Efficacy of Oral Nanoparticle-Encapsulated Insulin in Reducing Oxidative Stress and Enhancing Tissue Integrity in a Diabetic Rat Model

Abstract

Introduction: Diabetes mellitus, a chronic metabolic disorder, leads to systemic organ damage characterized by oxidative stress and structural alterations, contributing to increased morbidity and mortality. Traditional subcutaneous insulin therapy, while managing hyperglycemia, often falls short in addressing the oxidative damage and preventing organ-specific complications. This study evaluates the therapeutic efficacy of a novel oral nanoparticle-mediated insulin (nCOF/Insulin) against these diabetes-induced changes, comparing it with traditional subcutaneous insulin in a streptozotocin (STZ)-induced diabetic rat model.

Methods: We induced diabetes in Wistar rats, dividing them into four groups: standard control, diabetic control, diabetic treated with subcutaneous insulin, and diabetic treated with oral nanoparticle-mediated insulin (nCOF/Insulin). Assessments included organ and body weights, histopathological examinations, and oxidative stress markers (MDA and PCOs) across various organs, including the brain, muscle, intestine, spleen, heart, liver, kidney, and adrenal glands. Additionally, we evaluated antioxidant parameters (GSH and catalase) and conducted immunohistochemical analysis of E-cadherin to assess intestinal integrity.

Results: Our findings reveal that STZ-induced diabetes significantly impacts organ health, with subcutaneous insulin providing limited mitigation and, in some cases, exacerbating oxidative stress. Conversely, oral nCOF/Insulin treatment effectively restored organ and body weights, reduced oxidative stress markers, and mitigated histological damage. This suggests that oral nCOF/Insulin not only offers superior glycemic control but also addresses the underlying oxidative stress.

Conclusion: nCOF/Insulin emerges as a promising treatment for diabetes, with the potential to improve patient quality of life by ameliorating oxidative stress and preventing organ-specific complications. This study underscores the need for further investigation into the long-term effects and mechanisms of action of oral nCOF/Insulin, aiming to revolutionize diabetes management and treatment strategies.

Keywords: diabetes mellitus; diabetic complications; oral nanoparticle-mediated insulin; organ toxicity; oxidative stress.

Figure 1

Comparative Effects of Oral and Subcutaneous Insulin Delivery on Diabetic Complications in Rats. The left panel shows that subcutaneous insulin delivery in diabetic rats results in hypoglycemic episodes, non-optimal glycemic control, and glucose variability, leading to dysglycemia and subsequently exacerbating diabetes-induced oxidative stress, which may contribute to organ alterations and long-term diabetes complications. In contrast, the right panel demonstrates that oral administration of nCOF/Insulin has the potential to improve glycemic control with fewer hypoglycemic episodes and reduced glucose variability, thereby promoting euglycemia. This method reduces diabetes-induced oxidative stress in organs which could help mitigate the severity of organ-specific complications.

Figure 2

Overview of the Study Protocol. The diagram illustrates the ordered process employed in our investigation. The induction of type 1 diabetes (T1D) in rats was achieved through a single dose of streptozotocin (STZ). Five rats were randomly selected and set as the non-diabetic control group. The remaining 15 rats were administered STZ, and after confirming that their fasting glucose levels had exceeded 250 mg/dL, they were randomly assigned to three different treatment groups for subsequent analysis. These groups were subjected to various insulin treatment protocols. Post-treatment, following an overnight fast, the rats underwent weighing and were subsequently euthanized. A comprehensive collection of organs was undertaken, including the brain, heart, liver, spleen, kidneys with their adrenal glands, intestines, and soleus muscle, which were all washed with ice-cold 0.1 M phosphate-buffered saline. The four treatment groups’ heart, liver, spleen, and kidneys were weighed to determine relative organ weights. For histological evaluation, sections of each organ were dissected and preserved in 10% formalin. The intestine was specifically assessed via immunohistochemical analysis for E-cadherin, and the brain, heart, liver, spleen, kidneys, intestines, and soleus muscle were examined for oxidative stress markers, including MDA and PCOs, as well as antioxidant defense enzymes such as GSH and catalase. The statistical analysis of the data was executed using SPSS software.

Figure 3

Body and organ weight, and biochemical alterations in rat models. (a) Rat body weight changes across Control (C., blue), Diabetic Control (D.C., red), Subcutaneous Insulin-treated Diabetic (SC Ins, blue), and Oral nCOF/Insulin-treated Diabetic (Oral nCOF/Ins) groups, shown as a percentage of the Control. (b) Organ weight variations for spleen, heart, liver, and kidney. (c) Malondialdehyde (MDA), (d) Protein Carbonyls (PCOs), (e) Glutathione (GSH), and (f) Catalase concentration variations in intestine, spleen, heart, liver, kidney, brain, and muscle. Results are mean ± SEM, expressed as a percentage of the Control group. Comparisons: a vs C., b vs D.C. (*p < 0.05); (^†p < 0.001).

Figure 4

Histological Comparison of Rat Intestinal Sections Stained with Hematoxylin and Eosin across Four Experimental Conditions: Non-diabetic Control, Diabetic Control, Diabetic Treated with Subcutaneous Insulin (SC Ins), and Diabetic Treated with Oral nCOF/Insulin (Oral nCOF/Ins). Annotations: V for villi, M for muscular layer, with the villi borders highlighted by double dashed black arrows. The upper panel (X50) displays the cross-section of the intestine, demonstrating the general structure and condition of the tissue. The non-diabetic control shows healthy, a well-defined villus (V) and an underlying muscular layer (M), representing a normal intestinal architecture. Diabetic control sections reveal villi degeneration, as indicated by dashed red arrows. The middle panel (X500) provides a higher magnification of the villi within the lamina propria, where lymphoid elements (black arrows) are observed in the Control and insulin-treated specimens, suggesting preserved immune cell populations. The lower panel (X500) offers a closer look at the base of the villi. Healthy glandular structures (yellow arrows) are evident in the Control and Oral nCOF/Ins-treated groups. In stark contrast, the diabetic control and SC Ins-treated groups exhibit clear signs of pathology with necrotic glands (red arrows) and extensive necrotic zones (red asterisks), indicating significant histological damage associated with diabetic conditions.

Figure 5

Comparative Histological Analysis of Rat Spleen Sections Stained with Hematoxylin and Eosin across Non-diabetic Control, Diabetic Control, Diabetic Treated with Subcutaneous Insulin (SC Ins), and Diabetic Treated with Oral nCOF/Insulin (Oral nCOF/Ins) Groups. The upper panel provides an overview of the spleen’s architecture (X25), while the middle and lower panels offer high-magnification views (X500) of the red and white pulp, respectively. White dashed circles in the upper panel highlight the white pulp, showcasing the Germinal Zones (GZ) and Marginal Zones (MZ) which are particularly discernible in the Control and Oral nCOF/Ins specimens. The middle panel shows the red pulp, where both the Control and Oral nCOF/Ins samples display healthy cellularity. Conversely, the Diabetic Control and SC Ins groups exhibit a contracted tissue with thick Billroth cords, as indicated by red arrows. The lower panel zooms into the white pulp, revealing dilations (black asterisks) around the Central Artery (CA) in the Diabetic Control samples. However, the Control, Oral nCOF/Ins, and SC Ins groups exhibit typical lymphoid tissue (yellow arrows) surrounding the central arteries, while such tissue is notably absent in the Diabetic Control group, suggesting immune depletion.

Figure 6

Histological Analysis of Rat Heart Sections Stained with Hematoxylin and Eosin for Different Groups: Non-diabetic Control, Diabetic Control, Diabetic Treated with Subcutaneous Insulin (SC Ins), and Diabetic Treated with Oral nCOF/Insulin (Oral nCOF/Ins). The upper panel presents a wide-field view of the heart’s cross-section (X25), illustrating the layers from the endocardium (Ed) inward (indicated by the black arrow) to the epicardium (Ep) outward, including the myocardium (My) in the middle. Myocardial dilation, marked by the black asterisk, is observed prominently in the Diabetic Control specimens. The lower panel (X50) provides a focused view of the subendocardial area, showing the tissue’s condition and arrangement in each group.

Figure 7

Histological Sections of Rat Liver Stained with Hematoxylin and Eosin across four groups: Non-diabetic Control, Diabetic Control, Diabetic Treated with Subcutaneous Insulin (SC Ins), and Diabetic Treated with Oral nCOF/Insulin (Oral nCOF/Ins). Key identifiers include: cv representing the central vein and black arrows indicating hepatic sinusoids. The upper panel presents an overview of the liver’s architecture (X200), and the lower panel provides a higher magnification of the hepatic microstructure (X500). In the non-diabetic control and Oral nCOF/Ins treated rats, the liver architecture around the central vein (cv) appears normal with regular sinusoids (black arrows) between the cells. Pathological changes are evident in the hepatic sinusoids of both the Diabetic Control and SC Ins groups, as indicated by red arrows. In the diabetic group, the sinusoids appear dilated, while the hepatocytes exhibit signs of atrophy. In the SC Ins group, the sinusoids appear retracted, suggesting hepatocyte swelling. Higher magnification in the lower panel reveals necrotic hepatocytes (red asterisks) in both the Diabetic Control and SC Ins groups. Additionally, some cells exhibit pale, white nuclei, indicative of cellular necrosis (highlighted by red arrows). However, in the Control and Oral nCOF/Ins groups, healthy dark nuclei, which are indicative of high metabolism, are observed. Additionally, it is noteworthy that some hepatocytes in these groups appear binucleated, suggesting cellular regeneration or proliferation, as indicated by the double black arrows.

Figure 8

Histological Examination of Rat Kidney Sections Stained with Hematoxylin and Eosin (X500) across four groups: Non-diabetic Control, Diabetic Control, Diabetic Treated with Subcutaneous Insulin (SC Ins), and Diabetic Treated with Oral nCOF/Insulin (Oral nCOF/Ins). The Control and Oral nCOF/Ins sections display healthy glomeruli (G) and tubules with an intact epithelial lining, characterized by dark nuclei indicative of cellular integrity, along with a healthy brush border evident along the tubules (black arrows), indicating normal renal histology. In contrast, the Diabetic Control and SC Ins sections reveal signs of renal damage: glomerular necrosis (red asterisks), tubular dilation (black asterisks), and loss of the brush border (red arrows), suggesting pathological alterations in diabetic conditions.

Figure 9

Histological Sections of Rat Adrenal Glands Stained with Hematoxylin and Eosin across four groups: Non-diabetic Control, Untreated Diabetic Control, Diabetic Treated with Subcutaneous Insulin (SC Ins), and Diabetic Treated with Oral nCOF/Insulin (Oral nCOF/Ins). The upper two panels provide an overview of the adrenal glands (X25 and X50), showing the cortex with its three zones: Zona Glomerulosa (ZG), Zona Fasciculata (ZF), and Zona Reticularis (ZR), along with the medulla (M) at the core. In the Control and Oral nCOF/Ins groups, each zone is discernible, and the proportions of the different areas are similar between the two groups. However, in the Diabetic Control and SC Ins groups, the cortex exhibits altered proportions (highlighted by the double red arrows), with increased thickness, particularly in the ZF. The ZF in the SC Ins group shows a poorly defined structure, with regions resembling necrosis (indicated by red asterisks), suggesting severe impairment. Additionally, in the Diabetic Control rats, the medulla appears loosely and dilated compared to the Control and Oral nCOF/Ins groups, indicating potential stress or damage. In the second panel (X500), a high-magnification focus on the ZG reveals its structural integrity. The third panel (X500) focuses on the ZF, where the Diabetic Control and SC Ins samples show altered morphology and the presence of significant lipid droplets (highlighted by red arrows). These observations suggest metabolic disruptions and imply an overproduction of hormones by the ZF. Finally, the fourth panel examines the medulla (X500).

Figure 10

Immunohistochemical Analysis of E-cadherin Expression in Rat Intestinal Tissue across four groups: Non-diabetic Control, Untreated Diabetic Control, Diabetic Treated with Subcutaneous Insulin (SC Ins), and Diabetic Treated with Oral nCOF/Insulin (Oral nCOF/Ins). The staining highlights E-cadherin distribution in the intestinal epithelium across the different treatment conditions. The first row provides an overview at low magnification (X50), while the following rows offer detailed views at higher magnification (X500).