الفهرس 
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Abstract
Type 1 diabetes is a chronic condition, occurring as a result of autoimmune destruction of the β cells of pancreas, leading to total insulin absence. Patients suffering from T1D require exogenous insulin for their survival. Type 1 diabetes is induced in rats through streptozotocin injection. Type 1 diabetes can be diagnosed by increased FBG levels, as well as decreased C-peptide levels. The C-Peptide testing is considered a very reliable method for assessing endogenously released insulin, reflecting beta cells’ functionality.
The BCL-2 is a family of protein that regulate the initiation of intrinsic mitochondria pathway, which is a well-known apoptotic pathway. The BCL-2 family contains proapoptotic and antiapoptotic (pro-survival) members. The pro-apoptotic members promote apoptosis by facilitating the release of death-promoting proteins, including cytochrome c , from mitochondria to cytoplasm, while the pro-survival members inhibits that release. BAX is an example of pro-apoptotic members, while BCL-2 itself is an example of pro-survival members. It is not the total quantity but rather the ratio between proapoptotic & pro-survival BCL-2 members that define the cell susceptibility towards apoptosis. Decreased BAX with increased BCL-2 levels indicates cell survival and resistance to apoptosis, while increased BAX with decreased BCL-2 levels indicates a shift towards apoptosis. Diabetes mellitus is associated with pancreatic beta cells apoptosis, which can be indicated through increased BAX associated with decreased BCL-2 level.
High-mobility group box protein 1 is a nuclear DNA binding protein that possess a dual function depending on its localization. Inside the nucleus, where it is supposed to exist, HMGB1 stabilizes the nucleosomes, regulates gene transcription and DNA repair, whereas outside the cell, it serves as a proinflammatory cytokine, mediating inflammation. HMGB1 is released from cells by two main way, active secretion via stimulated immune cells, as well as passive release from necrotic and apoptotic cells, making HMGB1 DAMP mediating inflammation. All situations that increase extracellular HMGB1 is associated with diabetes, therefore high levels of HMGB1 were documented under diabetic conditions.
Thioredoxin reductase 1 (TrxR1) is the cytoplasmic isoform of TrxRs, which are a family of oxidoreductases catalyzing the NADPH-dependent reduction of redox proteins. Thioredoxin reductases acquired their name by being the only class of enzymes capable of reducing the oxidized form of thioredoxins into the reduced form, thereby, sharing the same importance as reduced thioredoxins. Thioredoxin reductase, together with thioredoxin and NADPH forms the thioredoxin system that functionally maintain a reduced environment in the cell by defending against oxidative stressors. The thioredoxin system reduction cascade starts when NADPH reduces thioredoxin reductase that consequently reduces oxidized Thioredoxin. The generated reduced thioredoxin will then reduce oxidized proteins produced by ROS, regenerating oxidized thioredoxin once again, that will be recycled again in the thioredoxin system reduction cycle, in order to maintain a reduced environment within the cell. Several studies reported an increase in the levels of thioredoxin and its enzyme (TrxR) under diabetic condition. This increase is considered a natural cellular response against diabetically induced oxidative stress.
Sirtuin 1, also known as SIRT1, is a deacetylase that withdraw acetyl groups from several proteins. Sirtuin 1 positively engages in the process of insulin secretion and sensitivity. In β-cells of pancreatic, SIRT-1 promotes insulin secretion by repressing UCP 2. In case of insulin sensitivity, SIRT-1 promotes post-insulin receptor signaling by repressing tyrosine phosphatase1 B transcription as well as deacetylating IRS-2 and AKT (insulin transduction pathway members) in order to facilitate their activation. Under diabetic condition, SIRT1 expression and activity is reduced by ROS. SIRT1 expression is declined by diabetic oxidative stress-responsive miRNAs ,including miR-34a, miR-217 and miR-181, while the SIRT1 activity is reduced due to decreased NAD+ levels under oxidative stress conditions. Therefore, SIRT1 levels are extremely reduced under diabetic conditions, and inducing its expression increases insulin sensitivity.
FOXO-1 is a is a multifunctional transcription factor whose activity is controlled by post-translational modifications. FOXO1 is negatively regulated by insulin via AKT-dependent phosphorylation, that translocated FOXO1 from the nucleus to cytosol, away from its site of action, where it is degraded. FOXO1 positively regulates the expression of proteins participating in cell cycle arrest ( p27Kip1 and p21), apoptosis (FasL, BIM,and PUMA), and gluconeogenesis (G6Pase and PEPCK). FOXO1 was also found to inhibit β-cell replication by suppressing PDX1 transcription, reducing β-cell proliferation. Under diabetic conditions, AKT activity is decreased, increasing FOXO1 transcriptional activity, in partly explaining the underlying causes of diabetically associated gluconeogenesis, apoptosis and inhibited β-cell proliferation.
Mesenchymal stem cells are self-renewal multipotent cells capable of and differentiation into endodermal lineage, including pancreatic beta cells. They are easily extracted from bone marrow and considered ideal for regenerative therapy due to their homing abilities towards sites of tissue damage, and their capability to differentiate and trans-differentiate to tissue specific cells, as well as their ability to secrete biological factors that modify the surrounding microenvironment in order to promote repair mechanisms , with minimum immune response.
MSCs mediates its action through paracrine secretion of EVs, including exosomes. Exosomes carry lipids, proteins, and nuclear material (DNA and RNA) from its parent cells (MSCs), protecting them from degradation, to recipient cells inside the damaged tissue, where it is released, initiating regenerative repair mechanisms. MSCs secreted exosomes mediated the same effect as MSCs, making exosomes a gold mine in regenerative therapy. Exosomes, being EV, have the advantage over stem cells in being completely non immunological, don’t cause graft rejection, and can easily cross biological barriers.
Chrysin is a natural flavone extracted from plants and natural produces. chrysin has a strong antioxidant and anti-inflammatory activities. chrysin has been reported to mediate hypoglycemic and antidiabetic effect on diabetic rats similar to metformin. A recent study reported that chrysin has a protective effect on MSCs by preventing their dysfunctioning under high glucose environment induced by DM. This study highlighted the potentiality of chrysin administration as a co-drug with regenerative therapeutic agents in case DM.
The aim of our study is to examine the effectiveness of BM-MSC derived exosomes alone or co-treated with chrysin in the treatment of STZ-induced type 1 diabetic rats. Thereby, determining their potentiality as a therapeutic agents for tissue repair and functional restoration of pancreatic beta cells in type 1 diabetic rats.
Our study was conducted on 40 Wistar strain rats. Five rats were sacrificed to obtain BM-MSCs and exosomes, the rest 35 rats were grouped into 7 groups, 5 rats in every group. group I consist of healthy control rats receiving standard diet. T1D was induced in the remaining 30 rats through STZ injection. group II is considered the diabetic control group. The rest of the groups (group III to VII) received treatment as the following: group III received chrysin monotherapy, group IV received MSCs monotherapy, group V received exosomes monotherapy, group VI received chrysin with MSCs combinational therapy, group VII received chrysin with exosomes combinational therapy.
Biochemical assessments as well as histopathological and immunohistochemical examinations were conducted on all experimental groups. Fasting blood glucose where measured weekly for four consecutive weeks. Blood samples were with drawn after four weeks for assessing serum C-peptide levels. Animals were then sacrificed and their pancreatic tissues were used to assess HMGB-1 and TrxR1 protein levels, as well as, SIRT-1 and FOXO-1 gene expression. Additionally, pancreatic tissues of all groups were subjected to histopathological examination, islets diameter measurements, as well as immuno-histochemical examination of anti- insulin, Ki67, BAX, and BCL-2 antibodies, in which their mean area percentage were calculated.
Our results reported a decrease in serum C-peptide levels, SIRT1 expression, islets diameter and mean area percentage of all of insulin , Ki67, and BCL-2, as well as an increased plasma FBG levels, pancreatic tissue TrxR1 and HMGB1 protein levels, FOXO1 expression, and mean area percentage of BAX in STZ induced diabetic rats. These reading were significantly corrected by regenerative therapy (MSCs and exosome alone or in combination with chrysin) in relation to diabetic control, with the best observed results in diabetic rats treated by combinational therapy of chrysin with exosomes.