الفهرس 
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Abstract
Type 1 diabetes is a chronic autoimmune disorder identified by hypoinsulinemia and hyperglycemia due to absolute insulin deficiency that results from destruction of pancreatic β cells. The immunopathogenesis of T1D is the results of a complex interaction between the pancreatic β-cells and infiltrating lymphocytes due to disturbance in balance between Teff and Treg cells as well as auto-reactive autoantibodies. The incidence rate of T1D rises with age peaking nearly at 10-14 years but symptoms can occasionally develop much later.
Knowledge about T1D has progressively increased during the last 25 years, improving the global understand of disease conditions including genetic behavior, environmental-associated factors (such as viral infections, vaccinations, drugs, psychological stress) as well as disease burden. Interventions aiming at conservation of pancreatic β cell mass improved the clinical management of T1D significantly.
Apoptosis is the foremost known form of programmed cell death that occurs via two main pathways; extrinsic (death receptor mediated) or intrinsic (mitochondria mediated). Caspase species play an important role in breakdown of cell nuclei during apoptosis where caspase-3 is the key element of both apoptotic pathways. Inhibitors of caspase-3 or knockout mice have been shown to protect from T1D.
Chitosan is a natural polysaccharide polymer prepared from chitin through an alkaline deacetylation process. It is biodegradable, biocompatible, and nontoxic rendering chitosan a good candidate for traditional and acceptable drug delivery systems in pharmaceutical race-track scince1990.
STZ is a widely used chemical model for induction of experimental diabetes in rodents through either single high dose ore multiple low doses. MLD-STZ diabetes induction is preferred according to its little side effects and its mechanism closely resembles the pathogenesis of T1D in human.
Balb/c is a murine model of chemical induction of T1D sharing many genetic, metabolic and pathophysiological properties with the human version of diabetes.
IL-2 cytokine is an autocrine growth factor that binds high affinity IL-2 receptors to induce T cell proliferation, differentiation and survival. The use of high-dose IL-2 therapy is associated with severe toxicity, coma and dehydration; side effects that can be avoided by the use of low dose IL-2 specially encapsulated onto controlled release drug delivery system such as Chitosan nanoparticles. The prospective use of IL-2 as candidate therapy for autoimmune diseases was extremely encouraged by the finding that IL-2 and/or IL-2R deficiency is characterized by severe systemic autoimmune disorders and lymphocyte infiltration. The present work was designed to evaluate the modulatory therapeutic effect of ultra-low dose of recombinant human IL-2 on lymphoid cell apoptotic changes (represented by caspase-3 activity) in an experimental model of STZ-induced T1D.
The current study tested several concentrations as novel formulation of IL-2 loaded onto chitosan nanoparticles. The study included a total of 129 male inbreed Balb/c mice with age range of 10-12 weeks were classified into five groups; healthy controls, diabetic non-treated mice, diabetic mice given empty chitosan nanoparticles and diabetic mice treated with either free or chitosan-loaded IL-2 at doses of 0.05, 0.1 and 0.33 MIU/m2/day. STZ was injected intraperitoneally into corresponding groups at a concentration of 50 mg/Kg/body weight for five consecutive days. Free rhIL-2 was intraperitoneally injected into diabetic mice once daily for five consecutive days, while its chitosan-loaded form was given via the same route at a single cumulative dose equivalent to the sum of five doses of corresponding free form.
• The present study showed the following results:
Diabetic status for all animal groups was monitored through measuring blood glucose, serum insulin and body weights once before and at three week intervals after diabetes induction and IL-2 treatment. Statistical analysis of the results showed a significant decrease in mice body weights after STZ injection as compared to healthy controls.
Concerning blood glucose, at week 1, the results revealed no statistically significant changes neither in diabetic non-treated mice nor following treatment with IL-2 regardless of the concentrations or the form. On the other hand, there was a significant decrease in glucose level in diabetic mice treated with chitosan loaded IL-2 at dose of (0.05) in comparison to those treated with the same dose of free IL-2, while the other two doses showed no significant difference. When these results were further expressed as % variation, data revealed that a remarkable elevation in glucose level was only recorded in diabetic mice given free IL-2 at all doses as compared to both healthy controls and diabetic non-treated mice. In addition, glucose levels were remarkably decreased in diabetic mice treated with chitosan-loaded IL-2 at all doses in comparison to their partners treated with free IL-2.
At week 2, there was absence of any significant variation among study groups except for diabetic mice treated with chitosan-loaded IL-2 at dose of 0.1 MIU, that recorded a significant increase in glucose level, when compared both to healthy controls and diabetic non-treated mice. On the other hand, glucose level in diabetic mice treated with different forms of IL-2 at all concentrations showed no significant variations as compared to each other. According to % variations, the remarkable rise in glucose level was regarded much more in diabetic mice treated with IL-2 in all concentrations and forms, but chitosan-loaded IL-2 in all concentrations were not as effective as free IL-2 in reducing glucose level.
At week 3, the results revealed no significant variations among all study groups when compared both to healthy controls and diabetic non-treated mice except for diabetic mice treated with free IL-2 at dose of (0.1 MIU) and chitosan loaded IL-2 at dose of (0.05 MIU) that registered a significant elevation in glucose level relative only to healthy controls. Furthermore, the results showed that the only significant improvement in glucose level was recorded in diabetic mice treated with chitosan loaded IL-2 at dose of (0.1 MIU) when compared to those treated with the same concentrations of free IL-2, while IL-2 at other two doses showed no significant changes upon comparing different forms of IL-2 with each other.
Referring to % variation, all mice groups showed remarkable increase in glucose level relative to healthy controls except for mice treated with chitosan-loaded IL-2 at dose of (0.1 MIU) that recorded a very mild increase. There was a relative decrease in glucose level in diabetic mice treated with both free and chitosan-loaded IL-2 as compared to diabetic non-treated mice except for those treated with free IL-2 at dose of (0.1 MIU) and chitosan-loaded IL-2 at dose of (0.05 MIU). Also, diabetic mice treated with chitosan-loaded IL-2 at doses of (0.1 and 0.33 MIU) showed a remarkable reduction in glucose level relative to those treated with free IL-2. In contrast, mice treated with chitosan-loaded IL-2 at dose of 0.05 MIU showed remarkable increase in glucose level when compared to those treated with free IL-2.
With respect to serum insulin levels, statistical analysis of the results throughout the three week durations showed that each of diabetic non-treated mice and those treated with IL-2 at all concentrations and forms, free or chitosan loaded, showed no significant variation in insulin level compared to both healthy control and diabetic non-treated mice. Further statistical analysis of these results revealed that diabetic mice treated with chitosan loaded IL-2 at all doses showed no significant variation in insulin level when compared to those treated with free IL-2.
To assess caspase 3, splenocytes were maintained in a short-term culture for in vitro assessment of basal against mitogen-induced production of caspase 3 as an irreversible apoptotic marker.
Regarding basal caspase 3, at week 1, the results revealed that both diabetic non-treated mice and those treated with free IL-2 at a dose of (0.33 MIU) showed a significant increase relative to healthy controls, while the remaining groups showed no significant change. Otherwise, there is no significant changes in diabetic mice treated with IL-2 at all concentrations and forms upon comparing with diabetic non-treated mice except for those treated with chitosan loaded IL-2 at a dose of 0.33 MIU that revealed a significant down regulation. Moreover, diabetic mice treated with chitosan-loaded IL-2 showed a significant reduction in basal caspase-3 when compared to those treated with free IL-2 but only at the highest dose, whereas the middle dose showed no significant variation, while it was significantly elevated at the lowest dose.
At week 2, the results revealed a significant elevation in basal caspase-3 following STZ-induction of diabetes relative to healthy controls. Otherwise, diabetic mice given empty chitosan and those treated with IL-2 at all concentrations and forms showed no significant changes when compared to both healthy controls and diabetic non-treated groups. A statistically significant down regulation in basal caspase-3 was recorded only in diabetic mice treated with chitosan-loaded IL-2 at the dose of 0.33 MIU when compared to those treated with free IL-2.
At week 3, the results revealed a significant up regulation in basal caspase-3 in diabetic non-treated mice as compared to healthy controls. In contrast, diabetic mice given empty chitosan and those treated with IL-2 at all concentrations and forms showed no significant changes. Profound, statistical analysis of the results revealed also that all diabetic mice given IL-2 at all concentrations and forms showed a significant down regulation in basal caspase-3 when compared to diabetic non-treated group. Furthermore, diabetic mice treated with chitosan-loaded IL-2 at all doses showed no significant changes when compared to their corresponding partners treated with the free form of IL-2.
Concerning mitogen-induced caspase 3, at week 1, the results showed no significant variations among all studied groups when compared both to healthy controls and diabetic non-treated mice. Also, the results showed that diabetic mice treated with chitosan-loaded IL-2 recorded significant reduction in mitogen-induced caspase-3 relative to those treated with free IL-2 only at two doses of (0.1 and 0.33 MIU), while those treated with the lowest dose of IL-2 (0.05 MIU) showed highly significant elevation.
At week 2, the results revealed no significant changes in mitogen-induced caspase-3 among all stud groups when compared both to healthy controls and diabetic non-treated mice except for both diabetic mice given empty chitosan and those treated with free IL-2 at dose of (0.33 MIU) that showed a significant elevation relative only to healthy controls. Profound statistical analysis of the results revealed significant reduction in mitogen-induced caspase-3 in diabetic mice treated with chitosan-loaded IL-2 at concentrations of (0.05, and 0.33 MIU) when compared to those treated with the same doses of free IL-2, whereas, IL-2 at a dose of (0.1 MIU) showed no significant change.
At week 3, the results registered no significant variations among all studied groups except for a significant reduction recorded in diabetic mice treated with chitosan-loaded IL-2 at dose of (0.05 MIU) when compared to their partners treated with the same concentration of the free form.
Conclusions
1. Streptozotocin at multiple low doses induced a state of mild, gradual, although sometimes insignificant, hyperglycemia associated with hypoinsulinemia and weight loss.
2. The pancreatic insulitis and/or the state of hyperglycemia induced by STZ treatment caused massive splenocyte cell-death manifested by an augmented cleavage of caspase 3 either spontaneously or following in vitro mitogenic activation.
3. Treatment with variable low doses of free rhIL-2 failed to contain both hyperglycemia and splenocyte apoptotic death. However, treatment with chitosan-loaded equivalent doses of the same cytokine caused milder insulitis (manifested by non-elevated glucose level especially at week 1) and repressed splenocyte apoptosis (manifested by down regulation of caspase 3 more remarkably at week 3).
4. Unfortunately, the potentially anti-diabetic effect of rhIL-2 did not persist at later stages and seems to characterize only early stages of diabetes induction. The persistence of anti-apoptotic effect of such therapeutic approach, however, was not sufficient to restore parallel normoglycemia.
5. The anti-diabetic effect of IL-2 was not uniform in treated groups where specific durations and/or IL-2 doses and forms exhibited relatively elevated glucose levels regardless of being treated with free or chitosan-loaded IL-2, in addition to missed significant correlations with caspase 3 expression status.
6. It seems likely that chitosan-loaded rhIL-2 at dose 0.1 MIU may be potentially promising in retrieving glucose homoeostasis in experimentally induced T1D. However, reaching the most effective modality necessitates an intensive optimizing rather than personalization researches.