Introduction: Excessive visceral adiposity is known to drive the onset of metabolic derangements, mostly involving oxidative stress, prolonged inflammation, and cellular senescence. N-acetylcysteine (NAC) is a synthetic form of l-cysteine with potential antioxidant, anti-inflammatory, and anti-senescence properties. This ex-vivo study aimed to determine the effect of NAC on some markers of senescence including β-galactosidase activity and p16, p53, p21, IL-6, and TNF-α gene expressions in visceral adipose tissue in obese adults. Methods: This ex-vivo experimental study involved 10 obese participants who were candidates for bariatric surgery. Duplicate biopsies from the abdominal visceral adipose tissue were obtained from the omentum. The biopsies were treated with or without NAC (5 and 10 mm). To evaluate adipose tissue senescence, beta-galactosidase (β-gal) activity and the expression of P16, P21, P53, IL-6, and TNF-α were determined. ANOVA test was employed to analyze the varying markers of cellular senescence and inflammation between treatment groups. Results: The NAC at concentrations of 5 mm and 10 mm resulted in a noteworthy reduction β-gal activity compared to the control group (p < 0.001). Additionally, the expression of P16, P21, and IL-6 was significantly reduced following treatment with NAC (5 mm) and NAC (10 mm) compared to the control group (All p < 0.001). Discussion/Conclusion: Taken together, these data suggest the senotherapeutic effect of NAC, as it effectively reduces the activity of SA-β-gal and the expression of IL-6, P16, and P21 genes in the visceral adipose tissue of obese individuals.

Overweight and obesity are major global health problems associated with morbidity and mortality [1]. Worldwide obesity prevalence of adult obesity, which was defined as body mass index (BMI) ≥30 kg/m2, was 13% in 2016 [2]. It almost tripled between 1975 and 2016 [3].

Obesity was characterized by the accumulation of abnormal or excessive fat leading to increased pro-inflammatory cytokines such as interleukin 6 (IL-6) and tumor necrosis factor alpha (TNF-α) in adipose tissues, which eventually lead to senescence-associated secretory phenotype (SASP) [4], particularly in severely obese individuals [5]. Systemic inflammation has been indicated to be necessary for the progressive adipose tissue dysfunction that occurs with aging, which can be associated with insulin resistance and ectopic lipid deposition [6]. Accordingly, weight gain by generating excess fat promotes a pro-senescent milieu [7].

Cellular aging refers to the overall process of cellular decline with time, while cellular senescence specifically refers to the state of irreversible growth arrest that cells enter into as a response to stress or damage [8]. While cellular aging is a gradual and continuous process, cellular senescence can occur at any stage of an organism’s lifespan, including during embryogenesis [8].

Cellular senescence is accompanied by SASP [8]. It is a state of permanent cell growth arrest as a tumor suppression mechanism to prevent damaged cells from potentially oncogenic stimuli including persistent DNA damage [8]. While many markers of senescence are currently being evaluated, no single marker is reliable to identify all senescent cells. Thus, a combinatorial panel of markers must be used to increase the specificity of detection of SASP [9]. Senescence-associated β-galactosidase (SA-β-gal) activity and the markers of p53-p21 and the p16-retinoblastoma pathways are regarded to be a biomarker of cellular senescence whether in culture or in mammalian tissues [9].

Previous research studies have expanded our understanding of the role of adipocyte senescence in obesity [10‒12]. Research shows that senescent adipocytes are susceptible to genome instability, leading to unhealthy adipose tissue remodeling and insulin resistance [11]. Additionally, it has been discovered that mature human adipocytes can unexpectedly display an active cell cycle program, which is associated with obesity and hyperinsulinemia [12]. These findings highlight the importance of targeting the adipocyte cell cycle program to influence adipocyte senescence and obesity-associated adipose tissue inflammation.

Since oxidative stress is a major contributor of senescence, antioxidant therapy could protect adipose tissue from inflammation and senescence [13]. An antioxidant drug known as N-acetylcysteine (NAC) has been approved by the Food and Drug Administration and designated an essential drug by the World Health Organization [14]. NAC has beneficial effects, including antioxidant, anti-inflammatory, and anti-senescence properties [14]. NAC’s senotherapeutic activity has been linked to limiting oxidative damage by free radical scavenging activity and attenuating an abnormal pro-inflammatory response [15]. Growing evidence supports the therapeutic potential of NAC treatment on obesity and its related complications [16]. Tsao et al.’s [17] study indicated the legacy effect of NAC in cellular senescence of obese mice. Based on a recent systematic review, most of the previous pre-clinical studies assessed the effects of NAC on preadipocyte cell line or on subcutaneous adipose-derived cells of rodent models [13]. Moreover, the effectiveness of NAC on human visceral adipose tissue in terms of senescence remains unclear. Therefore, our aim was to determine the effect of NAC on β-galactosidase activity and p16, p21, p53, IL-6, and TNF-α gene expressions in visceral adipose tissue in obese adults.

Design and Participants

This was an ex-vivo experimental study. The study includes 10 participants prospectively recruited between May and September 2022. Bariatric surgery candidates were selected by convenience sampling from the obesity clinic in Shahid Modarres Educational Hospital in Tehran, Iran. The study was reviewed and approved by the Research Ethics Committees of National Nutrition & Food Technology Research Institute (IR.SBMU.NNFTRI.REC.1401.002) in accordance with the seventh revised version of the guidelines of the Helsinki Declaration [18]. Written informed consent was obtained from each individual to participate in the study. All participants provided additional written informed consent prior to any surgical intervention.

The primary eligibility criteria included healthy, voluntary obese adults between the ages of 30 and 40, with a BMI greater than 40 kg/m2 and who had maintained a stable weight for at least 3 months prior to surgery. According to the bariatric surgery guidelines [19], participants underwent the standard bariatric surgery and were monitored throughout the process. Participants who smoked, drank alcohol, or used drugs; had a history of metabolic disorders such as diabetes mellitus, dyslipidemia, fatty liver, or cardiovascular disease; had used antioxidant supplements or drugs, weight loss supplements; or had surgery or an infectious disease within the past 3 months were excluded.

Adipose Tissue Biopsies

The adipose tissue collection protocol was approved by the Institutional Research Board of the National Nutrition & Food Technology Research Institute Ethics Committee. Duplicate biopsies from abdominal visceral adipose tissue, estimated weight 500–1,000 mg, were obtained from the greater omentum during bariatric surgery.

The biopsies were rinsed with a sterile PBS solution, freed from major blood vessels, cut into 100 mg pieces, and transferred to a plate containing DMEM (GIBCO, Life Technologies, USA) with 10% FBS (GIBCO, Life Technologies, USA) and 1% Pen/Strep (Sigma, USA) under a laminar hood. They were then treated with or without NAC (5 and 10 mm; Hakim Pharmaceutical Company, Iran) and incubated at 37°C in a humidified 95% air/5% CO2 incubator for 48 h, following a protocol from previous studies [5, 20]. The adipose tissue pieces were immersed in the NAC solution to ensure all tissue surfaces were in contact with the solution.

SA-β-Gal Activity

To investigate SA-β-gal activity, an optimized and standardized cytochemical assay was utilized for human adipose tissue biopsies. The treated whole tissue was used to measure SA-β-gal activity using a staining kit (Sigma, St Louis, USA). A total of 700 μL staining solution containing X-gal was applied to the treated adipose tissue which was then incubated overnight at 37°C. Afterward, the reaction was stopped using fixation buffer. The level of blue-green stain was measured in digital pictures of the tissue using Image J (NIH, USA) software, after converting the RGB images to CMKY format (https://imagej.net/RGB_to_CMYK). The cyan value of each pixel was calculated and the SA-β-gal values were determined as the ratio of cyan pixel intensity per biopsy area multiplied by 1,000 and presented in arbitrary units (AU).

Gene Expression Analysis

To evaluate adipose tissue senescence, the expression of SASP-related genes, specifically P16, P21, and P53, as well as cytokine genes such as IL-6 and TNF-α were analyzed. The treated adipose tissue was used to extract total RNA with RNX-Plus solution, following the manufacturer’s protocol (Cinaclone, Iran). The obtained total RNA (500 ng) was utilized for cDNA synthesis (Viragen, Iran), as directed by the manufacturer. The polymerase chain reaction (PCR) was applied for TNF-α, IL-6, P16, P21, P53, and 18 s rRNA (an internal control) in duplicate. Each PCR reaction was performed using 10 μL of BIOFACT™ 2X real-time PCR master mix (for SYBR Green I; High Rox, BIOFACT, South Korea), 7 μL of double-distilled water, 0.5 μL of forward primer (10 pmol/μL), 0.5 μL of reverse primer (10 pmol/μL), and 2 μL of cDNA in a final volume of 20 μL. Starting with an initial denaturation stage of 15 min at 95°C, 40 cycles of amplification were performed, consisting of a 25-s denaturation step at 95°C and a 25-s annealing step at 60°C (with the exception of TNF-α, which required a 30-s annealing step at 60°C). The melt curve was between 60 and 95°C, performed on a StepOnePlus Real-Time PCR (Applied Biosciences, Paisley, UK). The gene expression values were calculated as the fold change defined by 2−ΔΔCt, and the applied primers are listed in Table 1.

Table 1.

List of primers used for real-time PCR

Human geneSequence (5′ → 3′)Product lengths (base pair)
18 s rRNA F: CGG GGA GGT AGT GAC GAA 110 
R: ACC AGA CTT GCC CTC CAA 
p16 F: AAG CCA TTG CGA GAA CTT 125 
R: CAG AGG GCA GAA AGA AAA 
P21 F: GCC GAA GTC AGT TCC TTG TG 84 
R: TTC TGA CAT GGC GCC TCCT 
P53 F: TCA GTC TAC CTC CCG CCA TAA 86 
R: AGT GGG GAA CAA GAA GTG GAG 
IL-6 F: GGT ACA TCC TCG ACG GCA TCT 81 
R: GTG CCT CTT TGC TGC TTT CAC 
TNF-α F: GCT CCA GAC GGT GCT TGT G 95 
R: GCC GAT CAC CCC AAA GTG 
Human geneSequence (5′ → 3′)Product lengths (base pair)
18 s rRNA F: CGG GGA GGT AGT GAC GAA 110 
R: ACC AGA CTT GCC CTC CAA 
p16 F: AAG CCA TTG CGA GAA CTT 125 
R: CAG AGG GCA GAA AGA AAA 
P21 F: GCC GAA GTC AGT TCC TTG TG 84 
R: TTC TGA CAT GGC GCC TCCT 
P53 F: TCA GTC TAC CTC CCG CCA TAA 86 
R: AGT GGG GAA CAA GAA GTG GAG 
IL-6 F: GGT ACA TCC TCG ACG GCA TCT 81 
R: GTG CCT CTT TGC TGC TTT CAC 
TNF-α F: GCT CCA GAC GGT GCT TGT G 95 
R: GCC GAT CAC CCC AAA GTG 

NAC, N-acetylcysteine; IL-6, interleukin-6; TNF-α, tumor necrosis factor α.

Biochemical Measurements

Five cc of venous blood was drawn from participants. The serum was separated from the samples by centrifugation at room temperature for 15 min at 2,500 rpm. The extracted serum was stored in 1.5 cc microtubules in a freezer at −80°C until testing for biochemical variables. Serum total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), and triglyceride (TG) levels were assessed using Pars Azmoon commercial kits from Tehran, Iran, with a biochemistry autoanalyzer. Low-density lipoprotein cholesterol (LDL-C) levels were calculated using the Friedewald equation: LDL-C = TC − (HDL-C) − TG/5. FBS was measured with Pars Azmoon commercial kits by a biochemistry autoanalyzer, while serum insulin levels were measured by the immuno-turbidimetry method. HOMA-IR was calculated as fasting insulin (mU/L) × FBS (mmol/L)/405.

Statistical Analysis

The SPSS software (version 20, SPSS Inc., Chicago, IL, USA) was utilized to perform statistical analysis. The normality of continuous variables was ascertained using the Kolmogorov-Smirnov test, histograms, and Q-Q plots. When variables exhibited normal distribution, mean ± SD was used to express continuous variables. In order to compare mean values within the NAC treatment groups, Analysis of variance (ANOVA) was conducted, followed by Bonferroni’s test. A statistically significant difference was indicated by a p value <0.05.

Participants included 5 women and 5 men (mean age ± SD = 34.5 ± 3.03 years, range 31–40 years; mean BMI ± SD = 46.0 ± 3.70 kg/m2, range 41.1–52.9). Table 2 shows the cardiometabolic characteristics of the participants. The SA-β-gal activity assay exhibited highly variable blue-green staining in fat depots according to the treatment (shown in Fig. 1).

Table 2.

Baseline cardiometabolic characteristics of participants

Cardiometabolic variablesObese participants (n = 10)
FBS, mg/dL 101.1±6.1 
HOMA-IR 3.8±1.1 
Insulin, µIU/mL 15.1±4.1 
TC, mg/dL 179.5±2.5 
TG, mg/dL 169.7±22.8 
HDL-C, mg/dL 39.0±6.8 
LDL-C, mg/dL 106.6±24.1 
Cardiometabolic variablesObese participants (n = 10)
FBS, mg/dL 101.1±6.1 
HOMA-IR 3.8±1.1 
Insulin, µIU/mL 15.1±4.1 
TC, mg/dL 179.5±2.5 
TG, mg/dL 169.7±22.8 
HDL-C, mg/dL 39.0±6.8 
LDL-C, mg/dL 106.6±24.1 

Data are expressed as mean ± SD.

FBS, fasting blood sugar; HDL-C, high-density lipoprotein-cholesterol; HOMA-IR, homeostatic model assessment-insulin resistance; LDL-C, low density lipoprotein-cholesterol; TC, total cholesterol; TG, triglycerides.

Fig. 1.

Representative pictures of visceral adipose tissue biopsies used for quantification of SA-β-gal staining. Biopsies were classified in three groups according to the NAC treatment. NAC, N-acetylcysteine; SA-β-gal, senescence-associated β-galactosidase.

Fig. 1.

Representative pictures of visceral adipose tissue biopsies used for quantification of SA-β-gal staining. Biopsies were classified in three groups according to the NAC treatment. NAC, N-acetylcysteine; SA-β-gal, senescence-associated β-galactosidase.

Close modal

Mean (SD) of the SA-β-gal activity was 237.2 (62.9) AU in the control group, 109.1 (47.0) AU in 5 mm NAC group and 98.4 (37.9) AU in 10 mm NAC group. It was observed that the NAC treatments at 5 mm and 10 mm concentrations resulted in a decrease in SA-β-gal staining when compared to the control group (p < 0.001 in all cases) (shown in Fig. 2).

Fig. 2.

SA-β-gal activity in visceral adipose biopsies incubated for 48 h without or with NAC prior to SA-β-gal activity assay. *p value by analysis of variance test <0.001 followed by Bonferroni’s test. NAC, N-acetylcysteine; SA-β-gal, senescence-associated β-galactosidase.

Fig. 2.

SA-β-gal activity in visceral adipose biopsies incubated for 48 h without or with NAC prior to SA-β-gal activity assay. *p value by analysis of variance test <0.001 followed by Bonferroni’s test. NAC, N-acetylcysteine; SA-β-gal, senescence-associated β-galactosidase.

Close modal

The expression of SASP-related genes in visceral adipose biopsies is shown in Figure 3. Relative to the control, the expression of P16 and P21 was significantly decreased after both treatments with NAC (5 mm) and NAC (10 mm) (all p < 0.001). There were no observed differences in P53 expression in the visceral adipose biopsies between the 5 mm NAC and control (p = 0.066) as well as between the 10 mm NAC and control (p = 0.063).

Fig. 3.

Expression of SASP-related genes in visceral adipose biopsies according to the NAC treatment groups. Gene expression results were expressed as the fold change defined by 2−ΔΔCt. *p value by analysis of variance test <0.001 followed by Bonferroni’s test, compare with control. NAC, N-acetylcysteine; SASP, senescence-associated secretory phenotype.

Fig. 3.

Expression of SASP-related genes in visceral adipose biopsies according to the NAC treatment groups. Gene expression results were expressed as the fold change defined by 2−ΔΔCt. *p value by analysis of variance test <0.001 followed by Bonferroni’s test, compare with control. NAC, N-acetylcysteine; SASP, senescence-associated secretory phenotype.

Close modal

The expression of cytokine genes in visceral adipose biopsies is presented in Figure 4. When treated with the NAC drug, significant reduction in IL-6 expression was observed in the adipose tissue biopsies of the NAC (5 mm) and NAC (10 mm) groups, as compared to the control group (all p < 0.001). Although a decrease in TNF-α gene expression was noticed in both NAC groups when compared to the control group, it was not statistically significant (p = 0.884).

Fig. 4.

Expression of senescence-associated genes in visceral adipose biopsies according to the NAC treatment groups. Gene expression results were expressed as the fold change defined by 2−ΔΔCt. *p value by analysis of variance test <0.001 followed by Bonferroni’s test, compare with control. IL-6, interleukin 6; NAC, N-acetylcysteine; TNF-α, tumor necrosis factor alpha.

Fig. 4.

Expression of senescence-associated genes in visceral adipose biopsies according to the NAC treatment groups. Gene expression results were expressed as the fold change defined by 2−ΔΔCt. *p value by analysis of variance test <0.001 followed by Bonferroni’s test, compare with control. IL-6, interleukin 6; NAC, N-acetylcysteine; TNF-α, tumor necrosis factor alpha.

Close modal

The present study has demonstrated the senotherapeutic activity of NAC by reducing SA-β-gal activity, SASP-related gene expression, and cytokine gene expression in the visceral adipose tissue of obese individuals. This suggests a potential senotherapeutic nature of NAC in human obese visceral adipocytes. Thus far, few studies have investigated the effects of NAC on senescence markers in adipocytes or adipose tissue. Therefore, we compared our findings with previous literature that has examined this effect in other cells or tissues.

In the present study, we have demonstrated that treatment with 5 mm and 10 mm NAC resulted in a reduction of β-galactosidase activity, an anti-senescence marker, compared to the untreated group. Consistent with our finding, Chen et al. reported that administration of 1 mg/mL NAC in drinking water for 4 weeks attenuated adipocyte senescence by reducing SA-β-gal activity in DNA polymerase η knockout mice [21]. Our finding is also consistent with Ali et al., [22] who reported a significant decrease in SA-β-gal activity in diabetic-mouse-derived mesenchymal stem cells following treatment with 30 mm NAC, as compared to the control group. In line with our findings, Li et al. indicated that 10 mm NAC decreased the percentage of SA-β-Gal positive cells in cisplatin-induced senescence model in renal tubular cells [23]. Debeljak Martacic et al. [24] demonstrated that treatment with 0.1 mm NAC significantly reduced the percentage of SA-β-gal positive cells in deciduous teeth dental pulp stem cells, which is consistent with our findings. It is noteworthy that while 1.0 mm NAC showed no effect, 2.0 mm NAC resulted in an increase in the number of senescent cells [24]. Therefore, the response of NAC on SA-β-gal activity is a point of confirmation in the present study and that of Debeljak Martacic et al. [24]. On the other hand, Voghel et al. [25] observed that treatment with 10 μm NAC did not have a significant effect on the onset of senescence in endothelial cells obtained from arterial patients with coronary artery disease. The duration required to obtain 50% positive β-gal cells was similar between NAC-treated and control cells.

Our study indicated that NAC treatment decreased the levels of SASP-related gene expression including P16 and P21. However, the expression of P53 did not show a significant decrease. Our findings are in line with Chen et al.’s [21] study, which highlights that NAC can notably minimize DNA damage in adipocytes by reducing p21 levels in DNA polymerase η knockout mice. In contrast, Chen et al.’s [21] study also revealed that NAC can substantially decrease adipocyte senescence by lowering p53, which is not in line with our findings. Marthandan’s research contradicts our findings as it demonstrated that 7.5 mm NAC did not influence the expression of P16 and P21 in the human fibroblast strains that were sourced from embryonic lung and foreskin [26].

In the present study, it was observed that NAC significantly decreased the expression of the IL-6 gene compared to the control group. However, there was no significant difference observed between the treatment groups with regard to TNF-α gene expression. In line with our findings, Chen et al. [21] demonstrated that NAC attenuated the expression of IL-6 gene. However, contrary to our findings, they reported a reduction in TNF-α gene expression due to NAC administration. Tsao et al. [17] showed that NAC can improve chronic inflammation in a diet-induced obesity mouse model.

Based on the studies that were referenced and compared with the results of our study, it is evident that our current research significantly differs from other similar studies by utilizing an ex vivo model that involves whole tissue explants. This approach distinguishes us from the commonly used in vitro and in vivo models in the literature. It is important to acknowledge and consider these differences, including factors such as cell composition, tissue environment, and experimental conditions that vary among different research methodologies.

Manipulating adipose tissue has emerged as a promising strategy in combating obesity and its related conditions as it plays a significant role in regulating the entire body’s energy balance [27]. There is increasing evidence suggesting that p53 plays multiple roles in adipocytes and adipose tissues, making it a crucial regulatory center in this tissue [27]. One of its effects is elevating levels of p53 in white adipose tissue, which can lead to increased levels of senescence and chronic inflammation. The mechanism of NAC’s anti-obesity effect involves its ability to decrease oxidative stress and inflammation, enhance insulin sensitivity, and regulate lipid metabolism [16]. Li et al. [23] showed that NAC attenuates premature renal senescence by SIRT1 activation and p53 deacetylation. NAC’s senotherapeutic activity has been more linked to anti-oxidative stress effects [17, 28, 29]. Lee et al. reported that NAC increased adipose glutathione peroxidase 3 expression in obese and diabetic db/db mice [29]. Preclinical studies have shown that NAC can decrease senescence-associated secretory phenotype production in adipose tissue, leading to a reduction in inflammation and cell death [17]. Additionally, NAC has been found to attenuate adipose tissue fibrosis, leading to an improvement in adipose tissue function [30]. Moreover, adipose tissue is a complex and heterogeneous tissue that consists not only of adipocytes but also of immune cells, fibroblasts, and various other cell types [31]. It is important to consider how NAC may impact these diverse cell populations. By investigating the potential effects of NAC on the inflammatory milieu of visceral adipose tissue and its influence on different cell types, the current study can provide a deeper understanding of the observed ex vivo anti-senescence activity in obese volunteers. Therefore, further research in this area is strongly encouraged.

It is important to recognize that the levels of toxicity vary between the doses used in in vivo and ex vivo studies and those used in human studies. Wong et al.’s [32] study demonstrated that an increased dosage of NAC at 10 mm could potentially pose a higher risk of toxicity and injury to lung alveolar epithelial cells. Plein et al. [33] reported NAC toxicity at concentrations starting from 20 mm in endothelial cells derived from the human umbilical vein. Furthermore, it is crucial to highlight that concentrations of NAC exceeding 0.3 m in in vivo settings can result in lung ciliostasis and subsequent toxicity [34], and the doses used in our study were lower than the toxicity values mentioned in previous studies.

This study highlights both strengths and limitations. It is notably the first investigation into the effects of NAC on visceral adipose tissue in obese adults, while also simultaneously evaluating SASP and inflammatory gene expression related to cell aging and β-gal activity. However, limitations include lack of assessment of senescence in subcutaneous adipose tissue along with evaluations of visceral fat tissue, and failure to evaluate the antioxidant properties of NAC and its effects on adipose tissue. Additionally, the omission of adipocyte cultivation and examination of their morphology is a weakness of this study.

The current research has shown that NAC may have a senotherapeutic effect by decreasing the activity of SA-β-gal and the expression of IL-6, P16, and P21 genes in the visceral adipose tissue of obese individuals. This suggests that NAC could be beneficial as a treatment and preventative measure for age-related metabolic disorders and obesity. Further studies are needed to investigate the effect of NAC on protein expression levels of cytokine and SASP-related genes in adipose tissue. Furthermore, clinical trials should be conducted to further investigate the efficacy of NAC in addressing complications related to obesity.

The authors thank the participants for their contribution to this study. We also thank all the collaborators who helped with the data collection.

The study was approved by the Research Ethics Committees of National Nutrition and Food Technology Research Institute, Shahid Beheshti University of Medical Sciences, Tehran, Iran. (IR.SBMU.NNFTRI.REC.1401.002). Written informed consent was obtained from each participant included in the study.

The authors have no conflicts of interest that are directly relevant to the content of this article.

This work was supported by the National Nutrition and Food Technology Research Institute, Shahid Beheshti University of Medical Sciences, Tehran, Iran.

Conceptualization: G.E., A.G., and H.Z; data curation: D.B., G.E., A.G., A.S., M.H.S., S.F.T., and N.M.A; formal analysis: D.B., G.E., M.H.S., and S.F.T; funding acquisition and writing – original draft: G.E; investigation: G.E., N.M.A., and M.A; project administration: G.E., N.M.A., and H.Z; resources: N.M.A. and M.A; supervision: G.E., A.G., and M.A; visualization: A.S. and G.S; and writing – review and editing: D.B., A.G., H.Z., A.S., G.S., M.H.S., S.F.T., N.M.A., and M.A. All authors read and approved the final version of the manuscript.

Additional Information

Diba Behtaj and Arman Ghorbani contributed equally to this work.

The data that support the findings of this study are not publicly available due to ethical reasons but are available from the corresponding author request.

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