الفهرس 
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Abstract
The present study was planned to elucidate the effects imposed by stimulation of the A2A adenosine receptor on glycemic, oxidative and inflammatory states and bone integrity deduced by the expression of different bone regulating genes together with histopathological bone assessment in a diabetic induced osteoporosis rat model.
The study was performed on 40 adult male albino rats, allocated into 4 groups:
- group I: Control group (C), (n=8)
Rats in this group received a single intraperitoneal injection of the same volume of citrate buffer which was used as a solvent for STZ (1 mL).
- group II: Diabetic-induced osteoporosis group (DIO), (n=16)
This group was further subdivided into two subgroups:
a- Diabetic-induced osteoporosis group (DIO)a: (n=8)
Rats in this group were subjected to induction of diabetes by receiving a single intraperitoneal injection of STZ in a dose of 40 mg/kg/mL.
b. Diabetic-induced osteoporosis group (DIO)b, (n=8)
Rats in this group were subjected to induction of diabetes by receiving a single intraperitoneal injection of STZ at a dose of 40 mg/kg/mL. Four weeks after STZ injection, rats were daily injected intraperitoneally by the same volume of 10% DMSO used as a solvent for CGS-21680, A2AAR agonist, for another four weeks.
- group III: Insulin treated diabetic-induced osteoporosis (I-DIO) group, (n=8)
Rats in this group were subjected to induction of diabetes by receiving a single intraperitoneal injection of STZ at a dose of 40 mg/kg/mL. Four weeks after STZ injection, rats were injected with insulin (Humulin 70-30) once daily at a dose of 10 IU/Kg, subcutaneously in the inter-scapular region, for another four weeks.
- group IV: Adenosine receptor agonist treated diabetic-induced osteoporosis group (A-DIO), (n=8)
Rats in this group were subjected to induction of diabetes by receiving a single intraperitoneal injection of STZ at a dose of 40 mg/kg/mL. Four weeks after STZ injection, rats were subjected to daily intraperitoneal injection of the A2A adenosine receptor agonist (CGS-21680) at a dose of 0.1 mg/kg for another four weeks.
Fasting blood glucose was measured from rat tail vein using glucometer after streptozotocin injection to ensure development of diabetes. In all rats, initial and final body weight were measured, also initial and final body mass index were calculated. Retroorbital blood samples were collected at the end of the study and serum was separated for determination of glycemic state parameters; fasting serum glucose and insulin levels, the inflammatory marker; tumor necrosis factor-α (TNF-α), oxidative markers; malondialdehyde (MDA) and glutathione peroxidase (GPx), minerals; calcium and phosphorus and bone marker osteocalcin. Right tibias were dissected and homogenized for analysis of gene expression of receptor activator of nuclear factor kappa-B ligand (Rankl), osteoprotegerin (Opg), runt-related transcription factors-2 (Runx2), β-catenin (Catnb) and sclerostin (Sost) by quantitative RT-PCR (real-time polymerase chain reaction) with calculation of Rankl/Opg ratio. Histopathological and histomorphometric analysis of rat’s tibial bone tissue were performed.
The single intraperitoneal injection of 40 mg/kg of streptozotocin was able to develop type 1 diabetes in adult rats as evidenced by the significant elevation in fasting serum glucose level and reduction in fasting serum insulin level compared to the control group. Insulinopenia and reliance on fat fuels was accompanied by reduction in body mass index. STZ-induced cytotoxicity in pancreatic cells and prolonged exposure to hyperglycemia increased the generation of free radicals evidenced by the significant increase in serum MDA level and decrease in GPx activity. Hyperglycemia induced free radicles production leading to lipid peroxidation, enzymatic glycation of GPx and depletion of the antioxidant defense system. Also, Oxidative stress was accompanied by an inflammatory state denoted by the increased serum TNF-α level. A positive feedback loop relation exists between TNF and ROS.
At the end of the study (8 weeks after streptozotocin injection), diabetic induced osteoporosis was evidenced by bone porosity in trabecular bone microarchitecture and significant reduction in the tibial bone cortical and trabecular thickness compared to the control rats. This picture was accompanied by significant increase in Rankl gene expression, Rankl/Opg ratio denoting high resorption rate together with significant reduction in Opg gene expression. Elevated serum osteocalcin can be explained by its leak to the circulation during osteoclastic bone resorption. Runx2 overexpression goes with this high bone turnover state as it might increase Rankl expression in osteoblasts and inhibit the osteoblastic canonical Wnt pathway through depletion of β-catenin, causing reduction in bone mass and volume. The significant reduction in Catnb expression which can be related to hyperglycemia and the increase in Sost expression explain bone metabolism impairment and osteoblast activity deterioration in untreated diabetic rats, based on β-catenin importance in early osteoblast differentiation, proliferation and in the induction of osteoblastic genes as Opg. Serum MDA and TNF-α levels were positively correlated with Rankl, Rankl/Opg ratio and Sost and negatively correlated with Opg and both cortical and trabecular bone thickness highlighting the involvement of oxidative stress and inflammatory states in diabetic induced osteoporosis.
Insulin treatment for 4 weeks was accompanied by significant decrease in fasting serum glucose level and increase in fasting serum insulin level, this could be related to its ability to promote β-cell regeneration and alleviate glucotoxicity. Through its anabolic effects insulin treatment was able to decrease weight loss. In addition, insulin treatment reduced oxidative stress and inflammation denoted by the positive correlation between insulin and GPx activity and the negative correlation with MDA and TNF-α. This might explain the ability of insulin to restore bone microarchitecture and to increase tibial bone cortical and trabecular thickness. Bone improvement was also evidenced by the lowered bone Rankl and Sost expressions, as well as the increased Opg and Catnb expression. This was additionally explained by either insulin anabolic effect, good glycemic control or Catnb induction.
Adenosine A2A receptor (A2AAR) agonist treatment for 4 weeks decreased serum glucose level and increased serum insulin which might be related to either the enhancement of ATP sensitive K+ channels thus stimulating insulin secretion or to promoting β-cell proliferation. Improved insulin secretion led to a reduction in weight loss.
A2AAR agonist treatment implied partial improvement in the inflammatory and oxidative states as it decreased both serum MDA and TNF-α levels, with only MDA reaching the control values. This improvement might be involved in the inhibition of the inflammatory osteolysis and destruction in diabetes based on the positive correlation between both MDA, TNF-α and Rankl expression as well as the negative correlation between them and Catnb expression. A2AAR agonist promoted bone regeneration reflected in the well-organized bone lamellae together with an increase in the bone cortical and trabecular thickness compared to the untreated diabetic groups. This may be related to its ability to stimulate osteoblastogenesis and oppose osteoclastogenesis proved by the significant decrease in the ratio of Rankl/Opg which is an indicator of bone resorption, the decrease in Sost expression (the Wnt/β-catenin pathway inhibitor), restoration of Runx2 expression level and increase in expression level of bone Catnb which is critical for bone regeneration.
No significant changes were noted between the studied groups as regards serum calcium and phosphorus levels.
By comparing insulin treatment with A2AAR agonist treatment, both treatments similarly reduced the oxidative and inflammatory states. Insulin treatment implied better glycemic control. Meanwhile, A2AAR agonist treatment showed better bone regenerative effects.
Conclusion
A2A Adenosine receptor agonist treatment is more conductive to ameliorating bone health deterioration in diabetic rats compared to insulin treatment. This might be attributed to its anti-diabetic and anti-inflammatory effects as well as its anabolic and anti-resorptive effects on bone. Therefore, A2AAR stimulation can serve as a promising therapy for controlling progression of diabetic induced osteoporosis.
Recommendations
A2A adenosine receptor stimulation was able to mitigate the deleterious effects of diabetes on bone, but few A2A adenosine receptor agonists are approved in humans. So further research studies are needed to investigate their possible effects on other diabetic complications