Objective: Ulcerative colitis (UC) and Crohn’s disease (CD) are two subtypes of chronic inflammatory bowel disease (IBD). Differential diagnosis remains a challenge. Anti-TNFα treatment is an important treatment for IBD, yet resistance frequently occurs and cannot be predicted. Consequently, many patients receive ineffective therapy with potentially adverse effects. Novel assays are needed to improve diagnosis, and predict and monitor response to anti-TNF-α compounds.Design: Signal transduction pathway (STP) technology was used to quantify activity of STPs  in colon mucosa samples of CD and UC patients, based on transcriptome analysis. Previously described STP assay technology is based on computational inference of STP activity from mRNA levels of target genes of the STP transcription factor.Results: Results show that NFκB, JAK-STAT3, Wnt, MAPK, and androgen receptor pathways were abnormally active in CD and UC. Colon and ileum-localized CD differed with respect to STP activity, the JAK-STAT1/2 pathway being abnormally active in ileal CD. High activity of NFκB, JAK-STAT3, and TGFβ pathways was associated with resistance to anti-TNFα treatment in UC and colon-located CD, but not in ileal CD. Abnormal STP activity decreased with successful treatment.Conclusion: We believe that measuring mucosal STP activity provides clinically relevant information to improve differential diagnosis of IBD and prediction of resistance to anti-TNFα treatment in patients with colon-localized IBD, and provides new targets for treatment and overcoming anti-TNFα resistance. Inflammatory bowel disease (IBD) is an inflammatory chronic intestinal disorder. The two major types include ulcerative colitis (UC) and Crohn’s disease (CD). Crohn’s disease affects the small intestine and large intestine, as well as the mouth, esophagus, stomach and anus, whereas ulcerative colitis primarily affects colon and rectum. Differential diagnosis with respect to CD and UC remains a challenge, and in 15% of cases, definitive discrimination is not possible. Correct diagnosis is important to decide on the most suitable therapy including systemic drug treatment. Systemic therapy is directed at inhibition of inflammation and the auto-immune process, healing of the intestinal wall, and correction of the microbiome . MultiplWe show that our signal transduction pathway (STP) technology can be used to develop these clinically needed assays, and to identify new drug targets in patients with CD and UC.Development and validation of computational models to quantitatively measure activity of the androgen receptor (AR), MAPK, NFκB, WNT, TGFβ, JAK-STAT1/2, and JAK-STAT3 pathways have been described before and is simplified shown in PAS were calculated from Affymetrix expression microarray data obtained from the GEO database . For eacQuality control (QC) was performed on Affymetrix data of each individual sample, using standard methods as described before . In summGSE16879 . MucosalGSE23597 . 48 UC pGSE52746 , Colon mAll analyzed data were obtained in agreement with GDPR regulations, as implemented within Philips.1) On the same sample, log2 odds PAS cannot be compared between different signaling pathways, since each of the signaling pathways has its own range in log2 odds activity scores .2) The log2 odds range for pathway activity (minimum to maximum activity) may vary depending on cell type. Once the range has been defined using samples with known pathway activity, on each new sample the absolute value can be directly interpreted against that reference. If the range has not been defined, only differences in log2 odds activity score between samples can be interpreted.3) PAS are highly quantitative, and even small differences in log2 odds PAS can be reproducible and meaningful.4) A negative log2 odds ratio does not necessarily mean that the pathway is inactive.An important and unique advantage of the STP technology is that in principle analysis can be performed on each cell type. As described before , one shot-test (low sample numbers) or Mann-Whitney-Wilcoxon testing was used to compare PAS across groups. p-values are indicated in the figures, with p < 0.01 considered as significant. Mean log2 odds, mean delta between groups, p-values, u-stats and area under the curve (AUC) values have been summarized in Boxplots, individual sample plots and hierarchical clustering plots have been made using the Python data visualization library function seaborn; additional statistical annotations have been created using Python package statannot. Box-and-whisker plots show median values, interquartile range (IQR) between 25th and 75th percentile, extreme values, and outlier samples (diamond shape). Individual samples are presented as small circles . Two-sided p < 0.01.STP activity scores from colon mucosa biopsy samples from clinical studies on CD and UC are shown in To investigate which STPs are abnormally active in colon mucosa of IBD patients, and whether measuring STP activity can improve diagnostic accuracy, and can be used to facilitate differential diagnosis of CD and UC, STP activity was calculated for mucosa biopsy samples of IBD patients with different clinical manifestations. For CD, colon biopsy sample data were available from patients with specific involvement of either colon (CDc) or ileum (CDi) (dataset GSE16879), and of patients with disease present in colon, but not necessarily limited to colon (dataset GSE52746).In untreated CD , MAPK, NFκB, JAK-STAT1/2, JAK-STAT3, and Wnt PAS were increased compared to healthy controls . There wFor untreated UC patients (dataset GSE16879) the same STP PAS were increased as found in patients with CDc, with addition of the TGFβ pathway ; Table 1Summarizing these results  and UC  patients were analyzed.In both CDc and UC patients (GSE16879), NFκB, TGFβ, and JAK-STAT3 PAS were higher in non-responder compared to responder patients . CDi patIn the second UC study  two different dosages of anti-TNFα had been administered, and data were combined for our analysis. Again, high pre-treatment NFκB, TGFβ and JAK-STAT3 PAS were associated with resistance to remission-induction treatment (but not related to failure of maintenance treatment) .Summarizing these results, remission-induction response to the TNFα inhibitor Infliximab in patients with colon-localized CD and UC was reproducibly predicted by high pre-treatment NFκB, TGFβ, and JAK-STAT3 signa transduction pathway activity.Comparison of STP activity profiles between post-treatment responder and non-responder groups  revealed the same hyperactive STPs which had been identified as pathogenic by comparing healthy individuals with IBD patients , the decrease (delta) in AR and MAPK PAS was higher than in non-responder patients . In addiWe compared our STP analysis results with the data analysis results obtained by the investigators who had generated the Affymetrix data which we analyzed for the purpose of the current study. For their data analysis they used conventional public data analysis tools and compared groups of samples, aiming to identify differentially involved cellular biochemical processes. In contrast to the current analysis, their analysis was not performed on individual samples. Below we briefly summarize their results with the used data analysis tools, per dataset.GSE16879. Data was analyzed using Bioconductor tools, with focus on differentially involved antimicrobial peptides (AMP) genes. In non-responders to infliximab, differences in AMP gene expression were not found, neither before nor during treatment.GSE52746. Differential expression and clustering analysis was performed using Ingenuity Pathway Analysis (IPA). Top 10 biochemical processes identified were: Docosahexanoic Acid signaling; IL17A signaling in fibroblasts; tumoricidal function of hepatic natural killer cells; role of IL17F in allergic inflammatory airway disease; role of IL17A in psoriasis; granulocyte adhesion and diapedesis; differential regulation of cytokine production in intestinal epithelial cells by IL17A and IL17F; agranulocyte adhesion and diapedesis; role of Tissue Factor in cancer. Mechanistic networks: IL1B, IL17, NFkB, STAT3 are candidate upstream regulators. The investigators concluded that in non-responders an IL-17A-driven response was present which was not sensitive to anti-TNFα blockade. While involvement of NFκB and JAK-STAT3 pathways was identified, no information on activity status of these signaling pathways was obtained.GSE23597. Data was analyzed using differential expression and clustering analysis with IPA. Top 6 biochemical processes identified were: cell-to-cell signaling and interaction; cellular movement; hematological system development and function; immune cell trafficking; inflammatory response; skeletal and muscular disorders. No information on involvement of specific signaling pathways and their activity was reported.Despite many differences between the investigated studies, STP analysis results were generally consistent across patient groups, both for CD and UC. We identified the MAPK STP as a ‘core’ abnormally active STP in IBD, while NFκB, TGFβ, JAK-STAT1/2, JAK-STAT3, and possibly the Wnt pathway, seem to be differentially involved in the various clinical IBD presentations. High activities of NFκB, TGFβ and JAK-STAT3 pathways prior to treatment were predictive for resistance to anti-TNFα treatment in patients with colon-localized IBD, but not in ileal CD. Successful remission-induction treatment and maintenance was associated with a reset in pathway activity towards normal. For all STPs there was a large variation in STP PAS among patients within the various IBD subtypes.We previously reported a completely new method to quantitatively measure activity of clinically relevant signal transduction pathways (STP) on transcriptome data of individual cell-containing samples , Verhae, Stolpe, Canté-, Wessel, Van Oo, Bouwma, Wessel, Bouwma. Using tSince abnormal activity of STPs is informative on the pathophysiology of a disease (the “mechanism of disease”) we investigated which STPs are abnormally active in different forms of IBD. Activity of the MAPK growth factor pathway was consistently increased in colon biopsies of patients with either UC or CD, irrespective of colonic or ileal disease location. Clinically effective anti-TNFα treatment (partly) corrected this abnormal signaling pathway activity, further supporting a core pathogenic role for the MAPK signaling pathway in IBD, independent of disease location. Indeed, the MAPK pathway has been described as an important signaling pathway in a number of related auto-immune diseases, such as IBD and rheumatoid arthritis , Kumar , Coskun, Xu et .A few signaling pathways showed differential activity between colon- and ileum-located IBD. Activity of the NFκB, AR, JAK-STAT3, Wnt, and (less consistently) the TGFβ, signaling pathways was increased in patients with IBD disease localization in the colon, while the JAK-STAT1/2 pathway was specifically active in ileal localized CD, suggesting differences in pathophysiology depending on disease location. This is in line with current thinking regarding differences between ileal- and colon-localized IBD, including marked differences in clinical response to anti-TNFα treatment . The infOur results support the increasing evidence that the pathogenesis (or mechanism of disease) of ileal IBD is distinct from colon-located IBD , Naftal, Kredel, Howell, Meinze, Wu et , van de. InterfeIn patients with a diagnosis of IBD, either CD or UC, a biomarker assay to predict response to anti-TNFα treatment is needed because of potentially serious side effects of the treatment and significant treatment costs . In our NFκB pathway activity seemed to play a less dominant role in ileal-CD, clearly in line with anti-TNFα treatment being less effective in patients with ileal-restricted CD . Indeed,While considered highly necessary from a health economic standpoint as well as from a clinical point of view, currently clinical response to anti-TNFα treatment cannot be predicted in IBD patients , Lopetu, D’Haen, Targow. LiteratOur results suggest that to overcome resistance to anti-TNFα treatment, it may be needed to target the NFκB signal transduction pathway in a different manner than by inhibiting its ligand TNFα , Miller. Indeed,Potentially relevant for any treatment of IBD is the observation that activity of the signaling pathways showed large variations among patients with IBD. This suggests that clinical efficacy of a drug, including an anti-TNFα drug, in an individual patient may depend on the level of activity of the targeted signaling pathway, implicating that a personalized treatment approach is likely to be necessary to optimize treatment results. This requires an assay to measure STP activity in a sample of every individual patient to predict response to a drug. For example, a decision on treatment with an anti-TNFα drug such as infliximab ideally would be preceded by measuring NFκB pathway activity to guide this treatment decision.For each Affymetrix dataset for which we performed STP analysis, data-analysis results obtained by the original investigators were compared to our results. While gene expression profiles had been obtained for CD and UC patient groups , no gene profile had been identified by the previous investigators to separate anti-TNFα responder and non-responder group, and no information on STP activity had been extracted from the Affymetrix data. This emphasizes the difference between our STP activity analysis, which can be performed on individual patient samples and provides clinically actionable STP information for an individual patient , and other bioinformatics tools which are used to discover gene expression profiles as potential novel biomarkers for groups of patients.In conclusion, STP analysis provided a first-of-a-kind insight into the mechanism of disease of IBD and its relation to treatment resistance. Our results provide new treatment targets, for example, the MAPK pathway in all types of IBD, in ileal Crohn’s disease the JAK-STAT1/2 pathway, and in patients refractory to anti-TNFα treatment the NFκB, TGFβ, and JAK-STAT3 pathways.


Each cluster is assigned a meaningful name that highlights the main biological themes in the experiment. Our approach enables an automated and objective overview of the data without manual and time-consuming parameter tweaking.The interpretation of pathway enrichment analysis results is frequently complicated by an overwhelming and redundant list of significantly affected pathways. Here, we present an R package aPEAR is implemented in R, published under the MIT open-source licence. The source code, documentation, and usage instructions are available on https://gitlab.com/vugene/aPEAR as well as on CRAN (https://CRAN.R-project.org/package=aPEAR).The package  Pathway enrichment analysis (PEA) is indispensable when interpreting high-throughput omics data and identifying the underlying biological processes that are dysregulated in a particular condition or disease , which aids in the interpretation of the PEA results. The aPEAR package implements multiple metrics to calculate similarities between pathway gene sets, detects pathway clusters, and assigns biologically relevant names to them. Finally, aPEAR builds a visual representation of an enrichment network that can be explored interactively to elucidate the biological processes affected by the experimental conditions.Here, we present an R package aPEAR exports a single main function enrichmentNetwork which visualizes the PEA results as a network where nodes and edges represent the pathways and similarity between them, respectively. While it was created with the clusterProfiler , cosine similarity, or correlation similarity metrics.The similarity matrix is then used to detect clusters of redundant pathways using Markov (default) , hierarcPageRank (HITS (P-value can be used to select the most important pathway in the cluster. The description of this pathway is used as the cluster label.Each cluster is assigned a biologically meaningful name. Network analysis is used to determine the pathway with the most connections, using either PageRank  (defaultnk (HITS  algorithggplot2 (plotly (A ggplot2  graph is (plotly . Pathway (plotly .emapplot function from the R package enrichplot (Enrichment Map (emapplot has some limitations when handling large datasets and produces numerous small clusters with nonintuitive labels (aPEAR makes it easy to work with numerous pathways, highlights the biological context of the clusters and does not require additional manipulation of the graph, making it ideal for automated data visualization as well as interactive investigations (Currently, the most frequently used tools for gene set visualization include the richplot  and the e labels . Cytoscae labels . In contigations .aPEAR package (To determine which similarity metric and clustering algorithm is best suitable for pathway cluster analysis, 180 tests were performed using PEA results from 10 real-world datasets . Based o package . Note thaPEAR, that visualizes clusters of similar pathways as an enrichment network and, consequently, enables better interpretation of the PEA results.We developed an R package, btad672_Supplementary_DataClick here for additional data file.


Stypopodium zonale. We investigated stypoldione's mode of action in breast cancer using the network pharmacology method, and we confirmed our research by using a number of computational tools, including UALCAN, cBioportal, TIMER, docking, and simulation. The findings revealed 92 common targets between the chemical and breast cancer target network. Additionally, we found that stypoldione targets a number of unregulated genes in breast cancer, including: ESR1, HSP90AA1, CXCL8, PTGS2, APP, MDM2, JAK2, KDR, LCK, GRM5, MAPK14, KIT, and several signaling pathways such as FOXO signaling pathway, VEGF pathway, calcium signaling pathway, MAPK/ERK pathway and Neuroactive ligand-receptor interaction. The examined medication demonstrated a strong affinity for the major targets, according to a docking analysis. The best hit compound produced a stable protein–ligand pair, as predicted by molecular dynamics simulations. Our results are supported by the fact that when in-vitro assays were done on melanoma using stypoldione compound it was found that its mechanisms of action involved the PI3K/mTOR/Akt and NF-kB pathways. This study was set out to inspect the possible value of stypoldione as a breast cancer cure and to get a deeper understanding of the molecular mechanisms by which this drug acts on breast cancer.Breast cancer is the primary factor contributing to female mortality worldwide. The incidence has overtaken lung cancer. It is the most difficult illness due to its heterogeneity and is made up of several subtypes, including Luminal A and B, basal-like, Her-2 overexpressed and TNBC. Amongst different breast carcinoma subtypes, TNBC is the most deadly breast cancer subtype. The hostile nature of TNBC is mainly attributed to its lack of three hormonal receptors and hence lack of targeted therapy. Furthermore, the current diagnostic options like radiotherapy, surgery and chemotherapy render unsuccessful due to recurrence, treatment side effects and drug resistance. The majority of anticancer drugs come from natural sources or is developed from them, making nature a significant source of many medicines. Marine-based constituents such as nucleotides, proteins, peptides, and amides are receiving a lot of interest in the field of cancer treatment due to their bioactive properties. The role of stypoldione in this study as a prospective treatment for breast carcinoma was examined, and we sought to comprehend the molecular means/pathways this chemical employs in breast carcinoma. The most promising possibility for an anti-cancer treatment is stypoldione, a marine chemical produced from the brown alga  With mo as Skin , P388 ly as Skin . In addi as Skin . Additiopoldione . Therefon Brazil  we exploThe computational tools such as UALCAN, TIMER, cBioportal etc we utilized to confirm and validate our results further to reach to a conclusive target. A collaborative research strategy called network pharmacology can be utilised to identify complicated disease causes and develop effective treatment plans from a systems viewpoint . The mul22.1https://www.swissadme.chwebsite). This analysis makes it possible to calculate physical–chemical descriptors and predict the pharmacokinetic properties, drug-likeliness, and pharmacologic chemistry of one or more small molecules, which simplifies drug development in terms of ADMET analysis . The Swiss target prediction approach assesses some of a small molecule's most likely macromolecular targets and provides details on chemical compounds and their biological effects (https://www.genecards.org). This database gives inclusive and easily understandable information on both known and predicted genes that are of critical importance in human diseases (The target genes for the compound were identified using the Integrative Pharmacology-based Research Platform of Breast Carcinoma and SWISSPROT-TARGET-PREDICTION ( effects . The strdiseases . The dat2.3https://www.t2diacod.igib.res.in/), OMIM (https://www.omim.org/), and Gene Atlas of Breast Cancer (https://t2diacod.igib.res.in/) databases were employed to gather data concerning the mutual target genes associated with the disease and the compound (Stypoldione), which were visualized in a Venn diagram. Initially, data from the TCGA database were acquired for breast tumor samples exhibiting the peak 10 % of both over-expressed and under-expressed mRNA genes. Subsequently, potential targets of Stypoldione against genes implicated in breast cancer were identified and represented in a Venn diagram using information from the TCGA database.The TCGA (2.4https://stringdb.org) provides information on verified and anticipated protein interactions across different species, allowing us to access data on these protein–protein interactions (PPI)  . We empl2.5We employed Shiny GO to perform pathway enrichment and Gene Ontology (GO) analysis on shared target genes within the “Homo sapiens“ species, utilizing data obtained from the Kyoto Encyclopedia of Genes and Genomes (KEGG). The outcomes were visually depicted using bubble charts and bar graphs. Our study encompassed the enhancement of Gene Ontology terms associated with cellular components (CC), biological processes (BP), and molecular functions (MF). Additionally, we utilized bubble and bar graphs to showcase the results of the KEGG pathway enrichment analysis.2.6https://cytoscape.org/, version 3.8.0) was employed to construct an intricate network comprising interactions among a drug, a cancer-related target gene, and a gene pathway. The utilization of this freely available software, Cytoscape, facilitates the visualization of biological processes and molecular interaction networks . We established statistical significance using a P-value threshold of 0.05. To assess the expression of these crucial target proteins in both normal and breast cancer tissues, we relied on data from the Human Protein Atlas (HPA) (https://www.proteinatlas.org/). To determine the overall survival rates (OS) associated with these core targets, we used the TIMER cistrome database (https://timer.cistrome.org) and applied a significance threshold with a P-value of 0.05. We then presented the results visually, including hazard ratios. To investigate the genetic uniqueness and connections among the mRNA expressions of these crucial targets, we utilized the cBioPortal tool . We carried out a total of 2,746 analyses using breast cancer samples. Moreover, we employed the Genomic Identification of Significant Targets in Cancer (GISTIC) tool to examine the genomic characteristics of the 12 key targets.We evaluated the mRNA expression levels of essential genes by cross-referencing data from The Cancer Genome Atlas, which can be accessed through the UALCAN database  binding. Following the addition of non-polar hydrogens to both the protein molecules and the ligand, we saved them in the pdbqt format. The docking grid box has dimensions of 33 × 51 × 52 Å. The construction of the grid boxes had to be done with specific dimensions and 0.3 spacing. In each of the molecular docking studies, there were 50 solutions, 500 population members, 2,500,000 assessments, a limit of 27 generations, and all other parameters were kept at their default settings. The procedure included re-clustering at clustering thresholds of 0.25, 0.50, and 1 to produce RMSD clustering maps. The aim was to pinpoint the most desirable cluster characterized by the least energy change and the greatest concentration of population.2.92O molecules confined within a periodic boundary box measuring 10 Å × 10 Å × 10 Å  solvation model and the OPLS5 force field. The calculation of binding free energies in molecular mechanics followed an additive methodology, taking into account various energy components, such as hydrogen bonds, van der Waals forces, hydrophobic interactions, electrostatic interactions, self-interactions, covalent interactions, and solvation effects for both the ligand and the protein. These energies are measured in kilocalories per mole . The below equation may be used to determine 3Stypoldione was chosen for this investigation from the literature. The stypoldione's properties are listed in 44.1(a)).The top 30 most often occurring target genes were displayed in Fig. 2(b). ESR1, HSP90AA1, CXCL8, PTGS2, APP, MDM2, JAK2, KDR, LCK, GRM5, MAPK14, KIT and several target genes displayed elevated levels of protein interactions, suggesting their potential role as central proteins within the network, as indicated in Supplementary Files 2 and 3. The results suggest that the selected compound, “stypoldione,“ exhibited a high attraction to these proteins, suggesting its potential usefulness as a hopeful therapeutic candidate for breast cancer treatment.In the case of Stypoldione, a grand total of 104 potential target genes were identified, as illustrated in Fig. 24.2The gene ontology analysis of the mutual target genes, as shown in 4.3A). The protein nodes are arranged in a hierarchical manner. Additionally, Fig. 5B displays the primary pathway network from the KEGG analysis. In Fig. 5C, you can observe Stypoldione represented by a blue hexagonal node, along with the 92 shared targets and the connecting edges among them 4.4a). In turn, tumor tissues showed significantly lower levels of JAK2, KDR, PTGS2, APP, and KIT mRNA expression, but HSP90AA1, MDM2, or LCK mRNA levels did not decrease noticeably.To gain insights into their potential diagnostic and pharmacological significance, we assessed the mRNA and protein expression of 12 central targets selected due to their frequent occurrence in the study. Among patients with invasive breast carcinoma from the TCGA database in UALCAN, it was evident that tumor tissues exhibited significantly elevated levels of ESR1 and GRM5 mRNA, while lower levels of MAPK14 and IL-8 mRNA, both of which are known tumor suppressors, were observed a. In turb. In contrast, the mRNA levels of PTGS2, LCK, JAK2, KDR, MAPK14 and KIT compared to normal levels, were markedly reduced in 1 to 4 stages of breast cancer tissues.Afterward, using the UALCAN database, we inspected the association between 12 hub targets' mRNA levels and different breast cancer tumor phases. ESR1 and GRM5 expression levels were both noticeably higher than average, except that stage four BC tissues did not substantially exhibit increased levels of HSP90AA1, IL-8, APP, or MDM2 as represented in Fig. 6We next used the TIMER Cistrome database to examine the prophetic impact of 12 hub targets in breast carcinoma patients. a. Collectively, the overall alteration rate for these 12 central targets amounted to 42.5 %. Moreover, when considering diverse breast carcinoma types, the genetic variation rates spanned from 0 % to 100 %, as illustrated in Fig. 8b.We proceeded to analyze the incidence and kinds of genetic variations in the 12 central targets among a cohort of 2,746 breast cancer patients using the cBioPortal tool. The individual rates of gene modifications varied, ranging from 1.4 % to 12 %. Notably, PTGS2 exhibited the highest degree of variability at 12 %, while HSP90AA1 and KIT displayed the lowest modification rates at 1.4 %, as indicated in Fig. 8This was followed by molecular docking analysis of stypoldione with the respective 11 hub target genes represented in 4.5The binding energy ratings of all clusters were estimated from the best huddle (95 %) that is contained within the tightest RMSD of 0.25. Stypoldione has the highest binding potential for the KDR and the shortest binding affinity . 4.6A. Importantly, all Root mean square deviation values remained within the acceptable range of 3 or below. The consistent RMSD plot throughout the simulation indicated favorable convergence and persistent conformations. Considering the ligand's increased affinity, it suggests that the Stypoldione-KDR complex is highly stable. The RMSF plot of the KDR protein with Stypoldione displayed minor fluctuations in residues 15–25, 60–72, 145–153, and 170–220, which could be associated with greater flexibility, as shown in Fig. 11B. The RMSF plots show that the protein retains its flexibility in the conformations when bound to the ligand throughout the simulation. We evaluated the protein's compactness using its Radius of Gyration (Rg). In this study, the KDR C-backbone bound to Stypoldione consistently exhibited an Rg value ranging from 25.2 to 25.4, as illustrated in Fig. 11C. The consistently stable Rg value strongly indicates that the protein maintains a condensed structure while interacting with the ligand. Furthermore, the robustness and endurance of the complex were reinforced by the substantial count of hydrogen bonds identified between KDR and Stypoldione throughout the entire one hundred nanoseconds of the simulation, as depicted in Fig. 11D.To assess the constancy and union of the KDR + Stypoldione complex, we conducted molecular dynamics and simulation (MD) investigations. During a simulation lasting one hundred nanoseconds, we observed consistent conformations, as evidenced by comparing the RMSD values. When KDR was coupled to Stypoldione, the C-backbone showed a displacement of 2.5 in terms of RMSD, while the ligand exhibited an RMSD of 1.8, as shown in Fig. 114.7We employed the molecular dynamics simulation trajectory to assess the binding capacity, free energy, and other associated energies in the MM-GBSA format for the KDR + Stypoldione complex. The findings presented in 4.8The simulations of the molecular dynamics of the protein–ligand complex's trajectory were put through a PCA, allowing for the examination of the random, global movement of amino acid residue atoms . This an5https://www.midwestern.edu/departments/marinepharmacology/clinical-pipeline.xml]. Additionally, many articles have demonstrated the anticancer properties of compounds derived from marine sources in-vitro or in-vivo. The anticancer substances obtained from marine sources mostly come from cyanobacteria, sponges, tunicate, bacteria, and fungi, as well as mollusks and cyanobacteria  revealed consistent trends throughout the simulation, indicating that Stypoldione effectively maintains its position within the VEGFR2 binding site. The Rg graphs indicated that VEGFR2 remained compact during the simulation, reflecting a stable protein backbone. The examination of alterations in residues throughout the simulation demonstrated a decrease in the exposed surface area of the target following the binding of the ligand. This research underscores Stypoldione's potential as a promising anti-cancer drug for breast cancer treatment, substantiated by a range of molecular mechanisms. It's important to note that these findings are based on a theoretical approach and require further experimental validation. Notably, a Brazilian research team investigated this compound's efficacy against multiple myeloma and identified its mechanisms of action involving the NF-kB and PI3K/mTOR/Akt pathways Statistical data reveals that breast carcinoma is the majorly prevalent recurring carcinoma among females and ranks as the fifth leading reason of mortality on a global scale. In the year 2020, over two million women across the world were diagnosed with breast cancer for the first time, constituting 11.7 % of all cancer diagnoses . Surgicabacteria . Stypolde family . Researce family . Additioe family . To ascee family . The mose family . Vasculae family . In humae family . VEGFR-2e family . VEGFR-2e family . In thise family  study she family . Notablyaphs see A and B r6In our ongoing study, we utilized a network pharmacology approach to examine the molecular mechanism of a recently identified marine compound called Stypoldione, which was extracted from the brown seaweed Stypopodium zonale. Following that, we validated our findings through molecular docking and MD simulation experiments. According to the outcomes of the network pharmacology analysis, Stypoldione exhibits the potential to influence numerous top-ranking signaling pathways, which encompass, among others, the VEGF pathway, PI3K-Akt pathway, FOXO signaling pathway, MAPK pathway, neuroactive ligand-receptor interaction, and calcium signaling pathway. These findings align with prior research that employed molecular docking and computer simulations, demonstrating Stypoldione's proficient binding to its target. In summary, our study marks the inaugural comprehensive exploration of the potential molecular and pharmacological mechanisms underpinning Stypoldione's application in breast cancer treatment, employing a network pharmacology-based approach. These results imply that Stypoldione, due to its multifaceted impact on various targets and pathways, offers promise as an efficacious therapeutic choice for breast cancer.7The absence of stypoldione as a commercially available substance was the study's only drawback. We were unable to conduct the in-vitro experiments since the chemical was not commercially available. We will investigate this medication more in the future to learn more about this molecule, though. Nevertheless, because this substance had showed promising outcomes in our research, the major goal of our study was to at least share our findings with the public.Hina Qayoom: Data curation, Methodology, Project administration, Software, Validation, Visualization, Writing – original draft, Writing – review & editing. Bader Alshehri: Methodology, Project administration, Software, Validation, Visualization, Writing – review & editing. Burhan Ul Haq: Methodology, Project administration, Software, Validation, Visualization, Writing – review & editing. Abdullah Almilaibary: Project administration, Methodology, Software, Validation, Visualization, Writing – review & editing. Mustfa Alkhanani: Methodology, Project administration, Software, Validation, Visualization, Writing – review & editing. Manzoor Ahmad Mir: Conceptualization, Methodology, Project administration, Resources, Software, Supervision, Validation, Visualization, Writing – review & editing.The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.


You-Gui-Yin (YGY) is a classic prescription for warming up kidney-Yang and filling in kidney essence in traditional Chinese medicine, and has been used to treat osteonecrosis of the femoral head (ONFH) effectively. However, the underlying mechanisms are still unknown. This study is aimed at exploring the possible mechanisms of action of the YGY in the treatment of ONFH based on network pharmacology and molecular docking. TCMSP was used to screen the active components and targets of YGY. The disease targets of ONFH were collected in several public databases. The protein-protein interaction (PPI) Network was constructed using the STRING platform. The Metascape database platform was used for Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses. The key active components and core target proteins of YGY in the treatment of ONFH were verified by the molecular docking. 120 active components were obtained from YGY, among which 73 components were hit by the 117 drug-disease intersection targets. Key effective components included quercetin, kaempferol, beta-sitosterol, glycitein, beta-carotene, and so on. Core target proteins included ALB, AKT1, TNF, IL6, TP53, and so on. According to GO and KEGG analyses, there were 1762 biological processes, 94 cellular component, 138 molecular function and 187 signaling pathways involved. we selected the top 20 biological processes (BP), cellular components (CC), molecular functions (MF) and signaling pathways to draw the heat maps, showing that Lipid and atherosclerosis signaling pathway, IL-17 signaling pathway, HIF-1 signaling pathway, relaxin signaling pathway and MAPK signaling pathway and other pathways may play a key role in the treatment of ONFH by YGY. The results of molecular docking showed that key effective components and corresponding core target proteins exhibited the good binding activity. YGY can treat ONFH through multicomponents, multitargets, and multipathways, which provides a reference for the subsequent research, development of targeted drugs and clinical application. In the terminal stage of the disease, most patients must undergo artificial hip replacement. However, artificial hip replacement will make great damage to normal tissues, high economic cost, certain surgical risks and postoperative complications, and patients may still need to undergo another or even multiple revision after the operation. Therefore, there is an urgent need to find the effective conservative treatment in the early and middle stages of the disease.Osteonecrosis of the femoral head (ONFH) which leads to chronic pain, femoral head collapse and dysfunction of the hip joint is one of the outstanding problems in the field of orthopedics and difficult to treat. Its etiology and pathogenesis are still unclear and complex, and current theories related to etiology and pathogenesis involve in trauma, corticosteroids, alcohol use, vascular thrombosis, blood dyscrasias and miscellaneous factors.Jingyue Complete Works, is composed of 8 traditional Chinese medicines (TCM): Shudihuang (Radix Rehmanniae Praeparata), Shanyao (Dioscorea opposite), Shanzhuyu , Gouqi (Lycium chinense Mill.), Duzhong (Eucommia ulmoides Oliv.), Fuzi , Gancao  and Rougui (Cinnamomum cassia Presl.). Among them, Radix Aconiti Late-ralis Preparata and Cinnamomum cassia Presl warm and tonify the kidney-Yang, known as monarch drugs in the prescription; Radix Rehmanniae Praeparata, Dioscorea opposita, Cornus officinalis Siebold & Zucc. and Lycium chinense Mill. nourish the kidney-Yin, enrich essence and marrow, nourish liver and strengthen spleen, take the meaning of “seeking Yang in the Yin,” known as ministerial drugs; Eucommia ulmoides Oliv. nourishes liver and kidney, strengthens bones and muscles, known as the adjuvant drug; Glycyrrhiza uralensis Fisch. reconciles the medicines, known as conductant drug. It is a classic prescription for warming up kidney-Yang and filling in kidney essence in traditional Chinese medicine, and there is a synergistic effect between Yang tonic and Yin tonic to obviously improve or even correct the “kidney-Yang deficiency” state.,4 On the other hand, ONFH is often be differentiated by traditional Chinese medicine (TCM) as the “kidney-Yang deficiency” state. So YGY is a kind of effective prescription for treating ONFH, and many clinical and animal experiments have proved that YGY is effective in treating ONFH.–8 However, as YGY is a compound preparation, the mechanism of YGY in ONFH treatment has not been fully understood. Therefore, the article selected the monarch drugs, ministerial drugs and adjuvant drug  which played the major role in the prescription for further study.You-Gui-Yin (YGY), derived from  The application of this research method in the analysis of TCM prescription is in line with the characteristics of overall compatibility of TCM prescription and the comprehensive multi-way therapy of diseases. Molecular docking is a computer-aided theoretical simulation method based on the “lock and key principle” of ligand-receptor interaction, which can be used to simulate the interaction between small molecular ligands of drug and biological target proteins in body and predict their binding mode and affinity.,11 This effect is helpful for screening drugs, explaining the cause of the activity of drug molecules and optimizing the molecular structure of drugs.The occurrence and development of diseases in the human body are caused by the interaction of many comprehensive factors. Therefore, the effective treatment of many diseases requires multi-target and multi-pathway therapeutic regimens. The concept of network pharmacology which analyzes the interaction between the drug, disease and targets, proposed by Hopkins in 2007, offers a multi-pathway, multi-target approach to evaluate the intervention or effect of drugs on diseases.In this study, network pharmacology and molecular docking techniques were used to find and verify the active components, action targets, biological processes, cellular components, molecular functions and signaling pathways of YGY in ONFH treatment. This study aims to explore the potential mechanism of YGY in the treatment of ONFH, and further deepen the understanding of the pharmacological mechanism and clinical application of this prescription. The flowchart of the study design is shown in Figure https://tcmsp-e.com/). According to the standards of Oral bioavailability (OB) ≥ 30% and drug-likeness (DL) ≥ 0.18, we screened the active components of You-Gui-Yin in the TCMSP database and then obtained the corresponding targets for each active component. Meanwhile, other active components not retrieved were supplemented by related literature reviews. If the targets of active components cannot be obtained from the TCMSP database, the prediction of the relevant targets was conducted in Swiss Target Prediction (http://www.swisstargetprediction.ch). In order to standardize the all identified targets of active components, we put the targets into the UniProt database (https://www.uniprot.org/) and set the protein type as “Homo sapiens.”The active components of Radix Rehmanniae Praeparata(SDH), Dioscorea opposita(SY), Cornus officinalis Siebold & Zucc. (SZY), Lycium chinense Mill.(GQ), Eucommia ulmoides Oliv.(DZ), Radix Aconiti Late-ralis Preparata(FZ), and Cinnamomum cassia Presl(RG). were acquired from the new edition of Traditional Chinese Medicine Database and Analysis Platform (TCMSP) (https://omim.org/), GeneCards (https://www.genecards.org/), DisGeNET (https://www.disgenet.org/), DigSee (http://gcancer.org/digsee/), TTD (http://db.idrblab.net/ttd/) and Drugbank (https://go.Drugbank.com/) database search. These target genes were standardized through Uniprot database, and then the disease targets obtained from above databases were integrated to get the total target genes of ONFH after deleting the repeated ones.The search for target genes related to the disease was obtained using the keywords “osteonecrosis of the femoral head,” “femur head necrosis,” “avascular necrosis of femur head” and “femoral head necrosis” in the the OMIM (https://bioinfogp.cnb.csic.es/tools/venny/) to get the intersection targets and construct Venn diagram.The targets of active components of YGY and ONFH were introduced into online Venny2.1 platform , the minimum required interaction score was set to “highest confidence>0.9.” The species was selected as “Homo sapiens,” the disconnected nodes were hid in the network and the other settings were default to construct the protein-protein interaction (PPI) Network. Then the core target proteins were screened according to “Degree” value.The intersection targets of the active components of YGY and ONFH were imported into the STRING database  was used for Gene Ontology (GO) functional enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) signaling pathway enrichment by importing the intersection targets into this database. Results with values of P < .01 were selected, and “Homo species” was defined as the species. Finally, we screened out the first 20 categories of biological processes (BP), cellular components (CC), molecular functions (MF) and KEGG signal pathways, respectively. Then the bioinformatic online platform (https://www.bioinformatics.com.cn/) was used to visualize the result analyses as bubble charts. Based on the data obtained from KEGG signaling pathway enrichment analysis, the target- KEGG signaling pathway network was constructed by introducing intersecting targets related to the pathways and core signaling pathways into Cytoscape3.8.2 software. The KEGG Mapper tool (https://www.genome.jp/kegg/mapper/) was used to visualize the signaling pathway.The Metascape database platform (https://www.rcsb.org/).In order to further clarify and verify the internal molecular mechanism of YGY in treating ONFH, the 14 key active components and 14 core target proteins screened from above methods (mentioned in 2.4 and 2.5) were used for molecular docking. The 2D structures of the potential active components (*Mol2 format) in YGY were downloaded from the TCMSP database while the 3D structures of the target proteins (*PDB format) treated by YGY in ONFH were downloaded from the RCSB PDB database (https://www.pymol.org/) was used to remove waters and ligands from the target protein receptors. And AutoDock Tools was used to hydrogenate the receptors, calculate the charge and other pretreatments. The center coordinate and size of the box were set based on the position of the active site of target protein receptors and the area where the ligands might bind. Molecular docking of the receptors and ligands was conducted by AutoDock Vina. Affinity was the binding energy for the molecular docking, and when the affinity score value was lower, the binding affinity between the receptor and the ligand was stronger. The affinity score value ≤ 0 kcal/mol indicated that the active component could bind and interact with the target protein, whereas affinity score value <−5 kcal/mol demonstrated a high binding activity and strong force. The binding energy results obtained from the analysis were imported into Origin 2021 software (https://www.originlab.com/) to create 2D heat map. Finally, the docking models were visualized using the PyMol software (https://www.pymol.org/).The PyMol 2.5.2 software  and 613 edges  composed of ten TCM according to Chinese Pharmacopeia 2020 version. Most of the TCM components of YGW and YGY are the same which makes the 2 have a stable and close association, but the preparation methods are different. YGW is a Chinese patent medicine in pill form, while YGY is a Chinese medicine prescription in decoction form. The drug efficacies of Chinese medicine pills and Chinese medicine decoction are usually similar, but the drug efficiency of pills is milder and the action time is longer, while the drug efficiency of decoction is stronger, faster, and the action time is shorter. Despite the differences, both YGY and YGW can warm up and tonify kidney-Yang and correct the “kidney-Yang deficiency” state clinically.,4,13,14 YGW and YGY were commonly used to treat gynecological diseases of kidney-Yang deficiency syndrome such as polycystic ovary syndrome, vaginal atrophy,,17 infertility and premature ovarian failure. ONFH is also be differentiated by traditional Chinese medicine as kidney-Yang deficiency syndrome. Therefore, the underlying mechanism of YGY in treating ONFH is also to treat kidney-Yang deficiency syndrome. Some studies have shown that bone loss and osteoporosis occur in mice after ovary removal, and postmenopausal ovarian function decline is likely to lead to postmenopausal osteoporosis. All these indicated that the female reproductive system especially the ovary is closely related to bone, and YGY can inhibit bone loss by promoting osteogenic proliferation and differentiation and attenuating osteoclastogenesis.,21 In western medicine, ONFH is a disease involving changes in local stem cell activity of the femoral head. The researchers found a decrease in the number, activity, or osteogenic differentiation of mesenchymal stem cells in the bone marrow in patients with ONFH.,24 There was a close corresponding relationship between kidney essence and stem cells, and the decrease of the number and activity of stem cells happened in patients with kidney-Yang deficiency. The kidney-tonifying herbs could activate stem cells, increase the quantity of the stem cells, and adjust the microenvironment to a stable and coordinated state. Previous studies have shown YGY can effectively treat ONFH.–8Just as –31 In preclinical models of rheumatoid arthritis, gouty arthritis and osteoarthritis, quercetin showed significant joint protective effects, attenuated the synovial inflammation and hyperplasia, reduced the bone/cartilage destruction in joints, and decreased the secretion of inflammatory cytokine. Quercetin can also inhibit the macrophage polarization and the differentiation of osteoclasts, so that inhibit the inflammatory response, osteolysis, and osteonecrosis. Kaempferol is a flavonoid chemical with a wide range of medicinal properties such as anti-inflammatory, analgesic and anticancer effects. In vitro and in vivo experimental models, kaempferol have been acknowledged to have the bone-protective effect by inhibiting inflammation, oxidative stress, adipogenesis, osteoclastic differentiation, osteoclastic autophagy and osteoblastic apoptosis while activating osteoblastic autophagy, and to maintain the bone homeostasis and prevent resorption, loss and necrosis of bone.,37 Beta-sitosterol is a kind of phytosterol, which has the effect of immune-regulation on macrophages and attenuates the inflammation reaction in mice model of rheumatoid arthritis.By studying the effects of β-sitosterol on pleurisy, foot swelling and ear edema in mice, β-sitosterol was found to have anti-inflammatory effects.And β-sitosterol can play an anti-inflammatory role by inhibiting the activation the inflammatory body NLRP3 in epidermal cells and macrophages to inhibit the production of CAS1 and the activation of MAPK signaling pathway, resulting in the significant decrease of of TNF-α, IL-1β, IL-6, and IL-8.In addition, β-sitosterol also has antioxidant effect by reacting with organic acid to generate derivatives and inhibit the overexpression of TLR4 and NF-κB.Therefore, β-sitosterol may be play a role in the treatment of femoral head necrosis through its anti-inflammatory, antioxidant effect. There were studies showed that glycitein suppressed osteoclast generation and induced osteoclast apoptosis in vitro and might also exert bone beneficial effects in vivo. Glycitein can also promote the differentiation of osteoblasts from their progenitor. β-carotene is a natural anti-oxidant, and can suppress osteoclast formation and bone resorption by inhibiting NF-κB signaling pathway.Syringetin, a flavonoid derivative, can increase BMP-2 synthesis and subsequently activate SMAD1/5/8 and ERK1/2, and this effect may help to induce the maturation and differentiation of osteoblasts, thereby increasing bone mass.Diosgenin, a steroidal sapogenin, can increase the formation of calcium deposits and enhance bone formation by stimulating the synthesis and secretion of Type 1 collagen, ALP, Runx2 and osteopontin expression. Folwarczna J. et al showed that Diosgenin increased compact bone formation and probably inhibited cancellous bone resorption, which led to improvement of mechanical properties of compact and cancellous bone. Stigmasterol has good pharmacological effects on antioxidant and anti-inflammatory.,49 Chen WP et al found that Stigmasterol significantly inhibited the expression of matrix metalloproteinases and inhibited the degradation of cartilage. All the results of the above researches suggested that quercetin, kaempferol, beta-sitosterol, glycitein, beta-carotene, syringetin, diosgenin and stigmasterol had the potential effect in the treatment of ONFH. Other 6 components of the 14 major active components are also closely related to the key target genes associated with ONFH, and may also play important roles in the treatment of ONFH, which should be paid attention to and further studied.Based on the network pharmacological research method, a total of 14 key effective components with the highest degree values including quercetin, kaempferol, beta-sitosterol, glycitein, beta-carotene, tetrahydroalstonine, syringetin, (E)-3-[4-[-2-hydroxy-2-(4-hydroxy-3-methoxy-phenyl)-1-methylol-ethoxy]-3-methoxy-phenyl]acrolein, kadsurenone, (-)-tabernemontanine, diosgenin, hancinone C, 3-beta-Hydroxymethyllenetanshiquinone, stigmasterol in the treatment of ONFH were obtained in our study. Quercetin is a kind of natural polyphenolic flavonoid, and has been documented to possess the anti-inflammatory, cytoprotective, genoprotective and antioxidant effects in animal experimental models. It is also a unique signaling intermediate in osteoblasts that can regulate both the differentiation and formation of osteoblasts and osteoclasts to maintain bone mass. In addition, AKT1 contributes to the angiogenesis and ossification after injury at the end stage of endochondral bone formation, which might be helpful to the treatment of ONFH. EGFR, epidermal growth factor receptor, plays an important role in bone formation. There was a study showed that mice deficient in EGFR were growth retarded and exhibited severe bone defects, and signaling via EGFR stimulated osteoblast proliferation and inhibited their differentiation. Another research showed that down-regulated EGFR signaling leaded to the senescence of osteoprogenitors and the decline in bone formation on the endosteal surface of cortical bone. Stem cell function, a key contributor of bone remodeling, decreases in the process of ONFH and serum ALB is an effective activator of endogenous progenitor cells, so that ALB can successfully activate stem cell activity, support faster and functionally superior bone regeneration which is helpful for the treatment of ONFH. IL1B is a crucial inflammatory cytokine, can regulate the differentiation and activation of osteoclasts involved in the process of local bone erosiona, systemic osteoporosis and functional disabilities. Another study also showed that IL1B gene influenced the genetic susceptibility of steroid-induced ONFH. MYC, a highly efficient transcription factor, can promote the formation and differentiation of osteoclasts. Ni F. et al found that via the SLC7A5/c-MYC pathway IL-18 promoted the osteogenic differentiation of hBMSCs, which showed that MYC was also an important factor in osteogenic differentiation. TNF-α can regulate the early development of ONFH by mediating osteoblast autophagy and apoptosis and the onset of steroid-induced osteonecrosis of femoral head is influenced by TNF-α and hypoxia history, so TNF is an important target of ONFH. IL6 is an important cytokine and ONFH is accompanied by significant inflammatory response, so IL6 is an important target in the process of ONFH. There was a study showed that anti-IL6 therapy decreased hip synovitis and osteoclastic bone resorption and increased new bone formation after ischemic osteonecrosis. p53 protein, the product of TP53 gene, is a tumor suppressor that inhibits the growth of aberrant cells, sense and repair the damage of DNA. P53 can significantly co-regulate the mitophagy in BMSCs, decrease the accumulation of damaged mitochondria in cells, resist stress-induced apoptosis and senescence, and improve the repair of early steroid-induced ONFH.Target proteins with the top 14-degree values were screened as the core target proteins in the treatment of ONFH in our study. AKT1, a protein kinase, is a major mediator of angiogenic signaling, which have shown the important role in bone vascularization and trabecular bone formation. The lipoprotein level of >30 mg/dL was found in 16% of the Legg-Perthes disease group, which indicated the risk for atherosclerosis. Zhang Q. et al found that hyperlipidemia, fat hypertrophy, fat deposition within the femoral head intramedullary tissue, and fat embolism might cause ischemia by elevating intraosseous pressure and decreasing blood flow, eventually leading to ONFH. And dyslipidaemia also leads to a hypercoagulable state and aggravates ischemia in the process of ONFH. Zeng X. et al reported that avascular necrosis of the femoral head was significantly associated with blood lipid abnormalities in elderly patients with low-energy femoral neck fractures. Therefore, lipid and atherosclerosis signaling pathway is a very important pathway in the treatment of ONFH by YGY. IL-17, a kind of inflammatory cytokines, can stimulate macrophages and monocytes to produce proinflammatory factors and is mainly produced by T cells and monocytes. Through IL-17 signaling pathway, IL-17 performs the key function in regulating inflammatory processes in the inflamed synovium and peripheral blood in ONFH, which contributes to the development of ONFH. The relaxin signaling pathway is a potent stimulator of osteoclastogenesis from hematopoietic precursors, which regulate the activity of mature osteoclasts. Hypoxia and blood supply disruption are etiologies for ONFH, Hypoxia-inducible factor 1α (HIF-1α) is a master regulator of cellular response to hypoxia. And HIF-1α also plays a critical role in bone development, and contributes to regulate the bone regeneration and angiogenesis. HIF-1α, VEGF as well as apoptotic genes in the HIF-1α signaling pathway participate in the process of ischemic osteonecrosis.The HIF-1α signaling pathway plays a vital role in regulating the processes of osteoclast activation, bone loss and angiogenesis, and is closely associated with the treatment of ONFH in many previous studies.–77Via the HIF-1α/β-catenin pathway, hypoxia can stimulate angiogenesis and bone regeneration in BMSCs and contribute to treat the early stage of ONFH. In addition, IL-6 is produced in the hypoxic articular cartilage in the process of ONFH through HIF-1 signaling pathway, which can stimulate the inflammatory cytokine responses in synovial cells and develop the disease.MAPK signaling pathway can activate NF-κB to regulate osteoblast autophagy and apoptosis in ONFH.Activation of the MAPK signaling pathway can regulate osteoblast differentiation and inflammatory response, and can also reverse lipopolysaccharide – induced inhibition of osteoblast differentiation and bone resorption, so that promote the bone healing.The KEGG enrichment analysis in our study indicated that the main signaling pathways involved in the treatment of ONFH by YGY are lipid and atherosclerosis signaling pathway, IL-17 signaling pathway, HIF-1 signaling pathway, relaxin signaling pathway, MAPK signaling pathway, and so on. Lipid and atherosclerosis signaling pathway participates in many physiological processes associated with lipid metabolism and atherosclerosis. Increased lipids followed by dyslipidaemia in human blood lead to increased blood viscosity and slow blood flow, which is prone to the formation of small emboli, atherosclerosis and blockage of blood vessels. Therefore, blood flow to the femoral head can be reduced or even interrupted, resulting in ONFH. Thrombophilia closely associated with lipid and atherosclerosis signaling pathway has been implicated as a potential cause of Legg-Perthes disease which is caused by ischemia of the ONFH in children.The potential binding activity and interaction of key effective components and core target proteins were further elaborated through molecular docking analysis in our study, which provided a reference for the subsequent research and development of targeted drugs. According to Figure However, there are still limitations in this study. Firstly, there is less literature and lack of information on the TCM study in public databases such as PubMed. Therefore, there is selection bias in this study which leads to underestimate the usage of YGY. Secondly, network pharmacology and molecular docking are only the preliminary exploration of the possible mechanisms, the prediction results of our study should be further verified by animal experiments and clinical trials.According to the results in our study, quercetin, kaempferol, beta-sitosterol, glycitein, beta-carotene, tetrahydroalstonine, syringetin, (E)-3-[4-[-2-hydroxy-2-(4-hydroxy-3-methoxy-phenyl)-1-methylol-ethoxy]-3-methoxy-phenyl]acrolein, kadsurenone, (-)-tabernemontanine, diosgenin, hancinone C, 3-beta-Hydroxymethyllenetanshiquinone, stigmasterol are the key effective components, and ALB, AKT1, TNF, IL6, TP53, IL1B, CASP3, JUN, MMP9, EGFR, PTGS2, HIF1A, CTNNB1, and MYC are the core target proteins in the treatment of ONFH by YGY. The main signaling pathways involved in the treatment of ONFH by YGY are Lipid and atherosclerosis signaling pathway, IL-17 signaling pathway, HIF-1 signaling pathway, relaxin signaling pathway, MAPK signaling pathway, and so on. The study is the first to explore the mechanism of YGY in the treatment of ONFH via strategies of network pharmacology and molecular docking. And it reveals that YGY can treat ONFH through multicomponents, multitargets, and multipathways, which provides a reference for the subsequent research, development of targeted drugs and clinical application.Conceptualization: Zhi-Yuan Yao.Data curation: Shu-Yao Fan, Zhou-Feng Song, Zhan-Chun Li.Investigation: Zhan-Chun Li.Methodology: Shu-Yao Fan, Zhou-Feng Song, Zhan-Chun Li.Project administration: Zhi-Yuan Yao.Validation: Shu-Yao Fan.Visualization: Shu-Yao Fan, Zhou-Feng Song, Zhan-Chun Li.Writing – original draft: Zhi-Yuan Yao.Writing – review & editing: Zhi-Yuan Yao.


Chronic rhinosinusitis (CRS) is a multifaceted disease with variable clinical courses and outcomes. We aimed to determine CRS-associated nasal-tissue transcriptome in clinically well-characterized and phenotyped individuals, to gain a novel insight into the biological pathways of the disease. RNA-sequencing of tissue samples of patients with CRS with polyps (CRSwNP), without polyps (CRSsNP), and controls were performed. Characterization of differently expressed genes (DEGs) and functional and pathway analysis was undertaken. We identified 782 common CRS-associated nasal-tissue DEGs, while 375 and 328 DEGs were CRSwNP- and CRSsNP-specific, respectively. Common key DEGs were found to be involved in dendritic cell maturation, the neuroinflammation pathway, and the inhibition of the matrix metalloproteinases. Distinct CRSwNP-specific DEGs were involved in NF-kβ canonical pathways, Toll-like receptor signaling, HIF1α regulation, and the Th2 pathway. CRSsNP involved the NFAT pathway and changes in the calcium pathway. Our findings offer new insights into the common and distinct molecular mechanisms underlying CRSwNP and CRSsNP, providing further understanding of the complex pathophysiology of the CRS, with future research directions for novel treatment strategies. Chronic rhinosinusitis (CRS) is a debilitating chronic inflammatory disease affecting around 5–12% of the population ,4. CRSsNDysregulation of the immune response is essential to our present understanding of CRS. Identifying those mechanisms and their understanding might bring better insight into the multifaced illness. Using transcriptomic signatures to identify exact disease processes could shed light on complex molecular interactions in CRS ,11. The p = 0.03), asthma (p = 0.02), and gastroesophageal reflux disease (GERD) (p = 0.03). The CRSwNP group also showed predominant local eosinophilia in the tissue samples, with a mean number of 30 eosinophils in high-power field (HPF). We found no difference in the number of previous surgeries, time interval from the last surgical intervention, or Sinonasal outcome test 22 (SNOT-22) QoL score between subjects with CRSwNP and subjects with CRSsNP. The clustering using tissue eosinophilia levels into non-T2 and T2 subtypes significantly differed between phenotypes [The whole cohort involved 488 patients with CRS, and 37 controls. A total of 123 patients were eligible to be enrolled in the study. Due to exclusion criteria, 87 tissue samples were taken. Gene analysis was carried out on 22 subjects with CRSwNP, 11 with CRSsNP and 15 control subjects . Their aenotypes ,20.p < 0.001; We first compared differentially expressed genes (DEGs) in tissue samples of patients with CRSwNP/CRSsNP and control individuals, and found a high correlation between CRSwNP and CRSsNP ). Enrichment of DEGs shows immune regulation, cellular movement, and inflammation, suggesting their regulation is the most critical pathway in CRS. However, differences are notable between phenotypes, as in CRSwNP immune regulation through NF-kβ, Toll-like receptor signaling, and crosstalk, HIF1α regulation, Th2 pathway, IL-6, and IL-15 pathways recruitment, and the regulation of various immune cells. On the other hand, in CRSsNP, NFAT regulation of immune response, calcium-induced apoptosis of T cells were identified as highly activated pathways. CRSsNP specific were SPINK1 pancreatic cancer pathway and LXR/RXR activation ) uncovered common upstream regulators triggering the inflammatory processes and regulating the immune pathways, known innate immunity response molecules that have already proven to be involved in CRS, bacterial product (Staphylococcus aureus enterotoxin), and T-cell regulators. CRSwNP-dominant regulators are engaged in T2 inflammation or eosinophilia, such as ERBB, TNF pathway, inflammatory and host defense, cytokine regulators and their cytokines, such as CSF2 or IL4, and angiogenetic factors. CRSsNP expresses molecules involved in NFAT regulation, T-cell regulation, and stress-response genes , as a key upregulated gene [Interestingly, this was downregulated in athletes with a below-mean IgA and deletion involved in susceptibility to psoriasis ,26. The ted gene .human bone sialoprotein gene IBSP , extracellular phospoglycoprotein MEPE, and interferon-induced transmembrane protein 5 IFITM5 (both upregulated only in CRSsNP), which may play a potential role in viral infection [lipopolysaccharide-binding protein LBP in CRSsNP. LBP is a part of the cytokine induction pathway in immune cells, and is involved in the acute-phase immune response to gram-negative bacteria [C-C motif chemokine ligand 7 CCL7 as dominantly overexpressed in CRSwNP. It is a part of the chemokine-stimulating pathway associated with macrophages [calcium-activated chloride channel regulator 1 CLCA1, connected to the mucus cell metaplasia of chronic inflammatory airway diseases [interleukin 13 IL-13 gene and IL-13 as a highly active upstream regulator in CRSwNP  genes. Downregulated IL36A can activate NF-kappa-B and MAPK signaling pathways [IL36RN gene in CRSsNP. Interleukin 1β IL1B was an overexpressed gene and activated upstream regulator in both phenotypes. Its role in CRSsNP seems elusive, and might be explained as an intrusion of the T2 endotype in the CRSsNP subgroup [Downregulated CRS genes were physical-barrier protein genes of the  the CRS ,50. Trantermined . We havepathways . IL1RL2 pathways . IL36RN pathways . Our datsubgroup . In contsubgroup . Karjalasubgroup . lacritin LACRT, the proline-rich protein Haell subfamily 2 PRH2 [lipocalin 1 LCN1 [secretagogin SCGN gene, proven to be involved in early-onset inflammatory bowel disease; the alteration of their gene expression might reveal modified innate immunity, thus also showing a possible genetic basis for the inflammatory reaction [We have also confirmed a series of downregulated antimicrobial genes such as y 2 PRH2 ,58, or in 1 LCN1 . The latn 1 LCN1 . CRSwNP reaction . prolactin-induced protein PIP in CRSwNP. It has a role in binding to CD4 receptors on lymphocytes and Staphylococcus sp. bacteria [Cornulin gene CRNN was the most downregulated gene in CRSsNP. It was previously found to be upregulated in atopic dermatitis and eosinophilia [LCE3D. Specific downregulated barrier genes such as desmoglein-1 DSG1 have been previously implicated in CRSsNP pathophysiology [apoptosis-related protein gene CRCT1 [barrier protein IVL [antiprotease gene SPINK7, being key inhibitory regulators of the inflammatory response, barrier function, and differentiation, respectively [nitric oxide synthase 2 NOS2, arachidonate 5-lipoxygenase ALOX5 and the upstream regulator TFRC [Our data confirms downregulated bacteria ,62. Bothbacteria . Cornulinophilia . It coulysiology . Other dne CRCT1 , barriertein IVL , and antectively . To an etor TFRC . Our stutor TFRC . EMT is tor TFRC ,71. The limitation of our study is still a relatively small sample size, due to rigid inclusion criteria and the adequacy of the RNA extracted. Similarly to other transcriptome-based studies, we have not directly addressed possible discordances in transcriptomics and proteomics . AnotherThe study was designed as a prospective cohort study of 488 patients with CRS diagnosed and treated at a tertiary institution in Slovenia, responsible for managing the most burdensome patients. Ninety-eight eligible patients with a clear-cut phenotype of primary CRS (CRSwNP and CRSsNP) were identified ,2. All rThe control group’s inclusion criteria were a requirement for nasal surgery other than CRS or allergic rhinitis (AR) and an age of more than 18 years. All had previously received maximal medical therapy consisting of intranasal fluticasone propionate drops (CRSwNP) or mometasonfuroat nasal spray (CRSsNP). Prolonged antibiotic treatment with doxycycline or macrolides or amoxicillin with clavulanic acid was also implemented during the preoperative workout, but not for at least a month before the tissue biopsy. Systemic corticosteroids were also not administered at least six months before the biopsy .Exclusion criteria for controls were comorbid asthma, AR, benign or malignant tumor of the nose or paranasal sinuses, history of autoimmune diseases, and smoking. A detailed flowchart of patients and controls with inclusion parameters is presented in Exact locations were chosen, considering intranasal tissue differentiation ,72. In pTotal RNA was extracted using the miRNeasy Mini Kit  and TissueLyser , according to the manufacturer’s instructions. The purity of the isolated RNA was assessed using the NanoDrop 2000c spectrophotometer , and integrity was determined on the Agilent 2100 Bioanalyzer, using the RNA 6000 Nano kit . All samples used for library preparation had RNA integrity values greater than 6.0. Extracted RNA samples were stored at −80 °C before further processing. An entire graphical-study flowchart is presented in RNA sequencing (RNAseq) was performed on the Illumina NovaSeq6000 platform with a per-sample target of 15 million 100 bp paired-end reads, by Macrogen Inc. . According to the manufacturer’s protocol, sequencing libraries were prepared using the TruSeq Stranded Total RNA Library Prep workflow with Ribo-Zero Globin .Homo Sapiens (GRCh37.p13), and global TMM normalization of the reads, were performed. The CLC Genomics tool, applying multi-factorial statistics based on a negative binomial GLM for differential expression in two groups and the Wald test against the control group, were used to assess differences in gene expression. Gene-expression values were reported as RPKM (reads per kilobase of transcript per million mapped reads) and TPM (transcripts per million) [RNA-Seq data were analyzed using the CLC Genomics Workbench  as described previously . Brieflymillion) . Genes w® Pathway Analysis ).The functional analyses, which included diseases and functions, canonical pathways, and upstream transcriptional regulators, were generated using QIAGEN’s Ingenuityp value of less than 0.05 was considered significant.Statistical analysis and data preparation was performed using SPSS statistical software version 20 , GraphPad Prism 9 , and Microsoft Excel . The Kruskal–Wallis test, Pearson chi-square Monte Carlo, Fisher exact test, and Mann–Whitney U test were used. A This study identifies common and distinct genes and pathways involved in CRS. For the first time, we have identified new CRS-related genes such as ADGRG7, probably reflecting cellular-adhesion elements, LACRT, IBSP, MEPE, and IFITM5, probable antimicrobial genes with a potential role in viral infections. CRNN may be a link to other biologically related diseases such as atopic dermatitis or eosinophilia. The last group included genes with unknown functions, such as TMPRSS11B or PRR4. Our findings offer new insights into the molecular mechanisms underlying CRSwNP, with the involvement of the NF-kβ pathway, HIF1α regulation, and Th2 pathway, or the NFAT pathway and changes in the calcium-induced apoptosis in CRSsNP. Our findings augment our understanding of CRS and suggest implicit future research directions to elucidate candidates for novel treatment strategies.


In vitro experiment, the viability of ischemic neuron treated with Zuogui Pill was increased, and the ability of neurite outgrowth was significantly improved. Western blot assays shown that the pro-neurite outgrowth effect of Zuogui Pill on ischemic stroke may be relate to PTEN/mTOR signal pathway. The results of the study provided new insights into Zuogui Pill’s molecular mechanism in treatment of ischemic stroke, as well as clinical references for its use.Due to its high mortality, incidence and disability rates, ischemic stroke poses heavy economic burdens to families and society. Zuogui Pill (ZGP) is a classic Chinese medicine for tonifying the kidney, which is effective for the recovery of neurological function after ischemic stroke. However, Zuogui Pill has not been evaluated for its potential effects on ischemic strokes. Using network pharmacology, the research aimed to explore the mechanisms of Zuogui Pill on ischemic stroke, which were further validated in SH-SY5Y cells injured by oxygen and glucose deprivation/reperfusion (OGD/R). Network analysis of Zuogui Pill identified 86 active ingredients and 107 compound-related targets correlated with ischemic stroke. Additionally, 11 core active compounds were obtained, such as Quercetin, beta sitosterol, and stigmasterol. Most of the compounds have been proven to have pharmacological activities. Based on pathway enrichment studies, Zuogui Pill may exert neuroprotection through MAPK signaling, PI3K-Akt signaling and apoptosis, as well as enhance neurite outgrowth and axonal regeneration effect via mTOR signaling, p53 signaling and Wnt signaling pathways.  Chinese adults suffer from ischemic stroke most often, which leads to death and disability . It is eTraditional Chinese medicine categorizes ischemic stroke as “stroke” . The maiin vitro experiments, which provided a biological basis for treating ischemic stroke with “Bushen Shengsui” Chinese medicine.The technology of network pharmacology focuses on the relationship between drug molecules, action mechanisms, and disease targets based on the construction of biological networks . Integrihttp://lsp.nwu.edu.cn/tcmsp.php) and BATMAN-TCM (http://bionet.ncpsb.org/batman-tcm/). The rate and degree of drug absorption in the body is referred to as oral bioavailability (OB). A compound’s drug-likeness (DL) is defined as its similarity to a known drug. For the purpose of screening potential compounds, the active components with OB ≥ 30% and DL ≥ 0.18 were chosen. Compounds in ZGP  were search in TCMSP (http://www.uniprot.org/uniprot/). Get rid of targets that are repetitive, not human or standard. Cytoscape 3.8 software was used to import the ZGP’s primary active components and their targets to create a “Component-Target” network diagram. The targets and chemical components that were represented by the nodes in the network diagram. Interactions were represented by the edges. The degrees reflected the number of edges that combined a node, and function and degree values were positively associated. The information regarding degree centrality (DC), medium centrality (BC), and near centrality (CC) was obtained by evaluating the topological properties with the “CytoNCA” function.Search compound targets through TCMSP database, screen and eliminate the duplicate targets. The gene names and UniProt numbers of each target were converted into target abbreviations using the UniProt database (http://db.idrblab.net/ttd/), DisGeNET database (https://www.disgenet.org), GeneCards database (https://www.genecards.org), and OMIM database (http://omim.org/). With UniProt, duplicate targets were eliminated. The targets of the active ZGP components were compared to the targets of ischemic stroke by using the Venny platform (http://bioinfogp.cnb.csic.es/Tools/Venny/), and the targets of ZGP for treating ischemic stroke were determined.By utilizing the phrase “ischemic stroke,” disease targets were found in the TTD database . For the PPI network construction, the interacting proteins with a high confidence score of ≥0.700 were chosen. Cytoscape 3.8 was used to import the aforementioned data, create a target PPI network diagram, and screen the core network. In order to identify potential hub targets, the “CytoNCA” function was utilized to examine the topological properties.For the purpose of PPI analysis, the common targets of ZGP and ischemic stroke were imported into the STRING database  data, the Kyoto Encyclopedia of Genes and Genomics (KEGG) pathway. The biological process (BP), the cellular component (CC), and the molecular function (MF) are all included in the GO enrichment analysis. By setting the http://tcmspw.com/tcmsp.php), and the structure of target proteins were obtained from RCSB PDB (https://www.rcsb.org/). AutoDock Tools 1.5.6 was applied to perform molecular docking and predict the binding energy of compounds and proteins. Finally, PyMOL software was used to visualize the optimal docking results.Molecular docking aims to simulate the docking between small molecule ligands and large molecule proteins, and the docking results are evaluated by binding energy (affinity). Lower binding energy indicates better molecular docking binding effect. The 3D structure of the compounds were obtained from TCMSP database  was placed at the Central Laboratory of Jiangsu Province Hospital of Chinese Medicine. The detailed approaches for preparing and quality assurance of ZGP were as follows: A total of 10 prescriptions of ZGP (1050 g) were prepared, mixtures of components in ZGP were soaked with 1000 mL water for 30 min, then decocted twice for 30 min. The liquid was decocted twice, collected, concentrated to a density of about 1.3, and dried under vacuum at 60°C. Finally, the extract was crushed to obtain ZGP-dried powder (362 g). The powder was preserved in aliquots at −20°C and dissolved in a cell culture medium to create working solutions for bioassays. SH-SY5Y human neuroblastoma cells were supplied by the Chinese Academy of Sciences Stem Cell Bank. All-trans-retinoic acid (RA) was purchased from SIGMA (Unite State). Brain-derived neurotrophic factor . The anti- GAP43, p-S6, mTOR, PTEN, and GAPDH were purchased from CST (Unite State).6 and separated into control and all-trans-retinoic acid (RA) induction cohort. The control cohort was cultured normally, while the RA induction cohort was treated with 5 μmol/L RA to induce cells differentiation into mature neurons , differentiated SH-SY5Y cells induced by RA (RA), oxygen-glucose deprivation/reoxygenation (OGD), OGD + ZGP. For the ZGP cohorts, the cells were supplemented with ZGP at a density from 0.14 to 5.12 mg/mL for 24 h. The cells received 10 μL of CCK-8 reagent after undergoing a variety of treatments, and the optical density (OD) was measured at 490 nm after an additional 4 h of cultivation. Cell viability was obtained by calculating the ratio of OD value of each cohort to that of control cohort .The viability of the cells was determined with the help of the CCK-8 test. In 96-well plates, the differentiated SH-SY5Y cells were seeded at a density of 3 × 105 cells per well in 500 μL of media in 24-well plates. After inducing differentiation by ATRA and undergoing OGD/R injury, the cells were divided into the following groups: normal control (Control), oxygen-glucose deprivation/reoxygenation (OGD), OGD + ZGP . A treatment with 50 ng/mL of brain-derived neurotrophic factor (BDNF) was used as positive controls for neurite outgrowth  with skimmed milk powder. A primary rabbit anti-GAP43 (1:1000), rabbit anti-p-S6 (1:1000), mouse anti-mTOR (1:5000), GAPDH , and rabbit anti-PTEN (1:2000) were used to preserve the membranes overnight at 4°C. After being treated with secondary goat anti-rabbit IgG (1:5000) or goat anti-mouse IgG (1:2000) antibodies for 1 hour and electrochemiluminescence (ECL) reagents for 30 s to 2 minutes, the membranes were exposed to Kodak film (Japan). The IOD proportion was measured and evaluated employing the ImageJ program to display the data.p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.For statistical analysis, the software SPSS26.0 and Graphpad Prism 8 were utilized. All data were tested for normality using the Shapiro-Wilk test. Normally distributed parameters were expressed as mean±S.D. The one-way ANOVA and the Tukey multiple comparisons test were used to identify group differences. LSD-t test was used for pairwise comparison. The statistical significance was described as *Eighty-six active components of ZGP were retrieved from the TCMSP and TCMID databases in accordance with the two screening criteria of OB value (≥30%) and DL index (≥0.18). These components include 2 components in SDH (Rehmannia glutinosa), 16 components in SY(Fried yam), 20 components in SZY, 4 components in CNX (Sichuan Achyranthes), 48 components in GQ (Lycium barbarum), and 12 components in TSZ (Dodder seed). The six main components in GBJ (Tortoiseshell gum) and the two main components in CNX (Sichuan Achyranthes) were removed because they did not meet the screening requirements. The most important ZGP active components were shown in TCMSP and PubChem gathered the 86 active components for target gene prediction. SDH (29), SY (121), SZY (112), CNX (182), GQZ (311), and TSZ (286) were among the 1041 predicted targets that were discovered. 207 relevant human gene targets were found after eliminating gene targets for other species and duplicate values. The relationship between drug composition and target was shown in We continued to investigate their potential therapeutic targets after clarifying the key compounds of ZGP. 1020 ischemic stroke-related targets were gathered from the TTD, OMIM, GeneCards, and DisGeNet databases after duplicates were removed. The overlapping targets of ZGP-related targets and ischemic stroke-related targets were regarded as potential therapeutic targets for ZGP anti-ischemic stroke. Screening drug component targets and ischemic stroke targets yielded 103 intersection targets, as depicted in The disease-component-target network was set up in view of the strength of the 103 shared targets recognized as both potential ZGP targets and ischemic stroke targets. The PPI relationships of 103 target genes were obtained using the STRING platform to make clear the potential mechanism by which ZGP benefits ischemic stroke. The outcomes are depicted in p < 0.05) , 228 cellular components (CCs), and 350 molecular functions (MFs) were among the 4147 GO terms we obtained . Figures < 0.05) . The KEG < 0.05) , and 21  < 0.05) ; Table 3Eleven active compounds  were obtained by network pharmacological analysis as the core active compounds of ZGP in the treatment of ischemic stroke. Take mTOR as the potential target. Through docking simulations, the results were shown in p < 0.05, p < 0.05 and p < 0.01, respectively)  over 24 h; the highest nontoxic concentration was 1.28 mg/mL . The conctively) . These rp < 0.0001). As was to be expected, BDNF significantly improved the morphology of damaged cells and prolonged neurites growth. Meanwhile, the length of neurites was significantly longer after supplementation with various concentrations of ZGP  compared to that in the OGD cohort   . When cop < 0.0001), but gradually increased with the increase of ZGP concentration (p < 0.01), 0.32 mg/mL (p < 0.0001) and 0.64 mg/mL cohort (p < 0.0001) compared to that in OGD cohort (p < 0.05–0.0001). Compared to the OGD cohort, PTEN protein expression was significantly downregulated in the ZGP cohorts at various concentrations  . These findings suggest that ZGP increased the expression of the axon-related protein GAP43 and promoted the growth of neuronal axons. ZGP’s mechanism may involve increasing mTOR and p-S6 protein expression while decreasing that of the negative regulator PTEN protein.Based on the results of GO and KEGG enrichment analysis, we selected the mTOR signal pathway protein and its negative regulatory factor PTEN protein for Western blot detection to verify the mechanism of ZGP promoting neurite growth. The expression of axon growth marker protein GAP43 in induced SH-SY5Y cells decreased after OGD/R  were obtained by network pharmacological analysis. Quercetin exerts neuroprotection for acute ischemic stroke by improving neurological function score (NFS), reducing infarct volume, and protecting blood brain barrier (BBB) integrity . There aTo gain a deeper comprehension of the target genes’ interaction and action pathways, GO and KEGG pathway analyses were utilized. The following biological processes (BPs) were found to be strongly related with target genes through GO analysis: response to an organic substance, cellular response to a chemical stimulus, and oxygen-containing compound. Membrane raft, membrane microdomain, membrane region, endomembrane system, and cytoplasmic part were the most highly enriched CCs ontologies. Enzyme binding, identical protein binding, signaling receptor binding, and various other molecular functions were included in the enriched MF ontologies. The MAPK, PI3K-Akt, apoptosis, p53, Toll-like receptor, Jak-STAT, NF-kappa B, mTOR, and Wnt signaling pathways were the primary focus of the KEGG pathway analysis. The results shown that ZGP may act on multiple signal pathways.The mitogen-activated protein kinase (MAPK) and PI3K/Akt pathways play a significant role in the activation of a number of critical signal transduction pathways in response to stress . They nomTOR, p53, and Wnt signaling pathways are all associated with nerve regeneration and repair after ischemic brain injury. mTOR is a key signal pathway regulating the intrinsic growth ability of neurite outgrowth and axonal regeneration . The phoin vitro experiments in this study demonstrated that ZGP protected ischemic neurons by increasing the cell viability of differentiated SH-SY5Y cells following OGD/R. Several other investigations have revealed that the mechanism behind ZGP’s protective impact on ischemic stroke is related to inhibit oxidative stress and inflammatory reaction and regulate PI3K/Akt pathway (From the above results, we can see that ZGP not only has anti-apoptosis and neuroprotective effects on ischemic stroke, but also can promote neurite outgrowth and axonal regeneration after cerebral ischemia. Our  pathway . However pathway . We veriIn vitro experiment, ZGP treatment greatly boosted the survivability of ischemic neurons and enhanced their capacity for neurite outgrowth. Western blot assays shown that the pro-neurite outgrowth effect of ZGP on ischemic stroke may be relate to PTEN/mTOR signal pathway. All of the findings offered fresh explanations for the molecular basis of ZGP and served as a guide for its use in clinical settings. However, due to the limited database of traditional Chinese medicine and many network prediction results, the experimental verification is not comprehensive. Future research will still need to evaluate the therapeutic effects of ZGP in vivo and its potential mechanisms for ischemic stroke treatment.In conclusion, this research is the first time to use multiple network models to study the mechanism of ZGP on ischemic stroke, and provide new insights for the role of ZGP in ischemic stroke treatment. Network analysis of ZGP identified 86 active ingredients and 107 compound-related targets correlated with ischemic stroke. Additionally, 11 core active compounds were obtained, such as Quercetin, beta sitosterol, and stigmasterol. Most of the compounds have been proved to have pharmacological activities. Pathway enrichment demonstrated that ZGP may exert neuroprotective effect through MAPK, PI3K-Akt signaling pathway, and exert promoting neurite outgrowth and axonal regeneration effect via mTOR, p53 and Wnt signaling pathway.


Huangqi Guizhi Wuwu decoction (HQGZWWD) has been used to treat and prevent deep vein thrombosis (DVT) in China. However, its potential mechanisms of action remain unclear. This study aimed to utilize network pharmacology and molecular docking technology to elucidate the molecular mechanisms of action of HQGZWWD in DVT.We identified the main chemical components of HQGZWWD by reviewing the literature and using a Traditional Chinese Medicine Systems Pharmacology (TCMSP) database. We used GeneCards and Online Mendelian Inheritance in Man databases to identify the targets of DVT. Herb-disease-gene-target networks using Cytascape 3.8.2 software; a protein–protein interaction (PPI) network was constructed by combining drug and disease targets on the STRING platform. Additionally, we conducted Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses. Finally, molecular docking verification of active components and core protein targets was conducted.A total of 64 potential targets related to DVT were identified in HQGZWWD, with 41 active components; quercetin, kaempferol, and beta-sitosterol were the most effective compounds. The PPI network analysis revealed that AKT1, IL1B, and IL6 were the most abundant proteins with the highest degree. GO analysis indicated that DVT treatment with HQGZWWD could involve the response to inorganic substances, positive regulation of phosphorylation, plasma membrane protein complexes, and signaling receptor regulator activity. KEGG analysis revealed that the signaling pathways included pathways in cancer, lipid and atherosclerosis, fluid shear stress and atherosclerosis, and the phosphatidylinositol 3-kinases/protein kinase B(PI3K-Akt) and mitogen-activated protein kinase (MAPK) signaling pathways. The molecular docking results indicated that quercetin, kaempferol, and beta-sitosterol exhibited strong binding affinities for AKT1, IL1B, and IL6.Our study suggests that AKT1, IL1B, and IL6 are promising targets for treating DVT with HQGZWWD. The active components of HQGZWWD likely responsible for its effectiveness against DVT are quercetin, kaempferol, and beta-sitosterol, they may inhibit platelet activation and endothelial cell apoptosis by regulating the PI3K/Akt and MAPK signaling pathways, slowing the progression of DVT.The online version contains supplementary material available at 10.1186/s13018-023-03948-6. Deep vein thrombosis (DVT) is a disease in which blood in the deep veins of the limbs abnormally coagulates into clots due to damage to the venous wall and stagnation of blood flow, leading to narrowing or occlusion of the venous lumen , 2. The Hedysarum multijugum maxim, HM), Guizhi , Baishao , Shengjiang , and Dazao  [Traditional Chinese medicine has a long history of use for the prevention and treatment of DVT . Tradititus, JF) . HQGZWWDtus, JF) . Additiotus, JF) . Furthertus, JF) . DespiteThe complexity of traditional Chinese medicine stems from the lack of adequate quantitative evidence to assess its therapeutic efficacy. Typically, traditional Chinese medicine involves intricate formulas comprising multiple herbs, with their composition and dosage often based on ancient texts and empirical knowledge rather than on contemporary scientific research . Its pecNetwork pharmacology is a novel analytical approach that utilizes virtual computing and database retrieval to study the mechanisms of disease and drug action within a broader biological network. This method provides valuable insights into the pharmacological efficacy and mechanisms of the drugs . The prihttp://tcmspw.com/tcmsp.php) was used to identify the active components of HQGZWWD . Our selection criteria were based on the absorption, distribution, metabolism, and excretion (ADME) processes. Oral bioavailability (OB) is the amount of a drug that enters the circulation after entering the human body. However, drug-likeness (DL) refers to the degree of similarity between a compound and known drugs. OB is a representative pharmacokinetic parameter in ADME, and DL is used as a qualitative concept in drug design to estimate the molecular characteristics of drugs [The TCMSP platform (of drugs . Specifiof drugs –25.https://www.uniprot.org/). To obtain disease targets from the databases, we searched for “deep venous thrombosis” as a keyword in the OMIM  and Gene cards databases  [We identified the active components of HQGZWWD by applying predefined thresholds (OB ≥ 30% and DL ≥ 0.18) to the TCMSP database and individually confirmed the respective targets. To standardize the target and gene symbols, we used the UniProt database  for conversion  , 27. Nexhttps://string-db.org/) was used to predict protein–protein interactions . The PPI network was created by introducing overlapping targets [The Search Tool for Retrieving Interacting Genes/Proteins (STRING)  [P-value cutoff was established as P<0.01, where a lesser P-value signifies a higher likelihood of the current result being an authentic enrichment outcome rather than a random occurrence. The unit employed in this study was -log 10, and as this value increased, the reliability of the enrichment results increased.To elucidate the biological functions and signaling pathways associated with DVT, we conducted GO and KEGG enrichment analyses using the Metascape database  . GO analhttps://www.rcsb.org/, accessed on March 19, 2023) and TCMSP database [Molecular docking is a theoretical simulation method used in drug design to predict the binding modes and affinities of molecules by studying their interactions with receptors and ligands . AutoDocdatabase , followedatabase .According to the screening criteria, OB ≥ 30% and DL ≥ 0.18 were set in the TCMSP database for screening; the results showed that 41 active components of HQGZWWD were obtained, as shown in Additional file We searched the OMIM and GeneCards databases for potential targets related to DVT, removed duplicate targets, and identified 1731 potential targets. We then sorted the results from TCMSP, removed duplicate sites, matched the predicted drug targets with the corresponding disease targets, and obtained 64 core targets Fig. . The degThe core overlapping diseases and drug component targets were imported into the STRING database to generate a PPI network diagram Fig. . MoleculGO and KEGG enrichment analyses were conducted to better understand the potential pathways and biological functions of HQGZWWD in DVT. Figure We employed network pharmacology and PPI analyses to determine the top three compounds, quercetin, beta-sitosterol, and kaempferol, for docking with the top three proteins, AKT1, IL6, and IL1B. The best docking image of the receptor-ligand complex is presented in Fig. Our results indicate that beta-sitosterol can form hydrogen bonds with ALA-9 and GLY-101 through IL-1B protein Fig. a, while The tighter the binding between the ligands and receptors, the smaller the binding energy. Notably, binding energy < -5 kcal/mol indicates good affinity and binding activity between the receptor and ligand , 36. OurThis study investigated the mechanism by which HQGZWWD treats DVT. By constructing and analyzing databases and network diagrams of traditional Chinese medicine prescriptions, we identified quercetin, beta-sitosterol, and kaempferol as highly correlated with DVT. Previous studies demonstrated that quercetin and its derivatives possess anticoagulant, antiplatelet, and antifibrinolytic activities , 38, effThe results of the PPI network analysis suggested that AKT1, IL6, and IL1B could be potential core targets for treating DVT with HQGZWWD; IL-1B and IL-6 are proinflammatory cytokines with a wide range of biological activities . InitialWe conducted GO and KEGG analyses to investigate the mechanism of action of HQGZWWD in DVT. The results of the GO analysis showed that HQGZWWD treatment primarily involved responses to inorganic tolerance, positive regulation of phosphorylation, plasma membrane protein complex, and signaling receiver regulator activity. KEGG analysis revealed that multiple signaling pathways are involved in HQGZWWD treatment of DVT, including pathways in cancer, lipid and atherosclerosis, fluid shear stress, atherosclerosis, PI3K-Akt, and MAPK signaling pathways. The involvement of quercetin in cancer pathogenesis may be the main reason for its enrichment in cancer –54. A prAlthough atherosclerosis and venous thrombosis were once considered distinct pathological conditions, recent studies have challenged this notion . A studyThe PI3K/Akt signaling pathway is a crucial regulatory pathway playing a significant role in pathophysiological processes such as cell growth, differentiation, and proliferation . RecentlThe MAPK signaling pathway is a crucial component of the eukaryotic signal transduction network; it plays a key role in regulating cell proliferation, differentiation, apoptosis, and stress responses under normal and pathological conditions , 82. StuMolecular docking experiments revealed that quercetin, beta sitosterol, and kaempferol, the main compounds obtained from HQGZWWD, have binding energies with core target proteins AKT1, TP53, and TNF of < -5 kcal/mol  simulations to supplement molecular docking in determining the dynamic stability of receptor ligands , 90. AddIn summary, we adopted a new approach that combined network pharmacology and molecular docking to predict AKT1, IL1B, and IL6 as the most likely targets of HQGZWWD for treating DVT. Quercetin, kaempferol, and beta-sitosterol are the main active components of HQGZWWD; they may inhibit platelet activation and endothelial cell apoptosis by regulating the PI3K/Akt and MAPK signaling pathways, slowing the progression of DVT. Additionally, our results offer new insights into the pathogenesis of DVT and suggest potential avenues for developing innovative treatment strategies. The initial findings from network pharmacology have increased our confidence. However, further clinical experiments and in-depth basic research are necessary to fully understand the therapeutic effects and molecular mechanisms of traditional Chinese medicine on DVT; this will be the next area of our research.Additional file: 1. Effective compounds of Huang-Qi-Gui-Zhi-Wu-Wu Decoction.