الفهرس 
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Abstract
Diabetes mellitus (DM) is a chronic lifelong metabolic syndrome of multiple etiologies marked by long-lasting hyperglycemia that may result from insulin deficiency and/or insulin resistance. DM is associated with dysfunction and damage in various organs that eventually leads to development of dangerous diabetic complications. Current antidiabetic agents are based on synthetic drugs that may have limited efficacy, limited tolerability and/or severe side effects. Based on several in vitro and in vivo studies, bioactive phytochemicals were considered safe and effective alternatives for the management of diabetes. However, using of these phytochemicals in experimental studies revealed a gap between the in vitro and the in vivo actions that could be attributed to limited absorption and low bioavailability.
The present study designed to evolve a new formula of polydatin (PD), bioactive phytochemical, via its loading on chitosan nanoparticles (CSNPs) to improve bioavailability and therapeutic efficiency of PD against diabetes and followed by investigation the safety and antidiabetic efficacy of free and loaded PD using in vitro and in vivo models. Moreover, the study extended to assess the pancreatic and hepatic protective effects of PD and the possible mechanisms of action.
Firstly, the interaction mechanism between PD and CSNPs was studied in silico via Monte Carlo and molecular dynamics simulations followed by preparation and characterization of PD-CSNPs formula using XRD, FTIR, dynamic light scattering (DLS), transmission electron microscope (TEM), and in vitro release analyses. As well, in vitro cytotoxicity study on Vero cell line was conducted to evaluate the safety of the formula.
Next, experimental model of diabetes mellitus was induced in overnight fasted rats by single intraperitoneal injection of streptozotocin (50 mg STZ/kg b.wt.) after 15 minutes of nicotinamide injection (110 mg NA /kg b.wt., intraperitoneally). One week later, fasting blood glucose (FBG) was measured and rats with blood glucose level ≥ 200 mg/dl were considered stable mild diabetic. Rats were divided into the six groups (n=6) as follows: normal control rats (Ctrl) received vehicle, diabetes mellitus control rats (DM) received vehicle, diabetic rats treated with free PD (DM+PD, 50 mg/kg b.wt.), diabetic rats treated with PD-CSNPs (DM+PD-CSNPs, equivalent to 50 mg/kg b.wt. of Polydatin), diabetic rats treated with equivalent blank CSNPs (DM+CSNPs), and diabetic rats treated with metformin (DM+MET, 100 mg/kg b.wt.). All treatments were supplied daily for 28 days via oral gavage.
FBG was measured every week during the treatment period. By the end of treatment, oral glucose tolerance test (OGTT) was performed for all experimental groups, rats were sacrificed, and blood samples was collected for determination HbA1c and separation of serum. Further, Sera used for quantitative determination of, insulin, lipid profile (TC, TG, HDL-C, LDL-C, and vLDL-C), FFAs, protein profile (TP and albumin), ALT, AST, SDH, and PK. Pancreatic samples were collected and used for estimation of oxidative stress (MDA), antioxidant markers (SOD, CAT, GPx, and GSH), and inflammation marker (IL-1β). Moreover, we study the effect of PD on RINm5F β-cell line under oxidative stress in vitro via MTT assay, TUNEL assay, intracellular ROS detection by flow cytometry, Western blotting, and real-time PCR analyses. Later, liver samples were collected for biochemical analyses including glycogen content, oxidative stress (MDA) and antioxidant markers (SOD, CAT, GPx, G6PD, and GSH), gene expression analyses (GK, GLUT2, IL-1β, and TNF-α), and liver histopathological examinations.
The obtained data from the above-mentioned investigations revealed the following:
• Monte Carlo and molecular dynamics simulations revealed the suitability of chitosan as an efficient nano-carrier for loading PD to improve its therapeutic efficiency.
• characterization results of XRD, FTIR, DLS, and TEM confirmed the formation of nano-formula (PD-CSNPs), also the in vitro release study displayed that PD-CSNPs have a sustained release property.
• Cytotoxicity study revealed that PD-CSNPs at low and high doses do not produce any cytotoxicity on the Vero cell line indicating its safety and biocompatibility.
• In vivo results in diabetic rats showed that both free PD and PD-CSNPs have a hypoglycemic effect, however, PD-CSNPs exhibited better hypoglycemic action compared to free PD.
• Blood HbA1c level was obviously elevated in diabetic rats and was potentially decreased as a result of administration of the treatments.
• Serum insulin level and liver glycogen content significantly reduced in diabetic rats and significantly restored after treatment of diabetic rats with free PD or PD-CSNPs.
• The obtained results also indicated an improvement in the level of lipid profile parameters and of FFAs in PD or PD-CSNPs treated diabetic rats. PD-CSNPs showed a potent hypolipidemic effect compared to free PD.
• The current results revealed high level of oxidative stress (MDA) in diabetic pancreas and low activity of antioxidant enzymes (SOD, CAT, and GPx) as well as GSH content.
• PD or PD-CSNPs treatment displayed a significant reduction in the level of MDA in the pancreas of diabetic rats and enhanced the activities of CAT, SOD, and GPx as well as the content of GSH. PD-CSNPs showed more improvement effect relative to PD.
• The pancreatic level of pro-inflammatory cytokine IL-1β significantly elevated in diabetic control rats but markedly reduced upon PD and PD-CSNPs treatment, with more potent effect for PD-CSNPs.
• The in vitro results showed that PD also markedly decreased intracellular ROS accumulation in RINm5f β-cells exposed to oxidative stress in vitro.
• Furthermore, PD upregulated the protein expression of antioxidant enzyme HO-1 in H2O2- induced oxidative stress in RINm5f β-cells.
• The obtained results also showed an upregulation of pAkt protein level in PD co-treated RINm5F cells referring to the activation of PI3K/Akt pathway.
• The co-treatment of stressed β-cells with PD up-regulated the Bcl-2 mRNA expression and ameliorated Bcl-2/Bax ratio, indicating a protective effect of PD against oxidative stress-induced pancreatic β-cell death.
• Ins1 mRNA expression was down-regulated in β-cells under oxidative stress and markedly restored in PD co-treated cells, confirming the protective action of PD against oxidative stress-induced β-cell dysfunction.
• The levels of GLUT-2 and GK gene expression were downregulated in the liver of diabetic rats and restored to near the normal values after treatments, with a more positive effect in the PD-CSNPs group.
• Serum activities of key carbohydrate metabolic enzymes (PK and SDH) were markedly decreased in diabetic control rats while strikingly increased after administration of free PD and PD-CSNPs. Loaded PD showed better action compared to free PD.
• Treating diabetic rats with free PD or PD-CSNPs profoundly improved serum total protein and albumin concentrations.
• Lipid peroxidation level (MDA) showed a significant elevation in diabetic liver and significantly reduced after treatments with better action for loaded PD.
• The activities of antioxidant enzymes (GSH, GPx, SOD CAT, and G6PD) and GSH level significantly reduced in liver of diabetic rats and improved with supplementation PD or PD-CSNPs.
• The activities of serum ALT and AST significantly elevated in diabetic rats and markedly reduced after treatment with free or loaded PD.
• The current results showed that the IL-1β and TNF-α mRNA levels were upregulated in the liver of diabetic rats. Treatment of diabetic rats with free PD or PD-CSNPs downregulated hepatic levels of mRNA expression.
• Regarding the histological study, the liver of diabetic rats had a myriad of histopathological changes, including fatty changes, dilated hyperemic sinusoids, vacuolar degeneration of hepatocytes, and some hepatocytes with pyknotic and karyolitic nuclei. Treatment of diabetic rats with PD-CSNPs and PD causes marked improvement in most of the liver tissues, confirming the hepatoprotective effects. However, PD-CSNPs seemed to be more protective compared to PD.
To conclude, the present results demonstrated the loading of PD on CSNPs and formation of the novel PD-CSNPs nano-formula with sustained release properties and high loading efficiency. Moreover, the in vitro cytotoxicity results revealed the safety of the formula for in vivo applications. The in vivo results displayed that PD-CSNPs exhibited better antidiabetic efficacy relative to free PD that could be attributed to prolonged-release properties, improved absorption, and increased bioavailability. PD and PD-CSNPs also alleviated the damage of pancreatic β-cells and preserve cell function in vitro and in vivo. These actions of PD were mediated by decreasing oxidative stress, improving antioxidant status, inhibiting inflammation, and enhancing the expression levels of anti-apoptotic and cell function markers. Furthermore, the hepatoprotective effects of free PD and PD-CSNPs were markedly evidenced by ameliorating carbohydrate metabolizing enzymes, inhibition of oxidative stress status, enhancing antioxidant enzymes, and modulating pro-inflammatory cytokines. Therefore, the present study illustrated that PD-CSNPs formula is safe and biocompatible and displayed better bioavailability and efficiency against diabetes and its complications relative to free PD. Thus, this formula could be suggested as a promising candidate for management of diabetes.