Download Summary and Conclusion Facultyof Pharmacy Department of

Facultyof Pharmacy
Department of Biochemistry
Thesis Submitted for the Degree of Philosophy Doctor in Pharmaceutical
Sciences (Biochemistry)
BY
Nehal Mohsen Ahmed Elsherbiny
M.Sc. Pharm. Sci., (2010)
Under the supervision of
Prof. Dr.
Mamdouh M. El-Shishtawy
Professor of Biochemistry and Dean of faculty of Pharmacy
Mansoura University
Prof. Dr.
Prof. Dr.
Gregory I. liou
Laila A. Eissa
Prof. of Biochemistry and Molecular Biology
Department of Ophthalmology, School of Medicine
Georgia Regents University, USA
Professor of Biochemistry
Faculty of Pharmacy-Mansoura University
Dr.
Mohammed M. H. Al-Gayyar
Associate Professor of Biochemistry
Faculty of Pharmacy- Mansoura University
2013
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Summary and Conclusion
Summary and Conclusion
Part I: diabetic nephropathy
Diabetes is a major threat to global public that is associated with
many complications threatening life. Diabetic nephropathy (DN) remains
the most common cause for end stage renal disease. Adenosine is an
endogenous purine nucleoside released from various tissues and organs.
Adenosine controls the supply and demand of energy and modulates a
variety of physiological responses by interacting with specific cell surface
G-protein-coupled receptors Although, adenosine acts as local modulator
with a cytoprotective function, extracellular adenosine usually disappears
quickly due to rapid uptake into adjacent cells.
Therefore, the current study investigated the effect of 5′-(NEthylcarboxamido)-adenosine (NECA), stable nonselective adenosine
receptor agonist, on diabetes-induced increase in inflammatory cytokines
and adhesion molecules.
Enhancing adenosine receptor action by NECA was examined in
renal tissues of streptozotocin-induced diabetic rats. Daily IP injection of
0.3mg/kg/day NECA was given for two weeks to six-weeks diabetic rats
They were purchased from and kept in "Urology and Nephrology Center",
Mansoura University, Egypt. Serum creatinine was determined by kinetic
method. Serum Blood Urea Nitrogen (BUN) and urinary albumin were
determined by colorimetric method. Oxidative stress was assessed by
measuring tissue malondialdehyde by colorimetric method. Gene
expression of interleukin (IL)-18, tumor necrosis factor alpha (TNF-α) and
Intercellular Adhesion Molecule (ICAM)-1 was measured by real-time
PCR. Activation of apoptotic pathway was demonstrated by measuring the
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activation of c-Jun NH2-terminal kinases (JNK)–Mitogen-activated protein
kinase (MAPK) using enzyme linked immuno-sorbent assay (ELISA). In
addition, changes in kidney structure were examined to assess the severity
of histopathological changes in kidney sections.
This study found that diabetes-induced malondialdehyde formation
activated the production of TNF-α, IL-18 and ICAM-1 which in turn
activated the pro-apoptotic pathway in diabetic rats. Treatment with NECA
protected diabetic rats by exerting hypoglycemic and antioxidant effects as
well as reducing the gene expression of TNF-α, IL-18 and ICAM-1. These
effects were associated with inhibition of JNK-MAPK apoptotic pathway.
In addition, diabetic rats treated with NECA showed mild glomerular
affection and vacuolation of tubular epithelium.
These results demonstrated that the activation of adenosine receptors
is a potential therapeutic target for DN. NECA acts via multiple
mechanisms including: reducing diabetes-induced oxidative stress,
inhibiting gene expression of IL-18 and ICAM-1 and blocking activation of
JNK-MAPK pathway.
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Summary and Conclusion
Part II: Diabetic Retinopathy
Diabetic retinopathy (DR) is the most feared complication of
diabetes that is a leading cause of adult vision loss and blindness. As a
result, DR is becoming an increasing burden to society that needs improved
and preventive therapies. These therapies could be discovered by
unraveling the pathophysiology of DR which in itself presents quite a
unique challenge.
Retinal inflammation is a vision-threatening complication in diabetic
retinopathy. Amadori-glycated albumin (AGA), a risk factor for diabetes,
induces
microglia-mediated
retinal
inflammation,
suggesting
that
microglial activation causes diabetic retinopathy, and may be a target for
therapeutic intervention. Retinal inflammation is endogenously modulated
by extracellular adenosine via adenosine A2AAR signaling. During
inflammation, ATP released from stressed cells is converted to adenosine
by ectonucleoside triphosphate diphosphohydrolase1 (CD39) and ecto-5’nucleotidase (CD73). Extracellular adenosine re-uptake by the equilibrative
nucleoside transporter (ENT) allows for adenosine conversion to AMP by
adenosine kinase (AK). This study aimed to evaluate the relative
importance of CD73 and AK in regulating extracellular levels of adenosine
in the retina. As AK is the key enzyme for the regulation of ambient levels
of adenosine in the brain, this study hypothesizes that AK has a similar role
in the retina.
The hypothesis was tested by comparing retinal inflammation in wild
type (WT) and CD73-/- (CD73KO) diabetic mice, and in diabetic mice
treated and untreated with an AK inhibitor ABT 702 (of 1.5mg/kg IP twice
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a week). This study also compared TNF-α release in AGA-treated retinal
microglial cells ± AK or CD73 inhibitors.
In diabetic mice, Western, real-time PCR and immunoflourescence
analyses revealed up-regulation of retinal expression of A2AAR, ENT1,
Iba1, TNF-α, ICAM1, caspase3, TUNEL positive cells and reactive oxygen
species, but down-regulation of retinal AK expression. Treatment with AK
inhibitor attenuated all the above-mentioned alterations in diabetic animals.
In contrast, these retinal alterations remained unchanged in diabetic WT
and CD73KO mice. Moreover, treatment with AK inhibitor, but not CD73
inhibitor, blocked TNF-α release in AGA-treated microglial cells.
The data presented here provide experimental evidence that targeting
adenosine kinase can inhibit diabetes-induced retinal abnormalities that are
postulated in the development of diabetic retinopathy by potentially
amplifying the endogenous therapeutic effects of accumulation of
extracellular adenosine. Thus, ABT 702 appears to be a useful therapy to
possibly inhibit the development/progression of retinopathy, the sight
threatening complication faced by diabetic patients.
In conclusion, this study demonstrated that enhancing adenosine
signaling -by using adenosine receptors agonists or inhibiting endogenous
adenosine metabolism- suppresses inflammation which may be a
therapeutic option for the treatment of diabetic complications.
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