The current study was conducted to find out a therapeutic effects for each of the drug glimepiride (alone and in combination with selenium) and glimepiride nanoparticles loaded on chitosan on male white rats in which diabetes was created by using alloxan. In the beginning, the glimepiride nanoparticles was prepared and loaded on chitosan, then a procedure was conducted. The necessary tests to guarantee the success of the loading process and conversion the drug into a nanoparticles. The study was conducted in the animal house of the College of Science/University of Al-Qadisiyah during a period one month for the purpose of knowing the effect of the nanodrug glimepiride and selenium on male white rats in which diabetes was induced with alloxan. In the beginning the nanodrug glimepiride was prepared. And loading it on chitosan, then conducting the necessary tests to Make sure of loading process success and converting drug into a nan composite. The results of the particle size measurement test Show average drug particle size of glimepiride next loading on chitosan nano particles (CNP-GL.) was (80.1 nm) and in addition to the small size of the particles, the results showed the stability of glimepiride drug, as the polydispersity scale of the sample recorded (0.459). The study included (50) white male rats splited randomly to (5) groups, all group contained (10) animals after their weights were recorded. The healthy negative group (control-)(G1), (G2)positive control group in which diabetes develops, the group (G3) in which diabetes was induced was dosed with glimepiride nanodrug at concentration (1 mg/kg) and selenium(0.1 mg/kg), group (G4) in which diabetes was induced was dosed with glimepiride at a concentration of (1 mg/kg), group (G5) in which diabetes was induced was dosed with glimepiride at a concentration of (1 mg/kg) and selenium at a concentration of (0.1 mg/kg). At the end of the 30day experiment, weights of the animals were recorded. Then she was anesthetized with chloroform and blood samples were withdrawn from the heart directly to conduct tests of kidney function indicators (urea, creatinine). The results and indicators of kidney function showed a important increase (p<0.05) in the level of (urea, creatinine) in the (G2) group when compared with the negative control group (G1), while a significant decrease (p<0.05) was recorded in the groups that were treated with the drug. Nano- glimepiride combined with selenium compared with group (G2).
Diabetes mellitus: is one of a chronic diseases which occurs as a result of the inability of the pancreas gland to produce an adequate amount of the hormone insulin, or the body’s failure to respond to the effect of the insulin hormone that it forms and insulin is the hormone that regulates the level of glucose in the blood. The origin and causes of diabetes can vary greatly but it always includes defects in insulin secretion or response to it, or both. An increase in the level of glucose in the blood is common evidence of diabetes and over time it leads to serious problems in the body, especially in the nerves and blood vessels [1].
Glimepiride is the modernest sulfonylurea compound and it is sometimes divide as the 3rd generation of it. It was used as a clinical treatment for the first time in Sweden, where the US Food and Drug Administration (FDA) confirmed glimepiride in 1995 to therapy of type 2 diabetes as a single treatment or as a combined treatment with metformin [2]. Selenium is fundamental trace element to the body that is respectable in numbers of biological roles. Selenium-selenoproteins are included in a many of fundamental biological jops, ranging from organization the concentration of Reactive Oxygen Species (ROS) to the biological synthesis of hormones. Over the last years, a numeral of projectes focusing on a relationship between (Se) and such as diseases. In general, dietary status with reasonable levels of (Se) (selenium supplementation) has been linked with a lessen risk of numeral diseases. moreover, some studies have linked higher levels of (Se) in the body with a lower prevalence of diabetes or a lessen risk of hyperglycemia [3]:
Nano technology has played a main role in last years in a domain of biomedicine [4]
The use of nano particles raised an absorption and effects of curative materials and improved the level of efficiency and their effective dose [5]
The charge, geometry and size of nanoparticles have an important influence on a curative method, which influence loading ability of a treatment [6,7]
As balance between particle size and movement geometry is the main factor in the loading efficiency which is also reflected in the length of its presence and distribution in the remedial action [8].
Kidney Function Indicators
Measurement of Serum Creatinine Concentration
The colorimetric method is used using a kit prepared by the British company Randox to measure level of creatinine in serum, which depends on reaction of creatinine present in the sample with picric acid in a basic medium to give a red-colored complex of creatinine picrate, which has the highest absorbance at 520 nm [9].
The Principle of Experience 1
Creatinine reacts with picric acid in a basic solution to form a colored complex:
Reagents
The reaction solution is prepared by mixing an equal amount of two solutions: Picric acid and sodium hydroxide, shaking well and keeping the solution stable for 5 hours at a temperature ranging between (12-25) degrees Celsius (Table 1).
The Method of Work
Mix the tubes well and then measure the absorbance at wave length of 520 nm utilize a spectrophotometer (Table 2).
Calculating the Results
Serum creatinine activity was calculated using the following equation:
Standard Conc. = 177μmol /L
Estimation of urea level in the serum.
The Principle
The level of urea is estimated in a spectrophotometer before and the Urease Berthelot method relied on the enzymatic end point that enables the concentration of urea in the serum to be determined and the urease enzyme decomposes urea and produces ammonium ions [10]:
Ammonium ions react in a basic medium with salicylate and hypochlorite by sodium nitroprusside to form 2,2 dicarboxylindophenol, green in color, The intensity of the color is directly proportional to concentration of urea in the sample [11].
Solutions Used:
Standard reagent: It contains a fixed percentage of urea
The enzyme reagent contains the enzyme Ureas
Color reagent: It consists of EDTA, Sodium salicylate, Sodium nitroprusside and a phosphate buffer at a pH =8
Alkaline Solution: It contains Sodium hypochlorate and Sodium carbonate
The reaction solution is prepared by adding the enzymatic solution to the colorimetric reagent with good shaking and then the solution remains stable for two months at a temperature of (2-8) degrees Celsius.
The Procedure
Table 3 solutions and methods used to estimate the level of urea in serum.
Table 1: Show the reaction solutions
Reagents |
Contents | Initial concentration of solutions |
CAL | Standard | 177 µmol/L (2mg/dL |
RIa | Picric acid | 35 mmol/L |
RIb | Sodium hydroxide | 1.6 mol/L |
R2 | TA651(TCA) Trichloroacetic acid | 1.3 mol/L |
Table 2: Show how to estimate serum creatinine
| Substance | Blank | Standard | Sample |
| Standard | - | 0.5 ml | - |
| TCA | 0.5 ml | 0.5 ml | - |
| Serum | - | - | 1 ml |
| Working solution | 1 ml | 1 ml | 1 ml |
Table 3: Solutions and methods used to estimate the level of urea in serum
Substance | Blank | Standard | Sample |
Standard | - | 10 µml | - |
Sample | -- | - | 10 µml |
Working solution | 1 ml | 1 ml | 1 ml |
The tubes were shaken and kept at 37°C for 3 minutes | |||
Reagent 4 | 200 µml | 200 µml | 200 µml |
The tubes were shaken and stored at 37°C for 5 minutes, after which the absorbance was measured using a spectrophotometer at a wavelength of 580 nm | |||
Table 4: The effect of glimepiride and selenium on kidney function indicators (urea and creatine) in the serum of experimental diabetes white male rats
Creatinine (mg/dl) | Blood urea (mg/dl) | Groups |
0.310±0.021F | 30.6±1.66D | G1 |
0.813±0.014A | 66.21±2.4A | G2 |
0.343±0.007E | 33.9±2.23D | G3 |
0.373±0.008C | 46.6±2.91B | G4 |
0.356±0.024D | 41.7±1.29C | G5 |
0.062 | 4.56 | LSD |
Values represent averages ± standard error
Calculation of Results
The urea concentration in blood serum was calculated according to the following equation:
Conc. of standard = 8.33mmol /L
The Statistically Analysis
SPSS version 32 was used to statistically analyze all the results of the current study and for this purpose, one-way analysis of variance was used. test was applied, with the value of the Least Significant Difference (LSD) calculated. The confidence interval was equal to 95% and the probability level value was less. of 0.05 (p<0.05).
The Urea Level in Serum
The statistical to the current study results shown in (Tables 1-4), showed an important increase (p<0.05) in urea level due to type 2 diabetes development in (G2) when compared with negative control group (G1). (G4) A significant decrease compared to group (G2), An important decrease was also observed for the rest of the groups compared to group G2 (Figure 1-10).
The Creatinine Level in Serum
Inducing type 2 diabetes in experimental animals lead to an important (p<0.05) increase in level of creatinine group G2 (compared to negative control group G1) as shown in Table 4, while treatment of laboratory rats resulted in The experiment with the nano-drug Clamibird with selenium resulted in an important decrease (p<0.05) in the creatine level in group G3, while there are no apparent differences .were observed between groups G4 and G5:
Different capital letters indicate a significant difference (p<0.05) between the groups under study
Similar capital letters indicate that there is no significant difference (p>0.05) between the groups under study
Histological Study Results:
G1: negative control group
G2: positive control group
G3: nanodrug with selenium group
G4: Regular drug group
G5: Regular drug + selenium group
Figure 1: Section of the renal cortex tissue of from control group rat (G1) showing: (A) Normal renal glomerulus (B). Stained renal glomerulus capsule: Hematoxylin-eosin, 40X H&E
Figure 2: Section of the renal cortex tissue of a rat from the control group (G1) showing: (A) A normal renal glomerulus capsule (B) A normal renal glomerulus. Stained: hematoxylin-eosin, magnification power (40X)
Figure 3: Section of the renal cortex tissue of a positive control group rat (G2). Severe degeneration and necrosis of the cells lining renal convoluted tubules are observed, along with severe hemorrhage in the renal tissue. 10X H&E
Figure 4: Section of the renal cortex tissue of a rat from the positive control group (G2). Clear necrosis and sloughing of the cells lining the renal convoluted tubules with severe bleeding in the renal tissue, congestion and atrophy in the glomeruli with the presence of protein stones inside the lumen of the convoluted tubules 10X H&E
Figure 5: Section of renal cortex tissue of a rat from group (G3) treatment with nanodrug and selenium, show the glomeruli appear round and proliferated, with clear hyperplasia in the cells lining the renal convoluted tubules with very slight dilatation of the lumen of these tubules 10X H&E
Figure 6: Section of the renal cortex tissue of a rat from the (G3) group treated with nanodrug and selenium group. The glomeruli appear large, round and proliferated with marked hyperplasia of the cells lining the renal convoluted tubules. The tubule lining appears narrow and normal 10X H&E
Figure 7: Section of kidney cortex tissue of a rat from the (G4) group treated with the Regular drug group, showing a normal and slightly proliferated glomerulus with very little sloughing off of the tubule lining cells and slight hyperplasia in some other tubule lining cells, slight hemorrhage in the liver tissue 10X H&E
Figure 8: Section of the renal cortex tissue of a rat from group (G4) treated with Regular drug and selenium. Most of the renal glomeruli appear large, round and normal, and some of them show atrophied glomeruli, slight hemorrhage in the renal tissue with slight dilatation in the lumen of the renal convoluted tubules 4X H&E
Figure 9: Section of the kidney cortex tissue of a rat from the (G5) group treated with Regular drug and selenium, showing the presence of a large, round, proliferating glomerulus, a few tubules suffering from sloughing of the cuboidal cells lining them. Some of these tubules show hyperplasia of the cells lining them 40XH&E
Figure 10: Section of the renal cortex tissue of a rat from the (G5) group treated with Regular drug and selenium, showing some renal glomeruli slightly proliferated and rounded, others appearing atrophied glomeruli, dilatation of the renal convoluted tubules and slight hyperplasia of the cells lining these tubules 40XH&E
The results of the present study demonstrated an important increase in levels of both creatinine and urea in positive control group in which diabetes was induced when comparison to negative control group. These reasoning are consistent with many studies, including [12,13,14]. The reason may be due to the effects of alloxan on the pancreatic b cells, which are responsible for producing the hormone insulin, which works to regulate blood sugar levels [15].
High levels of blood sugar lead to damage millions of nephrons, making the kidneys disabled to preserve fluid and electrolyte balance. Then creatinine is filtered by the glomeruli, which leads to disruption and inhibition of the renal filtration mechanism [16].
The current study agrees with many studies such as [17]. Which stated that hyperglycemia is one of the main causes of progressive kidney disease. Approximately (20-30%) of diabetics suffer from abnormal kidney function, which is represented by a low glomerular filtration rate and high blood urea and creatinine [17].
The present study showed that there was a clear important decrease in levels of creatine and urea in the groups treated with free clamypirid, nano-clamypirid and selenium comparison to the 2nd group and that results were agreement with the study conducted by [18]. These results may be due to the main effect of sulfonyl. Ureas (including clampyrid) help enhance insulin secretion and improve metabolism by both the pancreas and extrapancreas by binding to sulfonylurea receptors that bind to the ATP-sensitive potassium channel [19], then stimulating the general metabolism of metabolites. This is through various body mechanisms that occur within the tissue [20].
The effect of selenium was clear in the groups in which it was used. The cause is the antioxidant and anti-inflammatory effects of selenoproteins [21]. Since type 2 diabetes (T2DM) is associated with oxidative stress, we find that Se has anti-diabetic and anti-insulin resistance effects [22].
One of the advantages of the nanomaterial is to increase its bioavailability and increase its therapeutic effectiveness, as well as reduce the side effects that result from some chemical treatments [23]. Or the reason may be because the nanostructures stay in the blood circulation for a long period and allow releasing of drugs according to specified dose. Thus, they cause less plasma changes with fewer adverse impacts [24].
Being ultrafine, these structures break through tissue system and facilitate drug uptake by cells, allow drug delivery effectively and guarantee action at target site. An absorption of nano structures by cells is extremely higher than the absorption of large molecules whose size ranges between (1 and 10) micrometers. So, they interact to treat diseased cells directly with advanced efficiency and low or minor side effects [25].
In terms of histological study, the results of laboratory examination of male rats in which diabetes was induced using alloxan for renal tissue sections showed the presence of many changes in the tissue, including: severe necrosis and degeneration in the cells lining the renal convoluted tubules, with the appearance of shedding in the cells lining the renal convoluted tubules and the presence of severe bleeding in the renal tissue, congestion and atrophy in the glomeruli, with the presence of protein stones inside the lumen of the convoluted tubules. The results of this study were consistent with a number of previous studies, including [26,27].
The most important effects of alloxan shown in these studies are the appearance of some convoluted tubules in atrophy with clear enlargement of glomeruli and increased thickness in some areas of the renal artery, as well as for the basement membrane of the renal glomerular cells. Damage was observed in the interstitial cortical tissue and disintegration of the membrane of some glomeruli with the exit of the cell nuclei and their internal contents, atrophy of the cellular components and the presence of a number of abnormal cells, in addition to the presence of bleeding in some renal glomeruli. Expansion of glomerular mesangium and raised thickness in Bowman's capsule were observed.
These changes could indicate high blood sugar levels., which caused damage to the renal glomerular mesangial cells, which are sensitive to blood sugar levels, which cause superoxide oxidation of fats, reduce antioxidant defense system and lead to increase generation of free radicals, as shown by [28]. It can be said that controlling metabolism is necessary to reduce oxidative stress in the kidney, which causes diabetic nephropathy that occurred in male animals treated with alloxan, due to the failure of blood sugar levels to return to what is known in the normal state and the inability to return the nephropathy that occurred in the kidney as in the normal state [29].
This explains the cellular pathological changes that occurred in the renal tissues and their components. Alloxan causes renal toxicity by generating free radicals in the oxidation-reduction cycle of uric acid, as shown by some studies such as [30], where these free radicals interact for cellular materials including nucleic acids, proteins and fats, which results in cell damage [31] and this is what likely happened in this study. As for the group treated with the nano-drug with selenium and the group treated with (, microscopic examination did not observe any pathological change in the kidney tissues of the treated rats, as the nano-drug and selenium may have provided efficient protection for renal mesangial cells in rodents with experimental diabetes and inhibited the increase or acceleration of diabetic nephropathy. This may be due to the increased solubility of the drug and the improved bioavailability of the drug using the drug delivery system based on nanotechnology [32], or it may be due to the properties that selenium has, such as possessing the synergistic pharmacological property and its role in raising the level of antioxidants [33].
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