Evaluation of antidiabetic activity of zinc oxide and chromium oxide nanoparticles alone or in combination on the pancreas of alloxan-induced diabetes mellitus in male albino mice (Mus musculus)

Introduction

Diabetes is a clinical metabolic syndrome characterized by high blood insulin levels because of a complete or partial decrease in insulin levels (1). This disease is caused when the pancreas can't perform its function or body cells are insensitive to insulin, making glucose unable to enter body cells (2). There are many complications of diabetes in all body systems, such as neurological, cardiovascular, reproductive, gastrointestinal, urological, ophthalmic, hematological, and biochemical complications (3). Nanoparticles are tiny particles with sole properties; their preparation could change the matter properties to make them interconnect with cell membranes and organelles (4). Nanoparticles are used in many fields of life: cosmetics, accessories, inks, drugs, etc. They were laden with therapeutic agents to supply the target cell (5). Zinc Oxide nanoparticles were from those bio-nanoparticles, an inorganic chemically active matter ranging from l to l00 nm (6). It is known that zinc is an essential element for some enzymatic reactions, with its structure permitting the proteins to change themselves for their functional form (7). So, zinc is achievable for insulin molecule structure, biosynthesis, secretion, and storage through zinc transporters (8). Studies established that ZnO nanoparticles might lead to cancer through protein and DNA damage as they generate free radicles in skin tissue (9). Other ones stated that some metals participate in the body's glycemic state and disease complication progression; one is zinc (10). Supplementations of chromium nanoparticles were very important in recovering body antioxidant activity in type ll diabetic patients, resulting from increased oxidative stress (11).

Materials and methods

Zinc oxide nanoparticles

Zinc oxide nanoparticles were prepared using stoichiometric quantities of zinc compound Zn (NO₃)₂.6H₂O were weighted and mixed well with ethylen glycol and distilled water. The resulting mixture was shaken by a magnetic stirrer on a hot plate until it reached dry; after autoignition, a fluffy white-yellowish powder was ground, heated, and then collected in clean test tubes. Using a transmission electron microscope characterized nanoparticles, the particles appeared as aggregated hexagonal platelets attached as a chain with 47nm size (12).

Chromium oxide nanoparticles

Chromium dioxide nanoparticles were synthesized by adding 10ml of potassium dichromate 0.2 M solution to 5ml cetyl-trimethyl ammonium chloride 0.96g/ml with 50ml of trisodium citrate at a concentration of 10mM. The vessel reaction was floodlit to lamp halogen under stirring for 3 hours (13). The solution color was changed from brownish-yellow to green, which showed Cr₂O3 nanoparticles being formed (14). Seventy adult, healthy, male albino mice were used there, weighing 20 to 25gm, aged between 12 to 15 weeks, brought from Babylon University animal house. Mice were habituated for one month before the experiment, ratified by the Institutional- Animal- Ethical- Committee, and kept under controlled conditions of light/ dark period and room temperature. Randomly, animals were divided into seven groups: Group 1 was given distilled water. Group 2 received 10mg/kg body weight daily orally of ZnONPs for a month. Group 3 received 1mg/kg body weight for a daily oral of Cr₂O3NPs for a month. Group 4 received a 150mg/kg body weight of alloxan (15). Group 5 received a single alloxan dose of 150 mg/kg and daily oral 10 mg/kg body weight of ZnONPs for a month. Group 6: received a single alloxan dose of 150 mg/kg and 1mg/kg body weight for a month daily oral Cr2O3NPs. Group 7: received single alloxan dose 150mg/kg and 1mg/kg body weight for month daily oral of Cr₂O3NPs and 10 mg/kg body weight of ZnONPs for month. Animals treated with alloxan are respected as diabetic when their fasting blood glucose is more than 200 Mg/d1 after seven days of alloxan injection. After one month, animals were sacrificed, and blood was withdrawn for estimation of serum for biochemical tests. Pancreases was removed from animals and fixed in buffered saline formaldehyde 10%, and processed histologically as stained with hematoxylin-eosin stain and studying pancreatic morphology (16). Glucose and HbAIc levels were estimated by spectrophotometer (17). At the same time, the ELIZA kit is used to assess insulin (18), and C-peptide levels (19).

Statistical analysis

Data were analyzed using SPSS version 20.0. Descriptive statistics mean ± standard error and ANOVA compared differences at P<0.05 (20).

Results

Results explained a significant increase in glucose and HbAlc levels and a significant decrease in blood insulin and C-peptide levels in the group of diabetic mice when compared with the control group. Also, there was a significant decrease in levels of glucose and HbAlc and a significant increase in insulin and C-peptide levels in the zinc oxide nanoparticles group and chromium oxide nanoparticles group compared with controls. Compared with the control group, the best result was the cooperation between the two nanoparticles types for decreasing blood glucose and HbAlc with increasing insulin levels (Table 1).

Table 1: levels of glucose, Hemoglobin A1 insulin, and C- peptide protein in the control and experimental mice groups

Different vertical letters refer to the significance at P<0.05.

Histopathological results

Cross section of the pancreas from control group mice exhibiting normal pancreatic architecture (Figure 1). Cross section of the pancreas from alloxan-receiving mice Exhibiting necrosis and degeneration of islets with telangiectasis (Figure 2). Cross section of the pancreas from ZnONPs receiving mice exhibiting normal histological organization (Figure 3). Cross section of the pancreas from CrONPs receiving mice Exhibiting normal histological organization (Figure 4). Cross section of the pancreas from diabetic mice treated with ZnONPs Exhibiting normal pancreatic islets with cellular vacuolation and slight necrosis with mild leukocyte infiltration and little congestion (Figure 5). Cross section of the pancreas from diabetic mice treated with CrONPs Exhibiting normal pancreatic islets with cell vacuolation and slight necrosis (Figure 6). Cross section of the pancreas from diabetic mice treated with ZnONPs and CrONPs, Exhibiting normal pancreatic islets with cellular vacuolation and slight necrosis with mild leukocyte infiltration and little congestion (Figure 7).

Figure 1: Cross section of the pancreas from control group mice exhibiting normal pancreatic architecture. Figure 2: Cross section of the pancreas from alloxan-receiving mice Exhibiting necrosis and degeneration of islets with telangiectasis. Figure 3: Cross section of the pancreas from ZnONPs receiving mice Exhibiting normal histological organization. Figure 4: Cross section of the pancreas from CrONPs receiving mice Exhibiting normal histological organization. Figure 5: Cross section of the pancreas from diabetic mice treated with ZnONPs Exhibiting normal pancreatic islets with cellular vacuolation and slight necrosis with mild leukocyte infiltration and little congestion. Figure 6: Cross section of the pancreas from diabetic mice treated with CrONPs Exhibiting normal pancreatic islets with cell vacuolation and slight necrosis. Figure 7: Cross section of the pancreas from diabetic mice treated with ZnONPs and CrONPs, Exhibiting normal pancreatic islets with cellular vacuolation and slight necrosis with mild leukocyte infiltration and little congestion. H&E, 400X.

Discussion

It is essential to find alternative methods for improving and managing diabetes and its complications for reducing or avoiding drug side effects (21). One of those methods is the use of nanoparticles as they were very tiny sized, and their biological features made them suitable for use in medical applications (22). Based on the present results, alloxan-induced diabetes is caused by an increase in glucose and HbAlc levels and a decrease in insulin hormone (23). It is known that alloxan destroys pancreatic B cells (24).

Blood glucose levels with glycated hemoglobin and insulin were respected as markers for diabetes and its progression (25). Their disturbances reflect the pancreatic dysfunction and the C-peptide that refers to the B cell function (26). To have different mechanisms in regulating gene expression of the factors involved in glucose homeostasis (27). Combining the two nanoparticles’ types, zinc oxide and chromium oxide, and alone administration exhibits a decrease in glucose and HbAlc levels and an increase in insulin hormone, proposing the antidiabetic activity of these nanoparticles’ types (28). Zinc manages glucose metabolism by augmentation glycogenesis in the liver via insulin signaling (29). Also, chromium oxide nanoparticles can increase the antioxidant capacity of cells resulting from diabetes by increasing the oxidative stress resulting from diabetes (30).

Moreover, zinc oxide nanoparticles' antidiabetic ability might have resulted from its regeneration effect on insulin-secreting cells of the pancreas, causing an increase in insulin levels supported by our histological results and the lowering in C- peptide levels that refer to the restoration of pancreatic B cells (31), as zinc oxide nanoparticles regenerate Langerhans s islets and B cell function (32). In the same way, Chromium oxide nanoparticles could conjugate with insulin receptors and increase insulin levels through its induction of substrate receptor phosphorylation, which plays an essential task in signal transmission such as Pl3K, which is critical in insulin function (33). This refers to the improved animal's glycemic state by the two nanoparticle combinations (34). Zinc oxide nanoparticles improved the destructed mass of B cells in diabetic mice, proposing their ameliorative effect on pancreatic tissue, especially Langerhans s Islets, and increased the degenerated B cells' restoration. These parameters were shown to be more improved in the group of nanoparticles combination (35).

Conclusion

ZnONPs and CrO3NPs exhibit synergistic cooperation on diabetes improvement, restoring the degenerated insulin-producing cells of Langerhans Islets.