Histopathologic effect and immunomodulatory of Indigofera oblongifolia with silver nanoparticles against splenic injury by Plasmodium chabaudi infection in mice

Abstract

Malaria, a globally prevalent disease caused by Plasmodium species, significantly impacts the immune system, particularly affecting splenic function. This study investigates the therapeutic potential of Indigofera oblongifolia leaf extracts (IOLE) with silver nanoparticles (AgNPs) against Plasmodium chabaudi-induced splenic damage in a female C57BL/6 mice model. Fifty female mice were infected with P. chabaudi and subsequently treated with IOLE AgNPs or chloroquine phosphate. Histopathological and immunohistochemical analyses revealed that IOLE AgNPs effectively restored splenic architecture, reduced inflammatory markers, and improved immune responses compared to the control and chloroquine-treated groups. These findings suggest that IOLE AgNPs may offer a novel therapeutic approach to mitigate splenic dysfunction associated with malaria and provide comparative analysis between established therapies like chloroquine and innovative combination of traditional medical plant and modern nano technology.

Keywords

Histomorphometic immunohistochemical malaria silver nanoparticles splenic dysfunction immune response

Introduction

Malaria is considered one of the most globally spreading diseases which can be transmitted through female mosquitos by various species of plasmodium causing fetal alteration and multiple disorders to various organs like the cerebral, spleen liver, and kidney.¹ P. chabaudi serving as an important model for researching malaria infections in laboratory settings.² Malaria infection stages can be the primary reason for the activation and suppression of body immunity.³ This parasite causes a complicated immunological response, mostly targeting the spleen, which is critical in the host's defense against infections.⁴ The immune response to P. chabaudi is characterized by both cellular and humoral components, with CD4+ T cells and B cells playing essential roles in regulating parasitemia and modulating tissue responses during infection.⁵

Impaired lymphocytes and numerous abnormalities appeared during plasmodium infection resulting in splenic dysfunction.⁶ P. chabaudi infection causes considerable changes in the spleen, including changes in leukocyte distribution and lymphocyte apoptosis, which can lead to reduced immune function and increased vulnerability to severe disease consequences.⁷ The inflammatory milieu established by the infection might cause splenic damage and dysfunction, prompting the development of therapeutic approaches to minimize these consequences.¹ The spleen was considered one of the most effective lymphoid organs that was responsible for initiating effective immune responses against several pathogens and bacterial series.⁸ Therefore, the microanatomical and circulatory regions that constituted splenic tissue, the spleen was considered a multiple-functional organ by stimulating the adaptive response of the immunity, reusing the iron product of RBCs phagocytosis, and selectively destroying several pathogens.⁹

Medical plants were considered a promising source in healing the parasitic infection disorders widely spread in recent decades.¹⁰ Indigofera oblongifolia is distributed over the world and has proven its effectiveness in the treatment of P. chabaudi parasites dysfunction of the liver and spleen.¹¹ I. oblongifolia, a plant recognized for its therapeutic characteristics, has received attention for its antimalarial and antioxidant potential.¹² Recent studies indicates that extracts of I. oblongifolia combined with silver nanoparticles (AgNPs) to produce a significant protection against P. chabaudi-induced splenic damage in animal models.¹³ These AgNPs have been demonstrated to efficiently decrease parasitemia and enhance histological results in infected spleens, implying a protective function against oxidative stress and inflammation.¹⁴ The injection of I. oblongifolia extracts not only increases antioxidant levels but also controls pro-inflammatory cytokine responses, ultimately promoting splenic health during infection.¹⁵

Given malaria parasites’ increasing resistance to current antimalarial medications, investigating alternative therapeutics derived from natural materials is crucial. The purpose of this study is to look at Indigofera oblongifolia's possible survival impact against P. chabaudi splenic infection, specifically its mechanisms of action in modulating immune responses and protecting against splenic damage. Therefore, the rationale for continuing this study is the urgent necessary for malaria treatments to various body organs and assessing the therapy effectiveness by histopathological analysis and immunohistochemistry assay to demonstrate its ability in restoring the histoarchitecture of studied organs and modulating the proinflammatory cytokines levels.

Material and methods

Study subjects and materials

Fifty female C57BL/6 mice, aged 9 ± 2 weeks and weighing 22 ± 3 g, were sourced from the King Faisal Hospital Research Unit in Riyadh, Saudi Arabia. The Plasmodium chabaudi strain was obtained from the Parasitology Laboratory at the College of Science, King Saud University. Cryopreserved P. chabaudi parasites were introduced into uninfected mice once. A phosphate buffer containing 100 μl and 10^⁵ erythrocytes parasitized by P. chabaudi was administered intraperitoneally to the mice.

Biosynthesis of silver nanoparticles

The biosynthesis of silver nanoparticles (AgNPs) was conducted following the method described by.¹⁶ Briefly, powdered leaves were stirred in methanol for 24 h, followed by filtration through Whatman No. 1 filter paper. A portion of the filtrate (5 ml) was utilized for AgNP synthesis while the remainder was evaporated using a vacuum evaporator (IKA, Germany). The residues were then dissolved in distilled water and stored at −20°C until further use. The nanoparticle preparation adhered to the protocol established by.¹⁷ In summary, 5 ml of the leaf extract was mixed with 8 × 10^{^−3} M silver nitrate in 45 ml methanol and heated at 50°C for 60 min, resulting in a dark brownish solution indicative of AgNP formation.

In vivo experimentation

The mice were divided into five groups, each comprising ten individuals. The first group served as a control and received distilled water orally for seven days. The second group was administered 50 mg/kg of IOLE AgNPs daily for seven days. The third, fourth, and fifth groups were injected ip with 10^⁵ phosphate buffer containing 100 μl and 10⁵ erythrocytes parasitized by P. chabaudi. One hour post-injection, the fourth group received 50 mg/kg of IOLE AgNPs daily for seven days,¹⁸ while the fifth group received chloroquine phosphate (Sigma-Aldrich, St Louis, MO) at a dosage of 10 mg/kg daily for four days.¹⁹ On day seven post-infection (p.i.), all animals were euthanized via CO₂ asphyxiation and subsequently dissected for sample collection. Histopathological analyses of splenic tissues were performed using standard histology techniques with hematoxylin-eosin (H&E) staining.

Spleen samples collection

Following CO₂ asphyxiation on day seven p.i., all mice were dissected to collect spleen. For histological examination spleen tissues were isolated and sectioned into small pieces which were fixed in neutral buffered formalin (10%).

Spleen histopathology

Spleen from both control and treatment groups were fixed in 10% neutral buffered formalin for 24 h at room temperature. Fixed organs were embedded in paraffin, sectioned to a thickness of 6 μm, and stained with H&E. Microscopic examination of spleen sections was conducted using a standardized non-linear semi-quantitative scoring system adapted from,²°comparing control group sections with those from treatment groups.

The staining protocol involved deparaffinization in xylene for 8 min followed by hydration through a descending series of ethanol concentrations (100%, 96%, 90%, 80%, 70%) and rinsing in distilled water for two minutes. Sections were stained with H for 12 min to visualize nuclei, followed by bluing with tap water for two minutes. Differentiation in 70% ethanol containing 1% HCl occurred for 1–5 s to enhance nuclear detail before re-washing in tap water until blue. Sections underwent a brief dip in ammonia water to accentuate nuclear staining before being stained with eosin solution for two minutes to visualize cytoplasm. Subsequent dehydration through an ascending series of ethanol concentrations (90%, 96%, 100%) was performed prior to clearing in xylene and mounting in DPX under coverslips.

Post-fixation in neutral buffered formalin, spleen specimens underwent paraffin embedding and H&E staining according to.²⁰ Morphometric analyses of various organ characteristics including alveolar wall thickness, necrosis intensity, and structural abnormalities were conducted using ImageJ software to assess histoarchitectural changes induced by P. chabaudi infection.

Morphometric analysis

The histomorphometric parameters such as white pulp diameter and density besides fibrosis, amyloidosis, and congestion intensity were calculated by image processing of image j software according to protocols of.^21,22 The results were analysed by SPSS to get an accurate comparison between the five experimental groups.

Immunohistochemistry

We used HRP/DAB Detection kit (Abcam) for immunohistochemistry. The proinflammatory cytokines’ primary antibodies were used in the following dilutions IL-6 1:400, and TNF-a 1:100. The sections were incubated with primary antibodies, followed by their incubation with horseradish peroxidase (HRP) conjugate and then with the DAB substrate. Finally, the sections were subjected to counterstaining with Mayer Haematoxylin for light microscopy examination. The detailed protocol of immunohistochemistry has been reported earlieR.²³

Semi-QuantitativeAnalysis: Immunostained sections were analyzed using a light microscope (Nikon, Tokyo, Japan). Five non-overlapping fields per section were randomly selected and evaluated at ×400 magnification. The staining intensity and percentage of positive cells were scored using a semi-quantitative grading system:

Staining Intensity:

0 = No staining

1 = Weak staining

2 = Moderate staining

3 = Strong staining

Percentage of Positive Cells:

0 = No positive cells

1 ≤ 10% positive cells

2 = 10–50% positive cells

3 ≥ 50% positive cells

Statics analysis

Using accepted statistical techniques, the means⁹ and standard error (S.E) of each set of data are determined.^24,25 After establishing the test's significance (p-value) at the 5% or 1% level, an ANOVA is utilized to assess the interaction effects in more detail. Throughout the whole statistical analysis, the program SPSS for Windows, version 10.0, is utilized.

Result

The current study was designed to investigate the therapeutic effect of Indigofera oblgofola AgNPs on severe inflammatory symptoms caused by P. chabaudi infection in the spleen.

Histopathological investigation

The normal organization of white and red pulps is represented in Figure 1 (Figure1(a) to (d)). Administration of indigofera oblgofola with silver nanoparticles didn't exhibit any alterations in comparison to the control tissues. On the contrary, Plasmodium chabaudi manifested multiple malformations in the histological structures such as a significant (p < 0.001) shrunk white pulp (129.17 ± 1.41 µm) in comparison to the healthy spleen (216.57 ± 1.451 µm). Similarly, whit pulp density was exposed to severe (p < 0.001) reduction (0.04 ± 0.002 pulp/µm) relative to the normal density (0.13 ± 0.007 pulp/µm) (Figure 2(a) and (b)). Moreover, the infected red pulp became more crowded with several atrophic compartments such as necrotic and depleted lymphocytes and spreading apoptosis which leads to contraction both of sinusoidal spaces (7.81 ± 0.33 µm) and thickness of marginal zone (7.83 ± 0.47 µm) in a significant way (p < 0.001) (Figure 2(d) and (e)). Furthermore, the atrophied markers appeared in the form of a dispersion of collagenous deposition in the splenic stroma and can be invaded into the marginal zone reaching the periarterial lymphatic sheath known as parenchymal fibrosis and registered significant intensity (p < 0.001) in the diseased spleen (7.93 ± 0.41%) more than the healthy ones (0.13 ± 0.009%) (Figure 1(e) and (f)). And also, the diseased red pulp indicated some amyloidosis features through amorphous proteinous precipitation adjacent to the marginal zone of white pulp with obvious intensity (6.78 ± 0.44%). The congestion was considered an accompanied indication of the inflammation of the Plasmodium-infected spleen (Figure 2(f), (g) and (h)) and Table 1.

Figure 1.

Photomicrograph of the histological section of splenic tissues in all groups. (a & b) a control spleen, (c & d) administrated group by IOLE AgNPs, (e & f) infected group by P. chabaudi, (G& H) group of P. chabaudi and 50 mg/kg of IOLE AgNPs, and (I& J) group of P. chabaudi and CQ 10 mg/kg. Red pulp (RP), sinuses (Si), macrophage (mc), marginal zone (Mg z), central artery (CA), maternal zone (Mn z), germinal center (Gc), congestion (Con), amyloid deposition (Am), apoptosis (Ap), depleted lymphocytes (Dly), necrotic cells (nc), fibrosis (Fb), Peri-arterial lymphatic sheath (PALS) (H&E X, A, C, E, G& I: 100 µm & B, D, F, H& J: 400 µm).

Figure 2.

Bar charts showing the morphometric analysis of histological parameters of spleen tissue which (a) white pulp diameter, (b) density of white pulp, (c) germinal center diameter, (d) diameter of sinusoidal spaces, (e) marginal zone thickness, (f) fibrosis intensity, (g) amyloid deposition intensity, (h) congestion intensity; among five groups: control group, administrated group by IOLE AgNPs, infected group by P. chabaudi, group of P. chabaudi and 50 mg/kg of IOLE AgNPs, and group of P. chabaudi and CQ 10 mg/kg. Values are represented as Mean ± STDV & n = 10 animals.

Table 1.

Shows the morphometric analysis of histopathological parameters of spleen tissue which diameter of white pulp, density of white pulp, germinal center diameter, sinusoidal spaces diameter, marginal zone thickness, fibrosis intensity, amyloid intensity, and congestion intensity.

ParametersGroups	Diameter of white puple (µm)	Density of white puple Pulp /µm	Germinal center diameter (µm)	Sinusoidal spaces diameter (µm)	Marginal zone thickness (µm)	Fibrosis intensity %	Amyloid intensity %	Congestion intensity %
Control	216.57 ± 1.45d	0.13 ± 0.007c	19.68 ± 1.09d	29.92 ± 1.84c	26.05 ± 0.65d	0.13 ± 0.009a	0.10 ± 0.005a	0.17 ± 0.004a
I. oblgofola AgNPs	159.59 ± 2.10b	0.09 ± 0.003b	16.24 ± 0.68c	15.76 ± 0.86b	11.73 ± 0.74b	0.94 ± 0.03a	1.40 ± 0.06b	0.93 ± 0.03b
Infected	129.17 ± 1.41a	0.04 ± 0.002a	3.16 ± 0.14a	7.81 ± 0.33a	7.83 ± 0.47a	7.93 ± 0.41d	6.78 ± 0.44e	5.87 ± 0.20ce
Infected + I. oblgofola AgNPs	230.18 ± 2.15e	0.06 ± 0.003a	16.35 ± 0.66c	15.45 ± 0.41b	17.12 ± 0.54c	3.043 ± 0.24b	4.05 ± 0.23c	2.64 ± 0.16c
Infectecd + CQ	189.95 ± 2.89c	0.11 ± 0.005c	7.36 ± 0.47b	18.73 ± 1.06b	9.16 ± 0.61a	6.29 ± 0.29c	5.65 ± 0.30d	4.73 ± 0.29d

Among five groups: The control; indigofera oblongifolia with silver nanoparticles (IOLE AgNPs) group; the infected group by P. chabaudi, the infected group by P. chabaudi and 50 mg/kg of IOLE AgNPs and the administrated group by P. chabaudi and CQ 10. Values are represented as Mean ± STD & n = 10 animals. Means within the same parameter and not sharing a common superscript symbol (s) differ significantly and values that are recorded with the non-significance difference (n. s) and p-value were calculated according to comparison with the control group.

The indigofera oblgofola with silver nanoparticles was found to significantly restore the splenic tissue to normal after Plasmodium infection. The normal organization of white and red pulps with their normal compartments was recovered in this group (Figure 1(g) and (h)). Although apoptotic markers were still registered, the healing features exhibited a significant (p < 0.01) reduction in fibrosis (3.043 ± 0.24%), amyloidosis (4.05 ± 0.23%), and congestion intensity (2.64 ± 0.16%) in comparison to the infected recordings. On the other hand, the treatment with CQ beyond the plasmodium infection displayed little recovery impact due to the atrophic signs that still present like depletion of the majority of lymphocytes and spreading of necrosis and fibrosis deposition. Despite suffering from the reduced diameter of the white pulp (189.95 ± 2.89 µm) relative to the normal one, their density recovered (0.11 ± 0.005 pulp/ µm) to its normal as illustrated in (Figures1(i), (j) and 2(a), (b)).

Immunohistochemistry investigation

The examination did not reveal the reactivity of the anti-TNF- α antibody immunostaining in the control spleen. Administration of indigofera oblgofola with silver nanoparticles demonstrated very faint immunostaining (0.51 ± 0.05%). However, Plasmodium infection caused intensive immunostaining to the splenic tissue (2.13 ± 0.07%). Indigofera oblgofola with silver nanoparticles displayed lower positive interaction with TNF- α immunostaining in a few cells over the whole tissue (0.99 ± 0.15%). CQ indicated mild expression of TNF- α (1.11 ± 0.10%) staining demonstrated in Plasmodium infection sections (Figure 3(a) to (j)).

Figure 3.

Photomicrograph of TNF-α- stained splenic tissue in all studies groups. (a & b) control group, (c & d) group of IOLE AgNPs, (e & f) infected group, (g & h) group of infected +50 mg/kg IOLE (i & j) group of CQ 10 mg/kg. (TNF-α ×, a, c, e, g & i = 100 X and b, d, e, h & j: = 400 X). TNF-α expression was indicated with black arrows. Stained by DAB -chromogen (Brown color) immunostaining.

Similarly, as expected, the anti-IL-6 antibody immunostaining did not react with any antigen in the control spleen. Sections taken from indigofera oblgofola with silver nanoparticles treated rats showed faint staining with the anti-IL-6 antibodies (0.47 ± 0.03%). However, a mild expression of anti-IL-6 antibody reactivity is shown in the sections from plasmodium-infected rats (1.87 ± 0.08%). On the contrary, the treatment agents neither indigofera oblgofola with silver nanoparticles nor CQ exhibit a fast recovery. The treatment of the indigofera oblgofola displayed moderate expression (0.79 ± 0.047%) while the local CQ (1.53 ± 0.06%) presented high expression of IL-6 immunostaining (Figure 4(a) to (j)). Our findings of a semi-quantitative analysis of immunostaining for TNF- α, and IL-6 proinflammatory cytokines are illustrated in Table 2.

Figure 4.

Photomicrograph of IL-6stained splenic tissue in all studies groups. (a & b) control group, (c & d) group of IOLE AgNPs, (e & f) infected group (g & h) infected group +50 mg/kg IOLE AgNPs, (I&J) group of CQ 10 mg/kg. lymphocytes (Ly), yellowish cytoplasm (arrows) in red pulp (Rp), brownish cytoplasm in major cells (arrows) of red pulp. (IL-6 ×, a, c, e, g & i = 100 x and b, d, e, h & j 400 x). IL-6 expression was indicated with black arrows. Stained by DAB -chromogen (Brown color) immunostaining.

Table 2.

Showing semi-quantitative analysis of TNF-α and il-6 immunostaining in spleen tissues across five experimental groups: control group, group of IOLE AgNPs, infected group +50 mg/kg IOLE AgNPs, group of cq 10 mg/kg.

Groups	Control	I. oblgofola AgNPs	Infected	Infected +50 mg/kg IOLE AgNPs	CQ 10 mg/kg
TNF-α	0.33 ± 0.01^a	0.51 ± 0.05^a	2.13 ± 0.07^c	0.99 ± 0.15^b	1.11 ± 0.10^b
IL-6	0.12 ± 0.006^a	0.47 ± 0.03^b	1.87 ± 0.08^e	0.79 ± 0.047^c	1.53 ± 0.06^d

Values are represented as Mean ± STDV & n = 10 animals. Different superscript letters (a, b, c, d, e) indicate statistically significant differences between groups, p < 0.05.

Discussion

The efficiency of the immune system of the body affects significantly the development and the healthy condition of the lymphoid organs, consequently, the continuous seeking for innovative therapy for parasitic infection became an urgent necessity.²⁶ As Malaria is considered a life-threatening infectious disease and responsible for high morbidity and mortality rates,²⁷ the present investigation explored the healing influence on deformed splenic tissue by malaria disease through a comparison between two administrative agents; one of them is traditional treatment (CQ10 mg/kg) as a powerful antioxidant and another new Nano-drug of a medical plant called I. oblgofola. The existing findings indicate the normal tubular construction that was formed by circular white pulp and the surrounding red pulp enables the splenic tissue from filtrating capacity, as well as the marginal zone that restricted between the two pulps was considered an eliminated area for the inert bacteria and various pathogens. Analogous studies confirmed the similar splenic construction.^28,29

Malaria parasite infection stimulated dramatic alterations on two levels; histological construction and immunohistochemistry saturation.³⁰ Many investigators referred to the robust hemolysis that was accompanied by malaria disease resulted in a strong release of heme that contributed to the generation of oxidative stress status, subsequently raising the intensity of fibrosis, amyloid deposition, and congestion.^31,32 Del Portillo³³ registered that the splenic construction witnessed red pulp expansion and loss of marginal zone during the malaria infection. Resemblant observations were recorded in the present Plasmodium infection. Several studies revealed the ability of P. chabaudi (rodent malaria) to replicate RBC_S and induce a strong inflammatory response after the creation of oxidative stress status and potent hemolysis.³⁴ In accordance with study,⁶ stated that the loss of the splenic marginal zone was responsible for the elevation of the intensity of proinflammatory cytokines such as TNF-α and IL-6 after Leishmania infection. The relevance to I. oblgofola with silver nanoparticles expressed mild expression with IL-6 and faint staining with TNF-α cytokines. A study³⁵ explained this expression by the high sensitivity of the organism's immunity to any size of nanoparticles of gold. Reinforcing this view,³⁶ indicated that the traces of golden nanoparticles stimulated the proinflammatory cytokines in different organs such as the brain, kidney, and liver. However, the histological examination showed no alteration from the normal splenic construction in this group.

Noteworthy, the administration of I. oblgofola on the infected spleen, the white pulp appeared more organized and restored its normal density and features, besides appearing faintly with TNF- α immunostaining. These findings were supported by¹¹ during the trypanosomiasis treatment due to the anti-malarial and inflammatory properties of I. oblgofola in delamination of the oxidative stress status and lipid peroxidation. Much evidence supported these findings, whereas the elimination of the acute parasitic inflammation revealed a significant drop in the TNF- α levels.

Alternatively, chloroquine 10 mg/kg has been used as a traditional antimalarial cerebral drug for the last decades according to³⁷ due to its potent antioxidant capacity in mitochondria and lipid membrane besides its modulatory impact on fibrosis, gene expression, and apoptosis. Although its putative influence in restoring the normal organization of white and red pulps was little significantly less than the I. oblgofola according to the histological and immunohistochemistry of the recent examination.^38,39 referred that the appearance of lymphocytes depletion during CQ treatment suggested that this therapy may not effectively recovery the immune system's functionality post-infection. A study to⁴⁰^, suggested that the total antioxidant activity of I. oblgofola was robust more than that was produced by CQ mg/kg.³⁷^,42 confirmed that the CQ may not be as effective in restoring immune function as previously due to the alterations in the organization of the white pulp and a notable depletion of lymphocytes consequently, the effective recovery for splenic tissue was observed through the medical plant.

The limitation of the present study is the absence of separate control groups treated with the IOLE alone and the AgNPs alone against P. chabaudi infection. While this limitation constrains the interpretation regarding the precise mechanism of action, we confirmed the safety of the composite agent by demonstrating that the non-infected IOLE AgNPs-treated group exhibited normal spleen architecture and minimal inflammatory markers, suggesting that the observed splenoprotective and anti-malarial effects are a true consequence of its interaction with the pathology induced by the parasite. Future investigations should incorporate these individual component control groups in a full dose-response study to precisely elucidate the synergistic or dominant role of each element in the overall therapeutic outcome.

Despite the study's significant findings, we must acknowledge certain limitations to provide a comprehensive interpretation of the results. The limitation of the present study is the absence of separate control groups treated with the IOLE alone and the AgNPs alone against P. chabaudi infection. While this limitation constrains the interpretation regarding the precise mechanism of action, we confirmed the safety of the composite agent by demonstrating that the non-infected IOLE AgNPs-treated group exhibited normal spleen architecture and minimal inflammatory markers, suggesting that the observed splenoprotective and anti-malarial effects are a true consequence of its interaction with the pathology induced by the parasite. Future investigations should incorporate these individual component control groups in a full dose-response study to precisely elucidate the synergistic or dominant role of each element in the overall therapeutic outcome.

Conclusion

The results of this study highlight the significant therapeutic effects of I. oblgofola extraction with AgNPs in ameliorating splenic damage caused by P. chabaudi infection. The treatment preserved the structural integrity of splenic tissues and modulated pro-inflammatory cytokine levels, suggesting a dual action of antioxidant and anti-inflammatory properties. In contrast, traditional treatments like chloroquine phosphate showed limited efficacy in restoring normal splenic architecture. Further research is warranted to explore the underlying mechanisms and optimize the therapeutic applications of I. oblongifolia in malaria treatment. So, we recommend future studies still need to precisely determine the exact contribution of both the extract and the silver nanoparticles individually.

Footnotes

Acknowledgments

Thank Researchers Supporting Project number (PNURSP2025R401), Princess Nourah bint Abdulrahman University, and Ongoing Research Funding Program (ORF-2025-1081), KSU, Riyadh, Saudi Arabia.

ORCID iD

Mutee Murshed

Ethical approval

Ethical approval for this research project was granted by the Animal Ethics Committees at King Saud University, Saudi Arabia (certificate number KSU-SE-21-86).

Author contributions

Conceptualization, J.T S.Q. and M.M.; methodology, M.M, J.T; software M.M, J.T; validation, J.T. and M.M.; formal analysis, J.T.; investigation and resources, M.M, H.Z; data curation, J.T.; writing original draft preparation, J.T, H.Z.; writing review and editing, M.M. and J.T.; visualization, M.M, supervision, S.Q.; project administration, S.Q.; funding acquisition, S.Q. All authors have read and agreed to the published version of the manuscript.

Funding

The authors received no financial support for the research, authorship, and/or publication of this article.

Declaration of Conflicting Interests

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

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