Investigating the potential role of T helper 17 cells among women experiencing overweight and obesity and their possible therapeutic targeting of the RORγt molecule

Abstract

Obesity is a growing healthcare problem globally. In Saudi Arabia, 24% of adults aged 15 years and above have been living with Obesity. It is considered a chronic inflammatory condition that is linked to a wide range of disorders including type 2 diabetes mellitus, insulin resistance and cardiovascular diseases. In this study, we aimed to assess the influence of obesity on the proportion of Th17 cells among healthy, overweight, and obese women in Saudi Arabia. Additionally, we aimed to explore potential ligands targeting the master transcription factor of Th17 cells: RORγt. A cross-sectional study was conducted, wherein their body mass index (BMI) and waist circumference (WC) were measured. The proportion of peripheral Th17 cells was determined using flow cytometry. We found a decrease in the proportion of peripheral Th17 among women with central obesity, though this was observed among overweight and obese participants. Interestingly, both BMI and WC were inversely correlated with the proportion of peripheral Th17 cells in women experiencing overweight or obesity, while no change was observed among healthy participants. Notably, the analysis revealed a significant moderate negative correlation between the proportion of Th17 cells and HbA1c levels, observed only among the overweight and obese participants. In this study, we identified three potential binding sites on RORγt molecules of Th17 cells, bound to 58 chemical ligands. The majority of the chemical structures (72.4%) were targeted binding pocket 1 of the RORγt molecule. These findings could provide a new insight to develop new pharmaceutical molecules targeting immune cells to combat obesity and related metabolic disorders.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-024-81070-1.

Introduction

Obesity is a growing healthcare problem globally. According to the World Health Organization (WHO), 16% of adults aged 18 years and over were obese¹. In Saudi Arabia, 24% of adults aged 15 years and above were living with Obesity². Obesity is considered a chronic inflammatory condition that is linked to a wide range of disorders, including type 2 diabetes mellitus, insulin resistance and cardiovascular diseases³. The development of diabetes is directly associated with obesity, often evaluated through glycated hemoglobin (HbA1c)^4,5. Indeed, HbA1c is considered a standard blood test that reflect an individual’s average blood glucose levels during the past three months and is commonly used for diagnosis and prognosis of diabetes⁶.

Additionally, Obesity is characterised by the accumulation of adipose tissue, composed of preadipocytes and adipocytes, as well as adipose tissue-resident immune cells, such as macrophages and lymphocytes^3,7. Although the main function of adipocytes is to store lipids, they also have a role in inflammation through the production of proinflammatory cytokines and adipokines⁷. Also, hormones dysfunction caused by obesity can drive inflammation by dysregulating the signalling pathways in immune cells⁸. Therefore, the state of obesity might influence the functionality of immune cells, including T lymphocyte subsets.

T lymphocyte is divided into two main subsets: T helper (CD4⁺) T cells and cytotoxic (CD8⁺) T cells⁹. T helper cells are further divided into different pro- and anti-inflammatory populations, including Th1, Th2, Th17 and Treg⁹. It has been found that obesity and accumulation of adipocytes can affect the functionality of T helper subsets^10,11. Th17 cells are known to be involved in the pathogenesis of a number of inflammatory and autoimmune diseases^12,13. The hallmark of Th17 cells is the production of pro-inflammatory cytokines IL-17 and IL-22 that are involved in the pathogenesis of different diseases and combat bacterial and fungal infections^12,13. The retinoid orphan receptor (RORγt) is the main transcription factor that regulates and activates the Th17 cells¹⁴.

Two isoforms of RORγ are known: RORγ and RORγt (also known as RORγ2). The focus of this paper is on RORγt, which differs from RORγ by having a truncated N-terminus and lacks the initial 21 amino acids. Moreover, the RORγt isoform is solely expressed in the cells of the immune system, whereas RORγ is expressed in many different tissues, such as the thymus and muscle. The activity of RORγt is essential for the proliferation and functionality of Th17 cells, and modulation of this activity has been studied for potential therapeutic benefits^15–17. In addition to mediating the differentiation of naive T cells into Th17 cells, RORγt regulates the production of pro-inflammatory cytokines such as cytokine IL17, IL-22, and Granulocyte-monocyte colony-stimulating factor (GM-CSF), contributing to inflammation in autoimmune diseases^18,19. Targeting the Th17/IL17 axis and IL-23/IL-17 pathway has been clinically successful, highlighting their significance in treating inflammatory diseases^20,21. In this disease-relevant pathway, small-molecule inhibition of RORγt has been suggested as a potential strategy for the treatment of autoimmune diseases^22,23. For example, small molecules targeting the ligand-binding domains of RORα and RORγ can suppress the development of autoimmune diseases in mice by inhibiting the production of IL-17 secreted by Th17 cells¹⁵.

RORγt has a highly-conserved ligand-binding domain (LBD), which is made of alpha helices that form a large hydrophobic pocket. The pocket allows the binding of small lipophilic ligands, including cholesterol, fatty acids, retinoid derivatives, and other lipophilic hormones and vitamins²⁴. Recent research has demonstrated that several cholesterol precursors, including oxysterols, lanosterol, and desmosterol, are endogenous ligands that naturally bind to RORγt^25–27. In the literature, only a few synthetic RORγt agonists have been described. The most advanced compound among them was LYC-55,716, which entered phase 2a clinical trials to treat patients with solid tumors (National Clinical Trial Numbers: NCT02929862 and NCT03396497)²⁸. Co-crystallization of the LBD of RORγt with several small-molecule antagonists has revealed an additional binding site other than the active site. This non-canonical allosteric binding pocket can induce a conformational change of helix 12 in the RORγt LBD, consequently blocking the cofactor binding²⁹. Several antagonists or inverse agonists, such as digoxin³⁰, SR1001¹⁵, and ursolic acid³¹, have been reported to bind either to orthosteric or allosteric sites, suppressing Th17 cell differentiation in animal models of autoimmune disease. Researchers have also performed a fragment screening of RORγt using a library containing 384 small molecule “fragments”³². This screening identified distinct subpockets and fragment hits that bind to RORγt, with most of these hits overlapping with previously published structures. However, these hits need to be elaborated into drug candidates, and their interactions with these sub pockets require further analysis.

Despite extensive research on obesity and its impact on health, there is limited data exploring the harmful effects of obesity on immune subsets among young women. We hypothesised that the proportion of Th17 cells is differentially regulated among participants stratified based on weight status. In this study, we aim to assess the influence of obesity on the proportion of Th17 cells among healthy, overweight, and obese women in Saudi Arabia. Additionally, we aim to explore potential ligands targeting the master transcription factor of Th17 cells (RORγt) with the goal of developing a future medication to combat obesity and related disorders. This investigation seeks to enhance our understanding of obesity’s impact on immune function and identify novel therapeutic targets for obesity and its complications.

Materials and methods

Study design and data collection

A cross-sectional study was conducted among young women recruited from January to March 2023 at Taibah University, Madinah, Saudi Arabia. This study included young females aged from 18 to 25 years old, healthy women with no history of chronic inflammation or autoimmune diseases and women who did not have previously received weight management interventions.

The ethical certificate to conduct this study was obtained from the Scientific Research and Ethics Committee of the College of Applied Medical Sciences, Taibah University, Madinah (project number 2023/154/105 MLT). All experiments performed in the study were following the guidelines and regulations at Taibah University. Informed consent was obtained from all subjects and/or their legal guardian(s) included in this study prior to data and sample collection. Sociodemographic data (i.e., age, marital status, employment status, nationality and family history of obesity) were collected through face-to-face interviews with the participants using a softcopy questionnaire (administered digitally).

Assessment of weight status

The weight (in kg) and height (in cm) of all participants were objectively measured following a standard procedure using a digital scale (Beurer, UK) and stadiometer (Seca, Germany), respectively. Weight and height were used to calculate the body mass index (BMI) for each participant. The World Health Organization (WHO) cut-offs were used to assess the weight status as follows: “underweight” BMI < 18.5 kg/m²: “healthy weight” 18.5 to 24.9 kg/m²: “overweight” 25.0 to 29.9 kg/m²: “obesity” ≥ 30.0 kg/m^233,34. Additionally, waist circumference (WC) was measured in centimeters where the tape measure was placed around the middle at a point halfway between the ribs bottom and hips top just above the belly button. The cutoff used for WC for women is > 88 cm to indicate abdominal obesity³⁵.

Flow cytometry

Peripheral blood mononuclear and polymorphonuclear cells were isolated from EDTA-blood samples collected from each participant. Briefly, 1 ml of red blood cell lysing buffer (Thermo Fisher, Massachusetts, USA) was added to 1 ml of whole blood sample for 10–15 min. Then, the lysed blood samples were centrifuged for 5 min at 1500 rpm to obtain the cell pellets for flow cytometry staining. All samples were stained with Live/Dead stain prior to surface and intercellular marker staining, with all antibodies from Thermo Fisher.

First, the resuspended cells were stained with anti-CD3 (PERCP-CY5.5) and anti-CD4 (FITC) surface markers to identify CD4⁺ T cells. Subsequently, the resuspended cells were fixed using Foxp3 fixation buffer (Thermo Fisher) before staining with the intracellular marker anti-RORγt (PE-CY7).

All stained samples were run on the Attune Flow Cytometer (Thermo Fisher) and the data were further analysed using FlowJo version 10 (LLC, USA).

HbA1c measurement

EDTA blood samples were sent to BanderGene Laboratories (Madinah, KSA) for glycated hemoglobin (HbA1c) level assessment using Finecare^™ FIA Meter (Wondofo, Guangzhou).

Structural analysis of RORγt-ligand complexes deposited in PDB

In order to retrieve protein structure, the Protein Data Bank (PDB) was queried specifically for RORγt proteins complexed with known ligands, excluding structures containing other RORγt family members. The retrieved PDB files were uploaded into ICM software (v3.9-3a)³⁶ for subsequent superposition analysis. K_d and IC₅₀ values reported in PDB were employed to assess ligand efficiency (LE) and binding site druggability. Duplicated ligands (the same ligand with different PDB ID) and K_d and IC₅₀values of more than > 3 mM were not considered. LE calculations were performed using DataWarrior 5.5.0 software³⁷. In the context of drug discovery, ligand efficiency (LE) is a metric employed to prioritize candidate ligands for further development. LE quantifies the biological potency of a ligand relative to its size and complexity. By calculating LE, we aim to identify ligands for RORγt that exhibit favorable pharmacological properties, potentially leading to the optimization of novel antidiabetic and anti-obesity therapeutics in the future.

Statistical analysis

All tests and graphical representations in this study were conducted using GraphPad Prism version 10 (San Diego, USA). The normality of the distribution of continuous variables was assessed with the Shapiro–Wilk test. Mean and standard deviation were calculated for all numerical data sets. Spearman correlation analysis was used to evaluate the strength of correlation between continuous variables, while the Mann–Whitney test compared median values between two different groups. Simple linear regression assessed the association between variables (BMI, WC, HbA1c and percentage of Th17 cells), with weight status used for data stratification. A 95%confidence level was applied to determine the significance of the data set.

Results

The sample characteristics

A total of 50 young females participated in this study, all aged between 18 and 25 years old. The majority of the participants were Saudis, singles and students (96%, n = 48). Only 24% reported a family history of obesity, yet 68% (n = 34) were experiencing overweight and obesity. Additionally, 36% (n = 18) had central obesity, indicated by a WC of > 88 cm. 80% of participants showed a normal range of HbA1c. The detailed characteristics of the participants including the mean and standard deviation of the numerical data are presented in Table 1.

Table 1.

Sample characteristics (n = 50).

	n	%	Mean ± SD
Age
18–20 years old	25	50	19.56 ± 0.6506
21–25 years old	25	50	21.84 ± 0.7461
Nationality
Saudi	48	96	-
Non-Saudi	2	4
Marital status
Single	48	96	-
Married	2	4
Employment status
Student	48	96	-
Unemployed	2	4
Family history of obesity
Yes	12	24	-
No	38	76
Weight status
Healthy weight	17	34	21.63 ± 2.035
Overweight	16	32	27.32 ± 1.535
Obese	17	34	37.37 ± 5.399
Waist circumference
≤ 88 cm	32	64	73.19 ± 9.667
> 88 cm	18	36	98.6710.77
HbA1c
Normal (< 5.7%)	44	88%	4.995 ± 0.3162
Pre-diabetic (5.7–6.4%)	5	10%	6.020 ± 0.2168

Both age and level of glycated hemoglobin showed no significant differences among both healthy and overweigh/obese participants (Table 2). Central obesity was not associated with all subjects who experiencing overweight or obesity (mean waist circumference 89 ± 14.01).

Table 2.

Comparison between healthy and overweight/obese participants.

	Healthy participants (n = 17)	Overweight/ Obese participant (n = 33)	P-value
Age (years)	20.47 ± 1.375	20.82 ± 1.334	0.3956
BMI (Kg/m2)	21.63 ± 2.035	32.5 ± 6.458	< 0.0001
WC (cm)	69.47 ± 10.56	89 ± 14.01	< 0.0001
HbA1c (%)	5.00 ± 0.3446	5.197 ± 0.5324	0.2757

Correlation of anthropometric parameters and glycated hemoglobin with the proportion of peripheral T Helper 17 cells

T helper 17 cells in the participants’ peripheral blood samples were identified by the expression of surface markers: CD3, CD4 and the transcription factor RORγt. The proportion of circulating Th17 cells was similar in both healthy women and those experiencing overweight or obesity (Fig. 1A). However, women with central obesity had a lower percentage of Th17 cells compared to healthy participants (Fig. 1B).

Fig. 1.

Central obesity but not weight status has influences on peripheral T helper 17 cells. T helper 17 were identified using flow cytometry by the expression of CD3, CD4 and transcription factor RORγt. (A) Percentage of Th17 cells based on body mass index. (B) Percentage of Th17 cells based on waist circumference.

Spearman correlation analysis was conducted to assess the correlation between BMI and WC with the proportion of circulating Th17 cells among healthy and overweight/obese participants. Interestingly, both BMI and the WC were negatively correlated with the proportion of circulating Th17 cells in overweight and obese participants, with r_s= − 0.446, and r_s= − 0.538, respectively (Table 3). On contrary, none of BMI, WC and HbA1c were correlated with percentage of T cells, CD4⁺ T cells and Th17 cell among healthy participants (Table 3). In overweight/obese group, BMI predicted a smaller proportion of Th17 cells, explaining 17% of the variation in Th17 cell proportion while no change was observed among healthy participants (Fig. 2A, B). Moreover, WC inversely affected the proportion of Th17 cells among the participants with a BMI of > 25 kg/m² but not healthy weight participants, explaining 30% of the variation comparing to healthy participants (Fig. 2C, D). These findings suggest that obesity may impact the proportion of circulating Th17 cells, potentially contributing to inflammatory diseases.

Table 3.

Correlation between frequencies of peripheral T cells, T helper cells and T helper 17 cells, and weight status and HbA1c.

	% of T cells	% of Th cells	% of Th17cells
Healthy weight (n = 17)
BMI	rs=0.216	rs=0.070	rs=0.302
	p = 0.404	p = 0.789	p = 0.237
Waist circumference	rs=− 0.028	rs=− 0.034	rs=− 0.162
	p = 0.915	p = 0.895	p = 0.530
HbA1c	rs=− 0.087	rs=− 0.236	rs=− 0.012
	p = 0.738	p = 0.358	p = 0.964
Overweight/Obese (n = 33)
BMI	rs=0.008	rs=− 0.008	r s =− 0.446*
	p = 0.963	p = 0.966	p = 0.009
Waist circumference	rs=0.022	rs=0.116	r s =− 0.538*
	p = 0.905	p = 0.522	p = 0.001
HbA1c	rs=0.141	rs=0.148	r s =− 0.477*
	p = 0.434	p = 0.411	p = 0.005

*Significant at 95% confidence level.

Fig. 2.

Both BMI and WC are negatively correlated with circulating T helper 17 cells in women experiencing overweight/obese population. T helper 17 were identified using flow cytometry by the expression of CD3, CD4 and transcription factor RORγt. (A) Association between percentage of Th17 cells and BMI in healthy participants. (B) Association between percentage of Th17 cells and BMI in overweight/obese participants. (C) Association between percentage of Th17 cells and WC in healthy participants. (D) Association between percentage of Th17 cells and WC in overweight/obese participants.

Association between Th17 cells and HbA1c based on weight status

Notably, the analysis revealed a significant moderate negative correlation between the proportion of Th17 cells and HbA1c levels, observed only among the overweight and obese participants (r_s= − 0.477, Table 3). In contrast to healthy participants, HbA1c predicted a smaller proportion of Th17 cells, explaining 26% of the variation in Th17 cell proportion among participants who experiencing overweight and obesity (P < 0.0022 Fig. 3A & B). Collectively, these findings suggest that obesity may influence the proportion of circulating Th17 cells, potentially contributing to the development of diabetes among young females. Therefore, Th17 cells could be an ideal target to prevent obesity and the onset of diabetes.

Fig. 3.

The decrease of proportion of T helper 17 in overweight/obese population associate with increasing percentage of HbA1c. T helper 17 were identified using flow cytometry by the expression of CD3, CD4 and transcription factor RORγt. (A) Association between percentage of Th17 cells and HbA1c in healthy participants. (B) Association between percentage of Th17 cells and HbA1c in overweight/obese participants.

Identification of potential binding sites and ligand efficiency

As illustrated in Fig. 4, the majority of ligands (42 out of 58) targeting Site 1 (orthosteric site) exhibited the most favorable LE profiles (LE = 0.17–0.43 kcal/mol/heavy atom). This region is a promising area for further drug development efforts through linking or growing overlapped molecules. Fifteen ligands bound to Site 2 (allosteric site) with LE = 0.26–0.39 kcal/mol/heavy atom, and only one ligand (chemical fragment) bound to Site 3 (LE = 0.27 kcal/mol/heavy atom) (Fig. 5). The chemical structures 2-(1-piperidinyl)−1,3-thiazol-4-amine and 4-{1-[2-chloro-6-(trifluoromethyl)benzoyl]−1 H-indazol-3-yl}benzoic acid showed the highest LE values and were thus more favorable among other ligands in site 1 and site 2, respectively (Fig. 5). The LE values for ligands and their characteristics have been listed in Table S1.

Fig. 4.

The crystal structure of RORγt (PDB ID: 4XT9) with superimposed 58 ligand structures. It reveals ligand occupancy at three binding pockets. (A) Front view: Site 1 is occupied by yellow ligands, while site 2 is complexed with orange ligands. (B) Back view: Site 3, located on the opposite face of the protein, harbors a red fragment.

Fig. 5.

LE of 58 ligands deposited in PDB and categorized based on their binding sites. LE was calculated using DataWarrior 5.5.0. software.

Discussion

The study aimed to explore the effect of obesity on the percentage of circulating Th7 cells in young women and how these cells could be targeted to mitigate inflammation and metabolic disorders associated with the Th17/IL-17 pathway. Interestingly, there was a decrease in the proportion of peripheral Th17 among women with central obesity, though, this was observed among overweight and obese participants. The decrease is consistent with previous studies. For instance, overweight adolescent males showed a decreased plasma IL-17 levels correlated with BMI and WC parameters³⁸. Additionally, mice on a high-fat diet exhibited a significant reduction in intestinal Th17 associated with metabolic disorders such as insulin resistance and glucose intolerance³⁹. Notably, the transfer of Th17 cells into these mice led to improvements in obesity and metabolic parameters, underscoring the critical role of these cells in obesity-related disorders³⁹.

However, it is important to acknowledge that the literature presents conflicting evidence regarding the relationship between Th17 cells and obesity. Some studies have reported an increase in Th17 cells and/or IL-17 levels in both adults and children with obesity and/or central obesity^40,41. For instance, Łuczyński et al. observed that weight reduction in obese children was accompanied by a decrease in circulating Th17 cells and improved metabolic parameters⁴¹. Similarly, Sumarac-Dumanovic et al. found elevated IL-17 levels in obese women compared to lean individuals, although this increase was not correlated with BMI or insulin resistance⁴². These discrepancies in the literature highlight the complex interplay between the Th17/IL-17 pathway and obesity, and further research is needed to elucidate whether obesity influences Th17 cells or if Th17 cells contribute to the development of obesity.

Although the function of Th17 cells in obesity is not fully elucidated, there is growing evidence indicating that Th17 cell serve as negative regulator of adipogenesis and can play a pivotal role in preventing obesity⁴³. This notion is supported by the observation that IL-17-deficient mice exhibit increased susceptibility to obesity, glucose intolerance, and insulin resistance⁴³. Moreover, the intestinal transfer of Th17 cells into obese mice has been shown to contribute to intestinal microbiota homeostasis, thereby aiding in the reduction of obesity and metabolic disorders³⁹. Consequently, a decreased proration of Th17 cells might promote adipogeneses, ultimately facilitate the development of obesity. Additionally, it is worth noting that gut microbiota plays a pivotal role in the development and function of Th17 cells⁴⁴. Thus, disruption of normal flora composition due to obesity may influence the proportion and functionality of both peripheral and intestinal Th17 cells.

In the present study, a negative association between the proportion of Th17 cells and the percentage of HbA1c, a biomarker of hyperglycemia⁴⁵, was observed exclusively among women who experiencing overweight and obesity. Notably, all participants in the study were healthy with no history of chronic inflammation, metabolic disorders, or autoimmune diseases. It has been shown that HbA1c and plasma glucose concentration are positively correlated with IFN-γ/IL-17 ratio in Th17 cells among children diagnosed with β cell autoimmunity⁴⁶. In contrast, Zeng, et al. demonstrated that HbA1c is not associated with peripheral subsets of T helper cells including Th17 cells, in type 2 diabetic patients⁴⁷. The inconsistency in the relationship between HbA1c and Th17 cells emphasizes the need for further studies to clarify the role of Th17 cells in the development of type 2 diabetes, particularly in the context of obesity.

All aforementioned reports have examined the association of HbA1c and Th17 cells in diabetic patients, however, there is a lack of studies investigating the association between glycated hemoglobin and the proportion of Th17 cells among healthy individuals based on weight status. Further studies must be conducted to assess the relationship of all T helper cell subsets, including both pro-inflammatory and anti-inflammatory subpopulations in the context of obesity. This would enhance our understanding of the functionality of theses subsets which could ultimately propose a new target to combat obesity and related disorders.

Generating pharmaceutical molecules targeting Th17 cells, specifically RORγt hold promise for combating obesity and related metabolic disorders such as diabetes. Future development of RORγt ligand should prioritize achieving a balance between lipophilicity, which is crucial for target binding, and physicochemical properties to ensure optimal bioavailability and drug-likeness. While previous studies have demonstrated the efficacy of certain ligands (PDB ID 7JYM, 6U25, 6VQF, and 7JTW) in treating psoriasis, these molecules may benefit from further optimization. Ligands with favorable ligand efficiency could serve as privileged scaffolds for structure-activity relationship (SAR) studies, ultimately leading to the development of novel therapeutics for diverse pathologies such as diabetes and obesity.

LE is a widely used parameter in the field of drug discovery, intended to quantify the binding affinity of a ligand relative to its molecular weight. The concept of LE was introduced as a tool to aid in lead compound selection⁴⁸, and it has been the subject of reviews^49,50, becoming a routine metric in established drug discovery projects. Future studies focusing in pharmaceutical molecules targeting RORγt are essential not only for developing novel therapies for obesity and related disorders but also for elucidating the role of Th17 cells using appropriate in vitro and in vivo models of obesity.

This study is the first in the region to assess the weigh status and glycated haemoglobin levels relative to the proportion of Th17 cells. However, several limitations should be considered. The sample size of the study was small, and only young women were included. Further investigation involving a larger and more diverse population, including both sexes and all age groups, would provide a more comprehensive understanding of the harmful effects of obesity on Th17 cells. Additionally, the current study also assessed only the proportion of Th17 cells, which may not accurately reflect the actual number of these cells. Future studies should also consider the cytokines signature of Th17 cells such as IL17 and IL-22 to better understand their relationship with weight status and HbA1c level. Also, additional parameters could be measured such as glucose and insulin intolerance along with HbA1c level to correlate them with Th17 cells and their cytokines based on weigh status.

Conclusion

Obesity is considered as a chronic inflammatory condition that is linked to wide range of disorders including type 2 diabetes mellitus, insulin resistance and cardiovascular disease. Therefore, investigating the association between the anthropometric parameters and immune subsets, including Th17 cells, is essential to understand the pathogenicity of such cells in obesity and related disorders. This could provide a new insight to develop new pharmaceutical molecules targeting immune cells to combat obesity and related metabolic disorders.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Author contributions

R.M.A. conceptualized the study. R.M.A., and S.D. designed the experiments and interpreted all data. R.M.A and B.A.S performed the experiments. R.M.A., S.D, N.A, Y.A.A and B.A.S wrote the manuscript. All authors reviewed and approved the manuscript.

Funding

No fund received for the project.

Data availability

The datasets used and/or analysed during the current study available from the corresponding author on reasonable request.

Declarations

Competing interests

The authors declare no competing interests.

Ethical approval

Ethical approval for the study was obtained from the Scientific Research and Ethics Committee of the College of Applied Medical Sciences, Taibah University, Madinah (project number: 2023/154/105 MLT).