Nada Fawzy Ibrahim AboElMagd Supervisors

Faculty of Pharmacy
Mansoura University
Department of Biochemistry
Presented by
Nada Fawzy Ibrahim AboElMagd
B. Pharm. Sci., (2012)
Demonstrator at Biochemistry Dept. Faculty of Pharmacy-Mansoura University
Supervisors
Prof. Dr.
Prof. Dr.
Mammdouh M. El-Shishtawy
Laila A. Eissa
Prof. of Biochemistry
Faculty of Pharmacy
Mansoura University
Prof. and Head of Biochemistry Dept.
Faculty of Pharmacy
Mansoura University
Thesis submitted as a Partial Fulfillment for
Master Degree in Pharmaceutical Sciences (Biochemistry)
2015
‫كلية الصيدلة‬
‫جامعة المنصورة‬
‫قسم الكيمياء الحيوية‬
‫رسالة مقدمة من الصيدالنية‬
‫ندى فوزى ابراهيم ابو المجد غازى‬
‫بكالوريوس العلوم الصيدلية دور مايو ‪ 2102‬بتقدير عام امتياز مع مرتبة الشرف‬
‫معيدة بقسم الكيمياء الحيوية كلية الصيدلة ‪ -‬جامعة المنصورة‬
‫المشرفون‬
‫أ‪.‬د‪/‬ممدوح محمد الششتاوي‬
‫أ‪.‬د‪/‬ليلى احمد عيسي‬
‫أستاذ الكيمياء الحيوية‬
‫كلية الصيدلة جامعة المنصورة‬
‫أستاذ و رئيس قسم الكيمياء الحيوية‬
‫كلية الصيدلة جامعة المنصورة‬
‫كجزء من متطلبات الحصول على درجة الماجستير فى العلوم الصيدلية (الكيمياء الحيوية)‬
‫‪2102‬‬
Summary and Conclusion
Summary and Conclusion
Chronic liver disease is an increasing cause of mortality and morbidity all over the
world. Most chronic liver disease like hepatitis B virus, hepatitis C virus and cholestatic
liver disease leads to liver fibrosis. Without early treatment of liver fibrosis, it may
progress to liver cirrhosis and hepatocellular carcinoma. Usage of anti-fibrotic drugs
may halt or reverse fibrosis progression.
Usage of anti-oxidant drugs that decrease oxidative stress is an effective strategy
to prevent liver fibrosis. So in this study, natural compounds which have anti-oxidant
properties have been used to protect against liver fibrosis.
The present study aimed to evaluate the hepatoprotective effect of glycyrrhizin
(GL) (the major bioactive component of liquorice extract), omega-3 fatty acids (ω-3) (a
combination of eicosapentaenoic acid and docosahexaenoic acid) and their combination
in thioacetamide (TAA)-induced liver fibrosis in rats. The present study elucidated the
effect of GL, ω-3 and their combination on liver lipid peroxidation, liver inactive toll
like receptor-4 (TLR-4) concentration, liver TLR-4 gene expression, liver nuclear factor
kappa B (NF-κB) concentration and liver NF-κB tissue expression.
The studied groups:
Fifty male Sprague-Dawley rats were randomized into 5 groups as following:
1) Control group (8 rats): rats received sterile saline i.p. injection twice weekly
for 8 weeks.
2) TAA group (12 rats): TAA-induced liver fibrosis. Rats injected 200 mg/kg of
TAA in sterile saline solution i.p. twice weekly for 8 weeks.
3) GL group (10 rats): rats received 25 mg/kg of GL in distilled water by oral
tube daily along with TAA for 8 weeks.
4) ω-3 group (10 rats): rats received 150 mg/kg of ω-3 by oral tube daily along
with TAA for 8 weeks.
5) GL + ω-3 group (10 rats): rats received GL and ω-3 with the same doses by
oral tube along with TAA for 8 weeks.
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Summary and Conclusion
Twenty-four h after the last dose of treatment, blood samples (around 4 mL) were
taken from the retro-orbital puncture under light ether anesthesia after a fast of 12 h.
Blood samples were left 20 min to clot, serum was separated by centrifugation then
aliquoted for analysis and stored at -80°C for determination of liver function tests:

Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) enzyme
activities by kinetic methods.

Total bilirubin, albumin and total protein concentrations by spectophotomertic
methods.
Rats were sacrificed by decapitation, and their livers were removed, rinsed in ice
cold PBS PH 7.4, blotted dry on a filter paper, weighed and divided into 4 parts as
follow:
1- Part was immediately immersed in liquid nitrogen and stored at -80°C for
quantitative real-time reverse transcription-polymerase chain reaction (qRTPCR) analysis of the expression of TLR-4 gene.
2- A small part from the right lobe of the liver was cut and fixed in 10% phosphate
buffered formalin (pH 7.2) for histopathological examination of:

Assessment of necroinflammatory activity grade.

Quantification of fibrotic areas.

Immunohistochemical detection of NF-κB.
3- 0.5 gm of the liver tissue were homogenized in 5 mL cold PBS, homogenized
with ultrasonic homogenizer, centrifuged then the supernatant removed and
stored at -80°C for ELISA assay of liver NF-κB.
4- 1 gm of the remaining liver tissue were homogenized in 10 mL cold PBS,
centrifuged then the supernatant removed and stored at -80°C in 2 parts, one for
spectophotometric assay of malondialdehyde (MDA), and the second for assay
of liver inactive TLR-4 by ELISA assay.
The most important results of the present study are:
1) Glycyrrhizin, ω-3 and their combination significantly decreased serum AST
activity and serum bilirubin level.
2) Glycyrrhizin and ω-3 combination significantly decreased serum ALT activity,
while GL or ω-3 individually have no significant effect on serum ALT activity.
- 91 -
Summary and Conclusion
3) Glycyrrhizin, ω-3 and their combination significantly increased serum albumin
and total protein levels so improve liver function and this indicate the protection
effect of GL and ω-3 for liver synthetic ability.
4) Significant decrease in both liver necroinflammatory scores and liver fibrotic
areas were observed in GL, ω-3 and GL + ω-3 (combination) groups.
5) Glycyrrhizin and ω-3 have anti-oxidant property as GL, ω-3 and their
combination significantly decreased liver MDA level so they decrease lipid
peroxidation.
6) Glycyrrhizin and ω-3 significantly increased liver inactive TLR-4 level, which
indicates inhibition of TLR-4 pathway, which confirmed by the significant
decrease of liver TLR-4 gene expression.
7) Glycyrrhizin, ω-3 and their combination significantly decreased liver NF-κB
level as they decreased the TLR-4/NF-κB inflammatory cascade pathway.
8) Expression of NF-κB was increased in TAA group that co-localized at the same
areas of increased necroinflammation and fibrosis which confirmed the relation
between inflammation and both necroinflammation and fibrosis.
9) There are significant negative correlation between liver inactive TLR-4 level
and NF-κB level which indicates the relation between TLR-4 activation and
inflammatory pathway.
10) There are also significant positive correlation between liver NF-κB and the
following parameters liver MDA level, liver necroinflammatory score, the liver
percentage of fibrotic area, serum AST activity and serum total bilirubin level
which indicates the relation between inhibition of inflammatory regulator, NFκB and the improvement of liver function and inhibition of lipid peroxidation.
11) Liver NF-κB has a significant negative correlation with both serum albumin
level and serum total protein level which confirms the relation between
inhibition of inflammatory regulator, NF-κB and the improvement of liver
synthetic ability.
From the results of this work, it can be concluded the following:

Glycyrrhizin and ω-3 can be used for protection against liver fibrosis.

Toll like receptor-4/NF-κB pathway has a potential role in the pathogenesis
of liver fibrosis.
- 92 -
Summary and Conclusion

The mechanism behind hepatoprotective effects of GL and ω-3 included
suppression of oxidative stress and inhibition of TLR-4/NF-κB pathway. So,
the hepatoprotective effects of GL, ω-3 and their combination against TAAinduced fibrosis appear to be mediated via its antioxidant, anti-inflammatory
and anti-fibrotic properties.

The protection potency of either GL or ω-3 alone is nearly the same as their
combination against liver fibrosis.

It is advised to fulfill some clinical studies on these natural compounds as
supplementary nutrient to protect against liver fibrosis.
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