The clinical efficacy of Chansu injection for COVID-19 treatment has been confirmed. Its mechanism of action remains unclear. We used network pharmacology and molecular docking technology to explore the potential material basis and mechanism of action of Chansu injection for COVID-19.  The main components of Chansu injection were determined using HPLC. The PharmMapper, SwissTargetPrediction, SEA, and TCMID databases were used to screen for the active ingredients and therapeutic targets of Chansu injection, while the OMIM and GeneCards Suite databases were used to search for COVID-19-related targets. The STRING database was used for protein-protein interaction (PPI) network construction and topological analysis, while DAVID was used for Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses of the core targets. The main active compounds of Chansu injection were docked with 3CL protease, ACE2, RdRp, and spike protein.  The three Chansu injection compounds were identified using HPLC. A total of 236 drug-related targets and 16,611 disease-related targets were identified, and 77 common targets were determined through mapping. The PPI mapping results revealed that 16 core targets were obtained through topological analysis and screening. Furthermore, GO and KEGG pathway enrichment analyses revealed that the PI3K and JAK-STAT signaling pathways are the major pathways. The molecular docking results suggest that the three Chansu injection components have high binding energies to the S protein.  The potential mechanism of Chansu injection for COVID-19 involves multiple targets and pathways, thereby providing a scientific basis for its clinical application and further research. Coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged in China in November 2019 . This isβ-subtype coronavirus [SARS-CoV-2 is the seventh coronavirus found to infect humans. Similar to SARS-CoV and MERS-CoV, SARS-CoV-2 belongs to the onavirus . Its genonavirus , 12. It onavirus , 14. Owionavirus .Although inactivated viral vaccines, adenoviral vector vaccines, recombinant protein vaccines, and mRNA vaccines have been intensively developed in various countries, they are also in use and have achieved good efficiency indicators . HoweverHowever, to date, no medicines specific for coronavirus treatment are available. Before the COVID-19 pandemic in China, the coverage and effectiveness of Chinese medicines were greater than 90% . ResultsBufo bufo gargarizans. Chansu injection is a prescription Chinese medicine, its main component is a derivative of indole base, and this product is a colorless to light yellow clear liquid, often used for clearing heat and detoxifying effect. It fights inflammation and infection and regulates immunity [Chansu is obtained from the excreta of the skin and postauricular glands of immunity , 21. Chaimmunity , 23. In immunity .Network pharmacology is an emerging discipline that integrates the technology and content of polypharmacology, computational biology, and network analysis . Becausehttps://lilab-ecust.cn/pharmmapper/), Universal Protein  database, similarity ensemble approach  database, Swiss database , STRING (https://string-db.org), the Database for Annotation, Visualization, and Integrated Discovery , GeneCards database platform (https://www.genecards.org), Comparative Toxicogenomics Database , and Therapeutic Target Database  were used.PharmMapper (μm) and the mobile phase containing acetonitrile and 0.1% ammonium acetate solution (50 : 50). The detection wavelength was 296 nm. Theoretical plate counts were calculated according to the bufalin level peak, which was not less than 3,000. To prepare the control solution, an appropriate amount of bufalin, resibufogenin, and cinobufagin was calibrated precisely and 1.6 μg each of bufalin, resibufogenin, and cinobufagin were added to 1 mL of 50% acetonitrile aqueous solutions. The control solution (20–50 μL) and 50 μL of the test samples were loaded into high-performance liquid chromatography (HPLC) equipment, and the calculations were derived with an external method. Each 1 mL of sample should contain 1.2–4.8 μg of bufalin (C24H34O4), cinobufagin (C26H34O6), and resibufogenin (C24H32O4).The Chansu injection sample was supplied by the Pujin Pharmaceutical Cooperation . The chromatographic conditions and system compatibility tests included the Agilent Zorbax SB-C18 chromatographic column . The crosstalk targets were considered potential targets for COVID-19 treatment with Chansu injection.A medicine-target-disease network diagram was prepared by introducing the acquired information of crosstalk targets and active ingredients into the Cytoscape software (v3.7.2). Thereafter, a topological analysis was performed.Homo sapiens. The free targets were deleted, and the resultant file, string_interactions.tsv, was introduced into the Cytoscape software for topological analysis. Core targets were determined according to the degree and centrality of the intermediate value.The acquired crosstalk targets were recorded in the STRING database using the genus The function of the core targets was described and annotated to investigate its pathway. Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) enrichment analyses were conducted using the DAVID database. For GO enrichment analysis, molecular function (MF), biological process (BP), and cellular component (CC) were used to confine and describe genes. The potential signaling pathway for COVID-19 treatment using Chansu injection was determined through KEGG enrichment analysis.The molecular structure of the active ingredients of Chansu injection was downloaded from PubChem, while those of 3CL protease, ACE2, RdRp, and spike protein were downloaded from the PDB database. The data were processed using MGLTools 1.5.6 and saved as a pdbqt file. The active sites and grid box coordinates of 3CL protease, ACE2, RdRp, and spike protein were defined according to their built-in ligands. The size of each box was 30 × 30 × 30 grids. Small-molecule proteins were docked using AutoDock Vina 1.1.2; molecular structures were stereotyped using PyMOL; and the top scoring conformations were mapped and analyzed using Maestro 11.9.https://www.rcsb.org), and we selected the 3D structures of RdRp (PDB ID: 6ld3), 3CL (PDB ID: 6LU7), ACE2 (PDB ID: 1R42), and spike (PDB ID: 6acc) were used as the proteins for docking in this project. Both proteins and compounds were converted to PDBQT format using AutoDockTools 1.5.6 [We used ChemBioDraw Ultra 17.0 to draw the structures of compounds, which were then converted to 3D structures using ChemBio3D Ultra 17.0 and optimized using MMFF94 force fields. The 3D structures of RdRp, 3CL, ACE2, and spike were downloaded from the RCSB Protein Data Bank . When the potential targets of Chansu injection were mapped to disease-related targets, a total of 77 crosstalk targets were identified. In particular, COVID-19 was found to have 96, 99, and 92 crosstalk targets with bufalin, cinobufagin, and resibufogenin, respectively .In the medicine-target-disease network diagram , the outHomo sapiens. Four free targets were deleted, and the resultant file “string_interactions.tsv” was introduced into the Cytoscape software (v3.7.2) to draw the protein-protein interaction (PPI) diagram. Thereafter, a topological analysis was performed. The nodes with retention value and the centrality of intermediate values above the mean values are the core targets  that also has Ser/Thr kinase and PIK activities, which participate in the regulation of various cellular functions, such as proliferation, differentiation, and glucose transportation , 42. TheκB (NF-κB), reduce the expression of inflammatory cytokines such as interleukin-6 (IL-6) and tumor necrosis factor alpha (TNF-α), inhibit the cascading reaction of an inflammatory outburst, and promote the absorption of inflammatory cells and tissue recovery.Iranian scholars found that viruses depend on two types of receptors to enter cells: ACE2 and CD147 [γ and IL-3) and inhibit virus duplication and amplification. The Mpro in SARS-CoV-2 could promote autophagic degradation of the transcriptomic factor STAT1 to weaken the entrance of STAT1 into the nucleus. This process inhibits the activity of type I interferon cells, reduces the expression of the downstream active ingredient of the JAK-STAT pathway, and helps COVID-19 patients acquire a natural immune escape.The JAK/STAT signaling pathway plays a crucial role in the regulation of cell growth, survival, and pathogen resistance –51. ProfA study  by June'In summary, in this study, we determined the binding efficiency of Chansu injection to the main protein of the novel coronavirus and explored the functioning mechanism of this Chinese medicine for COVID-19 treatment using network pharmacology and molecular docking based on highly effective clinical studies. The results showed that Chansu injection could regulate virus duplication, activate immune cells, and alleviate inflammatory responses through the PI3K-Akt and JAK-STAT signaling pathways. The Chansu injection treats patients with COVID-19 by acting on SARS-CoV-2 and ACE2. The results of this study provide theoretical and experimental evidence for COVID-19 treatment using Chansu injection.


Solanaceae family, i.e. SolanaCyc and the genus Nicotiana, i.e. NicotianaCyc as well as six species-specific databases for Nicotiana tabacum TN90, N. tabacum K326, Nicotiana benthamiana, N. sylvestris, N. tomentosiformis and N. attenuata. New pathways were created through the extraction, examination and verification of related data from the literature and the aid of external database guided by an expert-led curation process. Here we describe the curation progress that has been achieved in these databases since the first release version 1.0 in 2016, the curation flow and the curation process using the example metabolic pathway for cholesterol in plants. The current content of our databases comprises 266 pathways and 36 superpathways in SolanaCyc and 143 pathways plus 21 superpathways in NicotianaCyc, manually curated and validated specifically for the Solanaceae family and Nicotiana genus, respectively. The curated data have been propagated to the respective Nicotiana-specific databases, which resulted in the enrichment and more accurate presentation of their metabolic networks. The quality and coverage in those databases have been compared with related external databases and discussed in terms of literature support and metabolic content.Manually curated metabolic databases residing at the Sol Genomics Network comprise two taxon-specific databases for the  High-quality databases that store, manage and make large datasets readily accessible to a broad variety of biological analyses have taken a central place in bioinformatics , 2. The A number of metabolic databases have been established addressing this growing need for repositories that host and manage and curate high-quality data published by the scientific community. Such databases provide not only an overview of the latest state in the respective research field, but also offer the tools to supplement experimental findings with bioinformatics analyses. A broad variety of such databases with a different focus and functionality exist. Human metabolic databases such as Reactome  and Recohttp://www.plantcyc.org/) is a kingdom-level database, which contains the metabolic information from more than 350 plant species and is used, besides MetaCyc, as a supplementary reference database for the prediction of more plant-specific databases in the Plant Metabolic Network (PMN) [https://solgenomics.net/) [https://solgenomics.net/pages/solcyc/) and comprises, among others, the family-specific SolanaCyc database, containing all experimentally verified metabolic data of the Solanaceae family and the genus-specific NicotianaCyc database, which holds the corresponding metabolic information for the Nicotiana genus [MetaCyc is the principal pathway database, which contains all the reference pathways used for predicting metabolic networks for organisms with annotated genomes . Severalrk (PMN) . The latcs.net/) , 29 on tna genus .Metabolism is fundamentally important for understanding life processes. Numerous enzymatically catalyzed reactions convert a multitude of compounds within a defined network of primary and specialized metabolites that is similar but not identical between plant species. PMNs have evolved over time into a complexity unrivaled among any organisms . Those nAbies grandis is able to synthesize 52 sesquiterpene olefins [One of the reasons for the huge diversity of specialized metabolites lies in the multi-specificity or even promiscuity of the catalyzing enzymes , 41, whi olefins , and the olefins . The effhttps://solgenomics.net/pages/solcyc/). We added one more Nicotiana-specific database for Nicotiana attenuata to the SolCyc database collection, which brings the count of manually supervised databases under the umbrella of SolCyc to two taxon-specific databases, i.e. SolanaCyc and NicotianaCyc, and six Nicotiana-specific databases for N. tabacum TN90, N. tabacum K326, N. sylvestris, N. tomentosiformis, Nicotiana benthamiana and N. attenuata. Using the cholesterol biosynthesis in plants as an example, we illustrate curation progress made on pathways and, consequently, the associated complementation of the inherent metabolic grid, which extends toward related pathways such as the biosynthesis of the steroidal glycoalkaloids solasodine and alpha-tomatine. We also discuss the curation flow and exchange of data with the reference database MetaCyc, look at pathway curation quality and enzyme coverage between in-house and external databases and compare the metabolic networks of the Nicotiana-specific databases. The curation effort in our databases has contributed to a more complete, enriched and accurate representation of their metabolic networks and will continue to enhance the range and pathway predictability of MetaCyc to which all pathways will ultimately be submitted.In this publication, we describe the progress made in the manual curation of SolCyc databases (N. tabacum accession TN90 (GCA_000715135.1), N. sylvestris (GCF_000393655.1) and N. tomentosiformis (GCF_000390325.2) were downloaded from National Center for Biotechnology Information (NCBI) assembly (https://www.ncbi.nlm.nih.gov/assembly) [A. Bombarely, unpublished results, Nicotiana genomes using Maker-P [N. tabacum, N. sylvestris and N. tomentosiformis genomes, respectively. Functional annotation was performed searching annotated proteins by sequence similarity using BlastP  of the coding protein genes with the GenBank NR, TAIR10 and SwissProt databases (downloaded on 21 July 2014). Additionally, the protein domains were annotated using InterProScan. Functional annotations were integrated using the program AHRD v2.0.2 (https://github.com/groupschoof/AHRD).The assemblies for  Maker-P  with theN. attenuata assembly and annotation were used as provided by the N. attenuata sequencing project [N. benthamiana database was built with the N. benthamiana genome version 1.0.1 [The  project . The N. on 1.0.1 .Nicotiana species has been carried out, resulting in adding appropriate new pathways and removing incorrectly predicted pathways. A blacklist of known false-positive pathways generated from previous annotation efforts [Nicotiana-specific databases during the confirmation process to correct the predicted metabolic network of the particular species.The setup of PGDBs has been described in more detail previously . In shor efforts  was applhttp://bioinformatics.psb.ugent.be/webtools/Venn/).Figures and tables were created with the 2016 version of Microsoft Word, Excel or PowerPoint and saved in pdf and jpeg format. The Venn diagram was generated using an online tool at the Bioinformatics & Evolutionary Genomics webpage  [https://scholar.google.com/) are valuable resources for finding peer-reviewed original publications and review articles, which allow the retrieval and aggregation of new data that are then used for the design of a temporary pathway layout. In phase II the study of the more specific literature and tapping into external databases provides information about reactions, enzymes, genes and compounds. For the validation of chemical compound structures various resources such as ChEBI [Our main databases for curating pathways are the taxon-specific databases SolanaCyc and NicotianaCyc. In these databases, the creation of new and the revision of existing pathways take place, which subsequently are propagated to the corresponding organism-specific databases for  species . The cur/pubmed)  and Googas ChEBI , ChemSpias ChEBI , 59 or Pas ChEBI  are usedNicotiana-specific database, genes and enzymes with sequence information are blasted against the database and merged with the matching predicted counterparts of that database.For the final draft of the newly created pathway, MetaCyc is checked for existing reactions, which, if needed, are exported into the databases. New reactions are created in the curation databases SolanaCyc and NicotianaCyc, where also the final assembly of the pathway takes place. Published data for enzymes and genes obtained from the literature are cross-checked with external databases, for example GenBank  and UniPNicotiana species, which explains the overall higher numbers for the increase of metabolic data accrued in NicotianaCyc. A special effort was made in the curation of transporters responsible for the uptake of heavy metals, nutrients and specialized metabolites and their distribution within plants, which is reflected in the high number of curated transporters. Heavy metals are systemic toxicants and classified as known or probable human carcinogens [The SolanaCyc database was the first manually curated database created at SGN using the Pathway Tools suite software package , 14 and cinogens . Heavy mcinogens . Plants cinogens . Both asNicotiana-specific enzymes in SolanaCyc increased from 12.4 to 41.2%. In other words, although in version 1.0 about every 8th curated enzyme was contributed by Nicotiana species, version 2.6 shows that every 2.4th enzyme now belongs to Nicotiana-curated enzymes.The cumulative statistics for each release, for both curated pathways and enzymes in SolanaCyc and NicotianaCyc, are shown in N. tabacum, followed by Solanum lycopersicum pathways with a rise of 59.8%. Also, the increase of curated pathways associated with a number of economically important species gathered in the SolanaCyc database such as S. tuberosum (44.6%), Petunia x hybrida (52%) and Capsicum annuum (50%) is significant. Among the Nicotiana species, 20 new pathways have been added to the species-specific database of N. benthamiana and 13 new pathways to the newly created PGDB of N. attenuata. Moreover, the coverage of species in the two taxon-specific databases has also been significantly improved. Previously, 27 species were covered, which comprised 9 genera in SolanaCyc, and the 8 species recorded in NicotianaCyc in version 1.0 [The comparison of curated pathways between 2016 and 2019  shows thsion 1.0 ; these nNicotiana is demonstrated in N. tomentosiformis, a species for which no published pathway could be identified, the remaining 5 Nicotiana-specific databases were extensively curated and can, according to the BioCyc definition of curated databases, be considered as a Tier 1 category database [N. tabacum databases of TN90 and K326 for the version 1.2 and for the remaining Nicotiana databases in version 1.4. This is related to the fact that at those times, a blacklist of metabolic pathways was applied, containing pathways that had been previously found to be invalid for solanaceous species. Although those pathways had been predicted by Pathway Tools using MetaCyc as reference, 156 pathways were invalidated as they have not been reported to occur in N. tabacum and other members of the Solanaceae family [The impact of manual curation on the species-specific databases of database . The coue family . MetaCycSolanaceae and one lacks a key enzyme that is only found in lower photosynthetic organisms, but not in the highly evolved members of the Solanaceae family.The recent curation process for the SolCyc databases (version 2.6) includes updating this blacklist of invalid pathways, supplemented by new pathways from MetaCyc that were erroneously imported during the automated prediction process. Out of 27 new pathways, 7 were declared invalid, 5 are specific to bacteria or Mammalia, one produces a specialized metabolite not present in N. tabacum TN90 and K326, as well as for N. benthamiana. The progress of adding pathways to the N. attenuata database is not so obvious, due to the fact that N. attenuata specific pathways had already been curated before the creation of the database and deposited in NicotianaCyc. When the genome of N. attenuata became available [vailable , the corN. benthamiana [https://metacyc.org/META/NEW-IMAGE?type=PATHWAY&object=PWY18C3-1) and SolCyc databases (http://solcyc.solgenomics.net/SOLANA/NEW-IMAGE?type=PATHWAY&object=PWY18C3-1).In contrast to animals, plants generally contain only diminutive amounts of cholesterol of about 1–2% of total sterols. As a consequence, the pathway was well characterized in animals but poorly understood in plants . Howeverthamiana . It was thamiana . That wathamiana , 73. At thamiana , 75. ThiViridiplantae. To our knowledge, with the exception of enzymes and coding genes characterized in S. lycopersicum and N. benthamiana, no reports of enzymes from other plant species involved in the cholesterol biosynthetic pathway have been published. This is not entirely surprising since cholesterol is the starting substrate from which steroidal glycoalkaloid pathways such as the solasodine (http://solcyc.solgenomics.net/SOLANA/NEW-IMAGE?type=PATHWAY&object=PWY18C3-2) and alpha-tomatine (http://solcyc.solgenomics.net/SOLANA/NEW-IMAGE?type=PATHWAY&object=PWY18C3-3) biosynthesis are derived [Solanaceae family. Further research into cholesterol biosynthesis in plants outside the Solanaceae family is required to assess the general validity of the metabolic pathway in the plant kingdom or the classification of this metabolic pathway as a family or species-specific variant for the Solanaceae.Despite earlier beliefs that cholesterol may not be a plant metabolite, free cholesterol and cholesterol esters have been detected in leaves and organelles of various plants , a findi derived , 73 and Many of the identified cholesterol biosynthetic enzymes were shown to behave in a promiscuous way and are equally active in related pathways such as the C-24 alkylsterol biosynthesis. Enzyme promiscuity is often a hallmark of the formation of specialized metabolites. The operation of enzymes catalyzing reactions in both cholesterol and C-24 alkylsterol pathways agrees with the evolutionary ‘patchwork’ hypothesis, which proposes that duplication and diversification events propel the evolution of ancestral enzymes with low substrate specificity into enzymes of new catalytic properties, which leads to the genesis of new pathways , 77, 78.The pathway diagram of cholesterol biosynthesis provides the visualization of reactions, converting precursor compounds in a designated reaction sequence, associated enzymes and genes, and EC numbers and hyperlinks to related pathways. A ‘zoom-in’ functionality allows more detailed pathway information to be displayed and gives access to the detail pages of the various pathway elements. All of the curated enzymes are furnished with comments and links to external databases used for curation. The pathway summary finally provides a concise description of the salient points of the pathway, such as rate-limiting steps, key enzymes and regulation or stereochemistry of involved compounds, and lists the cited literature with links for access to the full publication .The release of MetaCyc (version 22.6)  listed 1Nicotiana-specific databases also reflects this current status. Among the species-specific databases for Nicotiana, proteins, predicted to function as enzymes, accounting for a share of all encoding genes in a range of 17.87% (N. sylvestris) to 32.93% (N. attenuata). Of those predicted enzymes, the percentage of curated proteins ranges between 0.079% (N. sylvestris) and 1.01% (N. tabacum TN90). A much higher percentage of curated enzymes versus predicted enzymes can be found in databases for the model organisms Saccharomyces cerevisiae (32.04%) and Homo sapiens (25.79%). It is important to note that the number of genes in the yeast genome is 5–12 times smaller than in Nicotiana species. Apart from being much better studied in the literature than any other organism, the human genome, with about 20 000 genes, is also significantly smaller than any of the Nicotiana genomes.The coverage of curated enzymes shown in Nicotiana database curation emerges when comparing the coverage of curated enzymes versus predicted proteins of other plant species  and complement the efforts of other metabolically oriented databases to get a more complete picture of plant metabolism.A similar picture as seen for the  species . Except  species . Althoug species , 87. SucS. tuberosum, S. lycopersicum and most of the Nicotiana-specific databases range an average of about 3–4 publications per pathway. The N. tabacum databases for the TN90 and K326 cultivar score a higher average number of 5–7 publications per pathway and also show a higher number of curated pathways of about 25% in comparison with at most 5% curated pathways in the remaining Nicotiana databases. The percentage of curated pathways refers to the number of paths with experimental evidence for which existence in the metabolic network of this organism is proven. Pathways predicted and propagated from MetaCyc to species-specific databases such as the N. tabacum databases are transferred without enzymes, genes or evidence codes curated in MetaCyc. After validating such pathways, findings from the published literature are used to furnish the pathways with the genes and enzymes of the respective species, which only then justifies marking the pathway as experimentally verified.The value of a database depends very much on the quality and reliability of the stored data, which in turn indicate the usefulness of a database equipped with bioinformatics tools that allow querying, visualizing and analyzing tasks , 89. OneNicotiana specific database started 2016. It can therefore be concluded that in the comparably short time, in which the databases of SolCyc have been curated, the approach of utilizing taxon-specific databases such as SolanaCyc and NicotianaCyc for the metabolic enrichment of Solanum and Nicotiana specific databases has been successful.The results summarized in https://biocyc.org/web-services.shtml). Downloads of BioCyc databases of interest are available but require the purchase of a license (https://biocyc.org/download.shtml). Programmatic interfaces for the Pathway Tools software at the SGN can be found here (https://solgenomics.net/downloads/index.pl). Data in all databases created by Pathway Tools, such as the SolCyc databases, can be accessed via a variety of search and analysis options. A comprehensive description of these tools has been published [Downloading, transportability and programmatic access to data in databases created with Pathway Tools are provided via Application Programming Interfaces (APIs), including web-based analysis tools. Many of those tools are available in desktop and/or web server modes and Pathway Tools maintains a webpage of those services (ublished  and is uublished . Many ofublished  and acceublished  and saveublished .N. tabacum and S. lycopersicum are well represented in UniProt and were among the six top-ranked plant species with the most protein entries at the time of this study [https://www.uniprot.org/) hosts more than 147 million protein sequences, among them close to 77 000 unreviewed protein data and 855 entries for reviewed proteins of N. tabacum. This resource of protein knowledge is invaluable for the curation in databases like the SolCyc collection of databases. Not only is UniProt a repository for the search of relevant enzymes published in the literature, but it also aids the process of authentication and matching of confirmed protein sequences with the predicted protein sequences embedded in the metabolic network of genome annotated species.The complexity of metabolic networks originates in the many thousands of enzymes, specifically functioning within the confines of the metabolic range that exists in individual plant species in a time and space dependent manner. A quantitative analysis of the proteome of pollen from tomato alone resulted in more than 1200 proteins , whereasis study . UniProtis study . At presNicotiana species has already been reported to be in good agreement with each other [Nicotiana species. The Venn diagram (http://bioinformatics.psb.ugent.be/webtools/Venn/) for the entirety of pathways in N. tabacum K326, N. attenuata, N. benthamiana, N. sylvestris and N. tomentosiformis highlights areas of shared metabolism and a number of pathways that are specific for each Nicotiana species. The intersection for the metabolic pathways of N. tabacum K326 and its parental species N. tomentosiformis and N. sylvestris is 59.3%, which further drops with the addition of N. benthamiana and N. attenuata pathways to 35.1%. The comparison of N. tabacum K326 pathways with the metabolic networks of N. benthamiana and N. attenuata shows an overlap that is about 20% lower than the one observed with the parents of N. tabacum. That indicates the greater evolutionary distance of those species to N. tabacum K326. The number of unique pathways not shared with any of the studied species is highest in N. tabacum (64), followed by N. benthamiana (34), N. attenuata (18), N. tomentosiformis (5) and N. sylvestris (1). A closer look at the nature of the pathways (results not shown) revealed that the majority of those pathways (34) belong to specialized metabolites. To a large extent, this also includes newly created pathways (23), which probably are valid for other Nicotiana species as well, but for which only enzymes from N. tabacum have been published. The existing gap in shared pathways will get smaller when metabolic information from the remaining Nicotiana species become available. However, a certain amount of divergence for some metabolic categories in domesticated and wild tobacco species is to be expected. The genus Nicotiana is known for its high molecular diversity due to the polymorphic variability of its species, often resulting in either gaining or losing biosynthetic flexibility [The metabolic composition of networks of ch other . Howeverxibility .Escherichia coli (EcoCyc) database and the Candida Genome Database, both manually curated, had individual error rates of 1.4 and 1.8%, respectively, [Plasmodium falciparum, manual validation was essential to accept or reject predicted pathways for correctly reconstructing the metabolic network of this species [Saccharomyces cerevisiae was also the subject of a study that manually curated the growth and cell cycle interaction network and provided detailed insights into the relationships between these developmental processes [Databases of all types have been widely accepted as efficient tools to aid researchers with bioinformatics analyses and visualization of stored and annotated data. The value of data directly depends on their overall quality, best achieved by the ‘gold standard’ manual curation by trained biocurators with the ability to distill the published literature to meaningful data through critical assessment . Biocuractively,  comparedctively, . When to species . Manual  species . Saccharrocesses .It has been projected that manual curation cannot achieve functional gene annotation in humans or keep up with the rate of newly sequenced genomes . Such coNew approaches need to be taken to solve the lack of trained biocurators and funding of manual curated databases versus the tremendous data input coming from the literature , 113. OnNicotiana-specific databases. Moreover, it has advanced SolanaCyc and NicotianaCyc to more comprehensive repositories for the biochemistry and molecular biology of the Solanaceae family and the Nicotiana genus. The data in those taxon-specific databases have been curated with a focus on narrower taxonomic ranges and are therefore more useful for exploring solanaceous metabolic networks and relevant bioinformatics analyses. These achievements can now be leveraged into new approaches, such as multi-species databases containing not only plant host but also their pathogens to more precisely and effectively model the interactions between host and pathogen.With the creation of manually curated taxon-specific databases and following an expert-led curation process, databases assembled under the umbrella of SolCyc have been significantly enriched with high-value data. The curation progress in SolanaCyc and NicotianaCyc has improved the completeness and accuracy of the metabolic networks in the six Streptococcus agalactiae [Nicotiana species and disease-causing microorganisms. We expect this MetaTobaccoCyc database to be helpful in exploring the metabolic diversity and commonality between Nicotiana and interacting species by defining metabolic subsets of core and specific pathways and their catalyzing enzymes, which may be indicative for adaptive evolutionary developments in individual metabolomes of Nicotiana species and known pathogens for the genus.The new emerging field of pan-genome research, first proposed in the genome analysis of pathogenic isolates of alactiae , opens talactiae , 116. WeMetabolic databases transform, evaluate and visualize Omics- and high-throughput data into the biological context of species and allow bioinformatics analyses in diverse areas of interest.Manually curated metabolic databases increase the accuracy and comprehensiveness of data and substantially lower the rate of misannotations and false-negative results in predicted metabolic networks.Solanaceae family and Nicotiana genus, respectively.SolanaCyc and NicotianaCyc are the first manually curated taxon-specific databases for the Taxon-specific metabolic databases are valuable and specific repositories for the biochemistry and molecular biology of defined taxonomic ranks.table_1S_bbaa136Click here for additional data file.tab_2_bbaa136Click here for additional data file.Tab_S3_bbaa136Click here for additional data file.legend_to_figures_and_tables_bbaa136Click here for additional data file.


In vitro, GUDCA reduced palmitic acid induced-ER stress and -apoptosis, as well as stabilized calcium homeostasis. In vivo, GUDCA exerted effects on amelioration of HFD-induced insulin resistance and hepatic steatosis. In parallel, ER stress and apoptosis were decreased in GUDCA-treated mice as compared with vehicle-treated mice in liver. These findings demonstrate that reduced GUDCA is an indicator of hyperglycemia. Supplementation of GUDCA could be an option for the treatment of diet-induced metabolic disorders, including insulin resistance and hepatic steatosis, with inhibiting ER stress.Recent studies reveal that bile acid metabolite composition and its metabolism are changed in metabolic disorders, such as obesity, type 2 diabetes and metabolic associated fatty liver disease (MAFLD), yet its role and the mechanism remain largely unknown. In the present study, metabolomic analysis of 163 serum and stool samples of our metabolic disease cohort was performed, and we identified glycoursodeoxycholic acid (GUDCA), glycine-conjugated bile acid produced from intestinal bacteria, was decreased in both serum and stool samples from patients with hyperglycemia. RNA-sequencing and quantitative PCR results indicated that GUDCA alleviated endoplasmic reticulum (ER) stress in livers of high fat diet (HFD)-fed mice without alteration of liver metabolism.  Metabolic disorders including type 2 diabetes mellitus (T2DM) and metabolic associated fatty liver disease (MAFLD), which is suggested to replace the current name of nonalcoholic fatty liver disease (NAFLD) as a more appropriate overarching term . T2DM anBacteroides fragilis species to antagonize intestinal farnesoid X receptor (FXR)-fibroblast growth factor 19 (FGF19) signaling, thereby improving insulin sensitivity in T2DM patients  × calibrator concentration ÷ sample protein concentration.t-test or Welch’s t-test. The chi-square test was used for categorical variables that are presented as numbers and percentages. A two-tailed, unpaired, Student’s t-test or one-way ANOVA were applied for the mouse experiments. Post-hoc comparison was performed using Bonferroni test. Data are reported as the mean ± SEM. Volcano plot was performed using R package “ggplot2” (https://CRAN.R-project.org/package=ggplot2). PCA analysis was performed using R package “ggfortify” (https://CRAN.R-project.org/package=ggfortify). Correlation network was performed using Cytoscape. The results were analyzed with GraphPad Prism 8.0  or R software (version 3.6.1). P<0.05 was considered significant.Human samples distribution was determined by the Kolmogorov–Smirnov normality test and were analyzed by Wilcoxon signed-rank test or two-tailed, unpaired, Student’s To explore the changes of the BA metabolite profile in patients with hyperglycemia, a total of 163 serum and stool samples from the patients in our metabolic disease cohort were collected. The average age of this cohort was 59.8 ± 0.8 years. These samples were divided into two groups according to whether the HbA1c was greater than 6.5 (High group) or less than 6.5 (Low group). Baseline characteristics of the cohort were summarized in The amount of total BAs was similar in serum and stool samples between the Low and High groups G,H. In sNext, we investigated the function of GUDCA in diet-induced metabolic disorders in mice. To explore the mechanism of GUDCA in regulating hepatic metabolism, RNA sequencing (RNA-seq) was analyzed to reveal the mRNA profiles in livers of HFD-fed mice with or without GUDCA administration. Principal component analysis (PCA) revealed that HFD-induced mRNA profile was substantially altered by GUDCA treatment A. TranscTo confirm the role of GUDCA in HFD-induced hepatic ER stress, the effect of GUDCA on saturated fatty acid PA-induced ER stress in HepG2 cells was examined . PA signEndoplasmic reticulum stress is one of the major mechanisms leading to apoptosis through prolonged activation of IRE or induction of CHOP . Therefo2+ ATPase (SERCA) is principally responsible for maintaining calcium homeostasis in ER [Endoplasmic reticulum provides a platform for the crosstalk among many cellular signaling pathways and metabolic regulatory events through calcium fluxes . To examis in ER , the GUDin vivo, we fed mice with HFD for 8 weeks to induce metabolic dysfunction and administered mice with either vehicle, GUDCA or TUDCA during the last 3 weeks. TUDCA was used as a positive control since it was an established ER stress inhibitor capable of improving metabolic dysfunction [To further verify the role of GUDCA in alleviating diet-induced metabolic dysfunction and ER stress function . AnalysiSince BAs are produced from liver cholesterol catabolism and the strong association between T2DM and MAFLD, the effect of GUDCA on ectopic lipid accumulation in liver was studied. Both H&E and Oil Red O staining showed a reduction in hepatic lipid accumulation in mice treated with either GUDCA or TUDCA as compared with vehicle-treated mice A. Both GFinally, the expression of UPR-related proteins and apoptosis in liver tissues of mice treated by GUDCA or TUDCA were measured. Generally, protein levels of p-IRE, and CHOP, p-p38 and p-JNK were reduced, along with a trend toward reduced p-eIF2α expression in GUDCA and TUDCA-treated mice as compared with vehicle-treated A. In addChanges in energy balance in mice fed a high-fat diet is associated with changes in the gut microbiota and increased BA metabolism . The α-gThe emerging importance of the intestinal microbiota in host health and pathogenesis of several metabolic disorders, including obesity, T2DM and MAFLD has been established . The hosIt is generally accepted that a surplus of nutrients and energy stimulates the biosynthetic pathways, which may lead to lipid and protein overloading in the ER. Several stimuli, including disturbances to the metabolic and calcium homeostasis, inflammation or oxidative stress ,38, can 2+ from the cytosol to the ER lumen in an ATP-requiring manner. Pioglitazone alleviates high glucose and apoptosis by increasing SERCA2b transcription [2+ depletion from the ER lumen and inhibition of ER stress, then alleviating metabolic diseases, which may be the mechanism throughout the present study.Maintenance of calcium homoeostasis is of crucial importance for the proper functioning of cells. Calcium dyshomeostasis is associated with many pathological conditions, including metabolic disorders, neurological and heart diseases ,46. Abnocription . Disturbcription . GUDCA tP value calculations are based on at least the two-dimensional information of “statistics” and “degrees of freedom”, both of them take into account the sample size of the two groups.In fact, age is a key factor for liver function, BA production and the crosstalk between BAs and metabolic functions . The prein vivo. The selection of taurine or glycine for BA conjugation is mainly controlled by the bile acid-CoA amino acid N-acyltransferase (BAAT) enzyme the BAAT enzyme is particularly inefficient at using glycine for conjugating bile acids in mice [It is noteworthy that the additional supplement of GUDCA increased the levels of GUDCA and TUDCA, but TUDCA treatment did not increase the relative concentration of GUDCA in the present study. This raises the possibility that the effect of GUDCA on metabolic disorders is related to the increase of TUDCA. The network of BAs transformation and interaction is highly complicated  in mice ,50. More in mice , and a rIn conclusion, our study provides the first evidence that GUDCA is decreased in patients with glucose metabolic disorders. Further, we found that GUDCA exerts protective functions against diet-induced insulin resistance and hepatic steatosis with inhibiting ER stress, might promise a new treatment option for metabolic disorders in patients.We explored the alteration of bile acid metabolism profiling in individuals with hyperglycemia and identified GUDCA was reduced dramatically.in vitro and in vivo.Our study provided the evidence that GUDCA inhibited ER stress and apoptosis both We suggested that small-molecule agents, GUDCA might have therapeutic potential for the treatment of diet-induced insulin resistance and hepatic steatosis.Click here for additional data file.


Yinchen Wuling powder is often used to treat clinical hyperlipidemia, although its mechanism of action remains unclear. In this study, we aimed to investigate the active ingredients found in Yinchen Wuling powder and find its mechanism of action when treating hyperlipidemia, using a combination of network pharmacology, molecular docking, and molecular dynamics simulation approaches.  The TCMSP database was used to obtain the principle active ingredients found in Yinchen Wuling powder and the NCBI and DisGeNet databases were used to obtain the main target genes involved in hyperlipidemia, and the intersectional targets were obtained by EXCEL. We also used Cytoscape 3.7.2 software to construct a “Traditional Chinese Medicine-Active Ingredient-Target” network and use STRING platform to conduct “protein-protein interactional” (PPI) analyses on the intersection targets. Bioconductor software and RX 64 4.0.0 software were then used to perform GO functional enrichment analysis and KEGG pathway enrichment analysis on the targets. Molecular docking of core protein-ligand interactions was modeled using AutoDock Vina software. A simulation of molecular dynamics was conducted for the optimal core protein-ligand obtained by molecular docking using Amber18 software.  A total of 63 active ingredients were found in Yinchen Wuling powder, corresponding to 175 targets, 508 hyperlipidemia targets, and 55 intersection targets in total. Cytoscape 3.7.2 showed that the key active ingredients were quercetin, isorhamnetin, taxifolin, demethoxycapillarisin, and artepillin A. The PPI network showed that the key proteins involved were AKT1, IL6, VEGFA, and PTGS2. GO enrichment analysis found that genes were enriched primarily in response to oxygen levels and nutrient levels of the vesicular lumen and were associated with membrane rafts. These were mainly enriched in AGE-RAGE (advanced glycation end products-receptor for advanced glycation end products) signaling pathway in diabetic complications, fluid shear stress, and atherosclerosis, as well as other pathways. The molecular docking results indicated key binding activity between PTGS2-quercetin, PTGS2-isorhamnetin, and PTGS2-taxifolin. Results from molecular dynamics simulations showed that PTGS2-quercetin, PTGS2-isorhamnetin, and PTGS2-taxifolin bound more stably, and their binding free energies were PTGS2-quercetin -29.5 kcal/mol, PTGS2-isorhamnetin -32 kcal/mol, and PTGS2-taxifolin -32.9 kcal/mol.  This study is based on network pharmacology and reveals the potential molecular mechanisms involved in the treatment of hyperlipidemia by Yinchen Wuling powder. Hyperlipidemia represents a major global health problem and is caused by abnormal fat metabolism or its transport throughout the body, resulting in a variety of lipid structural disorders in plasma. Hyperlipidemia is generally caused by elevated levels of total cholesterol (TC), triglycerides (TG), and low-density lipoprotein cholesterol (LDL-C) and a reduction in high-density lipoprotein cholesterol (HDL-C) and represents one of the major risk factors for cardiovascular diseases . AlthougTraditional Chinese medicine has accumulated a wealth of experience in the regulation of blood lipids during its long practical applications. Yinchen Wuling powder (YCWL) is a classical prescription medicine used clinically to treat hyperlipidemia, and its use was first recorded in “Synopsis of Prescriptions of the Golden Chamber” and consists of Artemisia capillaris herba, Alismatis rhizoma, Atractylodes lancea, Polyporus umbellatus, Poria, and Cinnamomi ramulus , 4.in vivo studies that YCWL can lower TCs, TGs, and LDL-Cs and increase the levels of HDL-C and improve hemodynamics and coagulation. Wu et al. [In recent years, there have been increasing clinical reports and experiments involving the treatment of hyperlipidemia with Yinchen Wuling powder; Zhang  reports u et al.  found thβ cells from its loss and exhibiting antidiabetic properties. Liu et al. [Modern pharmacological studies have shown that the scoparone and p-hydroxyacetophenone contained in the Artemisia capillaris herba play an important role in the process of regulating lipids, and scoparone contains peroxisome proliferators. Scoparone can antagonize peroxisome proliferator-activated receptors (PPARs), inhibit the formation of 3T3L1 preadipocytes, downregulate the expression of fat synthesis genes, and play a role in regulating fat metabolism . p-hydrou et al.  found thu et al. . Studiesu et al. . The alcu et al. –20.Network pharmacology is a method for predicting the composition of the effective components and disease targets of a compound at the systems level and the establishment of a multilevel network such as drugs-components-targets-disease. This then enables locking the key targets related to drugs and diseases, using bioinformatics methodology. The target point corresponds to the new method of the pathway , 22. MolThis experimental study uses network pharmacology molecular docking and molecular dynamics simulation technology to explore the relevant biological pathways associated with YCWL in treating hyperlipidemia. It achieves this by constructing a drug and disease target network and then verifying it through molecular docking technology thus providing a theoretical basis for clinical application. A workflow chart is shown in http://lsp.nwu.edu.cn/tcmsp.php), and the active ingredients were screened according to oral bioavailability (OB) ≥ 30% and drug‐likeness (DL) ≥ 0.18. The protein targets for the active ingredients were determined using the TCMSP platform [The active ingredients within YCWL were obtained from the TCMSP database (platform .https://www.ncbi.nlm.nih.gov/) and DisGeNet (https://www.disgenet.org/) databases, we screened targets related to hyperlipidemia, and “hyperlipidemia” was entered as the search keyword, and the gene targets retrieved were a result of two merged disease databases, and all duplicate values were deleted. After the disease target was obtained, both the disease targets and the drug targets were combined to screen out common targets and construct a “Chinese medicine-disease” intersectional target database, which was visualized using a Venn diagram (https://bioinfogp.cnb.csic.es/tools/venny/index.html).Using the NCBI (http://cytoscape.org/), a network of “Traditional Chinese Medicine-Active Ingredients-Target” for YCWL was constructed to help determine its pharmacological mechanisms of action. The degree value reflects the importance of the node in the network; the higher the value, the more important the node. And the core active ingredients were screened out by the degree [Using Cytoscape 3.7.2 (e degree .https://string-db.org) to construct a PPI network model using biological species set to “Homo sapiens”; the confidence > 0.4 and unconnected nodes were hidden. The rest of the parameters are set to default values. From this, a protein-protein interaction network (PPI) was obtained [The intersection targets of Chinese medicine and disease were imported into STRING 11.0 database , molecular function (MF), and cell composition (CC) .We also use Bioconductor software and RX 64 4.0.0 to perform GO functional enrichment analysis on the common target of YCWL and hyperlipemia and screened out the core pathways. After obtaining the KEGG pathway, Cytoscape 3.7.2 was used to construct a “target-pathway” network.http://zinc.docking.org) and the PubChem database (https://pubchem.ncbi.nlm.nih.gov), and protein structure was obtained from the PDB database (http://www.rcsb. org) [The three active ingredients with the highest degrees in the “TCM-Active Ingredients-Target” network diagram were molecularly docked with the four core proteins with the highest degrees in the PPI network. The structure of the compounds were obtained from the Zinc database (sb. org) . AutoDocThe amber18 software package was used to perform molecular dynamics simulation on the protein-active ingredients obtained by molecular docking. The protein uses the ff14SB force field parameter, and the active ingredients used the gaff general force field parameter and the ANTECHAMBER module to calculate its AM1-BCC atomic charge. Loading the protein, active ingredients were added into the leap module, and hydrogen atoms and antagonist ions to neutralize the charge were automatically added. The TIP3P explicit water model was selected, and periodic boundary conditions were set. The molecular dynamics simulation workflow includes four steps: energy minimization, heating, equilibrium, and production dynamics simulation. First, the heavy atoms of proteins  were confined, and 10,000 steps (including the 5000-step steepest descent method and the 5000-step conjugate gradient method) were performed on the water molecules to minimize the energy; then, within 50 ps, the system is slowly heated to 300 K. After the heating is completed, the system at 50 ps under the npt ensemble was balanced. Finally, the system is subjected to a molecular dynamics simulation of 50 ns  under the npt ensemble, with a time step of 2 fs, and the trajectory data is saved every 10 ps, and then, the CPPTRAJ module is used for the relevant analysis. The calculation of the binding free energy of active ingredients and protein is carried out using the MMPBA.py module.After screening, a total of 63 chemical constituents of Yinchen Wuling powder were found: 7 chemical constituents of Atractylodes lancea, 15 chemical constituents of Poria, 7 of Cinnamomi ramulus, 13 of Artemisia capillaris herba, 10 of Alismatis rhizoma, and 11 of Polyporus umbellatus, and these are shown in Supplementary Information Table https://bioinfogp.cnb.csic.es/tools/venny).Our results reveal that the CNKI database and DisGeNet had a total of 591 targets. After removing duplicate targets, 508 targets were finally obtained. To clarify the relationship between YCWL and hyperlipidemia, EXCEL was used to merge the targets found in YCWL with hyperlipidemia, and finally, 55 intersection targets were obtained, as shown in Using EXCEL to incorporate the intersection of Chinese medicine targets and disease targets, we obtained 55 target genes, and these were imported into Cytoscape 3.7.2 to construct a traditional Chinese medicine-active ingredient-target network. From this, 95 nodes were obtained, and 618 interactional relationships were found. The “TCM-Active Ingredients-Target genes” network is shown in By analyzing the network of the “TCM-Active Ingredients-target genes”, the five compounds with the highest degree values in YCWL were found to be: quercetin, isorhamnetin, taxifolin, demethoxycapillarisin, and artepillin A, and these are shown in Supplementary Information Table The specific names of the intersection targets were screened by EXCEL and imported into the STRING database for online PPI analysis. The interaction threshold was set to “0.4” to hide the episomal gene targets. The “edge” in the figure represents the extent of association between the intersectional genes and shows the degree of association. Thus, the greater the association, the greater the chance that the node represents the intersectional gene. There were a total of 55 nodes and 553 edges, as shown in The YCLW target network clustering analysis used the same method as the PPI network clustering analysis to obtain three different clusters. The clusters of the YCLW-Hyperlipidemia is shown in The results showed that the targets were mainly enriched in response to oxygen levels, nutrient levels, reactive oxygen species, metabolic processes, and responses to hypoxia, with a negative regulation of the apoptotic signaling pathway. Their main molecular functions were associated with the organization of vesicle lumens, membrane rafts, membrane microdomains, caveola, and membrane region, and they were mainly enriched in relation to nuclear receptor activity, transcription factor activity, direct ligand regulated sequence-specific DNA binding, steroid hormone receptor activity, heme binding, tetrapyrrole binding, and other biological processes. The top five enrichment results from each GO analysis are shown in Supplementary Information Table The KEGG results showed that the targets were mainly enriched in the following: the AGE-RAGE (advanced glycation end products-receptor for advanced glycation end products) signaling pathway involving diabetic complications, fluid shear stress and atherosclerosis, endocrine resistance, the TNF signaling pathway, bladder cancer, human cytomegalovirus infection, the HIF-1 signaling pathway, and the VEGF signaling pathway, and these represented 123 articles. The results of enrichment analysis from the KEGG pathway are shown in We used Autodock software to dock at the molecular level the three active ingredients  having the highest degree values with the four core proteins within the PPI network . The more stable the binding between the ligand and the receptor, the lower the binding energy of the two, and our results showed that the binding energy of the active ingredients and the four core proteins were all ≤-7 kcal/mol, the lowest binding free energy was PTGS2-quercetin , and the main forces involved were van der Waals forces, hydrophobic forces, and carbon-hydrogen bonds.This was followed by PTGS2-taxifolin , and there were pi-alkyl hydrophobic forces between LYS1137 and taxifolin; the conventional hydrogen bond with THR 331, LYS1137, CYS 1047, and the carbon hydrogen bond with THR549. Then, PTGS2-isorhamnetin  and the carbon hydrogen bond with THR549 and GLY 1135 were imported into Discovery Studio 4.5 software for visual optimization. Docking scores of isorhamnetin, quercetin, and taxifolin with potential targets is shown in Supplementary Information Table The molecular docking diagram is shown in The rmsd curve represents the fluctuations in protein conformation. It can be seen from The gyration radius curve represents the compactness of the overall structure of the protein. It can be seen from The rmsf curve represents the fluctuation of the protein amino acid residues. It can be seen from We take the trajectory of the rmsd stationary phase (10-50 ns) of PTGS2-quercetin and calculate the binding free energy. From this result, it can be seen that the binding free energy between the two is -29.5 kcal/mol. Van der Waals potential  and electrostatic interaction  are conducive to the combination of the two, while the polar solvation  is not conducive to the interaction of the two.We take the trajectory of the rmsd stationary phase (30-50 ns) of PTGS2-taxifolin and calculate the binding free energy. The result shows that the binding free energy of the two is -32.9 kcal/mol. Van der Waals potential  and electrostatic interaction  are conducive to the combination of the two, while the polar solvation  is not conducive to the interaction of the two.We take the trajectory of the rmsd plateau (25-50 ns) of PTGS2-isorhamnetin and calculate the binding free energy. The results show that the combination of the two are general, and the binding free energy is -32 kcal/mol. Van der Waals potential  and electrostatic interaction  are conducive to the combination of the two, while the polar solvation  is not conducive to the interaction of the two. Free energies of the binding are shown in the Supplementary Information Table It can be seen from It can be seen from It can be seen from κB and block the Toll-like receptor 4 signaling pathway. It can also reduce the production of reactive oxygen species and alleviate the inflammatory response of mouse microglia induced by lipopolysaccharide. Furthermore, Zhang et al. [In this study, based on network pharmacology, we found that YCWL principally regulates hyperlipidemia through quercetin, isorhamnetin, and taxifolin as well as other components. Studies have found that quercetin can reduce blood lipid levels by upregulating the expression of ABCA1, ABCG1, and other proteins and by increasing the cholesterol binding affinity of HDL and APOA1 . Low cong et al.  observedg et al. . Taxifolg et al. , 38. It g et al. . Sun et g et al. .β-cells and aggravates insulin resistance. Therefore, it regulates blood lipids and reduces the release of inflammatory mediators, which can effectively reduce the occurrence of atherosclerosis and acute myocardial infarction [The results from the PPI analysis showed that the targets for YCWL relating to the treatment of hyperlipidemia mainly involved AKT1, IL6, VEGFA, and PTGS2. There are three known subtypes of AKT, and it can play a regulatory role in glucose homeostasis, protein synthesis, lipid metabolism, cell proliferation, and survival. When AKT is activated by PI3K, it can then phosphorylate downstream substrates and these mechanisms are involved in the regulation of cell apoptosis, lipid metabolism, and cell proliferation. Studies have also shown that insulin secretions after food intake results in the activation of the PI3K/AKT signaling pathway  thus incfarction . VEGFA ifarction . PTGS isfarction , 46. It farction .Our molecular results showed that the binding energies of the three active ingredients, and the four core proteins were all ≤-7 kcal/mol, indicating that the abovementioned active ingredients had good binding for their target, while the binding energy of PTGS2-quercetin  was the lowest. This indicates that it may be the core target and active ingredient of YCWL when treating hyperlipidemia. In addition, molecular dynamics simulation results show that PTGS2-quercetin, PTGS2-isorhamnetin, and PTGS2-taxifolin bind more stably, and their binding free energies are PTGS2-quercetin , PTGS2-isorhamnetin , and PTGS2-taxifolin .κB, VCAM-1, tissue factor, and inflammatory factors and is one of the most relevant signaling pathways associated with chronic diseases such as diabetes, atherosclerosis, and cancer [α pathway can induce oxidative stress and increase transcytosis of LDLs across endothelial cells and promote the development of atherosclerosis [GO enrichment analysis showed that YCWL mainly regulates hyperlipidemia through biological processes such as response to oxygen levels, nutrient levels, reactive oxygen species, and other metabolic process, including hypoxia, and can also negatively regulate the apoptotic signaling pathway. KEGG pathway enrichment analysis showed that the main pathways that regulate hyperlipidemia are the AGE-RAGE signaling pathway in diabetic complications, fluid shear stress, and atherosclerosis, the TNF signaling pathway, and the HIF-1 signaling pathway. Studies have found that a high-fat diet and high blood sugar will increase the formation and accumulation of AGE. Increased AGE may cause inflammation, oxidative stress, and the activation of fat storage . The actd cancer . The HIFd cancer . Mylonisd cancer . Activatclerosis . Fluid sclerosis , 54.in vitro and in vivo studies are required to further elucidate its function and mechanism of action.Based on network pharmacology and molecular docking technologies and using PPI, GO, and KEGG analyses of novel targets, we were able to predict a role for YCWL in the regulation of the AGE-RAGE signaling pathway during diabetic complications, and this regulation was associated with AKT1, IL6, VEGFA, PTGS2, and other targets. We further found that fluid shear stress and atherosclerosis, the TNF signaling pathway, the HIF-1 signaling pathway, and other signaling pathways play a role in the regulation of hyperlipidemia. Therefore, this work has clearly highlighted some of the multiple components found within YCWL and found some of its targets which may be involved in its beneficial effects upon hyperlipidemia. These data supports or views that additional


Rhizoma polygonati  has been used for a long history with many chemical components in inducing anti-cancer, anti-aging, anti-diabetes, anti-fatigue, and more prevention of diseases or acts as nutrition sources in food. Here we investigated RP extract combination with kinase inhibitors in anti-cell growth and blockade in pathways targeting kinases. Experimental investigation and network pharmacology analysis were applied to test the potent kinase-mediated signaling. Herbzyme activity was determined by substrate with optical density measurement. Extract of processed RP inhibits cell growth in a much greater manner than alone when applied in combination with inhibitors of mTOR or EGFR. Moreover, processing methods of RP from Mount Tai (RP-Mount Tai) play essential roles in herbzyme activity of phosphatase suggesting the interface is also essential, in addition to the chemical component. The network pharmacology analysis showed the chemical component and target networks involving AKT and mTOR, which is consistent with experimental validation. Finally, EGFR inhibitor could be associated with nano-extract of RP-Mount Tai but not significantly affects the phosphatase herbzyme activity in vitro. Thus the processed extract of RP-Mount Tai may play a dual role in the inhibition of cell proliferation signaling by both chemical component and nanoscale herbzyme of phosphatase activity to inhibit kinases including mTOR/AKT in potent drug delivery of kinase inhibitors. Rhizoma polygonati  is a ‘medicine food homology’ (MFH) known to be used in Traditional Chinese Medicine (TCM) for thousands of years with many natural products to maintain proper blood sugar levels, and exhibit many more pharmacological functions . Cells were plated in 24 well plates with a density of 5000 cells/well for overnight followed by treatment with vehicle or agent and after 3–4 days cells were fixed and stained with crystal violet. Finally, cells were photographed and measured by optical density at 590 nm. The agents used for inhibition of differential signaling are olaparib (PARP inhibitor (i), PARPi (Sigma Aldrich), crizotinib , staurosporine (PKCi), SMI-4a , erlotinib Hydrochloride , rapamycin (mTORi), iCRT3 (β-catenin inhibitor) and verteporfin .HT-1080 cells were used for cell-based studies. HT-1080 cells are human cells originally derived from a 35-year-old White male patient with a disease of fibrosarcoma  database, NCBI (https://www.ncbi.nlm.nih.gov) gene database, and GenCLiP3 (http://ci.smu.edu.cn/genclip3/GeneAssociation.php) database using ‘kinase’ as the keyword while searching and selecting the species as ‘Homo sapiens’ to obtain kinase-related human genes. Venny 2.1.0 (https://bioinfogp.cnb.csic.es/tools/venny/) software  where the ‘Homo sapiens’ keyword was applied before running the Multiple Proteins service of the STRING. Further, Cytoscape 3.7.2  was used to construct a protein interaction network and identify the most important RP’s protein targets for molecular docking analysis.The potential targets were used to construct the target PPI network through the STRING database . The list of genes of RP targets against kinases was uploaded to the DAVID searching tool. Here, the species section was selected as ‘homo sapiens’, and all target gene names were corrected to their official names , and the threshold of p < 0.05 was set for GO enrichment before running the tool.To further understand the functions of core target genes and the main action pathways of the active substances of RP, the disease-related targets obtained from the above screening were entered into the DAVID database . Using p < 0.05 and sorting from small to large as the screening conditions, the enrichment analysis pathways were selected which then were visualized through online tool (http://www.bioinformatics.com.cn).1For KEGG enrichment pathway analysis the core target genes were imported into the KOBAS3.0 database (http://tcmspw.com/index.php) and the BenCaoZuJian database (http://herb.ac.cn/)  database. Additionally, the bioinformatics online mapping website (http://www.bioinformatics.com.cn)The active ingredients of RP were obtained by searching the Traditional Chinese Medicine System Pharmacology database (TCMSP) (https://pubchem.ncbi.nlm.nih.gov/) in the SDF file format which was then converted into a PDB file using the Open Babel 2.3.2 software. The receptor protein was retrieved from the Protein Data Bank database (http://www.rcsb.org/pdb). For AKT1 docking, the PDB structure of 4EKL was used which is bound with GDC0068 by amino acids of Phe 22, Lys 40, Ala91, Glu95, Asp 153 (https://www.rcsb.org/sequence/4EKL). GDC-0068 is a highly selective pan-AKT inhibitor. The PYMOL software was further used to perform operations, such as water removal and ligand removal on the receptor protein, while the Autodock 4.2.6, and AutoDock Vina 1.1.2  and absorbance measurement  and dehydrated for at least 24 h. SEM investigation was operated by loading RP extract on papers of foil and air-dry before putting it into the machine. Finally, the samples were investigated by Zeiss SEM microscope.An ancient way of RP processing includes many times steam and sun-drying, which is unique to RP, compared to most herbs processing like boiling, or fresh use. We applied for a RP from a mountain area of Mount-Tai, China for further studies. We first reproducibly found the characteristics of ancient ways of processing generated nanoparticles in RP extract. We found the density plays an essential role in nanoparticle accumulation and high density may produce cluster simulating polymers of the nanostructures investigated by SEM  targets . Then wep ≤ .05. Among them, there are 228 biological processes (BP), accounting for 73%; 30 cellular components (CC), accounting for 9%; and 54 molecular functions (MF), accounting for 17%. The important biological processes were recorded as a response to an antibiotic, protein kinase B signaling, fibroblast proliferation, response to a toxic substance, apoptotic process, response to hypoxia, response to estradiol, response to lipopolysaccharide, nitric oxide biosynthetic process, cellular response to hypoxia, intracellular receptor signaling pathway, aging, oxidation-reduction process, cell proliferation, and steroid hormone-mediated signaling pathway  against 31 sample genes . There, http://autodock.scripps.edu/faqs-help/manual/autodock-4-2-user-guide/AutoDock4.2_UserGuide.pdf)  indicates the binding between the docking molecule and the target . Also, similar to AKT inhibitor LY294002, the RP’s compound diosgenin has the strongest binding affinities with AKT1 . In addition, similar to mTOR inhibitor Rapamycin, diosgenin also showed strong binding to mTOR . See The molecular docking performed with diosgenin with AKT1 and mTOR target proteins displays that the binding pockets of both AKT1 and mTOR receptor proteins have many amino acid residues that form various bonds with diosgenin . AlthougAs shown in Overall, the obtained results of the paper indicate that the RP compounds can be effectively used to target the kinase signaling pathways regulating cell growth. The thorough examination of the RP compounds using network pharmacology and molecular docking demonstrated that one of its ingredients—diosgenin can fit well into the binding pocket of important target proteins, AKT1 and mTOR, which play a critical role in the cellular proliferation, survival, and apoptosis processes. This means that diosgenin is a good candidate for further experimental validation to design a potent drug that could be used against various diseases including cancer that uses kinase signaling, specifically, the PI3K-AKT-mTOR pathway.https://www.rcsb.org/sequence/4EKL)  on phosphatase herbzyme activity. First, we tested the binding of RP with EGFR inhibitor Erlotinib Hydrochloride by fluorescence quenching analysis. As shown in In summary, using network pharmacology we may identify the pathways that drug delivery carriers induced signaling and potent enzyme activity. The bioinformatics tool can provide an efficient approach to expedite the biochemical analysis to target the potent signaling and applying the drug delivery platform especially at nanoscale for enhanced efficiency (Liu et al., in vivo.RP contains a lot of natural compounds which may be used for drug delivery as natural product delivery has been technically effective with proper medium (Zhang et al., RP extract may against kinases through multiple pathways but one of the main signaling is AKT/mTOR. Network pharmacology and docking showed the consistent mTOR pathways targeted by RP. Thus, RP extract might be potentially used as a drug delivery carrier presented by a complex of the natural compound with kinase inhibitors to target kinases with intrinsic phosphatase herbzyme activity. Different from other types of drug delivery systems with potent combined kinase inhibitors, the RP extract compound may also exert a herbzyme of phosphatase function to target kinases. Thus the dual role of RP nano-extract shows the greatest potential of drug delivery by natural products complex.


Background: Gan–Dou–Fu–Mu decoction (GDFMD) improves liver fibrosis in experimental and clinical studies including those on toxic mouse model of Wilson disease (Model). However, the mechanisms underlying the effect of GDFMD have not been characterized. Herein, we deciphered the potential therapeutic targets of GDFMD using transcriptome analysis.Methods: We constructed a tx-j Wilson disease (WD) mouse model, and assessed the effect of GDFMD on the liver of model mice by hematoxylin and eosin, Masson, and immunohistochemical staining. Subsequently, we identified differentially expressed genes (DEGs) that were upregulated in the Model (Model vs. control) and those that were downregulated upon GDFMD treatment (compared to the Model) using RNA-sequencing (RNA-Seq). Biological functions and signaling pathways in which the DEGs were involved were determined by gene ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG) pathway analyses. A protein–protein interaction (PPI) network was constructed using the STRING database, and the modules were identified using MCODE plugin with the Cytoscape software. Several genes identified in the RNA-Seq analysis were validated by real-time quantitative PCR.Results: Total of 2124 DEGs were screened through the Model vs. control and Model vs. GDFMD comparisons, and dozens of GO and KEGG pathway terms modulated by GDFMD were identified. Dozens of pathways involved in metabolism , immune and inflammatory response , liver fibrosis (such as ECM-receptor interactions), and cell death  were identified as potential targets of GDFMD in the Model. Some hub genes and four modules were identified in the PPI network. The results of real-time quantitative PCR analysis were consistent with those of RNA-Seq analysis.Conclusions: We performed gene expression profiling of GDFMD-treated WD model mice using RNA-Seq analysis and found the genes, pathways, and processes effected by the treatment. Our study provides a theoretical basis to prevent liver fibrosis resulting from WD using GDFMD. Wilson disease (WD), also known as hepatolenticular degeneration, is a rare autosomal recessive genetic disease of the nervous system related to disorders of copper metabolism. The clinical manifestations of this disease include liver damage, extrapyramidal symptoms, pigmented corneal rings, and kidney damage. The worldwide incidence rate is about 1 in 30,000 individuals . It is rDespite concerns about the need to protect the liver of patients with WD, no major breakthrough has been achieved, and treatment of such patients is still very difficult. It is, therefore, important to find key molecular targets for prevention of liver fibrosis in WD. Both prevention and treatment have great clinical significance. Because the mechanism underlying the pathogenesis of liver fibrosis is complicated, the repair response after liver injury involves the whole body; for this reason, drugs developed for a single target are seldom clinically effective. There are no chemical or biological drugs with prominent curative effects for clinical application. In recent years, research on traditional Chinese medicine (TCM) and its use has revealed therapeutic advantages of these medicines in the prevention and treatment of liver fibrosis .Reynoutria multiflora (Thunb.) Moldenke , Lycium barbarum L , Panax notoginseng (Burkill) F.H.Chen , Curcuma aromatica Salisb , Smilax glabra Roxb , Bupleurum chinense DC , and Paeonia lactiflora Pall  , a formula of TCM herbs, has been clinically used to treat WD . It can ese, BS) . The antese, BS) . However−1 in vacuum at 50°C to obtain the GDFMD suspension. Thereafter, a 1.0 ml aliquot of GDFMD was added to 9.0 ml ethanol, and the mixture was centrifuged at 8000 × g for 5 min and filtered through a 0.22 µm membrane. Seven reference substances, namely gallic acid, resveratrol, quercetin, curcumin, paeoniflorin, saikosaponin A, and notoginsenoside (the purity of each compound was determined to be more than 98%), were obtained from Mansite Biotech Co., Ltd . The reference substances were weighed and dissolved in 90% ethanol to prepare a mixed reference solution. Two microliter aliquots of sample solutions and the mixed reference solution were used for LC-MS/MS analysis.The proportion of the seven herbs in GDFMD was as follows: ZHSW:GQ:SQ:YJ:TFL:CH:BS = 4:20:3:12:12:12:15. All the herbs were obtained from Anqing Huashi Chinese Herbal Medicine Beverage Co., Ltd . The steps in the preparation of GDFMD were as follows: First, all the herbal medicines were immersed in water  for 1.5 h. The two decoctions were combined and concentrated to 0.2 gmL−1. The mass spectrometry was carried by multiple reaction monitoring (MRM) in the ESI-mode. The optimum values of MS parameters were as follows: nebulizing gas pressure, 25 psi; drying gas (N2) flow rate, 10 Lmin−1; capillary temperature, 350°C. All the data were analyzed using the Mass Hunter workstation software .An Agilent 1290 HPLC system, coupled with an Agilent 6460 series QqQ/MS with electrospray ionization (ESI) source, was employed as the HPLC-MS/MS system for the quality control of GDFMD. The separation of complex components was performed on a BEH C18 column  maintained at 35°C. Formic acid (0.1%) in water (A) and acetonitrile (B) were used as elution solvents. The gradient elution procedure was as follows: 0–30 min, 5–25% B; 30–40 min, 25–45% B; and 40.01–45 min, 5% B. The flow rate was 0.3 mlminMale Toxic Milk (TX) mice  were purchased from the Jackson Laboratory (USA). All the mice were genotyped by Sanger sequencing. The mice were randomly divided into the following four groups: normal, model, GDFMD, and penicillamine groups, with 10 mice per group. The mice were anesthetized by intraperitoneal injection of sodium pentobarbital to minimize pain during surgery. The TX model was prepared as described previously  and the The liver was harvested at day 28 after GDFMD administration and fixed with 4% paraformaldehyde. Paraffin-embedded sections were stained with hematoxylin and eosin. Furthermore, we performed Masson staining of the sections and immunohistochemical staining for Col1 and α-SMA.A total RNA isolation kit  was used for extracting the total RNA from liver samples, according to the instructions provided by the manufacturer. The quantity and quality of RNA samples were determined using a NanoDrop one  and Agilent 2100 Bioanalyzer . The RNA library was prepared using the SureSelect chain-specific RNA library preparation kit . Thereafter, Qubit 3.0 Fluorometer and Agilent 2100 Bioanalyzer were used to quantify the purified libraries. The generation of clusters was performed using cBot, and the sequencing was achieved with Illumina NovaSeq 6000 . These procedures were outsourced to Origin-Biotech Inc .Mus musculus reference genome (mm10) was carried out using Hisat | > 1 . Fastp was used to trim the discovered Illumina TruSeq adaptor sequences, bad quality reads, and ribosomal RNA reads . The mapng Hisat . Thereafng Hisat . Trimmedng Hisat , and a Png Hisat . To visung Hisat , and sigge)| > 1 . The uprTo better understand the functions of DEGs, the R software package was used to analyze the signaling pathways enriched in Gene Ontology (GO)  and the STRING, a database of information on protein interactions including the data on predicted and experimentally determined interactions , was adoGAPDH as an internal control. Six GDFMD-regulated genes, also known to participate in liver injury, namely Timp1, Fbn1, Gas6, Alb, Apob, and Apoa2, were selected for qRT-PCR analysis. The expression levels in the liver samples from the control, model, and GDFMD groups were analyzed. The relative expression of mRNAs was determined using the 2−∆∆CT method . Statistical analysis of the data was performed using the GraphPad Prism . A To ensure the quality and stability of the GDFMD, HPLC-MS/MS was used to detect and identify the chemical components of GDFMD. Seven known components, namely gallic acid , paeoniflorin , resveratrol , quercetin , notoginsenoside , saikosaponin A , and curcumin  were identified by comparing the retention times with those of the reference standards. The MS parameters and the content of these seven components in GDFMD are shown in First, all the mice were genotyped. The mice with diploid mutation were tx-j mice (Model group), whereas the diploid wild type mice were used as control mice (control group) . To obseCollagen was stained blue in liver sections with Masson staining as well as with IHC staining for Col1a. The TX mice showed obvious collagen accumulation compared with those in the control group , and admPCA showed that control, tx-j, and GDFMD sample groups could segregate into different areas of the 2D plots  enriched in 1553 GO terms, 511 of which were related to biological processes (BP), 43 were related to cellular components (CC), and 152 were related to molecular functions (MF). The top five enriched BP terms were organic acid metabolic process (GO: 0006082), carboxylic acid metabolic process (GO: 0019752), monocarboxylic acid metabolic process (GO:0032787), lipid metabolic process (GO:0006629), and oxidation–reduction process (GO:0055114). Blood microparticle (GO: 0072562), extracellular space (GO: 0005615), endoplasmic reticulum (GO: 0005783), mitochondrion (GO: 0005739), and extracellular region (GO: 0005576) were the top five enriched CC terms. The top five enriched MF terms were oxidoreductase activity (GO: 0016491), catalytic activity (GO: 0003824), oxidoreductase activity, acting on paired donors, with incorporation or reduction of molecular oxygen (GO: 0016705), monooxygenase activity (GO: 0004497), and tetrapyrrole binding (GO: 0046906). The top 30 GO terms related to down-DEGs, according to enrichment factor, are listed in To further identify the 611 down-DEGs and 1513 up-DEGs related to GDFMD treatment in the Model group, GO enrichment analysis was performed using the clusterProfiler. The results of enrichment analysis showed that the 611 down-DEGs were significantly (p < 0.05) enriched in 1631 GO-terms, including 1375 BP, 124 CC, and 132 MF terms. The top five enriched BP terms were cell adhesion (GO:0007155), regulation of cell migration (GO:0030334), cell-cell adhesion (GO:0098609), cellular component movement (GO:0006928), and locomotion (GO:0040011). Moreover, we found that many of the significantly enriched terms were related to collagen, fibril, and fibroblast; these included negative regulation of collagen , fibrillar collagen , cellular response to fibroblast growth factor stimulus , and collagen type IV . Cell surface (GO:0009986), extracellular matrix (GO:0031012), proteinaceous extracellular matrix (GO:0005578), extracellular region (GO:0005576), and cell periphery (GO:0071944) were the top five enriched CC terms. The enriched MF terms were calcium ion binding (GO:0005509), glycosaminoglycan binding (GO:0005539), heparin binding (GO:0008201), growth factor binding (GO:0019838), and extracellular matrix structural constituent (GO:0005201). The top 30 GO terms related to up-DEGs, according to the enrichment factor, are shown in The 1513 up-DEGs were significantly , response to stimulus , immune and inflammatory response, etc. GDFMD can affect almost all cell components, including intracellular, membrane-bounded organelle, membrane, cytoplasm, cell periphery, vesicle, extracellular vesicle, nucleus, cytoskeleton, etc.p < 0.05. In particular, the top10 enriched pathways were metabolic pathways (mmu01100), steroid hormone biosynthesis (mmu00140), retinol metabolism (mmu00830), complement and coagulation cascades (mmu04610), chemical carcinogenesis (mmu05204), drug metabolism - other enzymes (mmu00983), glycine, serine, and threonine metabolism (mmu00260), primary bile acid biosynthesis (mmu00120), metabolism of xenobiotics by cytochrome P450 (mmu00980), and peroxisome (mmu04146). The top 30 KEGG pathway terms related to down-DEGs, according to the FDR-value, are shown in More information pertaining to the function of genes and interactions among them can be obtained through pathway enrichment analysis. ClusterProfiler was used for KEGG pathway analysis of 611 down-DEGs and 1513 up-DEGs, which were related to the GDFMD intervention in the Model group. The 611 down-DEGs were enriched in 230 pathway terms, including 65 significant terms with p-value < 0.05. The top10 enriched pathways were focal adhesion (mmu04510), ECM-receptor interaction (mmu04512), cell adhesion molecules (CAMs) (mmu04514), rheumatoid arthritis (mmu05323), cytokine-cytokine receptor interaction (mmu04060), toxoplasmosis (mmu05145), amebiasis (mmu05146), PI3K-Akt signaling pathway (mmu04151), tuberculosis (mmu05152), and asthma (mmu05310). The top 30 KEGG pathway terms related to up-DEGs, according to the FDR-value, are shown in The 1513 up-DEGs were enriched in 251 pathway terms including 73 terms with Thus, dozens of pathways, especially metabolic regulation, immune regulation, extracellular matrix, cell death, and other important pathways, can be affected by GDFMD.Alb , Plg , Fn1 , Fga , Apob , Agxt , Kng1 , F2 , Fgg , and Serpinc1 . The details of the top 10 down- and up-regulated genes upon GDMFD administration are provided in We constructed the PPI network related to DEGs altered by GDFMD, according to the data in the STRING database. The PPI network, consisting of 675 DEGs and 1,755 pairs of interactions, is shown in Using the MCODE plugin with default criteria, we obtained 39 modules, and these modules are presented based on MCODE scores in descending order. Following the MCODE scores, the top4 modules were designated as modules 1 to 4. These four modules were selected for visualization of the module network . SpecifiTimp1, Fbn1, and Gas6 were overexpressed in the Model group  was low and that of 2 genes (Cyp4a32 and Cyp4a10) was high in the Model group. A large body of evidence suggests that PPAR signaling pathway is involved in different liver diseases  and more than 120 DEGs are involved in the inflammatory response (p = 1.169e−12). It appears that both adaptive (p = 2.397e−06) and innate (p = 4.022e−06) immune responses are abnormal in WD. Several immune and inflammatory response pathways, such as complement and coagulation cascades, cytokine-cytokine receptor interaction, inflammatory mediator regulation of TRP channels, antigen processing and presentation, and T cell receptor signaling pathway, were enriched. We found that hundreds of immune factors, including 133 cytokines and cytokine receptors, 13 interleukins and interleukin receptors, 8 TNF family members and receptors, 10 TGF-β family members and receptors, and 34 chemokines and receptors, are dysregulated in WD, and that GDFMD could revert these dysregulations. Therefore, we hypothesize that WD can lead to the recruitment and induction of monocytes, T cells, eosinophils, and lymphocytes, among other immune cells. Previous studies have shown that the complement system is involved in a variety of chronic liver diseases , 5 integrins , 6 laminins , and 3 thrombospondins  were upregulated in WD, and GDFMD could effectively reverse their abnormal expression levels. Collagen has been used as an important marker of liver fibrosis, and a large number of drugs have been developed based on the regulation of collagen, which can play an effective role in inhibiting liver fibrosis (During liver injury, the expression of and changes in various ECM components in fibrous tissues may convey important pathophysiological information, which is valuable for the development of new biomarkers and antifibrosis interventions. We identified that more than 31 ECM-receptors, except for SDC4, VTN, and FN1, were upregulated in WD . In partfibrosis . We alsofibrosis . In geneLtbp1, Id3, Tgfb3, Thbs1, Dcn, Id4, Bambi, Tnf, Inhbc, Smad7, Bmp5, Inhbb, Gdf7, Bmp6, Id1, and Gdf6, could be enriched, consistent with our findings in a previous study (Besides, we also identified many cell death-related genes and pathways that could be affected by GDFMD, such as the PI3K-Akt signaling pathway, apoptosis, and TGF-beta signaling pathway. Sixteen genes related to TGF-beta signaling pathway, namely us study .In conclusion, GDFMD may attenuate tx-j WD by altering metabolism, immune and tissue remodeling, and cell death-related pathways. This is a bioinformatics study to explore the unknown mechanism underlying the effect of a TCM. The results provide valuable information for further research and can provide a new model for development of novel drugs. Although most of our data are consistent with those reported in other studies, some limitations of this study are worth noting. First, the progression of WD may be related to age. In this study, DEGs were examined in adult male mice. Further investigation is needed to verify the candidate targets in different age groups. Second, in view of the regulation of protein translation and post-translational modification, we do not know the extent to which altered gene expression affects protein function. The functions of proteins encoded by these genes need to be further studied to provide new ideas for the treatment of WD.Through genetic engineering mouse model and RNA-seq, we preliminarily verified the efficacy and mechanism of GDFMD in the treatment of WD. The pathogenesis of WD and the treatment mechanism of GDFMD were interpreted from the whole genome level. However, further experimental is needed to verify for identified genes and pathways.


The mechanism of Panax notoginseng in treating myocardial fibrosis (MF) was investigated using network pharmacology.  Effective ingredients and potential targets of Panax notoginseng were screened in relevant databases to construct a compound-target network. Targets of MF were then screened to select common targets and construct a protein-protein interaction network. This was followed by Gene Ontology and pathway enrichment analyses. Molecular docking then verified the results of network analysis.  A total of 14 effective ingredients and 119 potential targets for MF were predicted. Quercetin, beta-sitosterol, and gossypetin were speculated to be the main active ingredients. The mechanism of action may be related to AGE-RAGE, proteoglycans, and IL-17 signaling pathways. Five key targets  may be involved in the treatment of MF using Panax notoginseng.  This study embodies the complex network relationship of multicomponents, multitargets, and multipathways of Panax notoginseng in treating MF and provides a novel method for further research on this herb's mechanism. Cardiovascular disease (CVD) is the leading cause of mortality worldwide, and it is estimated that CVD will result in more than 23 million deaths by 2030 . DiabeteCompendium of Materia Medica (Ben Cao Gang Mu) compiled by Li Shizhen in the Ming dynasty. Modern research has revealed that the main components of Panax notoginseng include saponins, volatile oils, flavonoids, and polysaccharides. Its pharmacologic effects are mainly reflected in its actions on the circulatory and cerebrovascular systems. In murine experiments, Panax notoginseng has been found to have a therapeutic effect on MF [Panax notoginseng (Burk.) F.H. Chen (notoginseng) is an herb commonly used in Chinese medicine. Its traditional application is to promote blood circulation and dispel blood stasis. Records of Panax notoginseng date to the ct on MF –12. HoweNetwork pharmacology is a systematic research methodology that combines laboratory and clinical inquiries with data processing to guide drug discovery and development. It is an effective method for studying the complex relationship between Chinese herbal medicines and diseases . This cuhttp://www.symmap.org) and in the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP) database (https://tcmspw.com/tcmsp.php). The screening criteria were oral bioavailability (OB) ≥ 30% and drug‐like (DL) ≥ 0.18.The keywords “Panax notoginseng” were used to retrieve the compound components in the SymMap database (http://www.nrc.ac.cn:9090/ETCM/index.php/Home/Index/index.html), the SymMap database, the Similarity Ensemble Approach (SEA) database (http://sea.bkslab.org), and the STITCH database (http://stitch.embl.de). The UniProt ID of the target was then searched through the Universal Protein Resource (UniProt) database (https://www.uniprot.org), with the species defined as “Homo sapiens.” All gene names were assigned their official gene symbol. Then, targets that did not meet the screening criteria were eliminated. Next, the network mapping software Cytoscape 3.8.0 (http://www.cytoscape.org) was used to construct networks for the compounds and their targets. In the network, a node represents a target, gene, molecule, or protein, and the connections between nodes represent the interactions between the targets, genes, molecules, or proteins. The “degree” value of a node represents the number of connections between the nodes in the network; the larger the value, the more likely the target is to become the key target of compounds.The targets of the compounds were searched through the Encyclopedia of Traditional Chinese Medicine (ETCM) database (https://omim.org), GeneCards database (https://www.genecards.org), Drugbank database (https://www.drugbank.ca), and Pub-Gene database (https://www.ncbi.nlm.nih.gov/pubmed) to obtain the disease targets.The keyword “myocardial fibrosis” was searched in the Online Mendelian Inheritance in Man (OMIM) database (https://string-db.org) to obtain the protein interaction relationship. The results were then imported into Cytoscape 3.8.0 software to construct and analyze the interaction network.The potential targets of the retrieved compounds and disease targets were intersected, and the overlapping targets were selected and imported into the STRING database  to obtain the protein class of key targets.Cytoscape plugin MCODE was applied for cluster analysis, and the filter conditions were set as degree cutoff: 2, http://geneontology.org) includes various functions of genes including biologic process (BP), molecular function (MF), and cellular component (CC) and can be applied to the analysis of potential biologic molecular mechanisms. The KEGG database (https://www.kegg.jp) is used to identify biologic functions and candidate targets. In this study, ClusterProfiler (https://bioconductor.org/packages/release/clusterProfiler.html) in R package was applied to GO functional annotation and KEGG pathway analysis, and the enrichment analysis results were visualized.The Gene Ontology (GO) database (https://pubchem.ncbi.nlm.nih.gov/), and the three-dimensional structures of the key targets were downloaded from the Protein Database (PDB) (http://www.rcsb.org/pdb/home/home.do). The higher the absolute value of the docking score, the stronger the binding ability of small molecules to protease targets.The Ligand Docking module in Schrödinger software was used to verify the reliability of the results, and the binding activity of the compound to the key targets was evaluated by the docking score. The structures of all the compounds were downloaded from the PubChem database , AKT1 (docking score = −8.277), and TNF (docking score = −4.689) . DihydroThis study used network pharmacology to systematically analyze the mechanism of action of Panax notoginseng in the treatment of myocardial fibrosis (MF). The resulting PPI network has 119 targets, accounting for one-third of the target number of Panax notoginseng. Five key targets, IL6, ALB, AKT1, TNF, and VEGFA, have high network value. Thus, we speculate that the effective components of Panax notoginseng may have pharmacologic activities in the treatment of MF through these targets.β1 signaling pathway [α is also a proinflammatory cytokine with a wide range of biologic effects and is involved in the pathophysiology of various cardiovascular diseases. Low-level expression of TNF-α in normal myocardium has a protective effect on myocardial cells. However, its increased expression can cause myocardial fibroblast proliferation, myocardial cell death, systolic dysfunction, cardiac fibrosis, and ventricular remodeling [Fibrosis is the final stage of a chronic inflammatory response, which can be caused by many factors. IL6 is a potent proinflammatory cytokine involved in MF . Fibrobl pathway –20. TNF-modeling –23.In this network pharmacology study, we found that ALB, VEGFA, and AKT1 are also involved in MF. Jäntti et al. found a close relationship between the ALB level and cardiac function. When the plasma ALB level of hemodialysis patients was increased, cardiac function of patients improved, thus effectively raising their quality of life . StudiesThrough this network pharmacology study, we found that Panax notoginseng in treating MF mainly involves the AGE-RAGE signaling pathway in diabetic complications, proteoglycans in cancer, and IL-17 signaling pathways. Cardiovascular complications are the leading cause of death in diabetic patients. The AGE-RAGE signaling pathway of myocardial fibrosis in diabetic complications has been widely studied. It regulates the pathogenesis of cardiovascular disease and promotes increased collagen deposition leading to tissue fibrosis , 30. Theβ, a powerful fibrogenic factor [Recent studies have shown that proteoglycans are promising diagnostic biomarkers for cardiac fibrosis and may provide new treatment strategies for heart disease . Proteogc factor . Furtherc factor . In varic factor .β/Erk1/2/NF-κB (nuclear factor κappa B) pathway [IL-17 has also been found to be involved in MF. In diabetic mice, IL-17 can reduce MF and improve cardiac function by inhibiting long-term noncoding RNA-AK081284 . Studies pathway , 39. Thuα in myocardial tissues [We found that rutaecarpine and ginsenoside Rb1 are the prime binding compounds to the key targets through molecular docking. Rutaecarpine exhibits a number of pharmacologic effects on the cardiovascular system including cardiac protective, vasodilator, and antiatherosclerotic activities , 41. Rut tissues . Ginseno tissues –45. WhilPanax notoginseng has a wide range of clinical applications in treating MF, but there are few reports on its pharmacologic activities. In this network pharmacology study, a multicomponent, multitarget, and multipathway treatment of MF using Panax notoginseng was established and provides a theoretical basis for clinical treatment of MF. However, the results of this study are based on data analysis and have only a certain predictive effect, which needs to be verified by further in vitro and in vivo experiments.