Maternal oocyte aging is strongly contributing to age-related decline in female fertility. Coenzyme Q10 (CoQ10) exerts positive effects in improving aging-related deterioration of oocyte quality, but the exact mechanism is unclear.To reveal the system-level mechanism of CoQ10’s anti-aging effect on oocytes based on network pharmacology.This study adopted a systems network pharmacology approach, including target identification, data integration, network and module construction, bioinformatics analysis, molecular docking, and molecular dynamics simulation.A total of 27 potential therapeutic targets were screened out. Seven hub targets  were identified. Functional and pathway enrichment analysis indicated that these 27 putative targets exerted therapeutic effects on oocyte aging by regulating signaling pathways , and are involved oxidation-reduction process, mitochondrion, enzyme binding, reactive oxygen species metabolic process, ATP binding, among others. In addition, five densely linked functional modules revealed the potential mechanisms of CoQ10 in improving aging-related deterioration of oocyte quality are closely related to antioxidant, mitochondrial function enhancement, autophagy, anti-apoptosis, and immune and endocrine system regulation. The molecular docking study reveals that seven hub targets have a good binding affinity towards CoQ10, and molecular dynamics simulation confirms the stability of the interaction between the hub targets and the CoQ10 ligand.This network pharmacology study revealed the multiple mechanisms involved in the anti-aging effect of CoQ10 on oocytes. The molecular docking and molecular dynamics simulation provide evidence that CoQ10 may act on these hub targets to fight against oocytes aging. Aging leads to a gradual and continuous decline of physiological processes. Oogenesis is one of the first processes to fail during human aging. Human oocyte development begins in the early fetal life, reaches its peak at 20 weeks of gestation, and remains dormant before complete meiosis I . Over tiOocytes accumulate a large pool of maternal RNA and protein during meiotic maturation to support early embryogenesis before embryonic genome activation , 7. TherVarious interventions have been proposed to improve oocyte quality, thereby improving pregnancy outcomes in oocyte aging-related infertility. As the third most consumed dietary supplement, coenzyme Q10 (CoQ10) has attracted global interest due to its crucial role in mitochondrial bioenergy, anti-oxidation, anti-aging, and immune system regulation. In the meantime, the role of CoQ10 in human reproduction has received widespread attention, especially in improving oocyte quality and counteracting oocyte aging –14. TuriCurrently, although anti-oocyte aging activities exerted by CoQ10 have been reported, details of biomarkers and the biological pathways through which CoQ10 counteracts oocyte aging are limited. In previous studies, a network pharmacology-based approach was successfully used to reveal all candidate targets, functions, and potential therapeutic mechanisms of drugs –22. In thttps://www.ncbi.nlm.nih.gov/geo/), and the series (GSE96477 and GSE155179) were selected. The GSE95477 and GSE155179 include single-cell RNA sequencing (scRNA-seq) data of MII human oocytes from younger (≤30 years) and older (≥40 years) patients. DESeq2 software (version 1.32.0) was used to determine differentially-expressed genes (DEGs) in all datasets. Genes with a P value <0.05 and |log 2 (fold change) | >1 were considered to be DEGs, namely, oocyte aging-related genes.Oocyte aging-associated genes were searched in the GEO database (https://pubmed.ncbi.nlm.nih.gov) using human oocyte aging and scRNA-seq as keywords. We include Zhang’s research which isolated mRNA from human oocytes obtained from old and young patients for scRNA-seq and subsequently identified differentially expressed genes . PharmMapper server (https://lilabecust.cn/pharmmapper/), Drug bank (https://go.drugbank.com/),TargetNet (http://targetnet.scbdd.com/), and Comparative Toxicogenomics Database  were used to identify possible targets for CoQ10.Simplified molecular-input line-entry specification (SMILES) information and 3D structure of CoQ10 were obtained from PubChem database (http://jvenn.toulouse.inra.fr/app/index.html) and the protein-protein interaction (PPI) network of the common targets was obtained by using the STRING database (https://string-db.org/). The species was limited to Homo sapiens, and we selected the combined score >0.4. The Cytoscape 3.8.2 was used to construct the PPI network.The oocyte aging-rated genes and the CoQ10 action targets were intersected to obtain the genes of CoQ10 against oocyte aging. The Venn diagram was drawn by using the bioinformatics platform Jvenn (https://www.omicshare.com/), a free data analysis platform, were used to analyze the GO and KEGG enrichment of the potential therapeutic targets. The Go analysis includes biological processes (BP), cellular components (CC), molecular function (MF).The OmicShare tools (Two networks were constructed: (1) the PPI network was constructed using the genes of CoQ10 against oocyte aging; (2) the CoQ10-targets-pathways network, this network was divided into different functional modules using the Community Cluster (Glay) algorithm of clusterMaker2.https://www.rcsb.org/) (https://pubchem.ncbi.nlm.nih.gov/) (https://www.playmolecule.org/deepsite/). Molecular docking was completed by Autodock vina software (v1.1.2) (The 3D structures of protein receptors were obtained from the RCSB Protein Data Bank (sb.org/) . PyMol sih.gov/) . AutoDoc(v1.1.2) . The comhttps://www.bio2byte.be/acpype/) (The conformations with the highest scores obtained from the molecular docking (protein-ligand complexes) were further subjected to molecular dynamic (MD) simulations using Gromacs 2020.6. . The CHAacpype/)  was usedvia the same method.The GSE95477 data set derived from the GEO database was analyzed and 61 DEGs were obtained according to the cut-off criteria. 1455 DEGs of GSE155179 date are obtained .In addition, we downloaded the supplementary data of Zhang’s research . Then, w.A total of 242 CoQ10 targets were obtained after removing duplications from the PharmMapper, DrugBank, TargetNet and CTD databases .Based on the results above, 31 common target genes of CoQ10-putative targets and oocyte aging-related genes were identified after removing EDN1which was up-regulated in GSE155179 but down-regulated in Zhang’s research The PPI network of common target genes consisted of 27 nodes and 51 edges, and 4 free nodes (targets) were removed.. The CytoNCA, an app plugin in Cytoscape, was used to analyze the topological parameters of the network .The 27 nodes of the PPI network were identified as potential therapeutic targets . The Cyt. The seven genes were used for the subsequent molecular docking and molecular dynamic simulation.To find the hub targets in this complex biological network, the topological parameters including degree centrality (DC), betweenness centrality (BC), closeness centrality (CC), eigenvector centrality (EC), local average connectivity-based method (LAC), network centrality (NC) were analyzed. As a result, a total of seven genes , were obtained according to the median values of DC, BC, CC, EC, LAC, NC ). In terms of BP, the anti-aging effect of CoQ10 on oocyte mainly involves the regulation of reactive oxygen species, participation of immune process, and response to hormone etc. The MF mainly involves nuclear transcription, cytokine activity, and ATP binding etc. The CC is mainly manifested in cell membrane, cytoplasm, and mitochondrion etc. , apoptosis (has04210), necroptosis (has04217), IL-17 signaling pathway (has04657), Toll-like receptor signaling pathway (has04620), and NOD-like receptor signaling pathway (has04621). Modules 2 comprised 4 pathway, including the prolactin signaling pathway (has04917), T cell receptor signaling pathway (has04660), FaxO signaling pathway (has04068), and GnRH signaling pathway (has04912). Module 3 consisted of 2 pathways, including the Th1 and Th2 cell differential (has04658) and Jak-STAT signaling pathway (has04630). Module 4 comprised PPAR signaling pathway (has03320). Module 5 comprised 2 pathways, including the MAPK signaling pathway (hsa04010) and Ras signaling pathway (hsa04014).Then, the CoQ10-targets-pathways network was constructed after sorting out the genes enriched in each pathway . To furtThe binding energy can be calculated to predict their affinity. Generally, the lower the binding energy of the ligand and the receptor, the more stable the binding conformation. As presented in ).To further validate these molecular docking simulation results, MD simulations were performed. The RMSD and RMSF of the backbone were used to scrutinize the stability of the structure model of the CoQ10 and its targets complex. The trajectory files in MD simulation were made as movies  energy between CoQ10 and targets were calculated using g-MMPB/SA software. The results were shown in .To further explore the conformation changes of CoQ10 and its targets complex, we constructed the free energy landscapes of the complex and defined two reaction coordinates of the free landscape map: one is RMSD, which shows the stability of the protein structure; the other is the radius of gyrate (Rg), which reflects whether the complex is folded stably. The energy landscape was calculated along these two reaction coordinates using the gmx sham tools As shown in The MD simulation study further evaluated the stability of the interaction between the hub targets and the CoQ10 ligand predicted by the docking experiment. In addition, free energy calculation provides an assessment of the binding affinity of hub targets to CoQ10. In our study, MD simulations showed that the structures of all complexes are very stable. Among them, the PPARA-CoQ10 complex has the lowest binding free energy, indicating that its binding affinity is the highest and may play a major role in CoQ10’s anti-aging effect on oocytes.via CoQ10. In addition, the molecular docking of the hub genes and CoQ10 exhibited high affinities. Moreover, MD simulations showed that the structures of all complexes (seven hub genes and CoQ10 complexes) are very stable, implying that the seven hub genes may be highly correlated in against oocyte aging with CoQ10.The reproductive potential in women gradually declines but significantly beginning approximately in their early 30s. However, many young women choose to postpone marriage and/or childbirth driven by social trends. Aging-related deterioration of oocyte quality is one of the great challenges in reproductive medicine. As mentioned previously, increasing evidence has shown that CoQ10 effectively improve oocyte quality and counteract oocyte aging, but the mechanisms underlying these benefits are still not fully understood. In recent years, the emergence of scRNA-seq technology has dramatically expanded our knowledge of normal and disease-related physiological processes . Therefoin vitro-matured) aging through its antioxidant properties and mitochondrial protection  and two additional PPAR isotypes, PPARβ and PPARγ, are a family of nuclear hormone receptors belonging to the steroid receptor superfamily. PPARα is the first PPAR identified by Issemann and Green in 1990 . Since totection . Tiefenbotection . ConsistCAT, which encodes catalase, is a critical endogenous antioxidant enzyme defence against oxidative stress , 38. OxiMAPK14  is a member of the MAP kinase family. It is known that activated MAPK can regulate the expression of gonadotropin synthesis genes LHβ and FSHβ, and induce follicle growth and ovulation .However, numerous studies have shown that specific MAPK14 inhibitors could block FSH-induced MAPK 3/1 phosphorylation in granulosa cells (GCs). Consistently, the MAPK14 inhibitor SB203580, blocks cumulus cell-oocyte (COC) expansion and inhibits the induction of genes  critical for this process in a dose-dependent manner . In summvia up-regulating the expression of SQSTM1/p62, ameliorate the cellular senescence of FGSCs caused by OS . In addition, the mitochondrial genome only comes from the mother, mitochondrial DNA (mtDNA) copy number can be used as a potential biomarker to predict oocyte quality and embryo viability , 59. TheAccording to the free radical theory, oxidative stress caused by elevated intracellular levels of ROS is the most significant contributor to oocyte aging. However, mitochondria are the principal source of cellular ROS, especially through electron leakage from Complexes I and III, and the mitochondria themselves are the primary targets of ROS detrimental effects. In addition, mtDNA is particularly vulnerable to ROS-mediated oxidative damage due to the lack of histone protection. ROS is mainly produced in the matrix side of the inner mitochondrial membrane and overlaps with mtDNA positions. This condition promotes the formation of mtDNA-protein cross-linking mediated by ROS, which is one of the most serious forms of DNA damage. In turn, mtDNA damage directly impairs electron transfer chain and ROS production in the mitochondria, forming a “ROS vicious circle” , 62. TheInterestingly, there is a tendency to CoQ10 in a variety of mammalian tissues to be reduced with age  . This agFurthermore, the KEGG pathway analysis indicated that the PPAR signaling pathway was the most enriched. Other related pathways include the TNF signaling pathway, Apoptosis, Necroptosis, Prolactin signaling pathway, MAPK signaling pathway, etc. Previous studies have shown that the PPAR signaling pathway is involved in activating the most abundant fertility reserve in female mammals-dormant primordial follicles . In addiTo further understand the CoQ10 mechanisms in improving oocyte quality, five densely linked functional modules were identified based on the target pathway network, as shown in The last module includes pathways in the signal transduction related to GCs proliferation and oocyte meiosis. Among them, three-fifths of the modules involve the immune system. Specifically, immune system-associated cells and molecules exist in the hypothalamus-pituitary–ovarian axis, regulating the development of fetal and adult primordial follicles. Moreover, Bukovsky et al. demonstrated that immune cells and their secreting factors are an important part of the ovarian germline stem cells (OSC) niche. They participate in the asymmetric division of OSCs and promote the division, proliferation and migration of germ cells and the growth of follicles , 68. TheIn the present work, we found that the potential mechanisms of CoQ10 in improving aging-related deterioration of oocyte quality are closely related to antioxidant, mitochondrial function enhancement, autophagy, endocrine and immune system regulation, and anti-apoptosis , the Project of Zhejiang Province Scientific Research Foundation (2020ZA078), Science and Technology Projects of Zhejiang Province (2019C03086), the Project of Zhejiang Province Scientific Research Foundation (2022ZQ061), the Medical and Health Science and Technology Plan of Zhejiang Province (2021KY920), and Zhejiang Zhangqin Famous Traditional Chinese Medicine Expert Inheritance Studio Project (GZS2017014).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. 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The incidence of heart failure was significantly increased in patients with diabetic cardiomyopathy (DCM). The therapeutic effect of triptolide on DCM has been reported, but the underlying mechanisms remain to be elucidated. This study is aimed at investigating the potential targets of triptolide as a therapeutic strategy for DCM using a network pharmacology approach. Triptolide and its targets were identified by the Traditional Chinese Medicine Systems Pharmacology database. DCM-associated protein targets were identified using the comparative toxicogenomics database and the GeneCards database. The networks of triptolide-target genes and DCM-associated target genes were created by Cytoscape. The common targets and enriched pathways were identified by the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses. The gene-gene interaction network was analyzed by the GeneMANIA database. The drug-target-pathway network was constructed by Cytoscape. Six candidate protein targets were identified in both triptolide target network and DCM-associated network: STAT3, VEGFA, FOS, TNF, TP53, and TGFB1. The gene-gene interaction based on the targets of triptolide in DCM revealed the interaction of these targets. Additionally, five key targets that were linked to more than three genes were determined as crucial genes. The GO analysis identified 10 biological processes, 2 cellular components, and 10 molecular functions. The KEGG analysis identified 10 signaling pathways. The docking analysis showed that triptolide fits in the binding pockets of all six candidate targets. In conclusion, the present study explored the potential targets and signaling pathways of triptolide as a treatment for DCM. These results illustrate the mechanism of action of triptolide as an anti-DCM agent and contribute to a better understanding of triptolide as a transcriptional regulator of cytokine mRNA expression. Diabetic cardiomyopathy (DCM) is defined as abnormal cardiac structure and function in the absence of other cardiac risk factors . AlthougTripterygium wilfordii Hook. F [Traditional Chinese medicine (TCM), characterized by its multitarget nature, has been used to treat different types of diseases in China for thousands of years , 6. Rece Hook. F . The the Hook. F , 11.Network pharmacology-based drug discovery is a novel, promising, cost-effective approach for drug development. In contrast to the “one target, one drug” principle, network pharmacology uses the “network target, multicomponent” mode by combining systems biology, bioinformatics, and pharmacology. Network pharmacology has been widely used to explore the mechanism of the protective action of TCM on diseases. In the present study, the candidate targets of triptolide in the treatment of DCM were predicted by network pharmacology. The pathway enrichment analysis was performed to reveal potential therapeutic strategies for DCM. The procedures of this study are shown in https://pubchem.ncbi.nlm.nih.gov) is an open chemical database that provides information on compound structures and descriptive data. The PubChem database was used to retrieve the 2D chemical structure of triptolide (PubChem (iptolide .http://lsp.nwu.edu.cn/tcmsp.php) database is a systems pharmacology platform of TCMs and related compounds [The Traditional Chinese Medicine Systems Pharmacology  [http://www.genecards.org/) [The therapeutic targets of DCM were identified using the comparative toxicogenomics database   and the ds.org/)  with a rGeneMANIA is a website for generating hypotheses based on gene function, analyzing gene lists, and prioritizing genes for functional assays . In thishttp://www.webgestalt.org/option.php) is a powerful tool for functional enrichment analysis, supporting 12 organisms, 324 gene identifiers from various databases and technology platforms, and 150,937 functional categories from public databases and computational analyses [P value of less than 0.05 indicates statistical significance.The WEB-based Gene Set Analysis Toolkit  was used to construct a triptolide-target-pathway network and to analyze the relationships among compounds, targets, and diseases .http://www.pdb.org/) and modified using Autodock Tools (version 1.5.6) to add hydrogen, calculate Gasteiger electric charges, and combine nonpolar hydrogen. Autodock Tools was also used to add hydrogen and electric charges to triptolide. Autodockvina (version 1.1.2) was used to perform docking between triptolide and candidate target proteins.The crystal structures of candidate proteins were downloaded from the RCSB Protein Data Bank . A total of 30 targets of triptolide were predicted using the TCMSP database  , Table 2WWOX), secreted protein acidic and rich in cysteine (SPARC), TNF receptor superfamily member 1B (TNFRSF1B), receptor for activated C kinase 1 (JUNB), and mouse double minute 2 (MDM2).A gene-gene interaction network of the six common candidate targets was constructed, and their functions were analyzed using the GeneMANIA database . The 12 The targets of triptolide related to DCM were imported into the WebGestalt database for GO analysis. Ten biological processes were identified, including positive regulation of pri-miRNA transcription by RNA polymerase II, regulation of pri-miRNA transcription by RNA polymerase II, pri-miRNA transcription by RNA polymerase II, positive regulation of myeloid cell differentiation, positive regulation of peptidyl-tyrosine phosphorylation, regulation of peptidyl-tyrosine phosphorylation, myeloid cell differentiation, peptidyl-tyrosine phosphorylation, peptidyl-tyrosine modification, and cytokine-mediated signaling pathway . In addiA triptolide-target-pathway network was constructed by Cytoscape based on the targets and the results of pathway enrichment analyses. As shown in β1 (PDB: 4KV5), were collected from the RCSB Protein Data Bank (β1 with a binding pocket consisting of GLN-19 (3.3 Å) and SER-9 (3.0 Å).The crystal structures of potential targets, including STAT3 (PDB: 6NJS), VEGFA (PDB: 3V2A), FOS (PDB: 1A02), TNF (PDB: 5M2J), TP53 (PDB: 6RZ3), and TGFata Bank . TriptolCardiovascular complications are the leading cause of mortality and morbidity in patients with diabetes. While ischemia dominates the cardiac complications of diabetes, it is well accepted that the risk for developing heart failure is also increased in the absence of overt myocardial ischemia and hypertension or is accelerated in the presence of these comorbidities . Many poOB is one of the most important pharmacokinetic parameters . High OBα and TGF-β are important proinflammatory factor and fibrotic factor, respectively [After potential target screening, six DCM-associated targets of triptolide were identified , suggesting that these targets may play key roles in the treatment of DCM by triptolide. STAT3 has been shown to drive the development of DCM, while blockage of STAT3 and its upstream factor, epidermal growth factor receptor, attenuates DCM , 27. VEGectively –32. Althectively .WWOX, SPARC, TNFRSF1B, JUNB, and MDM2. It has been shown that STAT3 binds to the promoter of JUNB to induce inflammation and promote the progression of autoimmune diseases [α) and profibrotic factors  suppressed the synthesis of SPARC [α receptor related to inflammatory diseases, such as rheumatoid arthritis [To elucidate the functional relationship of these common targets in DCM, we constructed a gene-gene interaction network using the GeneMANIA database. The results revealed that these targets were correlated in terms of coexpression, implying intensive interactions among them. Moreover, these targets also interact intensively with other genes, such as diseases , 35. Antdiseases . SPARC rdiseases . Proinflof SPARC . FOS andof SPARC . The exprthritis . WWOX rerthritis .The GO analysis revealed that the target genes were enriched in positive regulation of pri-miRNA transcription by RNA polymerase II, regulation of pri-miRNA transcription by RNA polymerase II, and pri-miRNA transcription by RNA polymerase II, indicating that transcriptional regulation is related to the functions of these targets. In addition, the RNA polymerase II transcription factor complex and the nuclear transcription factor complex were identified as the major cell components involved in these biological processes. The major molecular functions of these target genes were core promoter sequence-specific DNA binding, RNA polymerase II transcription factor binding, cytokine receptor binding, and cytokine activity, suggesting that transcriptional regulation of cytokine mRNA expression is highly involved in the treatment of DCM by triptolide.α, TGF-β, and STAT3 synergistically increased the risk of developing cancers and promoted tumor growth and cancer-associated cachexia [α and TGF-β1) [κB signaling, leading to increased expression of the β-myosin heavy chain isoform in diabetic hearts. Dehydroepiandrosterone counteracts oxidative stress-induced activation of RAGE in rat models of DCM and normalizes the nuclear factor κB signaling and the upregulation of β-myosin heavy chain isoform, thereby contributing to the development of diabetic cardiomyopathy.The KEGG pathway analysis showed that triptolide regulated the activation of pancreatic cancer, rheumatoid arthritis, and AGE-RAGE signaling pathway in diabetic complications in the treatment of DCM. Systemic and local chronic inflammation may increase the risk of pancreatic cancer. Pancreatic cancer-associated inflammatory infiltrate in the tumor microenvironment promotes tumor growth and metastasis . The concachexia , 44. Thecachexia , and thecachexia , 47. Int TGF-β1) , 49. AGE TGF-β1) . AGEs ac TGF-β1) . The uprIn the present study, six core potential targets  of triptolide in the treatment of DCM are identified by network pharmacology. The GO and KEGG pathway enrichment analyses reveal the mechanism of action of triptolide, that is, triptolide ameliorates DCM through targets related to the inhibition of angiogenesis, synovial hyperplasia, and bone destruction. Our study provides a theoretical basis for further investigation of the therapeutic potential of triptolide for DCM and the use of network pharmacology in drug discovery for other inflammatory diseases. To verify the effects of triptolide and the molecular target genes of anti-DCM, further experimental studies need to be performed in the future. In addition, clinical trials should be conducted to identify the effectiveness of triptolide in humans.


Pathway models are an effective way to capture and share our current understanding of biological processes. A pathway model is defined here as a set of interactions among biological entities  relevant to a particular context, curated and organized to illustrate a particular process. Properly modeled pathways can be used in the analysis and visualization of diverse types of omics and other biomedical data ,2. The mBiological pathway information is often conveyed as published figures, and rules for better figures, in general, are also relevant when producing pathway models . HoweverHere, we describe a set of rules for constructing pathway models to optimize their use both as graphical representations for human consumption and as FAIR resources for computational analysis. The rules range from reusability and dissemination , intuitive visual concepts , to enabling computational analysis (Rules 3 to 6). We hope that these rules provide a simple framework for pathway model curators who want to create (re)usable resources for the scientific community.https://gladstone-bioinformatics.shinyapps.io/shiny-25years) and to facilitate curation by providing starter pathway models with extracted gene sets and by prioritizing pathway figures based on uniqueness against existing pathway databases.When constructing a pathway model, first, research existing content from the myriad of online databases containing pathway-related information. Searchable databases for biological pathways include Reactome , WikiPatOther valuable sources of pathway information are network and interaction databases such as BioModels , NDEx 22, Rhea , IntAct By researching these sources, one not only collects supporting evidence for their pathway model but also compiles a more complete and original model. All sources should be cited in the pathway model as part of the modeling process (see Rule 5). There are a variety of free tools available for pathway modeling, including PathVisio , CellDesTo illustrate a particular biological process, pathway models contain entities and their interactions relevant to the particular context. Thus, the scope of the pathway model, i.e., the entities and boundaries we choose, should be based on what we are trying to illustrate. When deciding on the scope of a pathway model, consider which reactions and entities are crucial to understanding the relevant process. As an example, comparing 2 “citric acid cycle” pathway models at WikiPathways, the canonical pathway  has a hiMany pathways reference or include (parts of) other pathways. If the inclusion of another pathway is not central to understanding the process, pathways or parts of pathways should be represented by a single pathway node instead of including many peripheral gene products or proteins. If a more detailed model representing part of the pathway is available, we encourage curators to add links to those pathways in their model. The number of nodes included in a model affects its usefulness in downstream analysis and data visualization. For enrichment analysis, pathway size affects the performance and interpretability indicating that meta pathways or other large pathways describing multiple processes should be split into individual smaller pathways for these types of analyses . SmallerUnderstanding biological processes and molecular entities can be a difficult job with the vast amount of synonyms in use. For example, the official gene name of neuroepithelial cell–transforming gene 1 protein (NET1) is also a synonym for the sodium-dependent noradrenaline transporter (SLC6A2). Also, well-known chemicals are commonly referred to by multiple names, for example, paracetamol/acetaminophen, which has well over 500 drug vendor–specific names worldwide . Initiatidentifiers.org [Names are often more descriptive and understandable to researchers with knowledge of the context; however, this is not trivial for computers. In pathway models, this problem is in part overcome by annotating molecular entities with both a human-readable label and a computer-readable identifier. These resolvable identifiers connect to online knowledge bases containing information about the individual molecular entities. Many of the pathway curation tools have integrated identifier resolution, and curators only need to define the identifier and database for the entity. Given the large number of online databases, choosing the proper identifier for a specific entity can be difficult. It is important to use the most precise identifier with the biological entity. For example, use UniProt for a specific protein, instead of the gene identifier, or RefSeq  if a speiers.org . For speiers.org . Similariers.org  for chemiers.org  or Swissiers.org . Annotatiers.org  providesiers.org  or Enzymiers.org  can be uIn general, consistency in labeling and annotation with identifiers is essential for users and analysis tools to interpret molecular pathway models. This is in line with the FAIR principles , which wPathway models need detailed interactions to describe how different biological entities influence each other. Therefore, specifying the meaning of interactions or the relationships between entities constitutes a core task in generating a pathway model. Pathway models use various line styles  to show which biological entities are interacting and how. Although the machine readability of pathway models is made possible by connecting entities, simply connecting entities is not enough. The lines and arrows in diagrams can have several biological meanings, e.g., metabolic conversion, stimulation, and modification, which should be easily distinguishable from one another. This warrants the necessity of a standardized set of biological interaction types used within a pathway model.Several established standards describe different types of biological interactions, all with a specific focus, for example, SBGN or Molecular Interaction Maps (MIMs) . We adviBy using a standardized set of interactions, pathway authors can specify the correct biological semantics for the interactions in the pathway model. Detailed annotation  of interactions facilitate their use to integrate pathway models with other resources . MappingFinally, if you decide to use nonstandard interaction types, always define their meaning in the pathway model in a visual legend.Information added to a pathway model, such as proteins or interactions, should be based on scientific evidence or at least a documented hypothesis. The origin of that information, also called provenance, and an evaluation of the evidence should be referenced such that people exploring the pathway model can easily access and verify the source of the information. Curators often add focused references for specific biological entities  or interactions . Additionally, many tools allow adding general literature references important for the understanding of the pathway model as a whole. In addition to the reference itself, by using the Evidence and Conclusion Ontology (ECO) , one canWhen provenance is provided in a machine-readable manner, this information can be used to automatically filter pathway content by the presence and type of supporting evidence. Furthermore, the coverage of a pathway model and pathway database can be assessed with more ease. Finally, linking the pathway model content to other databases and retrieving additional information thereof can be done effortlessly.To improve readability and reusability, accurately describing the pathway content on different levels is crucial. First, a descriptive and clear title is important to capture the overarching concept of the pathway model. The title should be concise and limited to a single line when possible. In general, abbreviations should be avoided and only included when the process refers to a specific gene or protein. Second, think of the pathway description as a textual representation of the pathway model, describing each main step as well as the overall process and outcome. While it might seem counterintuitive, writing the description before creating the model helps to make sure that the description is a proper representation of the model in the end. The description should be unique; do not simply copy the description from another source . Include literature references within your description, whether they refer to specific components in the model or provide the provenance from which the model was adapted . Finally, machine-readable metadata about the pathway diagram improves the findability and organization of the pathway models. Using existing ontologies like the Pathway Ontology , the GenIllustrating a biological process in a graphical format clearly and legibly is a challenge. The layout of reactions and entities is critical to readability. For most pathway models, a layout with top-to-bottom and left-to-right orientation is recommended. Cellular compartments and other graphical annotations are helpful to further organize the layout and should be labeled for clear identification. In general, labels on nodes describing proteins, gene products, and metabolites should have one consistent font, size, and placement throughout the pathway model. However, using a larger font size helps to highlight and draw attention to nodes of particular importance. To increase readability, the placement of nodes should avoid creating overlapping interactions as much as possible. Additionally, the layout should be optimized to avoid using multiple copies of the same node, unless there is a biological meaning. For example, when illustrating the transport of a protein into a compartment, 2 copies of the same node are necessary to describe the process properly. Similarly, illustrating 2 different complexes composed of an overlapping set of proteins requires multiple copies of the same node, as does illustrating multiple reactions involving the same common reactants, for example, ATP/ADP.Various types of data, like transcriptomics, proteomics, metabolomics, and fluxomics data, can be visualized on pathway models. Whereas data linked to molecular entities (nodes) are often depicted by using a color scheme to fill the nodes, data related to interaction types are often depicted by changing the width or color of the interaction linking the data nodes.To avoid interfering with such data visualization, the fill color for nodes and edges should be limited when designing your pathway, as they can interfere with data visualization and lead to misinterpretation at the process level. We recommended differentiating molecular entities using node shapes to designate state information  using dedicated glyphs and to depict different interaction types using standard arrowheads (see Rule 4), instead of using colors. If you decide to use colors in your pathway, consider readability for all users by choosing a visual accessible color palette.In Finally, when using different colors and line width is unavoidable, include a legend to define the color and line annotations. A legend can assist in differentiating between the data and the pathway entities themselves when visualizing data on the pathway model.Once your model is ready, you want people to reuse and cite it. With the above rules, you already ensured the easy reuse of the model. But full dissemination still starts with communicating to others that you have created a new pathway model. When your model is being developed as part of a research article, particularly if featured as a figure therein, include at least the machine-readable model as a supporting information. This simplifies the reuse of the knowledge by the readers of your article. You can even take this a step further and make your model available in an open pathway knowledge base, for example, WikiPathways or NDEx, or more general archives like figshare  or Zenodwww.wikipedia.org). You may also be able to include it on your website as an image linking to the machine-readable version or as a compact identifier in text, such as wikipathways:WP78 for WikiPathways WP78 [Once you are confident about the content, you can further communicate your work via social media , which allows rapid reuse of the content in other research workflows that use the semantic web . NetworkPathway models are never finished and should be considered living models, both in terms of content and graphical aspects, as seen in Updates are aimed at improving the graphical layout and content and should be described by the editor in accompanying comments. An example of a pathway timeline is illustrated in Another reason to keep your pathway updated is the identifier annotation for genes, proteins, metabolites, and other pathway elements (see Rule 3). Databases like Ensembl, ChEBI, and even WikiPathways phase out identifiers in data curation processes, and like broken website links, replacing phased out identifiers in your pathway ensures that links to external databases and general interoperability are preserved over time.Pathway diagrams are intuitive tools for collecting and communicating molecular details of biological processes. Every year, thousands of pathway diagrams are published in the literature often as static images, which slow down the distribution of the knowledge, limits reusability, and prevents computational analysis .Pathway diagrams, in general, should be made available, ideally in an online pathway database, as models created with appropriate pathway modeling tools and standards. By following the 10 simple rules described in this paper, pathway models maximize potential for computational analysis and reuse.


To clarify the therapeutic mechanisms of compound Xuanju capsule-treated rheumatoid arthritis (RA) based on network pharmacology tactics.  The TCMSP, TCMID and STITCH databases were used to screen the active ingredients and targets in the compound Xuanju capsule; the OMIM, TTD, PharmGKB and GeneCards databases were applied to screen the RA-related disease targets. Then, the obtained targets were imported into Cytoscape 3.7.1 software to construct the active ingredient-target network and the RA-related disease-target network. The active ingredient-target PPI network, the RA-related disease-target PPI network and the common target PPI network were built by using the STRING platform and Cytoscape 3.7.1 software. The GO and KEGG analyses of the common targets were analyzed by using the Metascape and Bioinformatics online tools.  A total of 51 active ingredients and 513 corresponding ingredient targets were harvested from the compound Xuanju capsule; 641 RA-related disease targets were obtained. After two PPI networks were constructed and merged, 116 RA-related targets of compound Xuanju capsules were identified and analyzed. 116 RA-related targets of compound Xuanju capsules are mainly involved in the biological processes and molecular functions, such as the cytokine-mediated signaling pathways, the response to lipopolysaccharide and the blood vascular development, the cytokine activity, the cytokine receptor binding and the receptor regulator activity. Furthermore, 116 RA-related targets of compound Xuanju capsules are concentrated in signaling pathways such as the IL-17, TNF, Th17 cell differentiation, Toll receptor and RA signaling pathway. α) and increase the production of anti-inflammatory factors (such as IL-10) by regulating inflammation-related signaling pathways (such as IL-17), thereby alleviating the inflammatory damage and improving the bone tissue repair. The compound Xuanju capsule had the action characteristics of multiple components, multiple targets, and multiple pathways in the treatment of RA, which might primarily reduce the release of proinflammatory factors (such as IL-6 and TNF- Rheumatoid arthritis (RA), a common autoimmune dysfunction disease characterized by the chronic inflammation of the joint synovium and the systemic vasculitis, can severely impair the physical function and the quality of life . WorldwiEpimedium brevicornu Maxim., YYH), Fructus Cnidii (Cnidium monnieri (L.) Cuss., SCZ), and Fructus Lycii . The black ants, as the primary drug, can nourish the kidney and liver, invigorate the circulation of blood and remove blood stasis, drive wind, and disperse cold [α and IL-6 and the inflammatory mediator NO [The compound Xuanju capsule, as a Chinese patent medicine, is composed of black ants (XJ), Herba Epimedii , Traditional Chinese Medicine Integrated Database , Natural Products Database of Traditional Chinese Medicine of Shanghai Institute of Organic Chemistry (http://www.chemcpd.csdb.cn) and the related literature of the compound Xuanju capsule [The active ingredients of black ants (XJ), Herba Epimedii (YYH), Fructus Lycii (SCZ) and Fructus Cnidii (GQZ) in the whole prescription were collected by the Traditional Chinese Medicine Systems Pharmacology (TCMSP) database and analysis platform . And then, the targets corresponding to the active ingredients screened from the two target databases were standardized in the UniProt database (http://www.uniprot.org), with the properties set to “reviewed” and “human” [To obtain the target of the each active ingredient in the compound Xuanju capsule, the potential targets were predicted from the TCMSP and STITCH databases ( “human” . Subsequhttp://bidd.nus.edu.sg/group/cjttd), the PharmGKB database (https://www.Pharmgkb.org), the Online Mendelian Inheritance in Man (OMIM) database (http://www.omim.org) and the GeneCards database (https://www.genecards.org) [With “Rheumatoid arthritis” to be the keyword, RA-related targets were retrieved from three public online databases, namely, the Therapeutic Target Database  , 26. TheThe active ingredient targets of the compound Xuanju capsule and the RA-related disease targets were analyzed using the STRING database, and “Homo sapiens” was limited. Two PPI networks of the active ingredient targets of the compound Xuanju capsule and the RA-related disease targets were built and merged through Cytoscape 3.7.1 software. Afterward, the common network (the intersection part of two PPI networks) was further analyzed by using Cytoscape 3.7.1 software, and the common targets were obtained. Additionally, we further screened targets whose degree value ranked in the top 20 as the core targets of compound Xuanju capsule-related RA.http://metascape.org), which got the potential signal pathways, related biological processes, cell components, and molecular functions of the active components in the compound Xuanju capsule. The results of GO enrichment analysis and the bubble diagram of KEGG pathway enrichment were drawn using the Bioinformatics online tools (http://www.bioinformatics.com.cn). The ClueGO plug-in in Cytoscape 3.7.1 software was applied for constructing the visual results of the target proteins involved in signal pathways.The Gene Ontology (GO)  biology Epimedium, 114 in Fructus Cnidii, and 188 in wolfberry. According to the screening criteria of the ADME, including the OB and DL, 87 active ingredients of the compound Xuanju capsule were obtained after duplicated targets were removed. In addition, three antirheumatic active ingredients of Xuanju were found from literatures, namely, citral, formic acid, and β-sitosterol [In the present study, we searched all the reported chemical components in the compound Xuanju capsule through TCMSP, TCMID, and Natural Products Database of Traditional Chinese Medicine of Shanghai Institute of Organic Chemistry. A total of 478 ingredients were collected, among which 46 was found in black ants, 130 in tosterol . IncludiSTITCH and TCMSP databases were used to predict the targets of the active ingredients. After the “Homo sapiens” targets were remained and the duplicated targets were removed, 51 active ingredients and 513 corresponding action targets were screened, as shown in Searching for RA-related targets through TTD, PharmGKB, OMIM and GeneCards databases, when “Homo sapiens” targets were remained and the duplicated targets were removed, a total of 641 targets were obtained. The above targets were submitted in the Cytoscape 3.7.1 software to construct the RA-related disease-target network .The STRING online database was used for PPI network topology analysis. The analyzed PPI relationship data results (.tsv format file) were imported into the Cytoscape 3.7.1 software, and then, the visualized PPI network of the active ingredient targets  and the β, JUN and CASP3. The 116 common targets were ranked according to the degree value, and the top 20 core targets of compound Xuanju capsule-related RA are shown in In the Cytoscape 3.7.1 software, the CytoNCA plug-in was applied to merge two PPI networks and obtain a visualized intersection target network , which cGO enrichment analysis includes molecular function (GO-MF), biological process (GO-BP), and cellular component (GO-CC). The top 10 GO analysis results were selected according to the degree of importance and significance, as shown in The KEGG pathway analysis was carried out to further explore which pathways were related to the screened common targets. Among the top 20 pathways , there were mainly the IL-17 signaling pathway, the TNF signaling pathway, the Th17 cell differentiation pathway, the Toll receptor signaling pathway, the RA pathway, the fluid shear stress and atherosclerosis pathway, and so on . Using tNetwork pharmacology is a research method to clarify the disease occurrence and development from the perspectives of systems biology and biological networks, through the collection of ingredients and action targets; an interaction network is constructed to further investigate the regulation effect of TCM on different signals, thereby revealing the action mechanism of TCM . Based oκB and JAK/STAT signal pathways [Among the 51 active ingredients selected in this work, formic acid, quercetin, kaempferol, luteolin, stigmasterol, anhydroicaritin and so on were the top 10 active ingredients according to the degree value, which might be the main active ingredients of the compound Xuanju capsule against RA. Researches have demonstrated that formic acid as one of the biologically active substances of black ants was the material basis for its anti-inflammatory, analgesic, antibacterial and antirheumatic effects and immune regulation and prevention of arteriosclerosis , 31. Quepathways . Stigmaspathways . Anhydropathways . Therefoβ, JUN, CASP3 and other targets showed an obvious correlation in the network relationship of compound Xuanju capsules in the treatment of RA, as shown in α was increased in the synovium and cartilage of RA patients, which could promote the cartilage destruction by inhibiting the collagen synthesis, stimulating the fibroblasts and chondrocytes to produce the prostaglandins and collagenase, stimulating the chondrocytes to secrete metalloproteinases, inducing the differentiation of peripheral blood mononuclear cells into osteoclasts, etc. [α can increase the production of the proinflammatory factors  and chemokines  through the cascade reaction and further aggravate the inflammatory response [β, MAPK8, JUN and other targets had higher degree values, indicating that these targets might be the core targets of the compound Xuanju capsule against RA, and the corresponding action mechanism was characterized by multiple components and multiple targets.Each node in the network represents the protein corresponding to the action target; thus, 116 common nodes involved in the intersection network indicated that there were a total of 116 potential action targets of the compound Xuanju capsule in the treatment of RA. Additionally, the size and color of the nodes represent the degree value of the target protein, and the connection between the nodes represents the potential interaction between the target proteins . The tarts, etc. . Meanwhiresponse . IL-6 isresponse . Thus, Iresponse . One of response , 44. AKTresponse . The MAPresponse . JUN, clresponse . The resβ, TNF and PTGS2 in the TNF signaling pathway [+ T cells. Among them, Th17 cells can highly secrete inflammatory cytokines such as IL-17, IL-6 and IL-22, mediating inflammatory reactions and autoimmune reactions in the body [The GO and KEGG pathway enrichment analysis were conducted on 116 common targets. The GO results found that the cytokine activity and the cytokine receptor binding were closely related to the occurrence of RA in terms of molecular functions. In addition, the signal pathways and vascular development mediated by the cytokines in biological processes had a certain impact on RA; this was also corroborated with the results that the compound Xuanju capsule might act on 166 potential core targets in the treatment of RA. KEGG pathway enrichment analysis results demonstrated that the main signaling pathways regulated by the compound Xuanju capsule were the IL-17 signaling pathway, the TNF signaling pathway, the Th17 cell differentiation pathway, the Toll receptor signaling pathway, the RA pathway, the liquid shear stress and atherosclerosis pathways, etc. It had shown that the TNF signaling pathway was the key pathway derived from the enrichment analysis and an important pathway in the inflammatory response . The occ pathway . IL-17 n pathway . Therefo pathway . The Tol pathway . The VEG pathway . Th17 anthe body . Abnormathe body . Cells ithe body . These mthe body , 15.RA is a chronic inflammatory autoimmune disease with the complex pathogenesis . The resIn conclusion, based on the network pharmacology methods, the current study found that the compound Xuanju capsule, in the treatment of RA, might mainly act on 116 targets based on IL-6 through 51 active ingredients such as formic acid, quercetin and kaempferol, and then regulated the IL-17 signal pathway, the TNF signal pathway, the Th17 cell differentiation pathway, the Toll receptor signaling pathway, the RA pathway, etc., to further affect the release of the proinflammatory factors and the anti-inflammatory factors, thereby alleviating inflammatory damage and improving bone tissue repair. The above results reflected the mechanism and advantages of the compound Xuanju capsule in treating RA with multiple components, multiple targets, and multiple pathways, and also confirmed that inhibiting the secretion of inflammatory factors was one of its major mechanisms.


The incidence of gout has been rapidly increasing in recent years with the changing of diet. At present, modern medications used in the clinical treatment of gout showed several side effects, such as gastrointestinal damage and the increased risk of cardiovascular disease. The traditional Chinese prescription Simiao Powder (SMP) has a long history in the treatment of acute gouty arthritis (AGA) and has a good curative effect. However, the mechanism and target of its therapeutic effects are still not completely understood.  Potential active compounds (PACs) and targets of SMP were found in the TCMSP database, and the disease target genes related to AGA were obtained by searching CTD, DisGeNET, DrugBank, GeneCards, TTD, OMIM, and PharmGKB disease databases with “acute gouty arthritis” and “Arthritis, Gouty” as keywords, respectively. The network of “Traditional Chinese medicine (TCM)-PACs-potential targets of acute gouty arthritis” was constructed with the Cytoscape 3.7.2 software, and the target genes of acute gouty arthritis were intersected with genes regulated by active compounds of SMP. The resultant common gene targets were input into Cytoscape 3.7.2 software, and the BisoGenet plug-in was used to construct a PPI network. The GO functional enrichment analysis and KEGG pathway enrichment analysis of the intersecting target proteins were performed using R software and corresponding program packages. The molecular docking verification was carried out between the potentially active compounds of SMP and the core target at the same time.  40 active components and 203 targets were identified, of which 95 targets were common targets for the drugs and diseases. GO function enrichment analysis revealed that SMP regulated several biological processes, such as response to lipopolysaccharide and oxidative stress, RNA polymerase II transcription regulator complex, protein kinase complex, and other cellular and molecular processes, including DNA-binding transcription factor binding. Results of KEGG pathway analysis showed that SMP was associated with AGA-related pathways such as interleukin-17 (IL-17), tumor necrosis factor (TNF), p53, and hypoxia-inducible factor 1 (HIF-1) signaling pathways. The results of molecular docking showed that active compounds in SMP exhibited strong binding to five core protein receptors .  Active components of SMP, such as quercetin, kaempferol, wogonin, baicalein, beta-sitosterol, and rutaecarpine, showed therapeutic effects on AGA. These compounds were strongly associated with core target proteins . This study reveals that IL-17, TNF, p53, and HIF-1 signaling pathways mediate the therapeutic effects of SMP on AGA. These findings expand our understanding of the mechanism of SMP in the treatment of AGA. Gouty arthritis (GA) is characterized by acute onset of single joint redness, swelling, heat, and pain at night or in the morning. The pain progressively worsens, and in severe cases, patients often suffer from cutting pain, which often reaches a peak within 24 hours . The numChengfang Biandu in the Qing Dynasty (1904 A.D.). SMP is composed of Rhizoma Atractylodis (Cang Zhu), Cortex Phellodendri (Huang Bo), Radix Vladimiriae (Niu Xi), and Semen Coicis (Yi Yi Ren), and it could clear heat, dry dampness, and promote blood circulation to invigorate the tendons. In the gouty arthritis model, Lin et al. revealed that SMP treatment could effectively relieve joint symptoms and improve laboratory indexes . Moreover, the anti-inflammatory effect of SMP was stronger than febuxostat [Simiao Powder (SMP) is a well-known prescription used in ancient China for the treatment of joint pain . This prbuxostat . A systebuxostat . Howeverbuxostat .The entire human body is a complex network, and the occurrence and progression of a disease are the results of the comprehensive action of multiple factors. Single-targeted therapy cannot meet the needs of multitarget and multipathway treatment of diseases. The network pharmacology concept put forward by Hopkins in 2007  providesThe present study employed network pharmacology and molecular docking techniques to establish the active components of SMP in AGA treatment and predicted the potential target and drug action pathways of SMP. Collectively, we aimed to explore the potential mechanism of action of SMP and provide a reference for pharmacological research and clinical application of this prescription. The specific flow chart is shown in Rhizoma Atractylodis (Cang Zhu), Cortex Phellodendri (Huang Bo), Radix Vladimiriae (Niu Xi), and Semen Coicis (Yi Yi Ren), SMP components, were acquired from the traditional Chinese medicine systems pharmacology database and analysis platform , capturing all the potential active components of drugs in SMP. We screened the active compounds in SMP following the standards of oral bioavailability (OB) ≥30% and drug-likeness (DL) ≥0.18. Then, a search was undertaken for corresponding targets of active compounds on the TCMSP platform. The identified drug targets were input into the UniProt database (https://www.uniprot.org/). To obtain the abbreviation of the human gene for each target, we set the protein type as “Homo sapiens.”https://www.genecards.org/), Online Mendelian Inheritance in Man , the Pharmacogenomics Knowledge Base database , DrugBank database (https://www.drugbank.ca/), DisGeNET database (https://www.disgenet.org/), the Therapeutic Target Database , and Comparative Toxicogenomics Database . The results of the above databases were integrated to obtain the target genes of AGA after deleting the repeated genes.The search for target genes related to AGA was achieved using the terms “acute gouty arthritis” and “Arthritis, Gouty” in the Human Gene Database  betweenness centrality (BC) of each node was calculated. A larger DC and BC of the node at the protein location implied that the protein was more important in the constructed PPI network . BecauseThe ID of the intersection target was obtained using R4.2.0 software and its “org.Hs.eg.db” package. Then, the “clusterProfiler” and other program packages were adopted for GO functional enrichment analysis of the target. A histogram was constructed after screening out the first 10 functional categories of biological process (BP), cellular component (CC), and molecular function (MF). The target proteins were subjected to the KEGG enrichment analysis, whereby the first 30 KEGG pathways were selected to construct the histogram. Information of the first 20 KEGG pathways was introduced into Cytoscape 3.7.2 to draw the KEGG pathway-gene network map. After that, we selected a signal pathway closely related to GA for visual display.https://pubchem.ncbi.nlm.nih.gov/). For small molecules, their energy was minimized using ChemBio3D 2014 software. The 2D structure was converted into a 3D structure. We downloaded the 3D structure of the core target protein from the Protein Data Bank (PDB) database (http://www.rcsb.org). Using the Pymol software, the target protein receptor molecules were processed, including dehydration and removal of ligand small molecules. The target protein receptor molecule was hydrogenated using Autodock Tools 4.2.6 software. The center coordinate and size of the box were set based on the position of the active site of the protein molecule and the area where it potentially acted on the ligand small molecule. Molecular docking was achieved using AutoDock Vina, whereby lower binding energy depicted a better affinity between the receptor and the ligand. The binding energy ≤0 kcal/mol indicated that the compound could bind and interact with the target, whereas the binding energy <−5 kcal/mol demonstrated a very strong binding force. The docking results with the best binding force between each core target protein and active compound were simultaneously presented in 2D images and 3D structures.The 2D structure of the potential compound was retrieved from PubChem (Rhizoma Atractylodis (Cang Zhu), 140 from Cortex Phellodendri (Huang Bo), 176 from Radix Vladimiriae (Niu Xi), and 38 from Semen Coicis (Yi Yi Ren). Besides, 52 active compounds were screened according to OB ≥ 30% and DL ≥ 0.18, including 4 from Rhizoma Atractylodis (Cang Zhu), 25 from Cortex Phellodendri (Huang Bo), 17 from Radix Vladimiriae (Niu Xi), and 6 from Semen Coicis (Yi Yi Ren). After eliminating repeated active compounds, 40 active compounds were obtained. From the TCMSP platform, 916 targets of the 40 active compounds were obtained, including 60 from Rhizoma Atractylodis (Cang Zhu), 430 from Cortex Phellodendri (Huang Bo), 384 from Radix Vladimiriae (Niu Xi), and 42 from Semen Coicis (Yi Yi Ren). After eliminating repeated targets, 203 targets and their abbreviations were obtained. Information of active compounds and the number of action targets of TCM in SMP are outlined in In total, 403 compounds of SMP were obtained from the TCMSP database, including 49 from The drug-disease intersection target  was obtained by intersecting the 203 drug targets with 1204 disease targets using R software, with 95 potential targets . NotablyWe drew and analyzed the potential targets of SMP in the treatment of AGA using Cytoscape 3.7.2 network drawing software. Then, a PAC-potential target network diagram of SMP for AGA treatment was constructed . The netAfter obtaining the potential target genes, they were imported into Cytoscape 3.7.2 software. We then drew the PPI network diagram using the Bisogent plug-in and obtained 5,264 nodes and 133,011 connections . For theP < 0.05 , etc.); 36 items via CC enrichment analysis ; 145 items via MF enrichment analysis . To draw the GO function histogram of SMP in AGA treatment, we selected the top 10 BP, CC, and MF results at P < 0.05 .To further elucidate the pathways regulated by the therapeutic target genes, we did a KEGG pathway analysis. Results revealed that these target genes were distributed in 155 pathways. To construct a histogram, the pathways with the top 30 enriched genes were selected . GA-relaUsing the AutoDock Vina software, PACs in the network map of “TCM-PAC-potential targets of AGA” were docked with five core target proteins  in PPI. A higher binding activity between the compound and the target protein receptor implied lower binding energy. Results demonstrated that the docking of 33 active compounds with the 5 core target protein receptors was less than 0 kcal/mol  . The molThe repeated attacks and protracted course of acute gouty arthritis can lead to joint swelling, deformity, and destruction of bone and joint cartilage. Tophi can be formed in many parts of the body, and it could seriously invade joints, kidneys, and even aorta and heart valves in severe cases –16. For Cortex Phellodendri (Huang Bo) and Radix Vladimiriae (Niu Xi), kaempferol is the active compound of Radix Vladimiriae (Niu Xi), wogonin is the active compound of Rhizoma Atractylodis (Cang Zhu) and Radix Vladimiriae (Niu Xi), baicalein is the active compound of Radix Vladimiriae (Niu Xi), beta-sitosterol is the active compound of Cortex Phellodendri (Huang Bo) and Radix Vladimiriae (Niu Xi), and rutaecarpine is the active compound of Cortex Phellodendri (Huang Bo). Among them, quercetin and wogonin are the common active components of two traditional Chinese medicines, which is similar to the compatibility and synergism of TCM compounds. Considering Semen Coicis (Yi Yi Ren) as an indispensable TCM in this prescription, the active ingredient stigmasterol, characterized by a great degree value in According to κB and some inflammatory factors, such as monocyte chemotactic protein-1 (MCP-1), interleukins, tumor necrosis factor, and cellular adhesion molecules. In addition, it also clears the reactive oxygen species (ROS) to show anti-inflammatory and antioxidant effects [κB and mitogen-activated protein kinase (MAPK) pathways in rutaecarpine-derivative R3 inhibits the NLRP3 inflammasome activation, which consequently lowers the expression of proinflammatory cytokine IL-1β [These PACs in SMP have a certain curative effect on inhibiting inflammation and lowering blood uric acid level . Among t effects . Moreove effects . Inhibitne IL-1β , 27. Thene IL-1β . In anotne IL-1β . Additione IL-1β , 31. Betne IL-1β , 33. Alsne IL-1β .β-induced chondrocytes [Herein, to obtain the core target proteins of SMP in the treatment of GA disease, the PPI network of intersecting target proteins was constructed. Also, we performed two topological screens on the SMP target for GA treatment based on DC, BC, CC, and EC. TP53, FN1, ESR1, CDK2, and HSPA5 were the five core target proteins. Based on the existing reports, TP53, also known as p53, contributes to transcriptional regulation of the cell cycle, DNA repair, cell survival, and cell metabolism . Inhibitdrocytes . The potdrocytes . The ovedrocytes . Cyclin-drocytes , and it drocytes  and inhidrocytes . Estrogedrocytes . In partdrocytes . Heat shdrocytes .Following the KEGG signal pathway analysis, the mechanisms of SMP in the treatment of AGA were mainly anti-inflammation and protection of the cartilage. The critical inflammatory signaling pathways included TNF and IL-17, whereas the apoptosis signaling pathways included p53 and HIF.α and IL-1β, which causes persistent gout episodes [β, TNF-α, and IFN-γ  and osteoarthritis (OA), which are hypoxia-associated diseases [α exerts a regulatory role in the function of immune cells. Activated LPS-induced macrophages can also express HIF-1α, which is crucial in glycolysis and the induction of proinflammation [The physiological dose of soluble uric acid exerts a specific protective effect on cartilage . Howeverdrocytes . Comparediseases , 55. HIFammation . The theAccording to the space matching and energy matching relationship between molecules, the core target protein of SMP in AGA treatment was docked with active compounds. This provides a reference for the subsequent research and development of targeted drugs. The 2D image of molecular docking depicts the binding mode between the core target protein and the compound as well as the interaction with the surrounding amino acid residues. Hydrophobic interaction best describes the main association of target protein TP53, FN1, and ESR1 with Inophyllum E. The target protein CDK2 residue Leu83 (A) forms a hydrogen bond with Inophyllum E, whereas the residues Asp34 (A), Lys96 (A), Thr37 (A), and Gly227 (A) of the target protein HSPA5 form five hydrogen bonds with baicalein. The main forces between ligand and protein are hydrophobic force and hydrogen bond, which both are chemical bonds with strong binding force. According to The present study systematically expounds the core compounds and molecular action mechanism of SMP in the treatment of AGA. The core compounds of SMP, including quercetin, kaempferol, wogonin, baicalein, and beta-sitosterol, were screened via network pharmacology, whereas the core targets of AGA including TP53, FN1, ESR1, CDK2, and HSPA5 were screened out via PPI. These core compounds are characterized by stable binding activity to the core targets. Also, SMP could regulate signal pathways related to AGA disease, for example, TNF, IL-17, p53, and HIF signaling pathways. These findings suggest the potential synergistic role of SMP in treating AGA via multiple components, multitargets, multiple biological functions, and multiple signal pathways.


Depression is a complex neuropsychiatric disease involved multiple targets and signaling pathways. Systems pharmacology studies could potentially present a comprehensive molecular mechanism to delineate the anti-depressant effect of emodin (EMO). In this study, we investigated the anti-depressant effects of EMO in the chronic unpredictable mild stress (CUMS) rat model of depression and gained insights into the underlying mechanisms using systems pharmacology and molecular simulation analysis. Forty-three potential targets of EMO for treatment of depression were obtained. GO biological process analysis suggested that the biological functions of these targets mainly involve the regulation of reactive oxygen species metabolic process, response to lipopolysaccharide, regulation of inflammatory response, etc. KEGG pathway enrichment analysis showed that the PI3K-Akt signaling pathway, insulin resistance, IL-17 signaling pathway were the most significantly enriched signaling pathways. The molecular docking analysis revealed that EMO might have a strong combination with ESR1, AKT1 and GSK3B. Immunohistochemical staining and Western blotting showed that 2 weeks’ EMO treatment (80 mg/kg/day) reduced depression related microglial activation, neuroinflammation and altered PI3K-Akt signaling pathway. Our findings provide a systemic pharmacology basis for the anti-depressant effects of EMO. Depression is a global health concern, affecting approximately 6% of the world population each year . As estiRheum officinale, known for its pleiotropic pharmacological actions, including anti-inflammation, anti-oxidant, anti-cancer and anti-virus effects [Emodin (EMO) is an anthraquinone isolated from  effects –14. Moun effects , 15–17.  effects . AlthougAs a powerful tool for drug discovery and development, systems pharmacology approach combines network biology and multipharmacology . To inveTo assess the anti-depressant effect of EMO, a CUMS induced depression model was employed –21. As sTo better understand the underlying mechanisms of depression, a total of 340 depression candidate targets were obtained from the Therapeutic Target Database (TTD) , GeneCarp < 0.01). The top 20 enriched KEGG pathways are presented in Furthermore, the enrichment analysis of KEGG pathways included 326 pathways (http://www.tcmip.cn/ETCM/) [http://lmmd.ecust.edu.cn/admetsar2/) [Compounds that complied with the requirements of Lipinski’s rule of five seem to be more likely to become drugs. SwissADME prediction , 25 shown/ETCM/) . In geneetsar2/)  showed tBy retrieving the Pathway Assembly from Literature Mining-an Information Search Tool (PALM-IST) database  and valiForty-three potential targets of EMO against depression were categorized into 6 different classes based on their cellular function, of which protein modifying enzyme  was the most enriched class . Among tTo obtain an overview of the 43 potential targets of EMO against depression, GO functional classification was investigated. Most of the potential targets existed on the cell and cell part with catalytic activity. Within the biological processes, the majority of potential targets were enriched in cellular process (GO:0009987), biological regulation (GO:0065007), response to stimulus (GO:0050896) and metabolic process (GO:0008152) . These rTo explore the interaction effect between 43 potential targets of EMO against depression, PPI analysis was performed using the STRING 11.0 database . In the The common targets were further analyzed for functional prediction by Metascape. The primary enriched GO biological process was positive regulation of transferase activity (GO:0051347), regulation of reactive oxygen species metabolic process (GO:2000377), cellular response to organonitrogen compound (GO:0071417), T cell activation (GO:0042110), negative regulation of cell differentiation (GO:0045596) and so on . The MolTo identify signaling pathways associated with targets of EMO against depression, Metascape was used to enrich pathways for these 43 potential targets. KEGG pathways mainly involved PI3K-Akt signaling pathway (hsa04151), longevity regulating pathway (hsa04211), insulin resistance (hsa04931), IL-17 signaling pathway (hsa04657), AMPK signaling pathway (hsa04152) and so on . DetaileMolecular docking analysis was used to validate the binding of EMO to core targets, and the lowest energy docking model was selected. Delta G is defined as the binding energy based on the ensemble free energy; the larger the absolute value of Delta G is, the more stable the binding. The molecular docking results of EMO to core targets are shown in Ligand–protein interactions were calculated using LigPlot . Figure Neuroinflammation is related to the pathogenesis of depression, and microglial activation is one of the characteristics of neuroinflammation , 32. MicWe further detected the cytokine levels in the PFC by ELISA, the DET+VEH rats had higher levels of interleukin-1β  and tumor necrosis factor α  than the CON+VEH rats , while DET+EMO rats had lower levels of IL-1β (57.4 ± 4.6 pg/mg/protein) and TNF-α (32.6 ± 3.7 pg/mg/protein) , 8D. Thep = 1.84E-19), and 16 target proteins were involved  could improve the depression-like behaviors of rats exposed to CUMS, as displayed by an elevated sucrose preference percentage in the SPT, an increased zone crossing in the OFT and a reduction of immobility time in the FST. It is worth noting that not all CUMS exposed rats appeared depression, and the incidences of depression were 41.54% and 46.15% in young female and male rats, respectively . RegrettThe PFC is a key brain region involved in the pathogenesis of depressive symptoms . DepressThe overexpression of GSK3β in neurons significantly promoted neuronal apoptosis . RegulatNeuroinflammation is strongly associated with the pathogenesis of depression, and anti-inflammatory agents may be a useful anti-depressant therapy or an adjuvant to the currently available conventional therapies . The levThe PI3K-Akt signaling pathway has been implicated in regulating the anti-depressant effects and plays an essential role in glutamate uptake, glutamate receptor trafficking, and synaptic neurotransmission –51. ThusAn important kinase downstream of PI3K is GSK3, which consists of the highly homologous GSK3α and GSK3β . The expEMO plays an anti-depressant role through a multi-target approach (including GSK3β and AKT1) compared with a single GSK3β inhibitor. Moreover, the PI3K-Akt signaling pathway also play a critical role on the neuroprotection and inhibiting apoptosis via enhancing the expression of SODs . EMO treThe gut-brain axis connects emotional and cognitive brain centers with gastrointestinal function and is associated with psychiatric disorders . DepressIn this study, EMO treatment for 2 weeks can significantly improve CUMS induced depression-related behaviors. We further employed a systems pharmacology strategy and molecular docking were used to uncover the multi-target mechanisms of EMO in depression treatment. A total of 43 targets of EMO against depression were screened out. PI3K-Akt signaling pathway and its downstream neuroinflammation and neuronal loss plays an important role in the anti-depressant effect of EMO. Although more studies are needed to confirm the current results, for the first time, the mechanisms of multi-target synergistic anti-depressant of EMO have been explored in a systemic approach. Taken together, our findings might provide a theoretical basis for the application of EMO as a therapeutic for depression.Emodin  was from Shanghai Base Industry . Carboxymethylcellulose sodium  was provided by Sinopharm Chemical Reagent Co. Ltd. . EMO was dissolved in 0.5% CMC-Na before use. All sandwich enzyme-linked immunosorbent assay (ELISA) kits such as for IL-1β (Cat# E-EL-R0012c), TNF-α (Cat# E-EL-R2856c) and SOD (Cat# E-BC-K020-M) were from Elabscience Biotechnology . Anti-Iba1  antibody was purchased from Wako . Anti-β-actin  antibody was obtained from Proteintech . Total glycogen synthase kinase 3β  and phosphorylated GSK3β , total Akt  and phosphorylated Akt , and total MAPK ERK1/2  and phosphorylated ERK  antibodies were purchased from Cell Signaling  and diluted 1:1000 for Western blotting. Anti-rabbit (Cat# 926-32210) or anti-mouse IgG (Cat# 926-32211) conjugated to IRDye® 800 CW used in Western blotting, was purchased from Li-Cor Bioscience .https://www.genecards.org/), TTD (http://db.idrblab.net/ttd/) [https://rgd.mcw.edu) using “depression” as a keyword, and duplicate targets were removed using Microsoft Excel software . The organism was set to Homo sapiens in the Rat Genome Database. Specifically, the top 300 relevance score target genes were selected for further study in the GeneCards database.Disease-related targets were screened by the GeneCards database (et/ttd/)  and the in vivo absorption abilities of the designed compounds [http://www.swissadme.ch) [Lipinski’s rule of five was used to assess the ompounds , 83. Speadme.ch) , 25 was http://www.hpppi.iicb.res.in/ctm/) [https://pubmed.ncbi.nlm.nih.gov/). The intersection of EMO-related targets and depression-related targets was analyzed by Venny 2.1 (https://bioinfogp.cnb.csic.es/tools/venny/index.html), and the common targets were the anti-depressant target of EMO. In addition, potential target proteins were categorized by the Panther classification system (http://pantherdb.org/) [Potential targets of EMO were collected from the PALM-IST (in/ctm/)  and validb.org/) .https://string-db.org/) [Homo sapiens, and only the minimum required interaction score > 0.4 was chosen as significant. The PPI network comprises nodes, which represent a target protein, and edges, which represent protein-protein interactions. The thickness of the edges represents the combined score. Degree represents the number of other nodes directly connected to a node. The higher the value of degree, the more important the node becomes. The core targets were identified through network analysis using Cytoscape software (v.3.7.1) [The PPI network was constructed by using the STRING database (db.org/) . The orgv.3.7.1)  and its https://metascape.org/gp) [p < 0.01, minimum count 3, and an enrichment factor > 1.5. Moreover, the MCODE algorithm has been applied to identify highly interconnected clusters and the criteria were set as follows: degree cutoff = 2, node score cutoff = 0.2, k-core = 2, and max depth = 100. The top 20 enriched terms were visualized using an online tool (http://www.bioinformatics.com.cn). Based on the results of KEGG pathway enrichment analysis, a KEGG pathway-target network diagram of EMO treatment of depression was created by Cytoscape software.GO biological process and KEGG pathway enrichment analyses were performed using Metascape (.org/gp) . The thrhttps://pubchem.ncbi.nlm.nih.gov/). Structure files of target proteins were obtained from the RCSB Protein Data Bank  [http://www.swissdock.ch/docking) [https://www.ebi.ac.uk/thornton-srv/software/LIGPLOT/) [To validate the binding of EMO to predicted core targets, the 3D molecular structure of EMO was downloaded from the PubChem database (sb.org/) . The Swidocking)  was usedIGPLOT/) .Male Sprague-Dawley rats  were from the Experimental Animal Central of Tongji Medical College, Huazhong University of Science and Technology (License No. SCXK-E 2016-0009). Rats were maintained in 12-hour-light/-dark cycle. The animal house was kept at a constant relative humidity (55 ± 15%) and room temperature (22 ± 2° C). Ethics approval was received from the Animal Care and Use Committee of Huazhong University of Science and Technology .The CUMS procedure was carried out as previously reported –21. EighThe SPT is a classic method for detecting the loss of pleasure due to depression . The SPTThe zone crossing times in OFT and the total duration of immobility in FST were recorded.Rats were sacrificed under isoflurane  anesthesia. The brain tissues of PFC were rapidly harvested and homogenized on ice. Western blotting was performed as previously described , 19, 20.The levels of IL-1β, TNF-α and SOD in the PFC were detected using ELISA kits, and the procedures were conducted strictly according to the instructions. The optical densities were measured at 450 nm using a BioTek Synergy 2 microplate reader .Animals were sacrificed through an over-dose of isoflurane. Immunohistochemical staining and Nissl staining were performed according to our previous studies , 20. Allp < 0.05.Data were expressed as means ± SEM. Graphic plots were presented by GraphPad Prism . SPSS 19.0 statistical software  was used for statistical analysis. One-way ANOVA procedure followed by Tukey’s multiple comparisons test was used for multiple comparisons. Statistical significance level was set at


Resveratrol is synthesized by the catalysis of resveratrol synthases (RS) in a limited number of higher plants. Resveratrol shows potential health-promoting properties, including as an antioxidant and in preventing cardiovascular diseases. Recently, resveratrol-enriched rice has been produced as a novel source of resveratrol. This study aimed to investigate the major agronomic characteristics of resveratrol-enriched rice, Iksan526 (I526) and compared them with those of a nontransgenic and commercial rice variety, Dongjin (DJ). Transgene (RS) integration was confirmed using Southern blot analysis, and homologous recombination was achieved after digestion with the SacI restriction enzyme. The phenotypic traits of I526 grown in Iksan were similar to those grown in Milyang but not similar to those grown in Suwon. In Suwon, I526 had slightly earlier heading dates [i.e., number of days from sowing to heading) and shorter culm lengths. When I526 was treated with 0.4% Basta in the seedling stage, no significant difference was observed among all the agronomic traits compared with nontreated I526; particularly, the culm length, panicle length, number of panicles per hill, 1,000 grain weight of brown rice, and brown rice yield of the Basta-treated rice were similar to those of the nontreated I526, regardless of their cultivation region. The resveratrol content of I526 grown in Suwon and Milyang was increased by 18% and 37%, respectively, than that of I526 grown in the Iksan area. Therefore, DJ and I526 are not significantly different in terms of major agronomic traits depending on variety/year and variety/cultivation region. The results indicated that I526 has the potential to become a commercialized variety in the near future. Staphylococcus gene to colon Bacillus, recombinant DNA technology can currently be used in microorganisms, animals, and plants. Since the commercialization of genetically modified (GM) herbicide-resistant beans in 1996, 526 types of events for 32 crops have been approved in 44 countries as of 2018. The region or area for the cultivation of GM crops across the globe has increased from 1.7 million ha in 1996 to approximately 191.7 million ha in 2018, which is equivalent to an increase of approximately 113 times. This region accounts for approximately 12.8% of the total 1.5 billion ha cultivation region across the globe. The aggregated cultivation region of GM crops with respect to countries is as follows: 71.5 million ha in the United States of America (US), 40.3 million ha in Brazil, 24.4 million ha in Argentina, and 1.10 million ha in India . Moreover, eight countries have a cultivation region of <50,000 ha. According to International Service for the Acquisition of Agri-biotech Applications, 18 million farmers in 26 countries cultivate GM crops and 16.5 million farmers (approximately 90%) have small-scale farms in developing countries. Recently, China and India are beginning to ease restrictions on the cultivation and import of GM crops to address their food shortage issues. In addition, five countries in the European Union, including Germany, have also initiated GM crop cultivation. As such, the governments of these countries have recently begun to allow the cultivation of GM crops despite opposition from consumers .Owing to the successful experiments of,2 and recently, vitamin A-enriched rice (golden rice) has been approved for commercial production in the Philippines. Moreover, research teams in Germany and India have developed GM rice showing a 22% increase in flavonoid content through the overexpression of anthocyanin synthase.3 In 2009, the Chinese Academy of Agricultural Sciences developed two kinds of rice, insect-resistant (Cry1Ac) Huahui-1 Bt rice and Shanyou-63 Bt rice; they also developed a marker-free event through crossbreeding after co-transforming a target gene and a selectable marker.4 In addition, in Switzerland, a new variety of rice containing six times more iron than existing varieties was developed by introducing two new genes.5GM rice, the first crop approved for commercialization, is a herbicide resistant rice variety approved by the US government in 1999,7 Resveratrol shows strong antioxidant activity and is being used in the treatment of diseases such as colon cancer, oral herpes, and ulcers. Moreover, its pharmacological efficacy in terms of health-promoting properties, such as the anticancer, antihyperlipidemic, and anti-inflammatory properties, has been reportedly observed to be effective for conditions including seizures and cardiovascular diseases.8–11 Recently, Iksan526 (I526), a resveratrol-enriched rice variety, was developed for the first time using the resveratrol synthase (RS) gene, I526 originates from Dongjin (DJ) rice, which is the most wildly cultivated rice variety in Korea. The RS gene used in the development of I526 is derived from Palkwang, a Korean peanut variety. Animal studies using mice models indicated that I526 is efficient in preventing metabolic syndromes and is effective in the treatment of conditions such as obesity, hyperlipidemia, and high cholesterol. 13Resveratrol is a phytoalexin substance with defensive properties that is produced in plants after exposure to strong ultraviolet (UV) rays or gray mold.14 To cultivate GM rice for commercial purposes, productivity and the surrounding environment should be considered and evaluated before being approved for commercial use. Thus, it is important to assess the conditions of the cultivation area and agronomic traits of transgenic crop by comparison with its wild-type. Transgenic crop adaptation and transgene expression stability should also be verified. A potential crop base is generally evaluated for its agronomic traits to select crop types that can grow for a long term in an agricultural system. Environmental interactions that affect the genotype, which are assessed in field experiments in different locations and years, are also important in crop improvement programs.15Genetic engineering is a tool for developing plants that produce specific metabolites as a novel source in functional food and in the pharmaceutical industry. In the past, GM rice was developed for improving the nutritional value of rice. Although GM rice is not approved for consumption in some countries, there is no evidence that GM rice consumption is harmful to humans.This study aimed to compare the major agronomic traits between I526 and its non-transgenic parent, DJ, in terms of cultivation year and region. This research was conducted to obtain data that can be used for the commercialization of resveratrol-enriched rice as a new pharmaceutical material.The genetic stability of RS gene in I526 was verified using Southern blotting following the method described by.17I526 and DJ rice were cultivated in GM organism-confined fields in the Suwon, Iksan, and Milyang regions of the National Institute of Crop Science from 2013 to 2015, with a planting distance of 30 × 15 cm. In addition, N–P–K at 9–4.5-5.7 kg/10a was used as fertilizer; all other cultivation conditions followed here were in compliance with the standard rice cultural practices of the Rural Development Administration. The investigation of major agricultural traits, including growth analysis  and yield response , was conducted following the standards of study, investigation, and analysis of Agricultural Science Technology of Rural Development Administration.TM, Bayer AG, Germany) at 2 weeks after planting. The major agronomic traits and heading dates  of transgenic DJ526 were observed by comparing the nontreated (I526) and glufosinate ammonium-treated (I526B) I526 rice. The weather information service  of Rural Development Administration was used to investigate the heading dates in the three regions.Transgenic rice was screened for the transformation of the RS gene by applying 0.4% glufosinate ammonium  equipped with stationary ACQUITYTM UPLC BEH C18 column  and a UV detector . Acetonitrile  and 0.1% formic acid (B) in water were maintained for 10 min at a ratio 10:90 (A:B), for 12 min with the ratio changed to 15:85 and changed to 25:75 for 0.4 min. Subsequently, the ratio was maintained at 90:10 for 0.6 min and rebalance the column at 10:90 for 1 min. The UV wavelength of the detector was set to 308 mm.The seeds were harvested from genetically modified organism-confined field trials in three locations, Suwon, Iksan, and Milyang. After dehusking seeds with mill grinder , fine powdered brown rice (300 mg) was used to examine resveratrol content; 80% methanol (90 µL) was added to the fine powder and the mixture was sonicated  at room temperature for 30 min. The extracts were centrifuged at 10,000 × Data were analyzed using ANOVA , and the mean of agronomic traits and yield trait in the three regions during the 3 y of investigation were compared using the least significant difference test (LSD) at 5% and 1% probability levels.In total, ten putative I526 plants were analyzed for the RS gene using Southern blotting hybridization. The genomic DNA of nontransgenic DJ plants was used as negative control (N) . A signaThe variation in heading dates of I526 and DJ rice by cultivation year and region is presented in 0.05 = 0.84). Further, no significance differences were noted in growth and yield characteristics between the Suwon and Iksan regions. Therefore, it was confirmed that Basta-treated and nontreated I526 showed no differences in terms of growth and yield characteristics.Analysis the heading dates of Basta-treated and nontreated I526 plants revealed that the heading dates of the two groups in the Iksan region were similar (0.4 d earlier in Iksan region). Further, there was no difference in the heading dates of the two groups in the other two regions , implyinThe evaluation of grain yield production of I526 and DJ rice by cultivation region and years is presented in The present study confirmed that I526 produces resveratrol glycoside (piceid) and trans-resveratrol with the retention times 7.4 and 13.6 min, respectively . I526 frI526 was produced from the original nontransgenic DJ rice with the aim of improving rice quality by establishing a resveratrol-containing rice variety via genetic modifications. Determining the adaptability of transgenic plants is crucial to control yield stability, and it is beneficial to manage genetic resources in sustainable agriculture. To confirm the uniformity and stability of the integrated DNA in transgenic plants, Southern blotting is generally used. Using this technique, it was determined that the RS gene was present only in I526 plants, indicating that the transformation was successful in putative I526 plants.18 DJ is a mid-to-late flowering rice cultivar that is generally cultivated in the Korean peninsula . Based on the results, mid-to-late August, which is the summer season in Korea, is suitable for heading of both DJ and I526 rice. Heading date or flowering time in rice is an important factor for breeders to evaluate environmental adaptability in a cultivation region. Information on heading dates helps breeders in cultivation management of future crop and improve crop yield.20 Both environmental and genetic factors have been reported to influence heading dates in rice. The two major environmental factors, i.e., photoperiod and temperature, are responsible for heading time.21 Therefore, changing the cultivation region can result in differences in climatic zones, which can induce earlier flowering in temperate japonica rice. Genetic factors have also been reported to affect heading dates; however, their function remains unclear.22 Our results show that DJ rice has earlier heading dates than I526 rice in three regions; this effect is supposedly controlled by a gene in the two varieties. Genes that regulate heading dates play a key role in determining rice crop yield. The genetic mechanism underlying heading dates in rice is complicated and is controlled by a combination of photoperiod and circadian clock in addition to regulatory epigenetic mechanisms.24One of important agronomic traits of rice is heading dates, which is related to the attributes of regional adaptability and yield.25 In Korea, higher production of both DJ and I526 rice was seen in the peninsula plain, including the Iksan and Milyang regions, than in with the central plain, including the Suwon region. However, the different cultivation regions did not affect the yield characteristics of I526 after treatment with Basta, indicating that I526 exhibits good geographical adaptation exhibiting better adaptation to multiple regions. However, the cultivation region and year are associated with environmental factors. Based on varieties, it is indicated that environmental factors also play a role in the expression of some agronomic traits (panicle-related attributes) in DJ and I526 rice cultivars.Because the yield characteristics of I526 and DJ rice in multiple environments did not differ during the 3-y study period, I526 can be commercialized in the market in a manner similar to that of DJ rice. Field-testing examinations of the transgenic crop reveal its agronomic performance, thus providing an opportunity to grow the plant in a local environment.26 In contrast, other sources, such as grapes, had varied total resveratrol contents ranging from 0.16 to 3.54 μg/g.27 Changes in resveratrol can be a consequence of glycosylation during growth and development stages. Some elicitors, such as yeast extract, upregulate glycosylation, thereby increasing the levels of piceid glycoside.29 The concentration of resveratrol varies on the basis of several factors, including weather conditions. In this study, drastic temperature changes or relatively high temperatures during the heading stage of rice resulted in increased resveratrol content in I526 rice. Among the cultivation regions in this study, I526 showed variation in resveratrol content, with the rice grown in Milyang showing the highest content. The difference in resveratrol contents of I526 among the three cultivation regions proved to be an interesting feature that must be analyzed for selecting the optimal cultivation region suitable for improving the resveratrol content of the crop in the future.The average total resveratrol content in I526 was 2.1 μg/g, which is similar to that in red wine (2.57 mg/L).Southern blotting of transgenic I526 confirmed that the RS gene was successfully transformed in the target plant. The cultivation year and region of the two rice varieties, DJ and I526, affected some agronomic traits , considering the interaction between different years and varieties. The herbicide Basta did not have any major effects on the agronomic traits, particularly those related to the growth and yield of I526 rice, across the three regions. Based on the data of agronomic traits during 3 y of observation in three different regions, I526 has the potential of being used as a commercialized variety in the near future due to its good yield stability and regional adaptability.