Guang-Ping Lang, Can Li, Ying-Ying Han

Abstract Microglial cells are important resident innate immune components in the central nervous system that are often activated during neuroinflammation. Activated microglia can display one of two phenotypes, M1 or M2, which each play distinct roles in neuroinflammation.Rutin, a dietary flavonoid, exhibits protective effects against neuroinflammation. However, whether rutin is able to influence the M1/M2 polarization of microglia remains unclear. In this study, in vitro BV-2 cell models of neuroinflammation were established using 100 ng/mL lipopolysaccharide to investigate the effects of 1-hour rutin pretreatment on microglial polarization. The results revealed that rutin pretreatment reduced the expression of the proinflammatory cytokines tumor necrosis factor-α, interleukin-1β, and interleukin-6 and increased the secretion of interleukin-10. Rutin pretreatment also downregulated the expression of the M1 microglial markers CD86 and inducible nitric oxide synthase and upregulated the expression of the M2 microglial markers arginase 1 and CD206. Rutin pretreatment inhibited the expression of Toll-like receptor 4 and myeloid differentiation factor 88 and blocked the phosphorylation of I kappa B kinase and nuclear factor-kappa B. These results showed that rutin pretreatment may promote the phenotypic switch of microglia M1 to M2 by inhibiting the Toll-like receptor 4/nuclear factor-kappa B signaling pathway to alleviate lipopolysaccharide-induced neuroinflammation.

Key Words: BV-2 cells; flavonoid; M1 microglia; M2 microglia; microglia activation; microglia polarization; neuroinflammation;proinflammatory cytokines; rutin; TLR-4 signaling

Introduction

Neuroinflammation is significant in the central nervous system(CNS) (Yang et al., 2016; Mo et al., 2020). Microglial cells are resident immune cells, accounting for 15–20% of cranial nerve gliacytes in the CNS, which primarily function to remove injured nerves, cellular fragments, and infectious substances(Schneble et al., 2017). Microglial cells in the CNS tend to react early following infection, inflammation, and other pathophysiological stimuli (Hopp et al., 2018). Microglial cells can be converted from a surveillant state into an activated state upon CNS injury due to infection, cerebral trauma, or ischemia (Salvi et al., 2017; Huang et al., 2020). Microglial activation results in alterations in cellular phenotypes and functions, and activated microglial cells typically demonstrate one of two distinct phenotypes, referred to as M1 and M2.M1 microglial cells can cause neurotoxicity by releasing cytotoxic substances and inflammatory factors, including matrix metalloproteinases that deteriorate the blood-brain barrier, further aggravating cerebral injury. Studies have found that M1 microglial cells can cause partial or extensive CNS injury, and the M1 phenotype serves as a histopathological marker for neurodegeneration, including Parkinson’s disease and Alzheimer’s disease (Hopp et al., 2018; Sternberg et al.,2019). Conversely, M2 microglial cells protect neurons by secreting anti-inflammatory components and neurotrophic factors (Frister et al., 2014; Pisanu et al., 2014; Meireles et al., 2016). By interacting with Toll-like receptors (TLRs) on the cell surface, pathogen-associated molecular patterns can initiate microglial activation, converting microglial cells into the M1 proinflammatory phenotype (Frister et al., 2014). The stimulation of TLR4, which is primarily expressed on the cell surface of microglia in the CNS, activates the nuclear factorkappa-B (NF-κB) signaling pathway, which is crucial for the activation of the M1 phenotype (Yin et al., 2018; Zhang et al.,2018b), resulting in the subsequent release of inflammatory factors that increase neuroinflammation and CNS injury.Previous studies have demonstrated that the conversion of microglia from an M1 to an M2 phenotype may serve as an effective approach to the control of neuroinflammation (Huang et al., 2016; Shabab et al., 2017; Li et al., 2018).

Rutin, which is also known as vitamin P, is a dietary flavonoid(Hollman and Katan, 1998). Clinically, rutin has been demonstrated to promote distinct pharmacological benefits,such as antioxidative, anti-inflammatory, and antiviral effects(Nafees et al., 2015). Rutin has been shown to protect the heart and kidney from injury caused by ischemia and inhibits apoptosis induced by myocardial ischemia (Jeong et al., 2009; Araújo et al., 2011). Importantly, rutin facilitates neuroprotection by activating antioxidative factors, including catalase, which reduces the formation of free radicals (Khan et al., 2009). Recent research has demonstrated that rutin can increase the expression of microglial cell phagocytic receptors,enhancing the clearance capability against β-amyloidosis,resulting in beneficial effects against the pathological progression of Alzheimer’s disease (Wang et al., 2012; Pan et al., 2019). Both rutin and microglial activation appear to be involved in the neuroinflammatory response; however,whether rutin plays a role in the regulation of microglial phenotypic conversion remains unclear.

In the present study, we attempted to investigate whether rutin serves as an effective candidate for the promotion of microglial phenotypic conversion from M1 to M2 to further ameliorate neuroinflammation initiated by lipopolysaccharide(LPS) treatment in BV-2 microglial cells.

Materials and Methods

Cell culture and treatment

BV-2 microglial cells (purchased from China Center for Type Culture Collection) were cultured in high-glucose Dulbecco’s modified Eagle medium (Thermo Fisher Scientific, Inc.,Waltham, MA, USA) supplemented with 10% fetal bovine serum (BioInd, Inc., Shanghai, China) and 1% penicillin/streptomycin (Solarbio Science & Technology Co., Ltd., Beijing,China) under conditions of 37°C and 5% CO2. Rutin (Solarbio Science & Technology Co., Ltd.) was dissolved in phosphatebuffered saline (containing 0.1% dimethyl sulfoxide, DMSO)and prepared as a 20 mg/mL stock solution. Rutin was diluted in Dulbecco’s modified Eagle medium to the specified concentrations used in each experiment. The control group was treated with 0.1% dimethyl sulfoxide, which served as the vehicle control. LPS (Sigma-Aldrich, St. Louis, MO, USA)was dissolved in normal saline, prepared as a 50 µg/mL stock solution, and diluted in Dulbecco’s modified Eagle medium to the specified concentrations used in each experiment. BV-2 cells in the control group were pretreated with DMSO, and cells in the experimental group were pretreated with rutin for 1 hour before stimulation with or without LPS (100 ng/mL) for 24 hours in the following experiments.

Cell viability assay

Cell counting kit-8 assay was performed to measure the viability of BV-2 cells. Cells (4 × 103cells/well) were cultured in 96-well plates and pretreated with rutin at various concentrations (0, 6.25, 12.5, 25, 50, and 100 µg/mL) for 1 hour, followed by the addition of 100 ng/mL LPS or normal medium and incubation for 24 hours. Subsequently, 10 µL Cell Counting Kit-8 reagent (Dojindo, Shanghai, China) was added to each well and incubated at 37°C for 3 hours. The optical density at 450 nm was measured using a microplate reader(Thermo Fisher Scientific, Inc., Waltham, MA, USA).

Enzyme-linked immunosorbent assay

Cells were pretreated with rutin (12.5, 25, and 50 µg/mL) for 1 hour, followed by stimulation with or without LPS (100 ng/mL)for 24 hours. The concentrations of inflammatory factors,including tumor necrosis factor-α (TNF-α), interleukin (IL)-6, IL-1β, and IL-10, in the cell supernatants were measured using enzyme-linked immunosorbent assay (ELISA) kits(Servicebio Technology Co., Ltd., Wuhan, China), according to the manufacturer’s instructions. The optical density at 450 nm was determined using a microplate reader.

Nitric oxide assay

The nitric oxide (NO) contents secreted by BV-2 cells were assessed using an NO test kit (Jiancheng, Nanjing, China).Briefly, cells were pretreated with 12.5, 25, and 50 µg/mL rutin for 1 hour, followed by stimulation with 0 or 100 ng/mL LPS for 24 hours. The concentrations of NO in cell supernatants were measured following the manufacturer’s instructions.

Quantitative real-time polymerase chain reaction

An Axyprep total RNA preparation kit (AXYGEN Biosciences,Union City, CA, USA) and PrimeScriptTMRT reagent kit (TaKaRa,Beijing, China) were used to perform total RNA extraction and reverse transcription, respectively. Relative mRNA expression levels were analyzed using the 2–ΔΔCtmethod and normalized against the expression level of glyceraldehyde 3-phosphate dehydrogenase (GAPDH), which was used as a housekeeping gene. Murine GAPDH primer was obtained from Sangon Biotech (Shanghai, China). The following murine primer pairs were used: CD86: forward: 5′-ACG GAG TCA ATG AAG ATT TCC T-3′, reverse: 5′-GAT TCG GCT TCT TGT GAC ATA C-3′; CD206 forward: 5V-CCT ATG AAA ATT GGG CTT ACG G-3′, reverse: 5′-CTG ACA AAT CCA GTT GTT GAG G-3′; inducible nitric oxide synthase (iNOS) forward: 5′-ACT CAG CCA AGC CCT CAC CTA C-3′, reverse: 5′-TCC AAT CTC TGC CTA TCC GTC TCG-3′.

Western blot assay

Cells were lysed in radioimmunoprecipitation assay buffer(Solarbio Science & Technology Co., Ltd.), the samples were centrifuged (12,000 r/min for 10 minutes at 4°C), and the protein concentrations were measured using a bicinchoninic acid protein assay (Generay Biotech Co., Ltd., Shanghai,China). After adding 5× protein sample buffer (Cat# P0015,Beyotime, Shanghai, China), lysates were heated to 95°C for 5 minutes. Protein samples were separated by 8% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then transferred to a polyvinylidene difluoride membrane.The membranes were blocked with 5% skimmed milk in phosphate-buffered saline containing Tween-20 for 2 hours at room temperature and immunoblotted overnight at 4°C with the following primary antibodies: rabbit monoclonal anti-CD86 antibody (1:1000, Cat# ab239075), goat polyclonal anti-arginase-1 (Arg-1) antibody (1:1000, Cat# ab60176), rabbit polyclonal anti-CD206 antibody (1:500, Cat# ab64693), rabbit polyclonal anti-iNOS antibody (1:1000, Cat# ab15323), rabbit polyclonal anti-TLR4 antibody (1:500, Cat# ab13556), rabbit monoclonal anti-inhibitor kappa B kinase β (IKK-β) antibody(1:500, Cat# ab32135), rabbit monoclonal anti-myeloid differentiation factor 88 (MyD88) antibody (1:1000, Cat#ab219413), rabbit polyclonal anti-NF-κB p65 antibody (1:1000,Cat# ab16502), rabbit monoclonal anti-p-IKK-β antibody(1:200, Cat# ab194519), rabbit polyclonal anti-p-NF-κB p65 antibody (1:1000, Cat# ab86299), and mouse monoclonal anti-β-actin antibody (1:5000, Cat# ab6276). All primary antibodies were obtained from Abcam (Shanghai, China). The membrane was washed with Tris-buffered saline-Tween-20 and incubated with the appropriate horseradish peroxidaseconjugated secondary antibodies (goat anti-mouse IgG,Cat# A0216, 1:2000, Beyotime; goat anti-rabbit IgG, A0208,1:2000, Beyotime; donkey anti-goat IgG, Cat# A0181, 1:2000,Beyotime) for 1 hour at room temperature. Protein bands were visualized with enhanced chemiluminescence substrate(Beyotime) and quantified using FujiFilm Multi Gauge Ver. 3.0 software (Fuji Photo Film Co., Tokyo, Japan). CD86, iNOS, Arg-1, and CD206 are critical markers for microglial polarization,whereas the remaining proteins are key components of the NF-κB pathway, which is closely associated with inflammation.

Immunofluorescence staining

Coverslips were coated for 2 hours by poly-L-lysine (0.1 mg/mL;Beyotime) at 37°C then placed into a 12-well plate. BV-2 cells were cultured on the coverslips at 75,000 cells/well and pretreated with rutin (12.5, 25 and 50 µg/mL) for 1 hour,followed by incubation with LPS (100 ng/mL) for 24 hours. The cells were fixed with 4% paraformaldehyde for 30 minutes at room temperature, followed by permeabilization with 0.3%Triton X-100 for 15 minutes. Cells were subsequently blocked with 2% bovine serum albumin in phosphate-buffered saline at 37°C for 1 hour and incubated with individual antibodies:anti-CD86 (1:50) and anti-Arg-1 (1:50) at 4°C overnight.Subsequently, the cells were incubated with the appropriate secondary antibody (cyanine 3-conjugated donkey anti-rabbit IgG, Cat# GB21403, 1:500, Solarbio Science & Technology Co.,Ltd.; fluorescein isothiocyanate conjugated donkey anti-goat IgG, Cat# GB22404; 1:500, Solarbio Science & Technology Co.,Ltd.) for 2 hours at room temperature, followed by incubation with 4′,6-diamidino-2-phenylindole for 2 minutes at room temperature. The coverslips were mounted and examined under a fluorescence microscope (Olympus, Tokyo, Japan) at 20× magnification.

Statistical analysis

All data are presented as the mean ± standard error of the mean (SEM) after analysis with GraphPad Prism software v5.01(GraphPad Software Inc., San Diego, CA, USA). Comparisons between groups were analyzed by one-way analysis of variance, andpost hoccomparisons were made using Tukey’s multiple comparison test. WhenP＜ 0.05, the difference was considered significant.

Results

Effects of rutin on LPS-induced BV-2 microglial cell morphology

To assess the potential cytotoxicity induced by rutin treatment in BV-2 cells, a Cell Counting Kit-8 experiment was performed.The viability of BV-2 cells was evaluated following treatments with various concentrations of rutin for 1 hour. The results indicated that BV-2 cell viability was significantly enhanced after rutin (12.5, 25, and 50 µg/mL) administration (P＜ 0.05)compared with cells treated with vehicle control. Therefore,these concentrations of rutin (12.5, 25, and 50 µg/mL) were selected for the subsequent experiments. No perceptible cytotoxicity was observed in BV-2 cells following 1-hour rutin treatment at concentrations ranging from 0 to 100µg/mL (Figure 1A). Upon incubation with 100 ng/mL LPS for an additional 24 hours, BV-2 cell viability continued to remain unaffected (Figure 1B). As shown inFigure 1C, cells stimulated with LPS demonstrated an activated phenotype,characterized by elongated branches, whereas BV-2 cells in the unstimulated control group showed small cell bodies,and rutin pretreatment at concentrations of 25 and 50 µg/mL attenuated LPS-induced morphological alterations in BV-2 cells (Figure 1C). These data suggested that rutin exerts an important function during LPS-induced BV-2 cell activation.

Rutin affects LPS-induced inflammatory mediator production from BV-2 cells

BV-2 cells were first pretreated with various rutin concentrations (12.5, 25, and 50 µg/mL) for 1 hour before incubation with 100 ng/mL LPS for 24 hours. The concentrations of inflammatory cytokines in the supernatants were measured by ELISA. Following LPS treatment, TNF-α, IL-1β, IL-6, and NO concentrations were significantly increased(P＜ 0.05) compared with the unstimulated control, which indicated the potential M1 proinflammatory polarization of BV-2 cells in response to LPS stimulation (Figure 2A–D). However, the IL-10 level was not greatly altered by LPS treatment (Figure 2E). As expected, the TNF-α, IL-1β, IL-6, and NO concentrations induced by LPS treatment were dramatically reduced by rutin pretreatment at certain concentrations or concentration ranges (Figure 2A–D).In contrast, 50 µg/mL rutin pretreatment significantly elevated the expression level of the typical M2-associated anti-inflammatory cytokine IL-10 (Figure 2E). These results indicated that rutin treatment might facilitate the microglial cell conversion from a proinflammatory M1 to an antiinflammatory M2 phenotype.

Rutin promotes the microglial phenotypic switch from M1 to M2

To further examine whether rutin contributes to BV-2 cell polarization to promote the anti-inflammatory M2 phenotype following LPS stimulation, we further evaluated the expression of iNOS, CD86, CD206, and Arg-1 in rutin-treated cells. Our immunofluorescence staining results revealed that BV-2 cells displayed enhanced CD86 immunopositivity after LPS stimulation compared with that in the control group, but the production of Arg-1 in BV-2 cells was not dramatically influenced by LPS stimulation (Figure 3AandB). The increase in CD86 immunopositivity induced by LPS stimulation was inhibited by rutin pretreatment, whereas Arg-1 immunopositivity was slightly increased in the rutin pretreatment group (50 µg/mL). In addition, when compared with those cells only exposed to LPS, western blot quantification data revealed that rutin-pretreated BV-2 cells expressed increased levels of Arg-1 and CD206 and decreased levels of CD86 and iNOS (Figure 3C). The corresponding representative western blotting image is shown inFigure 3D. We then examined the effects of rutin pretreatment on microglial polarization following LPS stimulation at the transcriptional level. The expression of CD86 and iNOS were significantly increased, whereas CD206 expression was significantly decreased in LPS-stimulated cells. Among these three markers, only iNOS mRNA expression was inhibited in the rutin pretreatment groups (Figure 3E). These results further suggested that rutin is capable of shifting LPS-induced BV-2 cell polarization from the M1 to the M2 phenotype.

Rutin inhibits activation of TLR4/NF-κB triggered by LPS in BV-2 cells

BV-2 cells were treated as described above. To explore which signaling pathways rutin acts on to promote the M1 to M2 polarization of BV-2 cells, the expression levels of TLR4,MyD88, IKK-β, NF-κB, p-IKK-β, p65, and p-NF-κB p65 were determined by western blot analysis. As expected, TLR4 and MyD88 protein expression levels were enhanced after LPS stimulation, whereas rutin treatment effectively reversed this upregulation in BV-2 cells. We subsequently tested the activation of IKK-β and NF-κB p65 by examining their phosphorylated states. Increased expression levels of p-IKK-β and p-NF-κB p65 can be observed following LPS stimulation,which was attenuated by rutin pretreatment (Figure 4). These results suggested that the activation of the TLR4/NF-κB,triggered by LPS stimulation in BV-2 cells, was limited by rutin pretreatmentin vitro.

Figure 1｜Effects of rutin on BV-2 microglial cells stimulated with LPS.

Figure 2｜Effects of rutin on LPS-induced inflammatory mediator production in BV-2 cells.

Figure 3｜Rutin switches microglial polarization from the M1 to M2 state in LPS-induced BV-2 cells.

Figure 4｜Rutin inhibits the LPStriggered TLR4/NF-κB activation in BV-2 cells.

Discussion

The present investigation discovered that rutin promotes the transition of microglial polarization from the M1 to the M2 state, which has the potential to ease neuroinflammation.Our findings showed that rutin inhibits the expression of CD86 and iNOS and increases the expression of CD206 and Arg-1; however, rutin has no regulatory effects on CD86 and CD206 at the transcriptional level, indicating that rutin may play a post-transcriptional regulatory for these two microglial polarization-related proteins. As expected, rutin significantly reduced IL-6, TNF-α, IL-1β, and NO expression in BV-2 microglial cells after LPS stimulation but significantly enhanced IL-10 expression, suggesting a protective effect for rutin during neuroinflammation. Studies have described the upregulation of iNOS when microglial cells switch to the M1 phenotype(Cunha et al., 2016; Zhao et al., 2019). The iNOS and NO concentrations were consistently decreased by rutin, which is significant in terms of microglial polarization. Moreover,our results suggested that rutin pretreatment significantly inhibited the activation of TLR4/NF-κB signaling, implying that NF-κB signaling might be an important target for rutin during this biological process.

A phenotypic switch between the M1- and M2-dominant inflammatory response can occur in both microglia and macrophages under certain conditions. To maintain homeostasis, a careful balance must be maintained between the M1 and M2 microglial states (Zhang et al., 2018a). In the tumor microenvironment, the proinflammatory cytokines TNF-α and IL-1β and the bacterial LPS can upregulate iNOS production, triggering high levels of NO production, which has been associated with tumor growth (Lamagna et al.,2006; Nath and Kashfi, 2020). In studies performed on cocultures containing glioblastoma and primary microglial cells,microglia-derived iNOS and NO levels were upregulated,resulting in enhanced microglial phagocytosis (Korbelik et al.,2000). Microglial phagocytosis capabilities were also improved by exogenous NO intervention (Kreutzberg, 1995). Similar to the outcome observed for tumor conditions, intracellular iNOS and extracellular NO concentrations increased when BV-2 microglia were stimulated with LPS in our study. The upregulation in both iNOS and NO production could be downregulated by rutin pretreatment, which suggested that rutin may inhibit microglia-mediated neuroinflammation via an NO-dependent mechanism. Other studies have shown that NO-mediated mechanisms are critical for M1 macrophage pathology in Duchenne muscular dystrophy and tumors(Villalta et al., 2009; Mukherjee et al., 2016). Consistently, our results suggest that rutin facilitated the microglial polarization from an M1 to an M2 phenotype through a mechanism associated with the downregulation of NO. Elevated NO was shown to provoke neuroinflammation in neurodegenerative diseases by regulating axonal degeneration and activating cyclooxygenases (Tse, 2017). In tumor biology, scientists have viewed the phenotypic switching of macrophages from M2 to M1 as a potential method for enhancing antitumor capabilities (Nath and Kashfi, 2020), which could be mediated by controlling NO-involved signaling. Similarly, our results indicated that rutin might be a promising candidate for the treatment of neurological disorders by promoting the phenotypic switching of microglia from the M1 to M2 state.

LPS has been well-established to activate TLR4 and induce microglial M1-phenotypic polarization with proinflammatory functions. Therefore, LPS-stimulated BV-2 microglial cells were employed to investigate whether rutin plays a role in microglial phenotypic switching. In the tumor microenvironment, the NF-κB pathway has been found to regulate macrophage polarization in mice (Kono et al., 2014).NF-κB signaling has been found to be critical for microglial phenotypic transformations bothin vivoandin vitroin Alzheimer’s disease (Wang et al., 2015; Ślusarczyk et al.,2018). Similar to previous work, our findings revealed that TLR4, MyD88, p-IKK-β, and p-NF-κB p65 expression levels were decreased by rutin pretreatment, resulting in microglial polarization directed toward the M2 state. Our data indicated that rutin might regulate microglial polarization via the suppression of TLR4/NF-κB signaling. However, our current data were unable to confirm whether rutin promotes the microglial M2 phenotype through the TLR4/NF-κB pathway because other pathways, such as Notch signaling and glycogen synthase kinase 3 signaling, could also be modulated by rutin during this process. Notch signaling inhibition has been shown to limit M1 marker expression and elevates M2 microglial polarization following LPS administration in BV-2 cells (Zhang et al., 2017). In addition, the expression level of the neuroprotective cytokine IL-10 was upregulated by rutin in our study, and regulatory T lymphocytes have been shown to ameliorate intracerebral hemorrhage-induced inflammatory injury by promoting microglial cell conversion to the M2 phenotype through the IL-10/GSK3β/phosphatase and tensin homolog axis (Zhou et al., 2017). Collectively, our results imply that rutin could represent a promising therapeutic candidate for neuroinflammatory diseases by promoting the microglia conversion from the M1 to M2 phenotype via effects on TLR4/NF-κB signaling. However, further investigations, including experiments performed in primary microglia and animal models, remain necessary to understand the role played by rutin on microglial polarization.

In summary, the present study illustrated that rutin is capable of shifting the microglial state from the M1 to M2 phenotypes,attenuating microglia-related neuroinflammation. Moreover,this study revealed that the TLR4/NF-κB signaling might be involved in the rutin-mediated phenotypic shift of microglia.Therefore, our findings offer evidence that rutin might represent an effective therapeutic candidate for the treatment of neuroinflammatory diseases. However, whether rutin acts directly on the TLR4/NF-κB pathway or indirectly regulates the expression of TLR4 or NF-κB to influence the microglial M1 to M2 phenotype polarization remains to be tested.

Acknowledgments:The authors thank Jiang-Man Liu and Ting Xie from Key Laboratory of Basic Pharmacology of Ministry of Education, China for their excellent technical assistance.

Author contributions:Study conception and design, manuscript preparation,editing and review: GPL, YYH; literature search and experimental implementation: GPL, CL, YYH; data acquisition and statistical analysis: GPL.All authors have read and approved the final manuscript.

Conflicts of interest:The authors declare that they have no conflict of interest.

Financial support:This study was supported by the Natural Science and Technology Foundation of Zunyi City, China, No. 201915 (to GPL); Doctor

Startup Foundation of Zunyi Medical University, Nos. [2017]5733-045 (to GPL),[2017]5733-044 (to YYH); and Natural Science and Technology Foundation of Guizhou Province, China, No. [2020]1Y292 (to YYH). The funding sources had no role in study conception and design, data analysis or interpretation, paper writing or deciding to submit this paper for publication.

Institutional review board statement:BV-2 microglia cell line is commercialized. The ethical approval is waived in this study.

Copyright license agreement:The Copyright License Agreement has been signed by all authors before publication.

Data sharing statement:Datasets analyzed during the current study available from the corresponding author on reasonable request.

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Peer review:Externally peer reviewed.

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