Influence of a combination of triptolide and ferulic acid on the activities of CYP450 enzymes and oxidative stress in HaCaT cells
- Authors:
- Published online on: October 6, 2020 https://doi.org/10.3892/etm.2020.9286
- Article Number: 157
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Copyright: © Zhang et al. This is an open access article distributed under the terms of Creative Commons Attribution License.
Abstract
Introduction
Triptolide (TP) is one of the main monomer constituents that can be derived from Tripterygium wilfordii (1). Previous clinical studies have shown that TP has significant anti-inflammatory and immunosuppressive activities, and it has been widely used in the treatment of rheumatoid arthritis (RA) in China (2-4). However, TP is also toxic in humans, and its multiorgan toxicity prevents further application in clinical practice (5). The development of external TP preparations has become more efficient, and percutaneously administered TP has been efficacious in the treatment of RA (6-8). However, our previous study revealed that transdermal administration of TP can produce specific toxicities in the skin (9).
Ferulic acid (FA) is a type of phenolic acid with medicinal values and is found in plant tissues (10). FA is a primary component of the traditional Chinese medicinal herbs Angelica sinensis and Ligusticum chuanxiong FA, and has been suggested to possess anti-inflammatory and antioxidative pharmacological activities, which contribute to its therapeutic effect on RA (11,12). Previous studies have shown that TP and FA (TP + FA) can be combined due to their component compatibility (13-15). Additionally, our previous study performed pharmacodynamic and toxicological experiments in a type II collagen-induced arthritis rat model and showed that the combination of TP + FA exerts significant anti-inflammatory effects and reduces skin irritation (data not published). However, to the best of our knowledge, the underlying mechanisms of TP + FA treatment in decreasing skin irritation have not been previously investigated. Oxidative stress can cause many diseases, and it is closely related to cell dysfunction, membrane structure, protein production, DNA changes and other cellular functions (16-18). Moreover, oxidative stress can result in loss of skin cells and degradation of the extracellular matrix (16-18). Cytochrome P450 (CYP) enzymes are important for controlling the normal physiological function and homeostasis of the internal environment of the skin. Moreover, CYP enzymes maintain the integrity and barrier role of the epidermis via biotransformation of exogenous and endogenous substances (19,20).
HaCaT cells are a cultured human keratinocyte cell line. The culture method for maintaining HaCaT cells is simple, and these cells can proliferate indefinitely (21). In addition, HaCaT cells exhibit similarities to primary keratinocyte cells in the activity of drug-metabolizing enzymes (22). In the present study HaCaT cells were cultured in vitro as a model of an active epidermis with the aim to investigate the underlying mechanisms of FA in reducing skin toxicity in relation to metabolism and oxidative damage to skin. The aim of the present study was to provide a theoretical basis for the potential clinical application and development of external TP preparations.
Materials and methods
Materials and reagents
HaCaT cells (cat. no. ZQ0044) were supplied by Shanghai Zhongqiao Biotechnology Co., Ltd. TP (purity >98%) and FA (purity >98%) were purchased from Chengdu Pufei De Biotech Co., Ltd. Chlorzoxazol, testosterone and fenacetin were provided by Chengdu Derrick Biotechnology Co., Ltd. Reactive oxygen species (ROS, cat. no. 20180815), glutathione (GSH, cat. no. 20180721), malondialdehyde (MDA, cat. no. 20180730), superoxide dismutase (SOD, cat. no. 20180721) and catalase (CAT, cat. no. 20180811) assay kits were obtained from Nanjing Jiancheng Bio-Engineering Institute Co., Ltd. A nuclear factor erythroid 2-related factor 2 (Nrf2) antibody was purchased from Abcam (cat. no. Ab92946) and a β-actin antibody (cat. no. AC026) from ABclonal Biotech Co., Ltd. DMEM was obtained from Beijing Solarbio Science & Technology Co., Ltd and FBS was obtained from Shanghai Jikai Ecox Biotechnology Co., Ltd.
HaCaT cell culture
HaCaT cells were grown in DMEM (4.5 g/l glucose) containing antibiotics (100 U/ml penicillin and 100 µg/ml streptomycin) and supplemented with 10% FBS at 37˚C in a 5% CO2 incubator. Medium was replaced every 2-3 days. The 7-20th generations of cells were used in subsequent experiments. Cell density was adjusted to 1x105 cells/ml. Cells were seeded into 96-well plates to carry out MTT assays and 6-well plates for all other experiments.
MTT assay
Cell cytotoxicity and the optimum ratio of TP to FA was determined using an MTT assay. HaCaT cells were seeded into 96-well plates (2x104 cells/well) in a complete growth medium [High glycemic DMEM (4.5 g/l D-glucose, L-glutamine) containing 10% fetal bovine serum, 1% double antibody (100 U/ml penicillin, 100 g/ml streptomycin)] for 24 h, and then treated for 6 h with TP at 37˚C, FA, mixed solutions of the two drugs at different concentrations (The concentrations of the TP groups were 19.54, 39.07, 78.13, 156.25, 312.5, 625 and 1,250 ng/ml, TP and FA equal to 1:12.5, 1:25, 1:50, 1:100, 1:120 in the TP + FA groups, respectively) or a control group with an equivalent volume of DMEM to that of the experimental drugs. Cells were washed twice with Hank's Balanced Salt Solution (HBSS) and incubated with 5 mg/ml MTT solution for 4 h at 37˚C. The resulting formazan crystals were dissolved in 150 µl DMSO, and optical density (OD) was measured at 490 nm using a microplate reader (MK3; Thermo Fisher Scientific, Inc.). Cell survival rate (%) was calculated using the following formula: [OD 490 (treatment)-OD 490 (blank)/OD 490 (control)-OD 490 (blank)] x100. OD 490 (treatment) was the mean OD value of cells treated with the target drugs, OD 490 (control) was the mean OD value of untreated cells and OD 490 (blank) was the mean OD value of cells treated with HBSS. The combination ratio of TP to FA was determined when the cell survival rate was at its highest.
Liquid chromatography-mass spectrometry (LC-MS) method validation
The chromatographic conditions were as follows. Solvent A was 0.1% formic acid (V/V) in water and solvent B a gradient elution with acetonitrile. The elution conditions of testosterone and fenacetin were as follows: 0-1 min, 70-45% A; 1-2.8 min, 45% A; 2.8-3 min, 45-70% A; 3-5 min, 70% A. The elution conditions of chlorzoxazol and carbamazepine were as follows: 0-1.5 min, 80-25% A; 1.5-3 min, 25% A; 3-3.1 min, 25-80% A; 3.1-5.0 min, 80% A. The flow rate was 0.3 ml·min-1 and the column temperature was 30˚C.
LC-MS conditions
A liquid chromatography-triple quadruple bar mass spectrometer (AB SCIEX, MODEL no. QTRAAP 5500) was used. Testosterone and fenacetin were detected in positive ion mode, and chlorzoxazol, in negative ion mode. An electrospray ion source was used and the ion source temperature (TEM) was 500˚C, ionized voltage was 5,500 V, curtain gas was at 35 psi, impact gas was at 7 psi, spray gas was at 50 psi at the auxiliary heater at 50 psi. The details of the multiple reaction monitoring transitions assessed were as follows: The quantitative ion pairs of testosterone were 289.1/109, DP (V) 103, CE (eV) 79 (positive); the quantitative ion pairs of fenacetin were 180.2/109.8, DP (V) 97, CE (eV) 26 (positive); the quantitative ion pairs of Internal standard carbamazepine were 236.8/194, DP (V) 120, CE (eV) 27 (positive); the quantitative ion pairs of chlorzoxazol were 167.9/131.9, DP (V) -60, CE (eV) -26 (negative); and the quantitative ion pairs of internal standard carbamazepine were 234.1/188.8, DP (V) -158, CE (eV) -23 (negative).
Specificity
Specificity of the method for measuring concentrations of the three probe drugs was achieved by selecting a precursor ion and then detecting and quantifying product ions. Specificity was evaluated by comparing the chromatogram of blank HBSS from HaCaT cells with that of blank plasma spiked with analytes (23), as previously described, as well as that of HBSS samples obtained from cells after administration of mixed probe drugs at a testosterone: Fenacetin:Chlorzoxazol dose of 120:6:6 ng/ml after treatment for 6 h at 37˚C.
Linearity
Linearity of the developed analytical method was investigated by analyzing the matrix-matched construction via an internal standard approach (24), using mixed probe drugs at a series of concentrations. Testosterone was used at the following concentrations 480, 240, 120, 60, 30, 15, 7.5 and 3.75 ng/ml at 25˚C. Fenacetin and chlorzoxazol were used at concentrations of 24, 12, 6, 3, 1.5, 0.75, 0.38 and 0.19 ng/ml at room temperature. Carbamazepine (National Institutes for Food and Drug Control; cat. no. 100142-201706) was used as an internal standard and at concentration of 0.01 ng/ml. Taking the ratio of the target component to the peak area of carbamazepine as the longitudinal coordinate and the concentration as the transverse coordinate, linear regression was performed via LC-MS analysis (SCIEX TRIPLE QUAD 5500; SCIEX; Analyst 1.6.2 software control and data processing system) and the standard curve was drawn with the reciprocal of the concentration as the weighted coefficient (25). The quantitative limit was set to a signal-to-noise ratio of ≥10.
Accuracy and precision
In total, six replicate analyses of the quality control (QC) samples (chlorzoxazol, testosterone and fenacetin) were prepared at three different concentrations (the concentrations of testosterone were 480, 120 and 30 ng/ml, and the concentrations of fenacetin and chlorzoxazol were 24.0, 6.0 and 1.5 ng/ml, respectively) on the same day to ensure inter-day accuracy and precision. The ratio of the actual measured concentration to the marked concentration was used to determine the recovery rate of the analytical method. The present study estimated intra-day precision by analyzing six replicate QC samples on three consecutive days. The relative standard deviation (RSD) was used to assess precision, and accuracy was defined as the percent ratios of the calculated concentrations to the nominal concentrations (26).
Stability
QC samples (n=6) at three concentrations (low, medium and high) were used to assess the freeze-thaw cycle stabilities of the mixed probe drugs chlorzoxazol, testosterone and fenacetin. All QC samples were stored at -80˚C and subjected to three freeze-thaw (at room temperature) cycles, each cycle lasted for 24 h, and the concentrations were determined using LC-MS.
Investigation of probe drug reaction concentration and duration of treatment
The mixed probe drugs were administered to HaCaT cells at testosterone: Fenacetin:Chlorzoxazol doses of 240:12:12, 120:6:6, 60:3:3, 30:1.5:1.5, 15:0.75:0.75 and 7.5:0.375:0.375 ng/ml. Cell survival rate (%) was calculated using the methodology described previously.
Mixed probe drugs were added to HaCaT cells at a density of 2x105 in 6-well plates at 37˚C. Then, 150 µl samples were collected from every group at 0, 0.5, 1, 2, 4 and 6 h, and the substrate concentrations of each probe was determined using the methodology described in the aforementioned description of LCMS validation.
Determination of CYP1A2, CYP2E1 and CYP3A4 enzymatic activities
In total, 150 µl mixed probe drug samples were collected from each group (TP high dose, 156.25 ng/ml, TP medium dose, 78.13 ng/ml and TP low dose, 39.07 ng/ml. TP + FA low dose, TP 39.07 ng/ml + FA 3.907 µg/ml, TP + FA medium dose, TP 78.13 ng/ml + FA 7.813 µg/ml, and TP + FA high dose, TP 156.25 ng/ml + FA 15.625 µg/ml) of HaCaT cells after 2 h of treatment at 37˚C, and were centrifuged at 26,400 g for 10 min at 4˚C. Then, the upper layers of the samples were analyzed via LC-MS (AB SCIEX, TRIPLE QUDA 5500). Testosterone and fenacetin were detected in positive ion mode and chlorzoxazol in negative ion mode. The conditions were electrospray ion source, ion source temperature was 500˚C. Ionization voltage was 5,500 V, curtain gas was at 35 psi, bump gas was at 7 psi, spray mist was at 50 psi and the auxiliary heater was at 50 psi in scan mode for multi-reaction monitoring. The specific methods were as described previously in this manuscript. The activities of cytochrome P450 (CYP) enzymes CYP family 1 subfamily A member 2 (CYP1A2), CYP2E1 and CYP3A4 were assessed using different concentrations of fenacetin (6.0 ng/ml), chlorzoxazol (6.0 ng/ml) and testosterone (120.0 ng/ml), respectively (27).
Measurement of intracellular ROS, GSH, CAT, SOD and MDA
HaCaT cells were seeded into 6-well plates (2x105 cells/well) in a complete growth medium for 48 h. Cells were treated for 6 h with TP, FA and TP + FA at different concentrations (Table V) in DMEM at 37˚C. Generation of intracellular ROS was assessed using 10 mM 2',7'-dichlorofluorescein diacetate (DCFH-DA), a fluorescent probe, which is converted to the highly fluorescent derivative dichlorofluorescin via oxidation by ROS and peroxides (28). At the end of the drug action (after 6 h), the residual liquid was discarded. Cells were incubated in the dark for 1 h at 37˚C with 1 ml DCFH-DA working solution (DCFH-DA:HBSS, 1:500) and then resuspended in HBSS (4.5 g/l glucose). Fluorescence was analyzed using a multifunctional enzyme labeling instrument (Thermo Fisher Scientific, Inc.) with excitation at 488 nm and emission at 530 nm. GSH, CAT, SOD and MDA activities in HaCaT cells were measured using commercially available kits following the manufacturer's instructions. Cells were collected by centrifugation (1,530 x g at 4˚C for 10 min) and suspended in HBSS (4.5 g/l glucose). Cells were then broken by ultrasonication (300 W; 5 sec/time; five times). The absorbance values were measured using an enzyme labeling instrument (MK3; Thermo Fisher Scientific, Inc.) to indicate the level of production of GSH, CAT, SOD and MDA.
Western blotting
HaCaT cells were seeded into 6-well plates (2x105 cells/well) in a complete growth medium for 48 h and were assigned into seven groups: 3 TP group (39.07, 78.13 and 156.25 ng/ml) and 3 TP (39.07, 78.13 and 156.25 ng/ml): FA (1:100) group and a negative control group (Blank solvent is DMEM). Cells were then harvested by scraping, collected in HBSS and centrifuged at 1,530 x g for 5 min at 4˚C before they were mixed with lysis buffer (RIPA; Beijing Solarbio Science & Technology Co. Ltd.) and placed on ice for 20 min. Following lysis the suspension was centrifuged at 12,000 x g at 4˚C for 15 min. Protein was collected and the concentrations determined using a bicinchoninic acid protein assay kit (cat. no. 23225; Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. A total of 20 µg of each protein was loaded for SDS-PAGE (12%) before transfer onto PVDF membranes. Membranes were blocked with 10% goat serum for 2 h at 4˚C before incubation with the primary antibodies (diluted in 5% skimmed milk) at 4˚C for 12 h. Nrf2 (1:1,000) and β-actin (1:100,000) were used as primary antibodies and anti-mouse immunoglobulin G (1:50,000) as the secondary antibody at 37˚C for 1 h. The blots were developed with ECL reagent (EMD Millipore; cat. no. WBKLS0010). The density of each protein band was analyzed and calculated using ImageJ version 1.44 (National Institutes of Health). Protein expression level was expressed as the ratio of Nrf2 to β-actin, and semi-quantitative analysis was performed.
Statistical analysis
Data are presented as the mean ± SD from 3 parallel experiments per group. Analyses were performed using SPSS software version 21.0 (SPSS, Inc.). Differences between the treatment groups and the control group were assessed by one-ANOVA followed by a least-significant-difference test. P<0.05 was considered to indicate a statistically significant difference.
Results
Determination of the compatibility ratio of TP to FA
It was found that TP inhibited the viability of HaCaT cells in a dose related manner, with higher doses reducing cell viability (Table I; Fig. 1). The cell survival rate was <90% when the concentration of TP >19.54 ng/ml. When the concentration of FA was <150 µg/ml, FA had no effect on the survival of HaCaT cells. However, the inhibitory effect on HaCaT cell survival was significantly decreased when TP + FA was administered. Moreover, the survival rate of HaCaT cells was increased at TP + FA combination ratios of 1:12.5, 1:25, 1:50, 1:100 and 1:120, compared with the TP alone group. The effect on cell survival of the TP + FA combination ratio 1:100 was >7 times higher compared with TP alone. Therefore, the present results indicated that the compatibility ratio of TP + FA was 1:100.
Influence of TP + FA on activities of CYP450 enzymes and method validation
Specificity. It was found that none of the reagents or disposables used for method setup interfered with detection or quantification of probe drugs. Furthermore, false-positive responses and co-eluting components were not detected in analyzed biomatrices, and no carryover was observed (Fig. 2).
Linearity
Calibration curves showed excellent linearity (r>0.999) at the following concentration ranges: 3.75-480 ng/ml for testosterone, 0.19-24 ng/ml for fenacetin and 0.38-24 ng/ml for chlorzoxazol (Table II).
Accuracy and precision
The present study validated the accuracy and precision, including both intra-day and inter-day precision, of the three probe drugs in HaCaT cells. It was demonstrated that the results were all acceptable (RSD, <15%; Tables III and IV).
Stability
The present study also evaluated the stability of the three probe drugs under various conditions by analyzing six replicates of QC samples at low, middle and high concentrations. The RSDs high, medium and low QC samples of testosterone were 3.04, 1.82 and 1.78%, respectively. The RSDs of high, medium and low concentration QC samples of fenacetin were 3.43, 1.63 and 3.57%, respectively. The RSDs of high, medium and low concentration quality control samples of chlorzoxazol were 5.26, 1.01 and 1.33%, respectively. It was demonstrated that the RSDs of all QC samples were within 6%, which was <15%.
Influence of TP + FA on the activities of CYP450 enzymes
Investigation of probe drug incubation, concentration and duration of treatment. MTT assay results indicated that no mixed probe substrate group had any significant effect on the growth of the HaCaT cells. Moreover, it was found that the probability of cell survival was greatest when the ratio of concentrations of testosterone: Fenacetin: Chlorzoxazol was 120:6:6 ng/ml (Fig. 3).
Mixed probe drug solutions containing testosterone, fenacetin and Chlorzoxazol were administered at a dose of 120:6:6 ng/ml to HaCaT cells in each group, and the concentrations of the samples were determined. It was identified that in the 1st h of administration, the concentration of chlorzoxazol (Fig. 4C) in the incubation system decreased, and after this time point the concentration remained relatively unchanged. However, the concentration of testosterone (Fig. 4A) and fenacetin (Fig. 4B) appeared decreased within 2 h of administration, after which the reductions were slower, thus the incubation time was determined to be 2 h.
Determination of CYP450 enzymatic activities
The present results suggested that the metabolic activity of fenacetin was reduced in the TP group compared with the control group. Furthermore, in the high dose 1:100 TP + FA combination group a decrease in fenacetin concentration was observed, compared with the TP group (Fig. 5). However, this difference was not significant, thus indicating that the compatibility of TP + FA had no effect on CYP1A2 activity in HaCaT cells. Moreover, the present results suggested that combined TP + FA treatment did not significantly change the activity of CYP2E1 or CYP3A4 (Fig. 5).
Protective effect of TP + FA against oxidative damage in HaCaT cells
Detection of antioxidant factors in HaCaT cells. It was demonstrated that the production levels of ROS, SOD and MDA were significantly higher, and those of GSH and CAT significantly lower, in the TP group compared with the control group (P<0.01; Table VI; Fig. 6). Furthermore, it was found that FA reversed the regulatory changes in oxidation factors induced by TP. The production levels of ROS, SOD and MDA in HaCaT cells were significantly decreased, and those of GSH and CAT increased significantly, in the TP + FA group compared with TP-alone group.
Expression of Nrf2
The present results indicated that, compared with the control group, there was no significant difference in the protein expression of Nrf2 in the high- or low-dose TP group. However, it was found that cells treated with TP + FA showed upregulated Nrf2 protein expression, which was significant in the high-dose group (P<0.05; Fig. 7).
Discussion
Skin is the first physiological barrier of the human body and is also the largest organ (29). It not only protects the body from adverse external factors, but also prevents loss of moisture and nutrients, and plays essential roles in defense (30). Moreover, skin is one of the primary locations for drug metabolism, in addition to the liver (29). The ‘cocktail’ probe substrates approach method has the advantages of high throughput, rapidity and simplicity, and it can reduce the influence of individual differences on the experimental results (31). This method has become an effective part of early high-throughput drug screening and research into mechanisms underlying drug interactions, and is also the main method of drug metabolism research (32). The present study used fenacetin, chlorzoxazol and testosterone as special probe drugs to investigate the activities of CYP1A2, CYP2E1 and CYP3A4, respectively (33). The present results indicated that TP + FA had no significant effect on the activities of CYP1A2, CYP2E1 or CYP3A4 in HaCaT cells. CYP3A4 is predominant in the metabolism of triptolide, while glycyrrhizin can significantly accelerate the metabolic elimination of TP from the body, mainly via induction of hepatic CYP3A activity and attenuation of the toxicity of TP (34,35); this is inconsistent with the present results. However, this discrepancy could arise for the fact that the content of CYP enzymes in epidermal cells is lower compared with liver cells, thus the results of the interaction between TP and CYP enzymes in epidermal cells were not significant. It is also possible that HaCaT cells were derived from human abdominal skin in the present study. However, most CYP450 enzymes have interspecies differences in the process of drug biotransformation, and in the affinity between active or toxic drug components (36-38). Therefore, these differences may help to explain why TP + FA did not exert significant effects on the three tested CYP450 enzymes in the present study.
ROS are a metabolic signals produced under normal physiological conditions, and play essential roles in maintaining normal oxidative stress (39). The normal state of redox equilibrium is compromised when ROS levels exceed the capacity of the antioxidant defense system (40), which in turn interrupts cellular activities and induces apoptosis, tissue damage and aging (41,42). Moreover, GSH, MDA, SOD and CAT are important for maintaining redox equilibrium, which is closely implicated in the occurrence and treatment of many diseases (43,44). The present results suggested that TP significantly reduced the survival rate of cells, increased production levels of ROS, MDA and SOD, and decreased GSH and CAT activities in HaCaT cells. These results are in line with previous studies investigating the activation of related genes and disruption of mitochondrial membrane potential, which compromises redox homeostasis, causes oxidative damage and promotes apoptosis in HaCaT cells (45). The present results indicated that FA reversed the regulatory changes of TP-induced antioxidant factors and protected HaCaT cells. Our previous study, performed on Madin-Daby Canine Kidney (MDCK) cells, showed that the toxicity of TP can also be reduced by combining it with FA, which increases the survival probability of MDCK cells (46). Moreover, isoliquiritigenin and glycyrrhetinic are antagonistic to TP-induced damage in HepG2 cells, which may be partly associated with their protective effects in TP-induced oxidative stress (47).
As Nrf2 is a master regulator of detoxification and antioxidative responses, under healthy conditions its expression is tightly regulated and controlled at the protein level (48,49). Furthermore, Nrf2 participates in the synthesis of the antioxidative enzymes GSH, SOD and CAT, by interacting with antioxidant-reaction elements and inducing the expression of downstream targets (50-52). The present study found that the expression of Nrf2 protein was not significantly affected by TP compared with the control group. However, the protein expression of Nrf2 was increased in the TP + FA group, and there was a significant difference in the high-dose group. Therefore, the present results indicated that FA may increase the protein expression of Nrf2 in HaCaT cells. However, it cannot be confirmed that the expression of this downstream antioxidant factor is affected by Nrf2 nuclear transposition (53).
In conclusion, the present results suggested that TP induced injury in HaCaT cells, whereas TP + FA alleviated this cytotoxicity. However, it was found that TP + FA had no significant effect on the activities of CYP1A2, CYP2E1 and CYP3A4 enzymes in HaCaT cells. Moreover, the underlying mechanisms may be related to the decrease in production of ROS in HaCaT cells, which increased the production levels of key antioxidant factors and the antioxidative ability of cells, and was associated with a protective effect against oxidative damage.
Acknowledgements
Not applicable.
Funding
This work was financially supported by the National Natural Science Foundation (grant no. 81460607), Jiangxi Natural Science Foundation (grant no. 20202BAB206081) and Jiangxi University of Traditional Chinese Medicine 1050 Youth Talent Project (grant. no. 1142001007).
Availability of data and materials
All data generated or analyzed during this study are included in this published article.
Authors' contributions
JZ, YG and LC were responsible for the conception and design of the study, acquisition, analysis and interpretation of the data, the drafting and writing of the manuscript, and revisions to its intellectual content. LH and LT were involved in the conception of the study, acquisition, analysis and interpretation of the data, and the drafting of the manuscript. WZ, ZZ and CJ contributed to acquisition and analysis of the data, and the drafting and revision of the manuscript. LC was responsible for the conception and design of the experiments, analysis and interpretation of the data, the drafting of the manuscript, and revisions to its intellectual content. All authors have read and approved the final manuscript.
Ethics approval and consent to participate
Not applicable.
Patient consent for publication
Not applicable.
Competing interests
The authors declare that they have no competing interests.
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