الفهرس 
Introduction and Aim of the workOnly 14 pages are availabe for public view
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Abstract
Introduction:
Anemia of chronic inflammation (ACI) is a common problem that complicates chronic inflammatory states and results from limited iron absorption in duodenum and iron retention in macrophages. The iron regulatory hormone ‘hepcidin’ is the central player in this hypoferremic response to inflammation. Infection and inflammation generate signals that dramatically increase hepcidin synthesis and release, resulting in the characteristic anemia of inflammation. RA is a systemic chronic inflammatory disease associated with cytokine network imbalance where the pro-inflammatory cytokines are excessively produced mainly, the tumor necrosis factor-alpha (TNFα). RA presents with anemia as the most common extra-articular manifestation.
Experimental RA induced in rats using FCA mirrors much of the pathology of RA including ACI. Hence, anemia in RA has served as a model for the anemia of chronic inflammation. TNFα inhibition using etanercept is an important modality in treatment of chronic arthritis.
Aim of the work:
The present study was designed to investigate the effect of TNFα inhibition using etanercept on hepcidin gene expression and consequent anemia progression in FCA induced anemia in rats.
Material and Methods
For induction of anemia of rheumatoid arthritis; an injection of CFA containing 1 mg of heat-inactive Mycobacterium bovis suspended in 0.1 ml paraffin oil was injected into the left hind paw. One month later, same injection was repeated twice within an interval of ten days.
Animal Groups: 32 male Wistar rats were divided into 4 groups:
Group Ι: control group
Group ΙΙ: FCA- group
Group ΙΙΙ: Naïve Etanercept- treated group
Group ΙV: FCA- Etanercept- treated group
Etanercept in the therapeutic dose of 0.3 mg/kg 3 times per week was administered in groups III and IV starting from day 40 after first injection of FCA (on day 0) till the end of the experiment on day 60.
Study design: On day 0 of the experiment, retro orbital blood samples were taken under brief anesthesia to measure hemoglobin Hb, MCV, MCH as a basic value to determine the development of anemia. The body weight was measured as an index of general condition and determined at the start on day 0, day 40 and at the end of the experiment on day 60 as an index of general condition. For evaluation of arthritis, paw volume and foot pad circumference were repeatedly measured on days 0, 40, 60. At the end of the experiment on day 60, blood samples were collected and analyzed for assessment of Hemoglobin (Hb) level, Mean corpuscular volume (MCV), Mean corpuscular hemoglobin (MCH), serum iron and ferritin and TNFα level. Animals were sacrificed and liver samples were taken for evaluation of hepcidin gene expression by semi-quantitative RT-PCR.
Results
(I) Effect on body weight change:
Comparing the changes in body weight between day 0 and day 40 as well as between day 40 and day 60 in the control animals and FCA treated animals, it is evident that FCA significantly (P<0.05) prevented the natural increase in the body weight. Although the animals significantly gained weight in etanercept group between day 40 and day 60, yet comparing this change in body weight to the change in the control group, it was evident that etanercept significantly (P<0.05) decreases the natural gain in the body weight. Comparing the change in body weight to that in FCA + etanercept group, it was evident that etanercept significantly (P<0.05) failed to increase the body weight in FCA injected animals.
(II) Effect on Foot pad circumference change
A) Effect on Right (un-injected) foot pads:
Comparing the changes in right foot pads circumference in the control animals and FCA treated animals between day 0 and day 40 as well as between day 40 and day 60, it is evident that FCA significantly (P<0.05) increased the right foot pads circumference in both time intervals.
Comparing the circumference change in FCA + etanercept group to circumference change in the FCA group, it was evident that etanercept significantly (P< 0.05) improves the increase in right foot pad circumference induced by FCA.
B) Effect on Left (injected) foot pads circumference:
Comparing the changes in left foot pads circumference in the control animals and FCA treated animals between day 0 and day 40, it was evident that FCA significantly (P<0.05) increased the left foot pads circumference in this time interval.
Comparing the circumference change in FCA + etanercept group to circumference change in the FCA group, it was evident that etanercept significantly (P<0.05) improves the increase in left foot pads circumference induced by FCA. Comparing the circumference change in FCA + etanercept to that in the etanercept group, it was evident that etanercept completely corrected the increase in left foot pad circumference induced by FCA.
(III) Effect on paw volume change
(A) Effect on Right (un-injected) paws volume
Comparing the changes in right paw volume in the control animals and FCA treated animals between day 0 and day 40, it was evident that FCA significantly (P<0.05) increased the right paw volume in this time interval.
Comparing the change in paw volume between day 40 & day 60 in FCA+ Etanercept group to the change in the FCA group, it was evident that etanercept significantly (P< 0.05) improves the increase in right paw volume induced by FCA. And comparing the change in paw volume in FCA+ etanercept to etanercept group, it was evident that etanercept did not completely correct the increase in paw volume induced by FCA (P<0.05).
B) Effect on Left (injected) paw volume:
Comparing the changes in left paws volume in the control animals and FCA treated animals between day 0 and day 40, it is evident that FCA significantly (P<0.05) increased the left paws volume in this time interval. However, between day 40 and day 60 the FCA effect failed to reach statistical significance.
Comparing the change in paws volume in FCA+ Etanercept group to the change in the FCA group, it was evident that etanercept significantly (P<0.05) improves the increase in paw volume induced by FCA. And comparing the change in paws volume in FCA + etanercept group to the change in the etanercept group, it was evident that etanercept is more effective in decreasing the left paws volume in presence of FCA.
(IV) Effect on Biochemical Measures
A) Effect on Hb level:
Comparing the changes in hemoglobin level in the control animals and FCA treated animals between day 0 and day 60, it was evident that FCA significantly (P<0.05) decreased the hemoglobin level.
Although the hemoglobin level significantly increased in etanercept group between day 0 and day 60, yet comparing this change in in hemoglobin level to the change in the control group, it was evident that etanercept significantly decreases the natural increase in the hemoglobin level (P<0.05).
Comparing the hemoglobin level change in FCA + Etanercept group to hemoglobin level change in the FCA group, it is evident that etanercept significantly (P<0.05) improved the DROP in hemoglobin level.
B) Effect on (MCV):
Comparing the changes in MCV in the control animals and FCA treated animals between day 0 and day 60, it was evident that FCA significantly (P<0.05) decreased the MCV. Comparing the MCV change in Etanercept group to the change in the control group, it was evident that etanercept significantly (P<0.05) increased the MCV. Comparing the MCV change in FCA + etanercept group to MCV change in the change in FCA group, it was evident that etanercept significantly (P<0.05) improved the decrease in MCV induced by FCA. And comparing the MCV change in FCA + etanercept group to that in etanercept group, it was evident that etanercept completely corrected the decrease in MCV induced by FCA.
C) Effect on (MCH):
Comparing the changes in MCH in the control animals and FCA treated animals between day 0 and day 60, it was evident that FCA significantly (P<0.05) decreased the MCH.
Comparing the MCH change in FCA + etanercept group to the change in The FCA group, it was evident that etanercept significantly (P<0.05) improved the decrease in MCH induced by FCA. And comparing the MCH change in FCA + etanercept group to the change in the etanercept group, it was evident that etanercept completely corrected the DROP in MCH that induced by FCA (P<0.05).
D) Effect on serum Iron:
Comparing the serum iron in the control group to FCA group, it was evident that FCA group serum iron was significantly (P<0.05) decreased.
Comparing the serum iron in the control group to Etanercept group, it was evident that Etanercept group serum iron significantly (P<0.05) increased. Comparing the serum iron in the FCA group to FCA+ etanercept group, it was evident that FCA/Etanercept group serum iron significantly (P<0.05) increased i.e. Etanercept over corrected the fall in serum iron induced by FCA.
E) Effect on serum Ferritin:
Comparing the serum ferritin in the control group to that in FCA group, it was evident that FCA group serum ferritin significantly (P<0.05) decreased.
Comparing the serum ferritin in the control group to etanercept group, it was evident that Etanercept group serum ferritin significantly (P<0.05) decreased. Comparing the serum ferritin in the FCA group to FCA+ etanercept group it was evident that FCA+ etanercept group serum ferritin significantly (P<0.05) increased .i.e. Etanercept over corrected the fall in serum ferritin induced by FCA
F) Effect on serum TNFα:
Comparing the serum TNFα in the control group to FCA, it was evident that FCA group serum TNFα significantly (P<0.05) increased.
Comparing the serum TNFα in the control group to etanercept group, it was evident that Etanercept group serum TNFα significantly (P<0.05) increased. Comparing the serum TNFα in the FCA group to FCA+ etanercept group, it was evident that FCA+ etanercept group serum TNFα significantly (P<0.05) decreased.
(IV) Effect on Hepcidin gene expression
Comparing the hepcidin RQ in the control group to FCA group, it was evident that FCA group hepcidin RQ is significantly (P<0.05) higher. Comparing the hepcidin RQ in the FCA group to FCA+ Etanercept group, it was evident that FCA+ etanercept group hepcidin RQ is significantly (P<0.05) lower i.e. Etanercept reduced the increase in hepcidin expression induced by FCA.
Conclusion
FCA-rats treated with etanercept exhibited a reduction in hepcidin expression with subsequent improvement in Hb, RBCs indices and iron profile. That could be explained in part by the amelioration of the inflammation rather than a direct effect of etanercept as an anti TNF-α.