Home Perinatal supplementation with selenium nanoparticles modified with ascorbic acid improves hepatotoxicity in rat gestational diabetes
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Perinatal supplementation with selenium nanoparticles modified with ascorbic acid improves hepatotoxicity in rat gestational diabetes

  • Ahmed M. Rady EMAIL logo , Hossam Ebaid , Mohamed Habila , Iftekhar Hassan , Jameel Al-Tamimi , Ibrahim M. Alhazza , Mohamed S. Moshab and Zeid A. ALOthman
Published/Copyright: November 10, 2023

Abstract

Because of the potential bioactivities, nanoparticles have engendered hope in scientific communities for developing novel therapeutic strategies. In the present study, it was tested whether selenium nanoparticles (Se-NPs) can protect the liver in mothers with gestational diabetes (DM). The gestational rats were divided into three groups (n = 8). Group 1 (CN) received the vehicle, Group 2 (DM) received a single intraperitoneal injection of 165 mg/kg of alloxan, and Group 3 (DM + Se-NPs) received a single intraperitoneal injection of 165 mg/kg alloxan and then treated with Se-NPs at a dose of 2.5 mg/kg twice a week for 6 weeks; 1 week before gestation and continued for 5 weeks. The structure of the fabricated Se-NPs modified with ascorbic acid indicated that nano-Se was associated with a carbon matrix. The body weight of diabetic mothers was lower compared to control animals. The use of Se-NPs as a treatment has led to significant restoration of the body weight in diabetic rat mothers compared to those diabetic animals without treatment. Concentrations of alanine transaminase, aspartate aminotransferase, LDH, malondialdehyde, cholesterol, triglycerides, and glucose were significantly increased in diabetic rats, while glutathione significantly declined in comparison to control gestational rats. Interestingly, Se-NPs in DM + Se-NPs rats were found to restore all these parameters to values close to the control levels. Se-NPs could improve the histological structure of the liver in gestational rats with diabetes (DM + Se-NPs). Our data demonstrate that Se-NPs shield the liver structure and function in gestational rats against alloxan-induced diabetes.

1 Introduction

Gestational diabetes is a frequently encountered pregnancy complication that affects the normal functioning of insulin. This condition can manifest in different levels of severity. Typically, it is identified for the first time during pregnancy. [1]. Insufficient insulin production and heightened insulin resistance are consequences of oxidative stress in some experimental models and play a significant role in diabetes pathogenesis [2,3]. Several liver enzymes were found to be substantially increased during gestational diabetes mellitus (GDM) in numerous metabolic disorders [4]

Demand for selenium (Se) has increased during pregnancy [5] as it is involved in the inhibition of cyclooxygenase-2 and P-selectin [6], and experiencing inadequate nutrition during pregnancy can elevate the risk of developing diabetes [7]. Hyperglycemia could increase nitrosative and oxidative stress [8]. Dysfunction in glycemic control is related to the diminution of serum antioxidant activity [9]. Dietary selenium (Se) in humans is obtained from consumable plant sources. Deficiency in human diets may reflect crop production on soils with low Se bioavailability or content [10,11,12]. The beneficial effects of Se in living organisms depend on its quantity in food and its chemical form [13,14]. Organisms have a higher absorption capacity for organic forms of selenium compared to inorganic forms [15].

Se is a fundamental nutrient essential for physiological functions in humans and other animals [16,17,18]. Selenium is a cofactor in selenoproteins and is necessary for enzymic function. In general, these selenoenzymes have selenocysteine in their active sites [19]. Selenium acts as a redox center. It is needed for the production of active thyroid hormone and also acts as an antioxidant and anti-inflammatory. Selenium is crucial for supporting the immune response and supports cell-cycle progression [15], and suitable levels of protection against protein fragmentation caused by oxidative stress and lipid peroxidation [20,21]. Increasing Se intake may be advantageous for reducing the risk of different diseases [22]. High levels of Se may give rise to problems in mechanisms of homeostasis and could even be fatal [23]. Lack of Se is associated with a weakened immune system and many other human diseases [11,24].

Recently, nanotechnology has been applied to allow the development of novel therapeutic strategies. Experimentally, silver nanoparticles were found to prevent carbon tetrachloride-induced hepatotoxicity in mice [25] and against nephrotoxicity in male mice. Selenium nanoparticles (Se-NPs) alleviate diabetic nephropathy and pancreatopathy in the offspring of rats [26] and in diabetic females during pregnancy [27] via inhibition of oxidative stress. Se-NPs provide relief from hepatotoxicity induced by carbon tetrachloride in Swiss albino rats [26]. Administration of biodegradable Se-NPs leads to the protection of myelination and axons in the hippocampus and the reversing of brain edema after cerebral ischemic stroke [28]. The application of Tet-1 peptide-coated Se-NPs may enhance the therapeutic potential for Alzheimer’s disease by inhibiting the aggregation of amyloid-β and promoting the healing process, as demonstrated in vitro [29]. Here, we investigated whether Se-NPs can shield the liver structure and function in gestational rats with alloxan-induced diabetes.

2 Materials and methods

2.1 Chemicals

Alloxan (C4H2N2O4) was acquired from S.d. Fine-Chem Ltd. (Boisar). It was then dissolved in 0.9% sodium chloride solution at pH 7.

2.2 Synthesis of nano-Se combined with an organic stabilizer

All substances used were of analytical-grade quality. During the synthesis process, deionized water was utilized. Se dioxide, citric acid, and ascorbic acid were purchased from Sigma. Nano-Se modified with ascorbic acid was prepared as previously described [30,31] with some modification. Se oxide was dissolved in deionized water and mixed with citric acid to form a clear solution with a Se oxide/citric acid ratio of 3:1. The ascorbic acid solution was introduced slowly, serving as a reducing agent to allow the slow formation of nano-Se with citric acid stabilization. Stirring of the reaction mixture continued for 15 h at 80°C. Nano-Se was then collected by centrifugation at 10,000 rpm. The product’s structure and morphology were examined using transmission electron microscopy (TEM), scanning electron microscopy (SEM), and X-ray diffraction (XRD). TEM images were captured using a JEOL JEM-2100F electron microscope(Japan), operating at 200 kV. SEM analysis was conducted using a Jeol GSM-7600F microscope (Japan). Fourier transform infrared spectra were recorded using a Bruker Vertex-80 spectrometer. Powder XRD patterns were obtained using a PANalytical X’Pert PRO MPD instrument (Netherlands), with Cu Kα radiation filtered through nickel (45 kV, 40 mA).

For the SEM sample examination, a piece of carbon tap is fixed on an aluminum setup, and then the portion of powder nano-Se is distributed on the carbon tap. The aluminum setup ensures that the nano-Se is spattered between 60 and 90 s to fix the platinum coating layer on the sample. Finally, the sample was examined by SEM. For TEM, 0.005 g of nano-Se was dispersed in water and exposed to ultrasonic waves for 5 min. Then, drops from the dispersed nano-Se were added slowly to the TEM grid and left until drying, and then the second drop was added, and the process continued until 5 drops were added.

2.3 Animals and ethical approval

A group of 40 physically sound and sexually mature male and virgin female Wistar albino rats with an average weight ranging from 130 to 150 g were selected for the study, obtained from the College of Pharmacy( King Saud University, Riyadh, Saudi Arabia). The animals were kept under a 12 h light and 12 h dark cycle, maintaining room temperature conditions (25 ± 5°C), humidity (55 ± 5.6), and good ventilation. The rats had unrestricted access to a standard diet and water throughout the study.

The study protocol in the Zoology Department of the College of Science at King Saud University (KSU-SE-20-38) received approval from the Animal Ethics Committee.

2.4 Diabetes induction

Alloxan was freshly prepared, and a dosage of 165 mg/kg was administered via a single intraperitoneal injection to induce diabetes in overnight-fasted rats as described previously [32]. Two days after the alloxan injection, fasting blood glucose levels were recorded using a blood glucose meter (BIONIME GmbH, Switzerland).

2.5 Experimental design

The rats were allowed to acclimatize in the laboratory for a period of 7 days after their arrival. During this time, the male rats were housed in cages together with three female rats, maintaining a ratio of 1:3 male/females. Vaginal smears were examined to ensure pregnancy. Group 1 (pregnant females, n = 8) was administered only the vehicle; Group 2 (pregnant females, n = 8) was administered a single intraperitoneal injection of alloxan; Group 3 (pregnant females, n = 8) was administered a single intraperitoneal injection of alloxan. In addition, rats of the third group were treated with Se-NPs, 2.5 mg/kg, twice a week for a period of 6 weeks. The treatment was initiated 7 days prior to gestation and continued for an additional 5 weeks.

2.6 Biochemical analysis

The Trinder method was employed to measure the blood glucose level [33]. The measurement of serum alanine transaminase (ALT) and aspartate aminotransferase (AST) in plasma was conducted using commercial kits obtained from Randox (UK) and Linear diagnostic kits (Spain), respectively. The method employed for these measurements was based on the approach developed by Gella et al. [34]. The levels of serum urea and creatinine were determined according to the method established in previous studies [35,36]. Serum urea and creatinine levels were assessed using kits obtained from Biosystems S.A. (Spain). Measurements of high-density lipoproteins (HDL), triglycerides (TAGs), and cholesterol (CHO) were conducted using kits obtained from Randox. The levels of reduced glutathione (GSH) and malondialdehyde (MDA) were determined following the established protocols described in previous studies [37, 38,39].

2.7 Histological investigations

The liver tissue from rats in the control, diabetic, and diabetic treated with Se-NPs groups was placed in 8% formalin for fixation. Tissue samples measuring 10 mm × 5 mm × 3 mm were immersed in paraffin. Sections of 7 μm thickness were sliced using a rotary microtome, followed by staining with hematoxylin and eosin. The sections were observed using a light microscope and imaged at magnifications of 100×, 200×, and 400× using an Olympus microscope (Japan).

2.8 Statistical analysis

The statistical analysis of the data was performed using the SPSS program, employing one-way analysis of variance with Tukey’s post hoc test. An asterisk (*) indicates a significant difference from controls (group I). A hashtag (#) indicates a significant difference from diabetic rats (group II). The data are reported as mean ± standard error of the mean (SEM), with a significance threshold set at p < 0.05.

3 Results

3.1 Structural characterization of nano-Se

The prepared nano-selenium stabilized with ascorbic acid is characterized with SEM, TEM, and XRD to assess the morphology and structure. The SEM (Figure 1a) showed spherical and agglomerated shapes with particle sizes between 88.4 and 349 nm. The spherical particulates are not uniform in size with different diameters (128, 164, 250, 244, and 349 nm). However, the agglomerated part appears as colonies that are labeled with blue circles, including small spheres of about 88.4 nm. The TEM examination (Figure 1b) showed spheres and sphere-like structures with particle sizes between 50 and 400 nm. The nano-Se spheres are accumulated to form larger structures with perforated tail-like colonies. In addition, the TEM showed that the nano-Se that appears in the carbon matrix originated from the presence of ascorbic acid. This combination is expected to enhance the stability of nanoparticles during biological applications.

Figure 1 
                  SEM (a) and TEM (b) examination of nano-Se.
Figure 1

SEM (a) and TEM (b) examination of nano-Se.

The formation of the crystalline nano-Se in the presence of an organic stabilizing agent is confirmed by XRD examination (Figure 2). Peaks related to nano-Se are detected at 24° and 30° and are attributed to lattice planes of 100 and 101 [30,31,40]. Se-NPS is present in a highly crystalline structure, as evidenced by the detection and indexing of all characteristic peaks of crystalline nano-Se at approximately 24°, 30°, 41.5°, 43.6°, 46°, 48°, 52°, 55.6°, 56°, 61.5°, 65.5°, 67° and 72°. These peaks are similar to peaks described by Angamuthu et al. [41], with some deviation that may be attributed to organic acid encapsulation [42].

Figure 2 
                  XRD peaks of nano-Se.
Figure 2

XRD peaks of nano-Se.

3.2 Effect on the body weight

The body weights of diabetic animals (DM) significantly decreased over time compared to the control animals. However, Se-NPs were found to significantly maintain the body weight of the diabetic pregnant rats to be close to the normal in DM + Se-NP rats (Figure 3).

Figure 3 
                  Body weights of female rats during gestation from control, diabetic, and diabetic treated with Se nanoparticles. Diabetes was induced at week 0, and diabetic rats were divided into diabetic-only and diabetic-treated with Se starting at week 6. The values are mean ± SEM (n = 10) of three independent experiments. *Statistically significant from controls at p ≤ 0.05. #Statistically significant from diabetic animals at p ≤ 0.05.
Figure 3

Body weights of female rats during gestation from control, diabetic, and diabetic treated with Se nanoparticles. Diabetes was induced at week 0, and diabetic rats were divided into diabetic-only and diabetic-treated with Se starting at week 6. The values are mean ± SEM (n = 10) of three independent experiments. *Statistically significant from controls at p ≤ 0.05. #Statistically significant from diabetic animals at p ≤ 0.05.

3.3 Effect on hepatic markers (ALT, AST, HDL)

The DM group rats showed an increase in ALT activity in plasma by 242.62%. In comparison, DM + Se-NP-treated animals displayed a significant recovery in this activity by 40.74% for CN and DM animals, respectively (Figure 4).

Figure 4 
                  Hepatic damage was decreased by Se nanoparticle treatment during the progression of diabetes. Serum levels of HDL, AST, and ALT, control (CN), diabetic (DM), and diabetic-treated rats with Se nanoparticles (DM + Se-NPs) were analyzed using a serum biochemical analyzer. The values are mean ± SEM (n = 10) of three independent experiments. **Statistically significant from controls at p ≤ 0.005. #Statistically significant from diabetic rats at p ≤ 0.05.
Figure 4

Hepatic damage was decreased by Se nanoparticle treatment during the progression of diabetes. Serum levels of HDL, AST, and ALT, control (CN), diabetic (DM), and diabetic-treated rats with Se nanoparticles (DM + Se-NPs) were analyzed using a serum biochemical analyzer. The values are mean ± SEM (n = 10) of three independent experiments. **Statistically significant from controls at p ≤ 0.005. #Statistically significant from diabetic rats at p ≤ 0.05.

The DM group rats exhibited an extreme increase in AST activity of 752.88%. DM + Se-NP treatment decreased this activity by 15.89% (Figure 4). In the DM group rats, the LDH activity increased by 110.67%, and DM + Se-NP decreased this activity by 37.93% (Figure 4).

3.4 Effect on redox parameters (GSH, MDA)

The GSH levels in the DM group animals exhibited a significant decline of 78.61%; DM + Se-NP treatment reversed this increase significantly with levels increased by 125.63% (Figure 5). The MDA levels in the DM group rats demonstrated a notable increase of 67.75%, and DM + Se-NP treatment reduced this increase by 35.59% (Figure 5).

Figure 5 
                  Effect of Se-NPs on reduced GSH in the liver of control (CN), diabetic (DM), and diabetic rats treated with selenium nanoparticles (DM + Se-NPs). Values are mean ± SEM (n = 10) of three independent experiments. *,**,***Statistically significant from the control at p ≤ 0.05, p ≤ 0.005, p ≤ 0.001, respectively. #Statistically significant from diabetic rats at p ≤ 0.05.
Figure 5

Effect of Se-NPs on reduced GSH in the liver of control (CN), diabetic (DM), and diabetic rats treated with selenium nanoparticles (DM + Se-NPs). Values are mean ± SEM (n = 10) of three independent experiments. *,**,***Statistically significant from the control at p ≤ 0.05, p ≤ 0.005, p ≤ 0.001, respectively. #Statistically significant from diabetic rats at p ≤ 0.05.

3.5 Effect on lipid profile (CHO, TAGs)

The DM group rats exhibited a notable increase in serum CHO levels by 74.38%, and DM + Se-NP treatment decreased this level by 10.23% (Figure 6). The DM group animals demonstrated a notable increase in TAG levels by 48.60%, and DM + Se-NP treatment decreased TAG levels by 26.99% compared with DM rats (Figure 6).

Figure 6 
                  The treatment with Se-NPs is effective in alleviating serum biochemical profiles related to lipid metabolism in diabetic rats. Blood was collected from the hearts of rats, and serum levels of CHO and TAGs were analyzed using a serum biochemical analyzer. Values are mean ± SEM (n = 10) of three independent experiments. *,**Statistically significant from controls at p ≤ 0.05, p ≤ 0.005, respectively. #Statistically significant from diabetic rats at p ≤ 0.05.
Figure 6

The treatment with Se-NPs is effective in alleviating serum biochemical profiles related to lipid metabolism in diabetic rats. Blood was collected from the hearts of rats, and serum levels of CHO and TAGs were analyzed using a serum biochemical analyzer. Values are mean ± SEM (n = 10) of three independent experiments. *,**Statistically significant from controls at p ≤ 0.05, p ≤ 0.005, respectively. #Statistically significant from diabetic rats at p ≤ 0.05.

3.6 Effect on serum glucose level

The DM group animals displayed an increase in the serum glucose level by 55.98% compared to controls. The administration of DM + Se-NP treatment resulted in a decrease of 31.54% in comparison to DM group rats (Figure 7).

Figure 7 
                  The serum glucose level is increased significantly in diabetic rats as compared to control rats. Treatment with Se-NPs decreased serum glucose levels, but levels remained significantly higher than those of control rats. Values are mean ± SEM (n = 10) of three independent experiments. **Statistically significant from controls at p ≤ 0.005, respectively. #Statistically significant from diabetic rats at p ≤ 0.05.
Figure 7

The serum glucose level is increased significantly in diabetic rats as compared to control rats. Treatment with Se-NPs decreased serum glucose levels, but levels remained significantly higher than those of control rats. Values are mean ± SEM (n = 10) of three independent experiments. **Statistically significant from controls at p ≤ 0.005, respectively. #Statistically significant from diabetic rats at p ≤ 0.05.

3.7 Liver histopathology

Liver histopathological assessment in diabetic rats showed notable differences from control and diabetic animals treated with Se-NPs, as shown in Figure 8, and are presented in the supplementary material. The control group (I) animals demonstrated normal sinusoids, Kupffer cells, and central veins. The hepatocyte cytoplasm appeared normal with a regular central nucleus. A normal hepatic architecture with intact triads was observed (Figure 8a). Diabetic group (II) rats showed alloxan treatment-related pathological changes. Liver sections displayed deformation of hepatocyte histology, with fibrosis, and the inflammatory infiltration was most prominent around the central vein, along with central vein congestion (Figure 8b). The Se-NP (group III) treatment reduced histopathological changes caused by alloxan, and hepatic architecture was typical of normal liver (Figure 8c).

Figure 8 
                  Histomicrographs of liver tissue of the three groups of rats. All sections were stained with hematoxylin and eosin and imaged at 100×, 200×, and 400× (Olympus, Japan) for the control group [CN] (a), diabetic group [DM] (b), and Se-NP-treated diabetic rats [DM + SeNPs] (c). The red arrows show the infiltration of the hepatic tissues with inflammatory immune cells.
Figure 8

Histomicrographs of liver tissue of the three groups of rats. All sections were stained with hematoxylin and eosin and imaged at 100×, 200×, and 400× (Olympus, Japan) for the control group [CN] (a), diabetic group [DM] (b), and Se-NP-treated diabetic rats [DM + SeNPs] (c). The red arrows show the infiltration of the hepatic tissues with inflammatory immune cells.

4 Discussion

Supplementing with selenium (Se) has been investigated as a potential treatment for various inflammatory conditions, including asthma, arthritis [43], and chronic lymphedema [44] as well as ameliorates hepatic damage [45]. Children diagnosed with food allergies exhibited lower selenium (Se) levels compared to children without any allergies. This decrease in Se levels was associated with reduced values of GSH peroxidase and superoxide dismutase, indicating the potential role of Se in the development of food allergies [46]. Se-NPs show superior biocompatibility when compared to organic and inorganic selenium compounds [47]. Se-NPs were found to produce higher levels of ROS for the treatment of cancer cells [48].

Se-NPs have been investigated under different conditions characterized by oxidative stress and inflammation, such as arthritis, cancer, diabetes, and nephropathy, showing promising therapeutic advantages [49]. Nano-sized antibacterial particles are expected to exert more potent effects on bacteria compared to their larger counterparts, indicating a potential for enhanced antibacterial activity. Therefore, Tran and Webster [50] suggested that the utilization of Se-NPs shows promise in the prevention and treatment of S. aureus infections, offering an effective approach. Generally, the administration of Se-NPs can significantly enhance the induction of the Th1 immune response platform by increasing levels of IFN-γ and IL-12. This effect may contribute to improved prognosis in mice with tumors [51].

The use of nanoparticles offers several advantages, including the reduction of toxicity, improved targeting, versatile control over the release profile of the encapsulated substance, and enhanced bioactivity. It was found that Se-NPs can form various shapes according to the solvents or compounds used for their preparation. [52]. Here, the structure of nano-Se is formed with citric acid in the presence of ascorbic acid for reduction. Se-NPs appear in the carbon matrix which is expected to enhance the stability of nanoparticles during biological applications. Results are similar to that described by Angamuthu et al. [41], with some deviation that may be attributed to organic acid encapsulation [42].

Selenium displays properties that imitate insulin, and our study aimed to explore the effects of perinatal supplementation with Se-NPs on diabetic complications during gestation. The intraperitoneal injection of rats with alloxan decimates a portion of pancreatic β-cells, causing insulin deficiency diabetes [53]. Untreated diabetic rats showed notably lower body weights compared with controls. This response could reflect impairment of glycemic indices. For instance, reduced weight gain in diabetic rats could be caused by exaggerated catabolism of protein to configure amino acids for gluconeogenesis [54]. Treatment of diabetic rats with Se-NPs likely led to an improvement in glycemic indices and largely reversed the effects of alloxan on weight gain. A decrease in food consumption in Se-NP-treated animals could explain the lack of complete reversal of effects on body weight compared with the control group. Such a mechanism might be similar to the anorexigenic impact seen in diabetic rats. Also, selenium accelerates the excretion of renal glucose in mice [55].

The liver functions in storage, excretion, metabolism, and detoxification, in addition to maintaining blood glucose levels. AST and ALT are sensitive biomarkers for hepatic toxicity and damage [56,57]. Liver damage-related alloxan toxicity leads to the release of ALT and AST from the cytosol of the liver into the bloodstream. This release leads to a detectable increase in the activities of these enzymes observed in the serum of diabetic rats [58]. Increased levels of AST and ALT caused by insulin shortage are related to elevated gluconeogenesis during diabetes [59]. This study shows that treatment with Se-NPs partially reversed the release of liver enzymes into the bloodstream. This action likely resulted from radical scavenging by Se and the overall importance of Se in the protection of tissue from oxidative damage [60].

Serum TAG and CHO levels are considerably influenced by liver function. HDL acts as a purifier of CHO in various tissues. The reduction in serum levels of TAGs and CHO indicates that Se-NP treatment may relieve symptoms of diabetes in rats. Elevated levels of alanine aminotransferase, AST, lactate dehydrogenase, TAG, and CHO in the blood serve as indicators of liver necrosis or disease [61]. Animals lacking antioxidant defense mechanisms have shown an increase in serum HDL activity, partially due to enhanced hepatic injury by lipid peroxidation [62,63].

The findings of this study reveal elevated levels of MDA and decreased levels of GSH in diabetic rats. Supplementation of Se-NPs in diabetic rats led to a reduction in serum MDA and an increase in serum GSH levels. Following a 12-week administration of 200 μg/day Se supplements in patients undergoing hemodialysis, a reduction in MDA levels was observed [64]. Similar results were observed after Se supplementation in animal models [65]. Se is a fundamental component of the red blood cell GPx system [66], and Se supplementation has the potential to reduce the generation of free radicals and lipid peroxidation by promoting the action of GSH peroxidase, particularly in conditions like GDM where free radical levels are elevated [67]. Oxidative stress can manifest as an increase in oxidants or a reduction in antioxidant capacity [68]. Diminished MDA and increased GSH levels after Se-NP supplementation may be indirectly related to diminished lipid peroxidation and free radicals [69].

Overall, our data demonstrate that Se-NPs shield the liver from diabetes-induced injury in alloxan-treated rats. The observed protective effect may be attributed to the metal’s capacity to scavenge free radicals and its ability to mimic insulin. Accordingly, functional foods might be developed with Se-NPs as a component for treating diabetes and decreasing its complications.

Acknowledgments

This work was funded by Researchers Supporting Project (RSP2023R225), King Saud University, Riyadh, Saudi Arabia.

  1. Funding information: This work was supported by the Researchers Supporting Project (number RSP2023R225), King Saud University, Riyadh, Saudi Arabia.

  2. Author contributions: I.A., H.E., and I.H. designed the study. A.R. and M.H. wrote the manuscript. H.E., I.A., and Z.A.A. supervised the study. A.R., J.A., M.M., I.H., and A.E. conducted experiments and acquired, analyzed, and interpreted the data. Z.A.A. and M.H. shared the preparation and characterization of nano-Se. J.A. and A.R. assisted in biostatistical and data interpretation. All authors have read and agreed to the published version of the manuscript.

  3. Conflict of interest: The authors declare that there are no competing interests.

  4. Ethical approval: The study was approved by King Saud University ethical committee under reference number KSU-SE-20-38.

  5. Data availability statement: All data generated or analyzed during this study are included in this published article.

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Received: 2022-11-20
Revised: 2023-10-23
Accepted: 2023-10-24
Published Online: 2023-11-10

© 2023 the author(s), published by De Gruyter

This work is licensed under the Creative Commons Attribution 4.0 International License.

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