PbO Nanoparticles Increase the Expression of ICAM-1 and VCAM-1 by Increasing Reactive Oxygen Species Production in Rat Choroidal Epithelial Line Z310 Cells

doi:10.21203/rs.3.rs-1956358/v1

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PbO Nanoparticles Increase the Expression of ICAM-1 and VCAM-1 by Increasing Reactive Oxygen Species Production in Rat Choroidal Epithelial Line Z310 Cells

https://doi.org/10.21203/rs.3.rs-1956358/v1

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published 06 Jan, 2023

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PbO nanoparticles (nano-PbO) are widely used in the production of electrode materials, but exposure to them can cause brain damage. The first barrier preventing nano-PbO from entering the brain is the choroid plexus. However, the effect of nano-PbO on the choroid plexus remains unclear. Thus, the purpose of this study was to explore the effect of nano-PbO exposure on the adhesion protein of the choroid plexus and the role of reactive oxygen species (ROS) during the process. Results showed that treatment of rat choroidal epithelial Z310 cells with 10 µg/mL nano-PbO upregulated the expression of surface adhesion proteins, including intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1). It also increased intracellular ROS production, and significantly decreased glutathione (GSH) content, glutathione peroxidase (GSH-PX) activity and superoxide dismutase (SOD) activity. Treatment with ROS inhibitor N-acetylcysteine (NAC) significantly downregulated the expression of ICAM-1 and VCAM-1expression. In conclusion, exposure of Z310 cells to nano-PbO increases the expression of ICAM-1 and VCAM-1 through oxidative stress.

nano-PbO

choroid plexus

ICAM-1

VCAM-1

ROS

NAC

With the rapid development of nanotechnology, nanoparticles are being used in various fields, such as cosmetics, biomedical products, and electronic products, owing to their unique material characteristics(Nikzamir et al., 2021; Selmani et al., 2022). Therefore, the impact of nanoparticles on health and the environment has drawn increasing attention. PbO nanoparticles (nano-PbO) are widely used in lead graphite composite and other electrodes, X-ray imaging detector, and so on, owing to their unique optical and electrical properties (Muhammad et al., 2019). Consequently, an increasing number of people are exposed to nano-PbO. Thus, the evaluation of the health effects of nano-PbO has received increasing attention. Nano-PbO accumulates in many organs, including the kidney and lung. Acute and sub-chronic exposure to nano-PbO decreases GSH levels in the lung and promotes lipid peroxidation in the kidneys (Lebedova et al., 2018). Six-weeks of inhalation of lead oxide nanoparticles in mice caused a significantly lower GSH concentration and a decrease in the GSH/ glutathione disulfide ratio in the blood (Tulinska et al., 2022). Nano-PbO can also accumulate in the brain, increasing the number dark, shrunken, damaged neurons in the hippocampal CA1 region (Dumkova et al., 2017). Given its small size, nano-PbO can cross blood brain barrier (BBB) and blood cerebrospinal fluid barrier (BCB). Recently, an increasing number of studies have focused on BCB, because of its rich blood flow, relatively loose tissue structure, and close connection with blood and CSF. However, few studies have investigated the impact of nano-PbO on the choroid plexus and its potential mechanism. Such an investigation is important to provide experimental support and theoretical basis for the safety evaluation of Nano-PbO.

The BCB consists of the choroid plexus, which is the target of exogenous neurotoxins(Ha et al., 2020; Verheggen et al., 2021). Immune monitoring is an important function of BCB. Adhesion proteins, such as vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule (ICAM-1), and P-selectin play key roles in regulating immune cells passing the BCB. ICAM-1 and VCAM-1 are important adhesion molecules belonging to the immunoglobulin superfamily. ICAM-1 is involved in the adhesion of leukocytes to vascular endothelial cells(Bui et al., 2020; Chen et al., 2022). VCAM-1 is a cell adhesion molecule that plays a key role in tumour metastasis, autoimmune disease, inflammatory response, and lymphocyte adhesion and migration (Schlesinger and Bendas, 2015; Troncoso et al., 2021). VCAM-1 can regulate the inflammatory response of endothelial cells in the whole body and promote the migration of leukocytes after binding to integrin receptor α4β1(Yousef et al., 2019).Under physiological condition, the expression levels of ICAM-1 and VCAM-1 are relatively low. Environmental stimuli upregulate the expression of these adhesion molecules (Poursaleh et al., 2021; Shen et al., 2021). ICAM-1 and VCAM-1 also participate in BBB permeability change(Walter et al., 2022). However, the time-effects of nano-PbO on the expression of VCAM-1 or ICAM-1 in the choroid plexus and the potential underlying mechanism remain unclear. Therefore, Therefore, we aimed to explore the effects of nano-PbO exposure on ICAM-1 and VCAM-1 expression in Z310 cells and elucidate the role of oxidative stress in this process.

2.1. Cell culture

The rat choroidal epithelial Z310 cell line was provided by Professor Zheng, Purdue University. Z310 cells were cultured in Dulbecco’s nodified Eagle’s medium (DMEM) (Gibco 11995, USA) supplemented with 10% fetal bovine serum (BI, Israel), 10 ng/mL epidermal growth factor (SIGMA, USA), and 1% penicillin-streptomycin (GIBCO 15140122, USA) and incubated at 37°C in a humidified atmosphere containing 5% CO₂.

2.2. Preparation of nano-PbO

Nano-PbO was synthesized by Pro Meng, School of Chemical Engineering, North China University of Science and Technology as follows. In brief, 6.6246 g of 0.02 mol Pb(NO3)2 and 1.60 g of 0.04 mol NaOH were placed in an agate mortar, with 5 mL of 100% ethanol, mixed, and then ground. The reaction solution quickly turned orange. After complete grinding for approximately 40 min, the reaction solution gradually became viscous, and the reaction proceeded until the color no longer deepened. The obtained product was ultrasonically washed twice in 100% ethanol, centrifuged, and then washed with deionized water until the pH was approximately 7.

Finally, the product was dried in an oven at 80°C for 10 h to obtain PbO samples. The obtained PbO samples were subjected to X-ray diffraction (XRD), transmission electron microscopy (TEM), and zeta potential characterization and detection (Lebedova et al., 2018; Mahmoud et al., 2020).

2.3. Exposure to nano-PbO

A 5 mg/mL nano-PbO suspension was prepared with PBS, autoclaved, and then diluted to 10 µg/mL with DMEM containing 10% FBS. Cells were treated with 10 µg/mL nano-PbO for 0, 1, 6, and 12 h. Cells treated for 0 h served as control. At the same time, the cells were pretreated with 5 mmol/L N-acetylcysteine (NAC; MedChemExpress, USA) for 1 h to inhibit reactive oxygen species (ROS) production before nano-PbO exposure.

2.4. Cell counting kid-8 cell viability assays

Cells were seeded in a 96-well plate. Upon reaching approximately 85% fusion, the cells were treated with nano-PbO at different concentrations (0, 5, 10, 20, 40, 80, 160, and 320 µg/mL) and then incubated for 12 and 24 h. Cells without nano-PbO treatment served as control. After treatment, the medium was removed, and each well was added with 100 µL of medium containing cell counting kit (CCK)-8 reagent (Dojindo, Japan) at a volume ratio of 9:1. After incubation for 1 h at 37°C, the absorbance of each well at 450 nm was obtained using a microplate reader (Thermo Fisher Scientific, USA). The cell survival rate was calculated as (OD of nano-PbO-treated cells /OD of control cells) × 100 (Bian et al., 2016).

2.5. Detection of intracellular ROS

Intracellular ROS were analyzed by measuring DCFH-DA fluorescence intensity with a flow cytometer. The cells were treated with 10 µg/mL nano-PbO for 0, 1, 6, or 12 h. After pretreatment with NAC (5 mmol/L), the cells were treated with 10 µg/mL nano-PbO for 12 h. The cells were collected and centrifuged at 2000 rpm for 5 min. A total of 0.5 mL of 10 µM DCFH-DA was added to each tube following the instructions of the ROS detection kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) and then incubated at 37°C for 1 h in the dark. The cells were washed twice, resuspended, and then analyzed using a flow cytometer (Beckman, USA) (Zhou et al., 2017).

2.3. Detection of GSH content, GSH-PX activity and SOD activity

GSH content, GSH-PX activity and SOD activity were measured using GSH kit, GSH-PX kit, and SOD kit (Nanjing Jiancheng Bio-engineering Institute, China) in accordance with the manufacturer’s instructions. RIPA lysis buffer was used to extract total protein from cells. Protein concentration was assessed using a BCA protein assay kit (Beijing Solarbio Science & Technology Co., Ltd., China) as previously described (Goc et al., 2017).

2.7. Western blot analysis

Z310 cells were lysed with lysis buffer and, then centrifuged at 13,000 rpm for 15 min at 4°C. The supernatant containing proteins was then collected. Protein concentration was determined using a BCA protein assay kit. Gel electrophoresis was performed using an 8% SDS-PAGE gel, and then the proteins were transferred onto polyvinylidene fluoride (PVDF) membranes. The membranes were incubated with 5% non-fat milk for 1 h at room temperature (20°C-28°C) and then incubated with diluted primary antibodies, including rabbit anti-ICAM-1 (1:1000; Proteintech Group, USA), rabbit anti-VCAM-1 (1:1000; Proteintech Group, USA), and rabbit anti-β-actin (1:3000; Proteintech Group, USA), at 4°C overnight. Subsequently, they were incubated with goat anti-rabbit IgG-horseradish peroxidase secondary antibody diluted at 1:3000 (Shanghai Universal Biotech Company, China) for 1 h at room temperature. After incubation, the membranes were stained with an enhanced chemiluminescence reagent. The gray value of each band was analysed using ImageJ software (Wang et al., 2022).

2.8. Immunofluorescence staining

The treated cells were fixed with 4% paraformaldehyde for 20 min, washed with PBS, and then blocked with 1% bovine serum albumin. The cells were incubated with diluted antibodies against VCAM-1 (1:400) and ICAM-1 (1:400) at 4°C overnight. The cells were washed thrice with PBS and then incubated with diluted fluorescein isothiocyanate (FITC)-labeled secondary rabbit antibodies (1:200; Shanghai Universal Biotech Company, China) at room temperature for 1 h in the dark. After washing twice with PBS, the cells were counterstained with 4′,6-diamidino-2-phenylindole for imaging. Semi-quantification was performed using Image J software (Wang et al., 2022).

2.9. Real-time polymerase chain reaction analysis

Cells were lysed with TRIzol reagent, and total RNA was extracted. Complementary DNA (cDNA) was synthesized using Transcriptor First Stand cDNA Synthesis kit (Roche, USA). The RT-PCR program consisted of initial denaturation for 10 min at 95°C, followed by 40 cycles of 10 s at 95°C, 15 s at 60°C and 10 s at 72°C. The relative quantification method (2ΔΔCt) was used to calculate the mRNA expression levels. β-Actin was used as an internal reference. The primer sequences used were as follows. ICAM-1 forward 5ʹ-CAGGTGGGCAAGAACCTCAT-3ʹ, reverse 5ʹ-TGTCGAGCTTCAGGACCCTA-3ʹ; VCAM-1 forward 5ʹ-ACAAGGCTACATGAGGGTGC-3ʹ, reverse 5ʹ-AACGGAATCCCCAACCTGTG-3ʹ; β-actin forward 5ʹ-GTGGATCAGCAAGCAGGAGT-3ʹ, reverse 5ʹ-CGCAGCTCAGTAACAGTCCG-3ʹ (Wang et al., 2022).

2.10. Statistical analysis

Data are presented as the mean ± standard deviation, and SPSS23.0 software was used for statistical analysis. Statistical analysis was carried out using one-way ANOVA with post-hoc comparisons through LSD-t and Dunnett’s tests, and the Dunnett T3 method was used when the variance was uneven. Differences were considered statistically significant atP < 0.05.

3.1. Characterization of Nano-PbO samples

The XRD patterns of the nano-PbO samples are shown in Fig. 1A. Analysis results of MDI Jade 6.5 software showed that the samples were α-PbO. The XRD patterns clearly show the diffraction peaks of the crystal planes of the samples. As shown in Fig. 1A, peaks measured over the diffraction angle range (2θ) of 20°–70° corresponded to 28.63(101), 32.59(200), 35.74(002), 48.59(112), 54.76(211), and 59.26(202) as per JCPDS pattern no. 05-0561, proving the crystalline nature of nano-PbO. This result is in agreement with the findings of a previous study.(Mahmoud et al., 2020)

The morphology and shape of nano-PbO were investigated via TEM. TEM micrographs showed that nano-PbO exhibited rough and irregularly shaped aggregates (Fig. 1B). Based on the TEM micrograph, the average size was calculated using ImageJ to be 101 ± 13 nm. The zeta potential of nano-PbO was 35.53 ± 7.72 mV (Fig. 1C), indicating good physical stability.

3.2. Nano-PbO exposure inhibited the survival rate of Z310 cells

CCK-8 analysis was used to determine the influence of nano-PbO on the survival rate of Z310 cells. As shown in Fig. 2, the cell survival rate decreased with increasing nano-PbO concentration. No significant differences in survival rates were found between the cells exposed to nano-PbO at concentrations less than 10 µg/mL and the control cells at 12 and 24 h (P > 0.05). However, the survival rates of the cells exposed to nano-PbO at 20, 40, 80, 160, and 320 µg/mL were significantly lower than those of the control cells (P < 0.05). Cell viability at 24 h was lower than that at 12 h, especially at nano-PbO concentrations of 160 and 320 µg/mL. Thus, the Z310 cells exposed to 10 µg/mL nano-PbO for 12 h were used for further analyses.

3.3. Nano-PbO exposure enhanced ICAM-1 and VCAM-1 expression in Z310 cells

Z310 cells were treated with nano-PbO for 0, 1, 6, and 12 h. The protein and mRNA expression levels of ICAM-1 and VCAM-1 were measured using western blot, immunofluorescence assay, and real-time PCR. Compared with the control cells, the cells exposed to nano-PbO for 6 and 12 h showed significantly higher expression of ICAM-1 and VCAM-1 (Fig. 3A–C, P < 0.05). The fluorescence intensities of ICAM-1 and VCAM-1 were stronger in the cells exposed to nano-PbO for 6 and 12 h than that in the control cells (Fig. 3D–F, P < 0.05).

3.4. Effects of nano-PbO exposure on ROS level, GSH content, GSH-PX activity, and SOD activity in Z310 cells

Flow cytometry, GSH kit, GSH-PX kit, and SOD kit were used to measure ROS level, GSH content, GSH-PX activity, and SOD activity, respectively. The intracellular ROS levels in the Z310 cells exposed for 1, 6, and 12 h were 1.20-, 1.26-, and 1.50-fold greater than those in the control cells (0 h), respectively (Fig. 4A–B, P < 0.05). Meanwhile, the GSH contents in the Z310 cells exposed for 6 h [(22.15 ± 1.51) µmol/mgprot] and 12 h [(20.75 ± 1.31) µmol/mgprot] were lower than those in the control cells [(26.86 ± 1.30) µmol/mgprot] (Fig. 4D, P < 0.05). The SOD activities in the Z310 cells exposed for 0, 1, 6, and 12 h were (39.43 ± 2.30), (36.55 ± 3.34), (35.62 ± 2.40), and (33.71 ± 1.90) U/mgprot, respectively (Fig. 4C). The GSH-PX activities in the Z310 cells exposed for 0, 1, 6, and 12 h were (133.52 ± 4.58), (125.63 ± 3.19), (118.16 ± 7.19), and (117.16 ± 9.42) U/mgprot, respectively (Fig. 4E). The change trends in SOD and GSH-PX activities were similar to that in GSH, suggesting that nano-PbO can affect oxidative stress in Z310 cells.

3.5. ROS production inhibition downregulated ICAM-1 and VCAM-1 expression in Z310 cells exposed to nano-PbO

Z310 cells were treated with NAC to inhibit ROS production. The oxidation and antioxidant levels and the expression levels of ICAM-1 and VCAM-1 were measured. The ROS and SOD levels in the cells with NAC treatment were not significantly different from those in the cells without NAC treatment. The cells treated with both NAC and nano-PbO had significantly lower ROS levels and higher SOD activities than the cells treated with nano-PbO alone (Fig. 5A–C, P < 0.05).

Treatment with 5 mmol/L NAC alone did not affect ICAM-1 and VCAM-1 expression in Z310 cells (Fig. 5D–F, P > 0.05), whereas treatment with 10 µg/mL nano-PbO significantly increased ICAM-1 and VCAM-1 expression (Fig. 5D–F, P < 0.05). In addition, 5 mmol/L NAC significantly inhibited nano-PbO-induced ICAM-1 and VCAM-1 expression, although the expression was still significantly higher than that in the cells without 5 mmol/L NAC treatment (Fig. 5D–F, P < 0.05). These results were verified by immunofluorescence (Fig. 5G–I)..

In current study, we investigated the effect of nano-PbO on the expression of adhesion proteins in Z310 cells and the potential role of oxidative stress. The results showed that nano-PbO treatment upregulated the expression of VCAM-1 and ICAM-1 and increased the production of ROS. The expression of VCAM-1 and ICAM-1 in the Z310 cells treated with nano-PbO was downregulated when ROS production was inhibited by NAC. Our findings suggested that oxidative stress participated in the up-regulation of adhesion protein expression in Z310 cells treated with nano-PbO, which might contribute to the immune imbalance induced by nano-PbO exposure.

ICAM-1 and VCAM-1, adhesion proteins in choroid plexus epithelial cells, play important roles in regulating immune cells and cytokines entering the cerebrospinal fluid through the choroid plexus (Demeestere et al., 2015). ICAM-1 is an adhesion molecule expressed by vascular endothelial cells, platelets, monocytes, neutrophils, and naive T lymphocytes, and its expression is downregulated under certain physiological conditions. During inflammation, ICAM-1 protein expression significantly increases and then regulates leukocyte transport (Peycheva et al., 2022). ICAM-1 participates in the process of adhesion between leukocytes and endothelial cells by interacting with CD11a/CD18 or CD11b/CD18 (Muller, 2011). VCAM-1 expression is significantly increased after cytokine stimulation. It is involved in the activation and migration of lymphocytes and plays important roles in inflammation and tumor metastasis (Corroyer-Dulmont et al., 2020; Troncoso et al., 2021). The present findings showed that the expression of ICAM-1 and VCAM-1 were increased in Z310 cells exposed to nano-PbO. ICAM-1 and VCAM-1can influence the endothelial cytoskeleton, transcription, and inter-endothelial junctions, all of which may play a crucial role in modulating endothelial disposition to infiltrating leukocytes; such an effect is important in controlling inflammation in the central nervous system (CNS) (Turowski et al., 2005). The expression of ICAM-1 and VCAM-1 is up-regulated in endothelial cells under inflammatory conditions, such as in individuals with Alzheimer’s disease (Janelidze et al., 2018). ICAM-1 directs the migration of circulating monocytes or neutrophils toward the activated endothelium and partly facilitates neuro-inflammatory responses (Yang et al., 2019). The nano-PbO-induced upregulation in the expression of ICAM-1 and VCAM-1 may be involved in the adhesion and migration of leukocytes to the choroid plexus epithelium, thereby affecting the immune balance in the CNS. The protein expression of ICAM-1 changed more than that of VCAM-1 possibly because of its structure, and the underlying mechanism warrants further investigation.

Exposure to Pb induces ROS overproduction, which leads to neurotoxicity (Lee et al., 2019; Qu et al., 2021; Xue et al., 2017). The generation and clearance of ROS are dynamic. Under pathological conditions, excessive ROS levels can cause oxidative damage. SOD is an ubiquitous antioxidant enzyme, that catalyzes the conversion of superoxide radicals to hydrogen peroxide (H₂O₂). Our data showed that ROS levels increased in the Z310 cells exposed to nano-PbO, which is in line with previous results on lead acetate exposure (Zhang et al., 2021). In addition, exposure to nano-PbO decreased SOD activity in the cells. Similarly, Cao et al. showed that nano-PbS can increase oxidative damage in the hippocampus and significantly inhibit SOD activity (Cao et al., 2020). Increased ROS production is a key factor contributing the toxicity of nanomaterials (Deciga-Alcaraz et al., 2020). However, whether or not ROS induce the upregulated expression of ICAM-1 and VCAM-1 in Z310 cells remains to be determined.

NAC is the precursor of L-cysteine, which stimulates the biosynthesis of glutathione and directly acts as a scavenger of free radicals, and it is commonly used as a ROS inhibitor. In the current study, treatment with 5 mM NAC inhibited the nano-PbO-induced production of ROS in Z310 cells. As second messengers, ROS play important roles in cell signaling pathways (Zhang et al. 2016). Lee et al. showed that 400 µM hydrogen peroxides can activate NF-κB signaling, leading to the expression of VCAM-1 in endothelial cells (Lee et al. 2007). In addition, HIV-1 Tat protein, one of the viral gene products necessary for viral genome expression and replication, can regulate monocyte adhesion by upregulating the expression of adhesion molecules, such as ICAM-1 and VCAM-1, through ROS- and NF-κB-dependent pathways in astrocytes, whereas NAC inhibits this process (Song et al. 2007). In the present study, the expression of ICAM-1 and VCAM-1 in the cells pre-treated with NAC was downregulated after nano-PbO exposure. Our findings indicated that nano-PbO exposure induced the expression of ICAM-1 and VCAM-1 through ROS in Z310 cells. ROS affect the activation of the NF-κB pathway and inhibit IκBα phosphorylation (Zhang et al. 2016). The expression of VCAM-1 is induced by tumor necrosis factor (TNF)-α and interleukin (IL)-1β. Overexpression of SOD can block the expression of VCAM-1 induced by TNF-α (Cook-Mills et al. 2011). During atherosclerosis treatment, antioxidants can reduce the production of ROS, inhibit the secretion of cytokines, and regulate the expression of adhesion molecules such as VCAM-1 and ICAM-1 in endothelial cells (Malekmohammad et al. 2019). In the present study, NAC treatment inhibited the nano-PbO-induced ROS production in Z310 cells, it might inhibit the secretion of the cytokine TNF-α, thus reducing the expression of ICAM-1 and VCAM-1.

However, our study has some limitations. For instance, other adhesion proteins aside from ICAM-1 and VCAM-1 should be analyzed in Z310 cells, and experiments in vivo are warranted. Further studies are necessary to verify the effects of ROS and ICAM-1 on the permeability of the BCB following exposure to nano-PbO.

In summary, our data showed that nano-PbO exposure upregulated the expression of VCAM-1 and ICAM-1 in the choroid plexus and that oxidative stress was involved in this process. Nano-PbO exposure might impact the immune surveillance function and permeability of the BCB and induce disorders of the central nervous system.

Funding

This work was supported by the Science and Technology Project of Hebei Education Department (ZD2020113), National Natural Science Foundation of China (82073598), and Natural Science Foundation of Hebei Province (H2020209250).

Ethical Approval

No involved.

Consent to participate

No involved.

Consent for publication

All authors have read and approved the manuscript and have accorded consent for publication.

Competing interests

The authors declare no competing interests.

Author’s contribution

YS Zhang and S Li designed this study; WX Wang and S Li interpreted the experimental results; X Wang, WX Wang, and JB Wang performed the experiments; X Wang, WX Wang, and S Li wrote the manuscript; and YS Zhang and WX Wang approved the final version of this paper.

Data availability

Supporting data set will be made available upon reasonable request.

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published 06 Jan, 2023

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PbO Nanoparticles Increase the Expression of ICAM-1 and VCAM-1 by Increasing Reactive Oxygen Species Production in Rat Choroidal Epithelial Line Z310 Cells

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Abstract

Figures

1. Introduction

2. Materials And Methods

2.1. Cell culture

2.2. Preparation of nano-PbO

2.3. Exposure to nano-PbO

2.4. Cell counting kid-8 cell viability assays

2.5. Detection of intracellular ROS

2.3. Detection of GSH content, GSH-PX activity and SOD activity

2.7. Western blot analysis

2.8. Immunofluorescence staining

2.9. Real-time polymerase chain reaction analysis

2.10. Statistical analysis

3. Results

3.1. Characterization of Nano-PbO samples

3.2. Nano-PbO exposure inhibited the survival rate of Z310 cells

3.3. Nano-PbO exposure enhanced ICAM-1 and VCAM-1 expression in Z310 cells

3.4. Effects of nano-PbO exposure on ROS level, GSH content, GSH-PX activity, and SOD activity in Z310 cells

3.5. ROS production inhibition downregulated ICAM-1 and VCAM-1 expression in Z310 cells exposed to nano-PbO

4. Discussion

Declarations

Funding

Ethical Approval

Consent to participate

Consent for publication

Competing interests

Author’s contribution

Data availability

References

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