Investigation of the Underlying Mechanism of Huangqi-Dangshen for Myasthenia Gravis Treatment via Molecular Docking and Network Pharmacology

The herbal pairing of Huangqi and Dangshen (HD) is traditional Chinese herbal medicine and has been widely used in China, especially to treat myasthenia gravis (MG). However, the mechanism of HD on MG is unclear. Aim of the Study. This study aims to investigate HD's possible role in MG treatment. Materials and Methods. The TCMSP database was used to identify the active chemicals and their targets. The GeneCards, DisGeNET, and OMIM databases were used to search for MG-related targets. The STRING database was employed in order to identify the common PPI network targets. We next utilised Cytoscape 3.8.2 for target identification and the DAVID database for gene ontology (GO) function analysis as well as Encyclopaedia of Genomes (KEGG) pathway enrichment analysis on the selected targets. The AutoDock Vina software was used to test the affinity of essential components with the hub gene before concluding that the primary targets were corrected through molecular docking. Results. 41 active compounds were screened from HD, and the number of putative-identified target genes screened from HD was 112. There were 21 target genes that overlapped with the targets of MG, which were postulated to be potential treatment targets. Through further analysis, the results showed that the active compounds from HD (such as 7-methoxy-2-methylisoflavone, quercetin, luteolin, Kaempferol, and isorhamnetin) may achieve the purpose of treating MG by acting on some core targets and related pathways (such as EGFR, FOS, ESR2, MYC, ESR1, CASP3, and IL-6). Molecular docking findings demonstrated that these active molecules have a near-perfect ability to attach to the primary targets. Conclusion. Through network pharmacology, the findings in this study provide light on the coordinated action of several HD formula components, targets, and pathways. It provided a theoretical basis for further study of HD pharmacological action.


Introduction
Antibodies attached to components of the neuromuscular junction, such as the acetylcholine receptor (AChR), are the primary cause of myasthenia gravis (MG). More than 700,000 individuals throughout the globe are thought to be afected by the condition, which has an incidence of 0.3 to 100,000 people, which is quite close to the MG incidence rate in Korea, which is also 0.69 per 100,000 people [2]. Te clinical manifestations include drooping eyelids, weakness of the limbs, muscle wasting, and dysphagia. Te symptoms worsen after exercise. Western medicine treatment mostly uses glucocorticoids, immunosuppressants, cholinesterase inhibitors, plasma exchange, thymectomy, etc [3]. Although it has a curative efect, there are certain adverse reactions to long-term application.
Traditional Chinese medicine (TCM) has been widely studied because of its stable curative efect, no drug resistance, and fewer toxic and side efects. Te herbal pairing of Huangqi and Dangshen (HD) is traditional Chinese herbal medicine and has been widely used in China, especially to treat myasthenia gravis (MG). Huangqi (HQ) is the root of Astragalus membranaceus (Fisch.) Bge. var. mongholicus (Bge.) Hsiao and has been found to be rich in favonoids, saponins, polysaccharides, and amino acids. Dangshen (DS) is the root of Codonopsis pilosula (Franch.) Nannf and is composed of several bioactive phytochemicals including lithospermic acid, protocatechuic acid, danshensu, cafeic acid, protocatechualdehyde, rosmarinic acid, salvianolic acid A-C, cryptotanshinone, dihydrotanshinone I, tanshinone IIa, and tanshinone I. However, it is not known how HD afects MG. HD is a very important supplement of TCM treasure house, both of which have the efect of invigorating the spleen and replenishing Qi. It can be used for diseases such as myasthenia gravis, heart failure, and hypotension caused by Qi defciency. Qi defciency syndrome is the main pathogenesis of myasthenia gravis, which runs through the whole process of myasthenia gravis. Terefore, invigorating Qi is the basic principle of treatment for MG with Qi defciency syndrome. Clinically, HD has been widely used for the treatment of myasthenia gravis [4]. Studies have found that the main active components in HQ are saponins, favonoids, and astragalus polysaccharides, which are rich in immune active substances. Because of their signifcant immune regulation, they can improve the body's immunity, thereby reducing the use of antibiotics [5,6]. DS extract can signifcantly increase the number of many cells, such as white blood cell, platelet count, reticulocyte, and bone marrow nucleated cell, and increase the ratio of CD4+/CD8+, spleen coefcient, thymus coefcient, and the secretion of TNF α. Terefore, DS extract can enhance the hematopoietic and immune function of mice [7]. Because of its wide range of pharmacological activities, DS extract also has neuroprotection, regulation of gastrointestinal function, regulation of endocrine function, antiaging, and antioxidation properties [8]. However, the specifc mechanism of action and related pathways related to HD treatment of MG remains to be further explored. Te immense pharmacological outcome associated with the use of HD makes us to undertake this study to explore the underlying mechanism of Huangqi-Dangshen for myasthenia gravis treatment via molecular docking and network pharmacology.
Network pharmacology is a complex system that describes the interaction of "drug (compound)-target (gene)disease." It analyzes the network of biological systems through bioinformatics databases, combined with systems biology, bioinformatics, pharmacological analysis, computational biology, and other multidisciplinary theories. It is a new method to analyze the target and mechanism of drug intervention in diseases from multiple perspectives [9]. When it comes to predicting the afnity and binding mechanisms of ligands and proteins, molecular docking is an invaluable tool. By predicting the binding mode and binding-free energy between receptor molecules and ligand molecules, the function and mechanism of action can be studied. It has been extensively employed in the research of TCM's particular targets and compound active elements from the recipe [10]. We employed network pharmacology and molecular docking technologies in this research to investigate the mechanism of HD therapy for MG, resulting in a novel idea for clinical implementation. Figure 1 shows the research fow diagram.

Composition of TCM Compounds.
Using the keywords "Astragalus" and "Codonopsis," the TCMSP (https:// tcmspw.com/tcmsp.php) [11] database was used to obtain information on the drug composition. Te bioavailability (OB) was greater than 30%, and the main active ingredients were screened out if the drug-like property (DL) was greater than 0.18.

Screening of Target Proteins and Disease Targets of Active
Ingredients of TCM. (1). Create a database of TCM active components using the TCMSP database to anticipate the target proteins of the active substances. (2). Search for myasthenia gravis (MG)-related genes in GeneCards (https://www.genecards.org/) [12], DisGeNet (https://www. disgenet.org/) [13], and the OMIM database (https://www. omim.org/) [14] using the search phrase "disease name." Te duplicated or invalid genes were eliminated from the disease target database, which was created by combining the data from the three databases.

Screening and Subnetwork Analysis of PPI Network and
Hub Genes. Te targets of each active component of TCM and the disease target were intersected. Tere were only a limited number of molecules that intersected in the collection; thus, the target molecules were uploaded to the STRING database (https://string-db.org) [15]. Cytoscape is an open platform with a variety of plugins to increase the visualization choices and the power of network analysis. Multiple layers of information, including protein function annotations, genome-wide studies, and large scale, can be placed on the interactome using Cytoscape, making it simple to access the network's graphical representation. Many Cytoscape plugins allow us to rank and grade the nodes according to network properties. For directed and/or undirected networks, CentiScaPe and NetworkAnalyzer, respectively, compute a number of topological network parameters. Tese plugins ofer more centrality metrics than other regularly used tools. Diferent approaches concentrate on various topological properties or comparable traits with various scoring schemes. More network properties are utilised to facilitate network analysis for biologists. Import the constructed PPI network information into the Cytascape software, and the PPI network associated with the intersection molecules were screened out. Ten, the cytoHubba plugin's topology algorithm is used to predict which protein nodes in the network are important and which of their subnetworks are important, with a confdence level of ≥0.700. Five parameters were used to jointly screen hub gene in this study, which was DEGREE, MNC, MCC, EPC, and CLONESS, and perform visual process. Te plug-in MCODE was used for cluster analysis. Clusters of genes were discovered, subnetworks were developed, and diferential genes were extracted from each cluster. Te primary biological processes of the targets in each subnetwork were also analyzed.

KEGG Pathway Analysis, GO Classifcation Enrichment
Analysis, and Disease Enrichment Analysis. Import the screened Hub gene into DAVID 6.8 database (https://david. ncifcrf.gov) [16], and selected "Humo space" for sepecies, then analyzed with GO analysis and KEGG pathway analysis (P < 0.05). Key targets' signalling pathways and important biological processes were examined. Te signifcance of an association between the target gene set and a given gene ontology or biological pathway was determined using a hypergeometric distribution model, as shown in the following equation: Specifcally, n is the number of genes identifed as being targets of HD, M is the number of genes annotated to specifc GO terms or pathways, N is the total number of genes in the reference set, and k is the number of genes that are shared between HD-target genes and the reference set.
With the parameters set at P 0.5 and a large number of target enrichment, 247 molecules were added into the DAVID database for GO enrichment study and analyzed for GO enrichment. Key biological processes include the positive regulation of gene expression and transcription from the RNA polymerase II promoter, as well as the response to drugs, the negative regulation of the apoptotic process, cell proliferation, and so on. Finally, the fndings of the disease enrichment were exported.

Molecular Docking of Key Targets and Components.
An AutoDock [18] programme is used to examine and dock molecular docking target molecules. Te AutoDock tools are used to construct the docking grid box of the crystal structure for the target. AutoDock Vina is used to select the combination in the docking structure. Molecules with the lowest energy and the binding efect can be observed by comparison with the original ligand and intermolecular interactions. Discovery Studio is used for docking, preprocessing, and visualization. TMC's small molecule compounds' 3D structures may be obtained from PubChem (https://pubchem.ncbi.nlm.nih.gov/) using the PubChem ID. From the PDB (http://www.rcsb.org/pdb/home/home. do) database [19], the high-resolution crystal structures of important targets can be obtained. Evidence-Based Complementary and Alternative Medicine (HD) were obtained through the TCMSP database, including 20 from HQ and 21 from DS. Te corresponding information for the screened active ingredients is shown in Table 1.

Screening of Targets Related to Myasthenia Gravis in TCM
Compounds. A total of 97 HQ active ingredient targets and 51 DS active ingredient targets were identifed using the target database's prediction fndings. A total of 112 targets were gathered from the merged and deduplicated data sets. Te GeneCards database yielded 918 disease-related targets; the DisGeNET database returned 336; the OMIM database returned seven; all based on the search keywords. After integrating and deduplicating, we had a total of 1067 targets. In order to create the Venn diagram, 112 TCM components and 1067 disease targets were mapped out (Figure 2(a)). A TCM-drugtarget-disease interaction network was established with the help of 21 crossover compounds uncovered ( Figure 2(b)).

Construction of Protein-Protein Interaction (PPI) Network and Screening of Key Targets.
Tere are only 21 intersection molecules between the action targets of TCM ingredients and disease targets, which are relatively small. Tese target compounds were then submitted to STRING to acquire information on their interactions with proteins. And a total of 21 molecules from the intersections were isolated. Ten, the linked protein interaction information was obtained and formed a PPI network map (Figure 3(a)), in which there were a total of 247 molecules. Cytoscape was used to create a PPI network map based on the imported protein interaction data. Screening was based on MNC, MCC, CLONESS, DEGREE, and EPC criteria ( Figure 3(b), Table 2). Select the key genes of each algorithm and intersect them with the top 30 results of each algorithm. 23 key targets were obtained ( Figure 3(c), Table 3), fnally. As the color of the red area is darker and the corresponding degree value is larger, there are additional potential targets that can work along with the projected disease-related targets.

Subnetwork Analysis.
MCODE subnetwork analysis is used to fnd more closely connected genomes in the network. Te point with the highest weight obtained by weighted calculation is set as the seed. Starting from the seed, it moves outward, recursively, to fnd nodes that can join the subnetwork. Te closer the target is to the center, the more important it is. Subnetwork 1 is centered on EGFR, FOS, ESR2, and MYC, and its important targets, such as MAPK14 and RAF1, are targets related to the MAPK pathway, indicating that subnetwork 1 is closely related to it (Figure 4(a)). Te core of subnetwork 2 is ESR1, CASP3, and IL6, which are closely related to the TNF signalling pathway and the hepatitis B pathway (Figure 4(b)).

GO Classifcation and Enrichment Analysis Results.
A total of 247 molecules were entered into the DAVID database for GO enrichment analysis and evaluated for GO enrichment under the parameters of P < 0.5 and a large number of target enrichments, as shown in Figures 5(a)-5(c), Table 4. In the biological process, the key targets are concentrated in the positive regulation of gene expression, and transcription from RNA polymerase II promoter, response to drug, negative regulation of the apoptotic process, and cell proliferation, and positive regulation of cell proliferation, etc. Among the cell components, the most targets are in the cytoplasm, followed by the plasma membrane, cytosol, extracellular space, the nucleus, etc. Molecular functions mainly involve protein, enzyme binding, cytokine activity, transcription factor binding, etc.
3.6. KEGG Pathway Analysis. Potential targets were subjected to KEGG pathway enrichment analysis (P < 0.05) through the DAVID 6.8 data platform, as shown in Figure 5(d) and Table 5. Hepatitis B, the PI3K-Akt signalling pathway, cancer-related and proteoglycan cancer pathways, infammatory bowel disease, Chagas disease, prostate cancer, osteoclast diferentiation, colorectal cancer, and the TNF signalling pathway are the top ten pathways.

Results of Network Construction of "Active Ingredients-
KeyTargets-Action Pathways" of TCM. As a starting point, the herbs in the recipe were loaded into Cytoscape 3.8.2 software, together with possible targets, active components, and signalling pathways evaluated in the compound ( Figure 6(a)). Further calculations were performed using cytoHubba's MCC algorithm to obtain the closest association of components to key targets ( Figure 6(b)).

Molecular Docking to Simulate the Interaction between the
Target and Related Compounds. Te fve compounds with the most key target genes were docked with their key target genes, and it was found that their docking binding energies with the target were all <−5, indicating that the two had a better binding efect. Among them, quercetin and FOS, luteolin and EGFR, isorhamnetin, and ESR2, the lowest binding energies are possessed by 7-methoxy-2-methylisofavone and ESR2, and the binding mechanism is shown in Figure 7 and Table 6.

Discussion
In this study, 41 major chemical components and 21 targets related to myasthenia gravis from HQ and DS for HD were selected. Quercetin, kaempferol, lignan, isorhamnetin, and 7-methoxy-2-methylisofavone were the most important active components in the treatment of MG through active component screening and complex target network analysis.
Studies have shown that quercetin, kaempferol, lignan, isorhamnetin, and 7-methoxy-2-methylisofavone have active efects, such as anti-infammatory, immunomodulatory, antioxidative stress, and neuroprotective [20][21][22][23]. Te JAK-STAT pathway may be blocked by quercetin, which inhibits the release of IL-12 by T cells, as well as the proliferation of activated T cells and the development of T1  [24]. Kaempferol can inhibit the release of infammatory factors by inhibiting MAPK pathways activated by extracellular signal-regulated kinases 1 and 2 (Erk1/2) [25]. It can also efectively interfere with the reverse transcription of STAT3 and inhibits the activation of infammatory factors by blocking the Tyk-STAT signalling pathway [26]. Lignocaine can signifcantly reduce the levels of IL-6 and TNF-α cytokines in serum and act as an anti-infammatory and neuroprotective agent [27]. Isorhamnetin pretreatment can inhibit caspase-3 activation and signifcantly increase AKT serine/threonine kinase 1 (AKT) and phosphorylation of phosphatidylinositol 3kinase (PI3K) in cells [28]. 7-methoxy-2-methylisofavone belongs to the group of favonoids. And favonoids can reduce the expression level of infammatory mediators. In addition, favonoids can also modulate the imbalance of T1/T2 cytokines, thereby playing a role in regulating immunity [29]. An unbalanced ratio of T1 to T2 cytokines is thought to have a role in immune-mediated illness genesis and progression [30]. In summary, HD may treat myasthenia gravis through anti-infammatory, immunomodulatory, and neuroprotective efects and may inhibit infammatory cytokines through a variety of infammatory signalling pathways. In order to predict the mechanism of action of HD for MG, the intersecting targets were screened in the PPI network by cytoHubba and MCODE plugins, and the 7 core targets that were obtained were EGFR, FOS, ESR1, ESR2, MYC, CASP3, and IL-6. It was found that HD could infuence the occurrence and progression of MG through biological processes such as the MAPK cascade, the extrinsic apoptotic signalling pathway in the absence of ligand, immune response, infammation response, and negative regulation of apoptotic processes, as shown by GO enrichment analysis. Recent studies have reported that EGFR can be expressed in thymoma, which is an important cause of myasthenia gravis, and that EGFR overactivates the downstream PI3K/Akt/mTOR signalling pathway, inhibits apoptosis, and stimulates tumor growth [31,32]. EGFR can also activate the MAPK signalling cascade, which in turn stimulates transcription factors that drive the expression of genes associated with tumor invasion and metastasis [33]. Fosl1 and Fosl2 are members of the Fos family. INKT cells were shown to grow when Fosl2 was targeted specifcally to CD4+ T lymphocytes. And Fosl2 regulates the normal development and cellular function of iNKT cells and is involved in the process of iNKT cell selection [34] and signifcantly decreased after treatment [35]. In autoimmune myasthenia gravis, which is more frequent in women than in men, the ESR1 gene expressing estrogen receptor α (ERα) and the ESR2 gene expressing estrogen receptor β (ERβ) can mediate multiple physiological efects of estrogen [36]. A recent study carried out by Chao et al. has reported that EGFR and MYC are key targets in MG on performing molecular docking and systems pharmacology analysis considering nux vomica [37]. Immune cells include B cells, CD4+ T cells, CD8+ T cells, NK cells, and plasma cell-like DCs, which have been shown to express high levels of the ESR1 and ESR2 genes. And estrogen receptors regulate autoimmune responses through positive or negative regulation of proinfammatory cytokines [38,39]. Cystathione-3 (CASP3) is a key executive enzyme of apoptotic function, and research has shown that CASP3 expression increases with the progression of MG staging, and apoptosis in the thymus of MG patients is closely associated with CASP3 activation [40,41]. Lack of CASP3 prevents skeletal muscle atrophy by inhibiting apoptotic signalling in denervated muscles [42]. Plasmacytoid dendritic cells (pDC) mainly produce IFN-1. When cells are induced by viral infection or tumor cells, the body produces endogenous IFN-1, which enhances innate and acquired immunity, stimulates the activity of cytotoxic T cells and NK cells, and promotes antibody production. Downregulation of Myc in pDC cell lines can lead to a signifcant increase in the secretion of IFN-1 which in turn triggers infammatory and adaptive autoimmune responses [43,44]. Te proinfammatory cytokine interleukin-6 (IL-6) has a wide range of applications. As Tfh, T17, and regulatory T cells' abilities are suppressed by IL-6, the development of autoimmune disorders is facilitated (Treg) [45]. Studies have shown that anti-IL-6 antibody treatment can reduce the level of anti-AChR antibody in EAMG model rats. It can inhibit T1, T17, and B cell responses and reduce the severity of MG as well. Muscle atrophy and elevated levels of histone proteases were the results of transgenic mice overexpressing IL-6. Immunity and muscle   Evidence-Based Complementary and Alternative Medicine proteolysis seem to be regulated by IL-6, according to these studies [46,47]. Clinical studies have shown that the level of IL-6 in serum is signifcantly elevated in MG patients positive for anti-AChR antibodies [48,49]. In addition, tocilizumab monoclonal antibody blocks the binding of IL-6 to the IL-6R receptor, thereby inhibiting classical and trans-signalling and proinfammatory factor activity. It is expected to be an alternative therapeutic option in cases where rituximab monoclonal antibodies are inefective [50]. Te results showed by Bahauddin et al. revealed that histone deacetylases (HDACs) had diferent efects on infammation depending on the isoform, and they also uncovered a large number of genes involved in several infammatory like IL-6 and IL-21 and autoantibody pathways like acetylcholine receptor (AChR)-specifc autoantibodies, in EAMG that are controlled by HDACs [51]. So the above-mentioned intersecting targets EGFR, FOS, ESR1, ESR2, CASP3, MYC, and IL-6 are key targets of action in the treatment of MG with HD. HD may serve a therapeutic function in MG via the PI3K-Akt signalling pathway, infammatory bowel illness, according to the fndings of the KEGG enrichment study. Cell proliferation, diferentiation, metabolism, and death are all regulated by the PI3K-Akt signalling system, according to research. Diferent hormones, growth factors, and cellto-cell contacts frequently trigger this regulation [52]. It is believed that the loss of Treg function and T17 diferentiation and activation are responsible for the onset of MG in humans. It is possible to suppress the development of Treg   Degree  EPC  1  EGFR  EGFR  EGFR  EGFR  EGFR  2  FOS  CASP3  CASP3  CASP3  ESR1  3  ESR1  IL6  ESR1  IL6  CASP3  4  MYC  FOS  FOS  FOS  FOS  5  IL6  ESR1  IL6  ESR1  IL6  6  CASP3  AR  AR  AR  AR  7  ESR2  MYC  MYC  ICAM1  MYC  8  TP53  JUN  ICAM1  MYC  STAT3  9  NR3C1  STAT3  ESR2  CRP  TP53  10  STAT3  TP53  NR3C1  CHRM1  ESR2  11  AR  MAPK14  CRP  ESR2  NR3C1  12  ICAM1  TNF  BIRC5  BIRC5  ICAM1  13  SRC  NR3C1  STAT3  NR3C1  JUN  14  CRP  MAPK1  TP53  BCL2  BIRC5  15  BIRC5  MAPK3  BCL2  STAT3  MAPK14  16  MAPK14  ESR2  TNF  TP53  TNF  17  HSPB1  SRC  RELA  ACHE  RELA  18  JUN  VEGFA  MAPK14  HSPB1  CRP  19  MAPK1  INS  SRC  GRIA2  SRC  20  MAPK3  MAPK8  JUN  SRC  MAPK1  21  TNF  HIF1A  MAPK1  JUN  HSPB1  22  MAPK8  NGF  VEGFA  TNF  MAPK3  23  VEGFA  MMP9  HSPB1  RELA  BCL2  24  BCL2  HSPB1  MAPK3  VEGFA  MAPK8  25  RELA  RELA  MAPK8  MAPK14  VEGFA  26  CHRM1  PTEN  RAF1  MAPK1  HIF1A  27  PTEN  ICAM1  RB1  RAF1  RB1  28  SMAD3  PRKCA  HIF1A  MAPK3  IGF1  29  SMAD4  PTGS2  INS  MAPK8  SMAD4  30 RUNX2 BIRC5 SMAD3 RB1 SMAD3  Evidence-Based Complementary and Alternative Medicine cells by activating PI3K and mTORC1 in CD4+ T cells while promoting the diferentiation and expression of T17 cells [53,54]. As a downstream target of mTORC1, HIF-1α enhances T17 expression and regulates T17 diferentiation by increasing aerobic glycolytic activity required for rapid Tcell expansion [55,56]. Myasthenia gravis patients' immune systems may be infuenced by the PI3K/Akt/mTOR signalling pathway, according to these studies. Infammatory bowel illness has been linked to signal transducer and activator of transcription 3 (STAT3), according to research.

Evidence-Based Complementary and Alternative Medicine
When STAT3 or STAT3 phosphorylation expression is increased in human IBD, STAT3 is activated in an IL-6dosedependent relationship. Phosphorylated STAT3 can upregulate transcription factors in EAMG model rats' Tfh cells and improve humoral immune response [57,58]     After docking the fve major components of HD with their key target genes, it was found that their docking binding energy with the target was all <−5. It indicated that the fve key components showed a strong afnity with the key target genes. Terefore, HD is expected to be a major component of novel natural drugs for the treatment of MG.

Conclusions
In summary, the results of the network pharmacological analysis indicate that HD mainly acts on EGFR, FOS, ESR1, ESR2, MYC, CASP3, IL-6, and other genes through various active components, such as quercetin, kaempferol, lignan, isorhamnetin, and 7-methoxy-2-methylisofavone. Tese   components treat myasthenia gravis through antiinfammatory, immunomodulatory, and inhibiting muscle cell apoptosis. Molecular docking verifed the treatment efect of HD on MG. Te mechanism of action of HD in the treatment of myasthenia gravis is through the combined action of various major active ingredients from the formula with potential core targets. Tis study laid the foundation for the in-depth study of the pharmacological mechanism of HD in the treatment of myasthenia gravis.

Limitations and Recommendations
Our study results showed signifcant efcacy of HD against MG by interacting and altering several gene expressions. Besides these wonderful results, our study lacks practical experimentation. To prove these facts as an outcome of network pharmacological analysis, experimentation is recommended and could be a step forward.

MG:
Myasthenia gravis HD: Huangqi and Dangshen AChR: Acetylcholine receptor TCM: Traditional Chinese medicine OMIM: Online mendelian inheritance in man PPI: Protein-protein interaction TCMSP: Traditional Chinese medicine systems pharmacology database and analysis platform TTD: Terapeutic target database PDB: Protein data bank.

Data Availability
Te data used to support the fndings of this study are included within the article.

Conflicts of Interest
Te authors declare that there are no conficts of interest regarding the publication of this paper.