 24 weeks.
(2) Obese control group received interaperitoneal injection of saline twice a week for 6 weeks after feeding HFD for 24 weeks.
(3) Hepatic fibrosis lean group received interaperitoneal injection of TAA (200 mg/kg b.wt) twice a week for 6 weeks after feeding a standard chow diet for 24 weeks.
(4) Hepatic fibrosis obese group which received interaperitonial injection of TAA (200 mg/kg b.wt) twice a week for 6 weeks after feeding HFD for 24 weeks.
(5) Lycopene prophylactic group was administered daily with an oral dose of 20 mg/kg b.wt for six weeks simultaneously with the interaperitoneal injection of TAA (200mg/kg b.wt) before feeding HFD for 24 weeks.
(6) Resveratrol prophylactic group was administered daily oral dose of 30 mg/kg b.wt of reseveratrol for six weeks simultaneosly with the interaperitoneal injection of TAA (200mg/kg b.wt) before feeding HFD for 24.
(7) Lycopene therapeutic group was fed with HFD for 24 weeks and then received interaperitoneal injection of TAA (200mg/kg b.wt) for six weeks, after that it received 20 mg/kg b.wt of lycopene by oral administration daily for other 6 weeks.
(8) Resveratrol therapeutic group fed with HFD for 24 weeks and then received interaperitoneal injection of TAA (200mg/kg b.wt) for six weeks, after that it received 30 mg/kg b.wt of resveratrol by oral administration daily for other 6 weeks.
At the end of the experiment, all rats were fasted overnight then, anesthetized by sodium pentobarbital. The blood samples were collected into plain tubes and centrifuged to obtain sera. After blood collection, all animals were rapidly sacrificed and the liver specimens were excised, rinsed with saline solution, blotted dry on filter paper and fixed in formalin saline solution (10%) for Immunohistochemical and histological procedures.
Biochemical assays
Serum aspartate transaminase (AST) and alanine transaminase (ALT) activities, cholesterol (Chol), LDL- cholesterol (LDL) and serum triglycerides (TG) levels were quantified colorimetrically. Serum TGF-β1, leptin and fibronectin levels were detected by enzyme linked immunosorbent assay (ELISA).
Immunohistochemical investigation
The immunohistochemical study was performed on the liver tissues paraffin blocks for detection of α-SMA, CD31 and CD34 expression using ati-α-SMA, anti-CD31 and anti-CD34 respectively.
Histological examination
The liver tissues paraffin blocks were also used for the histological examination of liver tissue using the Hematoxylin and Eosin (H&E) staining procedure.
Results
(1) The results of the current study showed significant elevation (p˂0.05) in the body weight, body length, thoracic circumference (TC), abdominal circumference (AC) and body mass index (BMI) in obese control rats versus the lean control counterparts. Also, the obtained data revealed significant enhancement (p˂0.05) in plasma glucose and insulin levels as well as HOMA-IR value in obese control rats by contrast with the lean control one.
(2) The gathered findings denoted that the obese control group exhibited significant raise (P<0.05) in serum ALT and AST activity by contrast with the lean control group. Of note, a prominent enhancement (P<0.05) in serum ALT and AST activity is noticed in hepatic fibrosis lean group as compared to the lean control group. Importantly, a pronounced augmentation (P<0.05) in serum ALT and AST activity is demonstrated in hepatic fibrosis obese group versus the obese control group. On the opposite side, the administration of lycopene or resveratrol in hepatic fibrosis obese group led to significant inhibition (P<0.05) in serum ALT and AST activity in contrary to the hepatic fibrosis obese group.
(3) The current results demonstrated that obesity produced significant rise (P<0.05) in serum cholesterol, triglycerides (TG) and low density lipoprotein (LDL) levels when compared to the lean control group. Noteworthy, hepatic fibrosis lean group experienced significant heightening (P< 0.05) in serum cholesterol, triglycerides and LDL levels relative to the lean control group. Notably, hepatic fibrosis obese group displayed significant aggravation (P< 0.05) in serum cholesterol, triglycerides and LDL levels in respect with the obese control group. On the other hand, lycopene or resveratrol administration in hepatic fibrosis obese group brought about significant decline (P<0.05) in serum cholesterol, triglycerides and LDL levels as compared to the hepatic fibrosis obese group.
(4) The present findings evidenced a significant accretion (P<0.05) in serum levels of TGF-β, fibronectin and leptin serum levels in obese control group versus the lean control group. Remarkably, hepatic fibrosis lean group disclosed significant enhancement (P<0.05) in serum TGF-β, fibronectin and leptin levels opposed to the lean control group. Significant expansion (P<0.05) in serum levels of TGF-β, fibronectin and leptin levels is recorded in hepatic fibrosis obese group contra to obese control group. In contrast, the administration with lycopene or resveratrol in hepatic fibrosis obese group yielded significant DROP (P<0.05) in serum TGF-β, fibronectin and leptin levels in opposition to the hepatic fibrosis obese group.
(5) Immunohistochemical observations in the current study indicated that, the severity of immunohistochemical reactions in the hepatic tissue differ significantly among the various experimental groups. The immunohistochemical reactions for α-SMA, CD31 and CD34 were severe and prominent in the hepatic fibrosis obese group followed by the hepatic fibrosis lean group, then, obese control rats and lastly, the prophylactic lycopene group. Whereas, the mild immunohistochemical reactions for α-SMA, CD31 and CD34 were noticed in resveratrol prophylactic group followed by lycopene therapeutic group, then resveratrol therapeutic group.
(6) Microscopic examination of section in liver tissue of rat in the lean control group showed no histopathological change and the normal histological feature of the central vein and surrounding hepatocytes in the hepatic lobules are noticed. Microscopic investigation of section in liver tissue of rat fed with HFD (obese control group) showed fatty changes in the hepatocytes underneath the hepatic capsule accompanied by congestion in the portal vein and few inflammatory cells infiltration in the portal area. Microscopic examination of section in liver tissue of rat in the hepatic fibrosis lean group showed fibrosis with inflammatory cells infiltration that divided the hepatic parenchyma into nodules associated with congestion in the portal veins and formation of increasing numbers of newly formed bile ductules at the portal area with karyocytomegaly as well as degeneration and necrosis in the hepatocytes. Microscopic investigation of section in liver tissue of rat in the hepatic fibrosis obese group showed that the hepatic parenchyma is divided by proliferative fibroblastic cells and inflammatory cells into nodules associated with appearance of prominent nucleoli in the hepatocytes and dilatation in the portal vein. Microscopic examination of section in liver tissue of rat in the lycopene prophylactic group showed that there is a congestion in the portal vein and few inflammatory cells infiltration surround
the dilated bile ducts at the portal area. Microscopic investigation of liver tissue section of rat in the resveratrol prophylactic group showed a congestion in the portal area including portal vein with few inflammatory cells infiltration. Microscopic examination of section in liver tissue of rat in the lycopene therapeutic group showed numerous numbers of newly formed bile ductules with inflammatory cells infiltration in the portal area. Microscopic investigation of section in liver tissue of rat in the resveratrol therapeutic group showed fibrosis with inflammatory cells infiltration divided the hepatic parenchyma into nodules.