Avena sativa (AS) known as potent dietary grain to identify the combinatorial efficacy through network pharmacology.Non-alcoholic fatty liver disease (NAFLD) is a serious public health issue globally, currently, the treatment of NAFLD lies still in the labyrinth. In the inchoate stage, the combinatorial application of food regimen and favorable gut microbiota (GM) are considered as an alternative therapeutic. Accordingly, we integrated secondary metabolites (SMs) from GM and We browsed the SMs of AS via Natural Product Activity & Species Source (NPASS) database and SMs of GM were retrieved by gutMGene database. Then, specific intersecting targets were identified from targets related to SMs of AS and GM. The final targets were selected on NAFLD-related targets, which was considered as crucial targets. The protein–protein interaction (PPI) networks and bubble chart analysis to identify a hub target and a key signaling pathway were conducted, respectively. In parallel, we analyzed the relationship of GM or AS─a key signaling pathway─targets─SMs (GASTM) by merging the five components via RPackage. We identified key SMs on a key signaling pathway via molecular docking assay (MDA). Finally, the identified key SMs were verified the physicochemical properties and toxicity in silico platform.The final 16 targets were regarded as critical proteins against NAFLD, and Vascular Endothelial Growth Factor A (VEGFA) was a key target in PPI network analysis. The PI3K-Akt signaling pathway was the uppermost mechanism associated with VEGFA as an antagonistic mode. GASTM networks represented 122 nodes  and 154 edges. The VEGFA-myricetin, or quercetin, GSK3B-myricetin, IL2-diosgenin complexes formed the most stable conformation, the three ligands were derived from GM. Conversely, NR4A1-vestitol formed stable conformation with the highest affinity, and the vestitol was obtained from AS. The given four SMs were no hurdles to develop into drugs devoid of its toxicity.In conclusion, we show that combinatorial application of AS and GM might be exerted to the potent synergistic effects against NAFLD, dampening PI3K-Akt signaling pathway. This work provides the importance of dietary strategy and beneficial GM on NAFLD, a data mining basis for further explicating the SMs and pharmacological mechanisms of combinatorial application (AS and GM) against NAFLD.The online version contains supplementary material available at 10.1186/s12967-023-04122-6. Non-alcoholic fatty liver disease (NAFLD) is characterized by excessive fat accumulation in liver tissues, especially, in liver parenchyma . Its patAvena sativa  and gut microbiota (GM) to identify the key SMs in both AS and GM for the treatment of NAFLD. Furthermore, AS has a wide spectrum of pharmacological activities such as antioxidant, anti-inflammatory, antidiabetic and anticholesterolemic efficacy [In the incomplete project, we pioneered the secondary metabolites (SMs) from efficacy . The AS efficacy , 11. Curefficacy .Bifidobacterium and Spirillum [Specially, the flavonoids from AS ameliorated hyperlipidemia caused by high-fat-diet via modulating bile acid and GM in mice . The AS pirillum . Furtherpirillum . TherebyIn parallel, GM in human intestine is significant community to control the physiological responses for host . Some faIt is believed that NP might be a key to decrypt the therapeutic issue in dilemma, ending up with combinatorial application. As aforementioned, our study has established that the combinatorial application of AS and GM is to be expected as an alternative therapeutic strategy for NAFLD. Thus, this approach might be given critical hints to further clinical trials and advancement of the combined applications with AS and GM. The process of this study is displayed in Fig. At present, the development of data science provides a great deal of information related to biological pathways between compounds and targets, which can be a platform by merging the important databases. Based on it, we gathered valuable data to utilize NP as a drug discovery methodology. We profiled the available bioinformatics resources in Table http://bidd.group/NPASS/) (accessed on 28 September 2022) was utilized to select the significant SMs from AS [https://sea.bkslab.org/) (accessed on 28 September 2022) [http://www.swisstargetprediction.ch/) (accessed on 28 September 2022) [Natural Product Activity & Species Source (NPASS) database  . With ther 2022) . Apparener 2022) .http://bio-annotation.cn/gutmgene/) (accessed on 29 September 2022) [The gutMGene database was used to obtain the SMs converted by GM (er 2022) . The obthttps://www.disgenet.org/) (accessed on 30 September 2022) [https://www.omim.org/) (accessed on 30 September 2022) [The intersecting targets were identified between AS-related targets and GM-related targets, which were considered as significant targets for combinatorial therapeutics. The NAFLD- related targets extracted by DisGeNET (er 2022)  and OMIMer 2022) . Finallyhttps://string-db.org/) (accessed on 01 October 2022) [We utilized String database (er 2022)  to identThe construction of bubble plot was established by Kyoto Encyclopedia of Gene and Genomes (KEGG) pathway enrichment analysis. The signaling pathways on the bubble plot were depicted, according to Rich factor value. We discerned a key signaling pathway for the treatment of NAFLD, suggesting that the mechanism might be inhibitive effect on NAFLD. The bubble plot was constructed by R package.We described GASTM network to know the relationships of each component: GM or AS, a key signaling pathway, targets, and secondary metabolites. The GASTM network was constructed by utilizing R Package. Taken together with GM or AS, a key signaling pathway, targets, and SMs as nodes, matching associations above components were assembled with Microsoft Excel, then input into R package to identify the interaction network of GASTM against NAFLD.https://pubchem.ncbi.nlm.nih.gov/) (accessed on 02 October 2022), changing into.pdb format via Pymol tool. The.pdb format was transformed into.pdbqt format to prepare for the MDA on targets. The targets were selected by the Protein Data Bank (PDB) (https://www.rcsb.org/) (accessed on 01 October 2022) for.pdb format, which were switched into.pdbqt format by setting parameter in AutodockTools-1.5.6. The MDA was conducted on.pdbqt format by preparing for conformer between SMs and targets. The docking site was set in cubic box  in a central point of each target.The Molecular docking assay (MDA) was implemented with AutodockTools-1.5.6 to understand what the most significant SMs in both GM and AS are. Commonly, the threshold of AutodockTools-1.5.6. was fitted as -6.0 kcal/mol  or SM wihttp://www.swissadme.ch/) (accessed on 02 October 2022) [2) or Moriguchi octanol–water partition coefficient (MLogP) (≤ 4.15) or Hydrogen Bonding Acceptor (HBA) (< 10) or Hydrogen Bonding Donor (HBD) (≤ 5). To accept the rule, the molecules should not be violated more than 2 parameters out of 5 parameters. The toxicity of the uppermost SMs was confirmed by ADMETlab 2.0 [The properties of drug-likeness on the uppermost SMs were performed by SwissADME (er 2022) . The filTlab 2.0  and ProTTlab 2.0 , its parTlab 2.0 ; Human HTlab 2.0 ; CarcinoTlab 2.0 ; CytotoxTlab 2.0 ; and EyeTlab 2.0 .The number of 12 SMs from AS was documented from NPASS database, all of which were accepted by Lipinski’s rule , the final 16 targets were identified as bona fide targets to be expected to exert combinatorial efficacy on AS-based and GM-based application .The number of 23 targets obtained from the combined AS-related targets and GM-related targets was compared with NAFLD-associated targets 1836)  out of 16 targets were not interacted with one another s sp. 45 , rutin bs ovatus , 48, avis sp. 46 , myricitliflavus , can conThe bubble plot provided by PPI shows three signaling pathways: Prolactin signaling pathway, T cell receptor signaling pathway, and PI3K-Akt signaling pathway Fig. . In para, green circle), PI3K-Akt signaling pathway , targets , and metabolites , consisting of 122 nodes and 155 edges  or AS . Likewise, myricetin on GSK3B had the highest affinity with -10.6 kcal/mol  shows what the promising SM(s) are on the PI3K- Akt signaling pathway, and the key SM(s) are derived from either GM or AS. As mentioned previously, the key SMs  of VEGFA were derived from GM  was accepted by Lipinski’s rule, thus, which could be important agents to develop therapeutics. Accordingly, the parameters of toxicity were all confirmed: hERG, Human Hepatotoxicity, Carcinogens, Cytotoxicity, and Eye corrosion. Thus, the identified four SMs are promising candidates against NAFLD Table . The fouThe PPI network shows that VEGFA is the uppermost target to regulate other 10 targets. The key SMs  have been revealed by MDA, which could form the most stable conformers on VEGFA. The myricetin diminishes the lipid synthesis in liver cell and inflammatory response by tuning GM . AdditioThe three signaling pathways confirmed by PPI are related to occurrence and development of NAFLD, which concisely discussed in Table The most stable SM on IL2 was diosgenin, suggesting that the diosgenin is a representative compound in saponin derivatives . A reporEscherichia sp. 12, Escherichia sp. 33, Enterococcus sp. 45, Bacteroides sp. 45, Bifidobacterium dentium, Bacteroides uniformis, Bacteroides ovatus, Bacillus sp. 46, and Enterococcus casseliflavus. However, the GM that converts diosgenin is still veiled. Although there were no evident relationships either positive or negative feedback between AS and nine GM, we could expect the high possibility as relievers on NAFLD, obtaining the four SMs: (1) myricetin, (2) quercetin, (3) diosgenin from GM, and (4) vestitol from AS .Overall, this study shows that dampening of PI3K-Akt signaling pathway might be a potential mechanism to relieve NAFLD. In detail, the key effectors that we suggested are myricetin, quercetin from All in all, these findings shed light on the importance of GM as therapeutics, and AS with auxiliary role in the context NAFLD. Despite that, our study is required to do clinical tests and extensive investigation with rigorousness. The key findings of this study are represented in Fig. From the incorporation of NP, this study exploratory organizes on the key GM, signaling pathways, targets, and SMs with the application of AS in NAFLD treatment, suggesting theoretical evidence for further clinical verification. As limited borderline of NP, the pharmacological pathway of integrating analysis on AS or GM against NAFLD is only dependent on data-driven analysis, and its combinatorial effects, the interaction between AS and GM in vivo were ignored, which is integral to validate the authenticity through preclinical and clinical tests. Hence, the performance of our analysis needs to be advanced more, for instance, by integrating new dataset continually. At this point, our approach platform is easy to merge new data to improve the performance and might be a hallmark to elucidate the relationships between diet and GM. In addition, our study provides a rationale for how to improve accuracy prior to clinical trials.Avena sativa (AS), indicating antagonists  to inhibit PI3K-Akt signaling pathway. These findings provide a new insight to utilize the endogenous species (gut microbiota) and exogenous species (Avena sativa) on microbiome-based therapeutics. However, this study should be taken in vitro or in vivo experimentation into consideration to uncover bona fide pharmacological efficacy.In conclusion, our study highlights the therapeutic effects and mechanisms of the treatment on NAFLD via combinatorial application: gut microbiota (GM), and Additional file 1: Table S1: The number of 208 SMs of gut microbiota from gutMGene; The number of 1256 targets from SEA; The number of 947 targets from STP; The number of 668 targets between SEA (1256) and STP (947); The number 1836 targets related to NAFLD.Additional file 2: Table S2: The binding energy and coordinated amino acid bonds of SMs on each key target.


Objective: Kang-ai injection (KAI) has been a popular adjuvant treatment for solid tumors, but its anti-tumor mechanism in intrahepatic cholangiocarcinoma (ICC) remains poorly understood. This study applied a network pharmacology-based approach to unveil KAI’s anti-tumor activity, key targets, and potential pharmacological mechanism in ICC by integrating molecular docking and in vitro validation.Methods: The KAI-compound-target-ICC network was constructed to depict the connections between active KAI compounds and ICC-related targets based on the available data sources. The crucial ingredients, potential targets, and signaling pathways were screened using GO, KEGG enrichment analysis, and the PPI network. Molecular docking was performed to visualize the interactions between hub targets and components. In vitro experiments were carried out to validate the findings.Results: Among the 87 active components of KAI and 80 KAI-ICC-related targets, bioinformatics analysis identified quercetin as a possible candidate. GO and KEGG enrichment analysis indicated that the PI3K-AKT signaling pathway might be essential in ICC pharmacotherapy. The PPI network and its sub-networks screened 10 core target genes, including AKT1 and IL1β. Molecular docking results showed stable binding between AKT1 and IL1β with KAI active ingredients. The in vitro experiments confirmed that KAI might suppress the proliferation of ICC cell lines by inhibiting the PI3K/AKT signaling pathway, consistent with the network pharmacology approach and molecular docking predictions.Conclusion: The study sheds light on KAI’s biological activity, potential targets, and molecular mechanisms in treating ICC and provides a promising strategy for understanding the scientific basis and therapeutic mechanisms of herbal treatments for ICC. This research has important implications for developing new, targeted therapies for ICC and highlights the importance of network pharmacology-based approaches in investigating complex herbal formulations. Intrahepatic cholangiocarcinoma (ICC) is a type of liver cancer that is second in prevalence only to hepatocellular carcinoma (HCC). It accounts for approximately 5%–10% of all primary liver cancers and has seen a rising incidence rate in recent years . While sTraditional Chinese Medicine (TCM) has been a cornerstone of health and wellness for millennia, embracing a holistic approach to promoting optimal balance, preventing ailments, and treating conditions. In particular, TCM holds great promise in managing complex conditions such as cancer tumors, immune disorders, and cardiovascular diseases . TCM canIn response to the declining rates of drug discovery, mounting difficulties in research and development, and high rates of failure in phase II and III clinical trials, Hopkins introduced the concept of network pharmacology . For theKang-ai Injection (KAI) is a traditional Chinese medicine that combines the properties of Astragalus, Ginseng, and sophora flavescens. It is famous for its beneficial effects, such as enhancing immune function, reducing side effects of chemotherapy, and improving chemotherapy sensitivity . It is uin vitro cellular research . Initially, we screened the active ingredients of Astragalus membranaceus, Ginseng, and Sophora flavescent in KAI through TCMSP, and set the oral bioavailability (OB) ≥ 30% and drug-like (DL) ≥ 0.18 as the active compound ADME screening conditions . These tWe screened targets related to intrahepatic cholangiocarcinoma by integrating information from OMIM, the Drug-Bank database, the DisGeNET database, and the GeneCardsSuite database platform.The targets of the active ingredients in KAI were mapped to coincide with the disease targets of intrahepatic cholangiocarcinoma to acquire the core target proteins for medication therapy. The STRING database was used to build the shared target-gene-protein interaction network; the protein interaction results were loaded into Cytoscape 3.7.2, and two algorithms were used to screen for critical genes. First, we used the cytoHubba function to perform a topological analysis of the network nodes to screen the top ten potential genes; then, we used the cytoMCODE function and the PPI network to screen the sub-networks and selected genes with scores above the median in the sub-network to intersect with the critical genes in the cytoHubba screen; finally, the Hub gene is screened according to the score .The intersection targets of the KAI and ICC disease target network were analyzed for GO and KEGG pathway enrichment through the application of the biological information annotation database (DAVID). The GO analysis notes are divided into three parts, namely, molecular function (MF), cellular component (CC), and biological process (BP). The results were finally visualized by R software .After searching the Uniprot database for the receptor protein encoded by the selected gene, we downloaded the crystal structure of the receptor protein in the RCSB PDB database and the 3D structure of the active ingredient of KAI in the PubChem database. ChemBio 3D software calculated the minimization energy and output 3D structure. The receptor protein was dehydrated using PyMOL 2.4.0 software, and it was hydrogenated and charge calculated by Autodock software. This receptor protein docking site’s parameters were configured to incorporate the active pocket site for small molecule ligand binding. Ultimately, the receptor protein is docked to the active compound’s small molecule ligand using Auto-dock Vina. Lesser the docking score, the more securely the ligand binds to the protein .2  and RBE . Cell lines were incubated in RPMI 1640, with 10% FBS (Gibco) and antibiotics , at 37°C in an incubator containing 5% CO2 .Utilizing CCK-8 , the inhibitory impact of cell growth was detected. HCC9810 and RBE were cultured at a density of 5,000 cells per well in 96-well plates. They were incubated for 8 h before being treated for 24 h, 48 h, or 72 h, respectively, with or without KAI at the stated doses. The tumor cells were washed twice with PBS, treated with a 1:10 dilution of CCK-8 reagent, and incubated for two hours at 37°C. Each day, the absorbance of the cells at 450 nm was observed .Cell lysates were collected using RIPA buffer containing 0.1% PMSF . SDS-PAGE performed separation on a 10% gradient gel. The separated proteins on the gel were transferred to a PVDF membrane of 0.45 μm . Refer tt-test or Wilcoxon signed-rank test. The categorical data comparison was performed using the χ2 or Fisher’s exact test. Statistical significance was set at p < 0.05. All experimental results were independently replicated three times  and GraphPad Prism 7 . Continuous variables were expressed as mean ± SEM; comparisons between groups were made using Student ee times .By using the TCMSP database, following the active compound ADME screening conditions, we obtained the active compound as a critical component in the drug: Astragalus membranaceus was screened for 20, Ginseng for 22, and Sophora flavescens for 45 active compounds; details are shown in After discovering the compound-target disease-associated genes, we visualized the drug-active ingredient-target interaction network with 121 nodes and 264 edges using Cytoscape 3.7.2 . TypicalThrough KEGG enrichment analysis, 80 target genes enrichment pathways were identified. We selected Hub genes  for molecular docking validation. Following identifying the docking components based on the target molecules, we got three active compounds against the AKT1 protein and two active compounds against IL1β from the compound-target interaction network for molecular docking. The affinity is judged by binding energy, less than 5.0 kcal/mol, indicating excellent affinity. According to the findings of molecular docking, the binding energies of the core target proteins AKT and IL1β with the active components of KAI are less than 5.0 kcal/mol, respectively . The outThe bioinformatics predictions suggest the crucial role of AKT1 as a target and the essential role of the PI3K/AKT Pathway in drug therapy for ICC. Thus, we used CCK-8 assay to detect the proliferation of ICC cell lines treated with different concentrations of KAI and found that it significantly inhibited the growth of tumor cells in a time- and dose-dependent manner . Figure We used western blotting to evaluate the expression levels of PI3K/AKT signaling pathway. The results showed that the expression of P-AKT, p21 Waf1/Cip1, and BAX was significantly reduced, while proteins such as Cyclin D1 were downregulated after treatment of HCCC9810 and RBE with KAI . These fIntrahepatic cholangiocarcinoma is the most common type of primary liver malignancy after hepatocellular carcinoma. Due to its heterogeneity and aggressiveness, effective treatment has been lacking . TCM hasin vitro were verified using HCCC9810 and RBE, two intrahepatic bile duct-derived cell lines.Nevertheless, as the second largest tumor in primary liver cancer, ICC is far more malignant than HCC. There is a lack of research on the efficacy and mechanism of KAI in intrahepatic cholangiocarcinoma. In this research, we utilized network pharmacology and molecular docking to predict the potential targets and mechanisms of the effect of KAI in ICC. Concurrently, the drug’s anti-tumor activity and pharmacological mechanism The main ingredients of KAI are Astragalus membranaceus, Ginseng, and Sophora flavescent, which are isolated from Chinese herbs . About 8in vitro experiments both support the significant role played by the PI3K-AKT pathway in the mechanism of drug action in ICC (KAI, one of the traditional injections of Chinese medicine, exhibits excellent anti-tumor activity and synergistic effects of chemotherapeutic drugs in various solid tumors . Howevern in ICC . PI3K/AKn in ICC . PI3K/AKn in ICC . WesternIn addition, our research also found that IL1β, another critical target molecule screened out, may also exert a potential role. As a cellular inflammatory factor secreted by various immune cells, such as macrophages, IL1β is often released together with proinflammatory factors after hepatocyte apoptosis, such as IL-6 and TNF-α . We specin vivo; secondly, this study lacks further verification of these phenotypes of immune infiltration, which may be of value for future anti-tumor collaboration with immunotherapy. Further data should be collected in our subsequent preclinical studies to verify anti-tumor activity, toxic effects, and adverse events.This study also has several limitations. Firstly, our study lacks further validation of the efficacy and safety of the drugs in vitro validation. It also revealed that the anti-tumor effect of KAI is mainly via inhibition of AKT phosphorylation levels, thereby inhibiting PI3K/AKT signal, leading to changes in tumor cell proliferation of tumor cells.In conclusion, our study demonstrated for the first time the anti-tumor efficacy of KAI in ICC, and its key components, target proteins, and signaling pathways were predicted using network pharmacology, molecular docking, and


Background: Endometrial cancer (EC) is the most common gynecologic tumor and the world’s fourth most common cancer in women. Most patients respond to first-line treatments and have a low risk of recurrence, but refractory patients, and those with metastatic cancer at diagnosis, remain with no treatment options. Drug repurposing aims to discover new clinical indications for existing drugs with known safety profiles. It provides ready-to-use new therapeutic options for highly aggressive tumors for which standard protocols are ineffective, such as high-risk EC. Methods: Here, we aimed at defining new therapeutic opportunities for high-risk EC using an innovative and integrated computational drug repurposing approach. Results: We compared gene-expression profiles, from publicly available databases, of metastatic and non-metastatic EC patients being metastatization the most severe feature of EC aggressiveness. A comprehensive analysis of transcriptomic data through a two-arm approach was applied to obtain a robust prediction of drug candidates. Conclusions: Some of the identified therapeutic agents are already successfully used in clinical practice to treat other types of tumors. This highlights the potential to repurpose them for EC and, therefore, the reliability of the proposed approach. Endometrial cancer (EC) is the most frequent gynecologic tumor . Its incIn recent years, thanks to the acquired deep knowledge about genetic and molecular assets of many types of cancer, a growing number of targeted therapies have become available but, at present, few are approved for EC ,4. Drug In this work, we introduce a new robust integrated repurposing approach identifying new ready-to-use therapeutic options for metastatic EC.The drug repurposing integrative pipeline used in the present work is shown in Transcriptomics data used in this work belong to the Uterine Corpus Endometrial Carcinoma (UCEC) project of The Cancer Genome Atlas (TCGA) and were downloaded by the R package “UCSCXenaTools” . Since oAll data were downloaded from TCGA, which is open to the public under certain restrictions, therefore no ethical approval was needed.Differential gene expression analysis was performed in the R environment (v3.6.3)  comparing the mean of the expression of each gene in M and NM groups by Kruskal Wallis statistical test and calculating the false discovery rate (FDR) using the Benjamini-Hochberg correction for multiple hypothesis testing to identify statistically significant deregulated genes (FDR < 0.05). For the definition of a consistent dGES, only protein-coding genes with a minimum mean expression higher than 0.5 log2 (FPKM + 1) in both groups were selected. The same statistical analysis was performed comparing Stage I vs. Stage II and Stage III vs. Stage IV.Enrichment pathways analyses were performed exploiting Gene Ontologies Biological Process (GO_BP) and Molecular Function (GO_MF) as references using the EnrichR R package . Pathwayhttps://clue.io) (accessed on 9 November 2020) [p-value corrected for multiple hypothesis testing using the false discovery rate method [The obtained dGES was used as input of the cMap  ,33. cMape method . Since ne method ,37,38,39We applied the Pathifier algorithm  using thwww.dgidb.org) (accessed on 13 July 2021). The DGIdb was queried using Application Programming Interface (API) within the Python environment . Results are returned in JSON format, which was parsed to extract all DEGs associated with one or more drugs and the related type of interaction.The lists of DEGs mapping into the most altered biological processes and molecular functions were then merged for the following drug-target association analysis using the DGIdb database  Identifier, to avoid loss of information during the merging step, we used the PubChem Identifier Exchange Service to convert compound names from DGIdb query in other identifiers. This mapping step was performed using an ad-hoc Python pipeline that returned a final dataset of drugs common to cMap and DGIdb query results.https://www.cancerrxgene.org/, accessed on 1 July 2021) [For each of our previously defined drugs, present in the GDSC database  , IC50 rehttps://www.fda.gov/, accessed on 1 July 2021), Inxight Drugs .The experimental information about the validated compounds were manually curated through the literature mining and querying several databases: DrugBank , clinicaDifferential gene expression analysis between metastatic (M) and non-metastatic (NM) EC identified a robust dGES composed of 212 genes, 29 UP-regulated and 183 DOWN-regulated . FunctioTo deeper investigate the molecular processes underlying metastatization, we expanded the information obtained from the dGES by comparing the transcriptomics profiles of M and NM patients, exploiting the Pathifier algorithm . It tranThese results define a complex molecular landscape of biological alterations leading to EC metastatization and unveil perturbed pathways never considered before as EC vulnerability which can be exploited to target the most aggressive EC.Using the results of these analyses, we searched for already approved drugs that could be repurposed on EC by employing two different approaches: (i) querying the cMap to obtain matched drug signatures and, (ii) using the Drug Gene Interaction Database (DGIdb) to derive drug-gene associations.For the Signature-matching Approach, we used the dGES as input for the cMap which returned a list of perturbagens ranked by the Connectivity Score (CS). By applying the reversal method, we identified 115 compounds and six main PCL comprising most but not all the compounds A, potentFor the Pathway-based approach, we used Pathifier to establish the most altered pathways. From these pathways, we obtained two lists of differentially expressed genes (DEGs), 113 genes for GO_BP, and 78 genes from GO_MF. Then, we combined them to define a unique list of 121 most perturbed DEGs to be considered as potential pharmacological targets. They were used to query the DGIdb, which integrates about 15 pharmacological databases with the purpose to identify, for a gene of interest, drugs able to interact with its functions. We obtained a dataset comprising 575 compounds , for whiTo get a robust prediction of drugs potentially able to restrain EC metastatization, we merged the results derived from cMap and DGIdb. To validate the ability of our predicted drugs to restrain EC metastatization, we took advantage of the GDSC database (24) and searched for the predicted compounds. Eleven out of 18 were found. For each of them, we queried GDSC to evaluate their effect on a list of endometrial cancer cell lines considering only endometrial adenocarcinoma to best mimic EC samples used for our analysis . InteresTo our knowledge, this is the first time that drug repurposing is applied to EC by integrating different approaches.We reasoned that reverting tumor phenotype to “normal” status is unlikely to be achieved in patients, but it would be more feasible to restrain tumor aggressiveness, by inhibiting its ability to metastasize, which is the deadliest feature of cancer cells. We identified a list of drug candidates using two drug repurposing approaches, one signature-matching and the other pathway-based, starting from transcriptomics data of EC patients. The combination of these two different computational approaches allowed us to exploit the strengths of both and minimize their weaknesses. Indeed, while the pathway-based method has a wider spectrum of candidate drugs, the signature-based method is more stringent. As a result, outputs overlap identified 18 drugs, pointing at these compounds as robust candidates to restrain EC metastatization and enhancing the reliability of our combined approach. In-silico validation highlighted eight of these compounds as very promising.Pictilisib is the most promising among the identified PI3K/AKT/mTOR pathway inhibitors. It has been tested in several Phase 2 trials on different types of tumors both hematopoietic and solid, but it was never specifically used to treat endometrial or other gynecological cancers. To date, clinical trials with these PI3K/AKT/mTOR pathway inhibitors have shown few encouraging results, but still, high expectations and efforts are put into this class of drugs . In thisSelumetinib, a MEK inhibitor, is registered in a total of 118 trials on different solid tumors, including one trial with EC patients (recurrent/resistant) . The stuAlisertib is an AURKA inhibitor used in several clinical trials for many types of cancers, in particular hematopoietic tumors. Despite non-homogeneous results, its anti-tumor effect is compelling, and it remains under investigation both as monotherapy and in combination with chemotherapy ,50.Dactinomycin and Palbociclib are probably the most promising compounds since they are widely used in clinical practice. Dactinomycin is an RNA-polymerase inhibitor, thus a classical chemotherapy drug, but it has never been used for treating EC. Its extensive use in clinics ensures confidence in its scheduling and side effects management.Palbociclib is a CDK inhibitor, approved for metastatic breast cancer . Notably, among the 174 currently active trials, one is testing Palbociclib in combination with Abexinostat and Fulvestrant in gynecological tumors  (NCT04498520). Another interesting trial is evaluating the combination of Palbociclib with Letrezole in metastatic-ER+ EC (NCT02730429). Of interest, this second trial almost mimics part of our results, since we observed that ESR1 expression is significantly increased in M compared to NM EC. Thus, we expect that the vast majority of metastatic EC are ER+ and we suppose that these tumors are the best candidates for our predicted drugs, including Palbociclib. The results of this trial will be of great interest for the clinical validation of our research, and it will enhance the reliability of our repurposing approach.Our integrated approach enabled the identification of five drugs predicted to be highly efficient in reducing EC metastatization. Notably, they are already used in clinical practice which makes them excellent candidates to be tested as ready-to-use compounds to treat specifically metastatic EC. Moreover, our results suggest a new enrollment criterion for trials testing PI3K/AKT/mTOR pathway inhibitors, a class of drugs in which the medical community has high expectations, but that are still providing little results.Collectively, these results serve as proof of principle demonstrating the robustness and reliability of our new, integrated repurposing approach. Further studies on larger and more balanced cohorts of samples will be needed to challenge this method which can be successfully applied not only to EC but also to other cancer settings.


Ischemic stroke (IS) is the most common cardiovascular and cerebrovascular disease in clinic. Qiangli Tianma Duzhong Capsule (TMDZ capsule) has significant therapeutic effect to IS. Therefore, it is great significance to explore the mechanism of action of TMDZ capsules in the treatment of IS.The potential active components and possible targets of TMDZ capsule were obtained from TCMSP and The Encyclopedia of Traditional Chinese Medicine databases. IS related targets were collected by Genecard database, OMIM database, TTD database and DisGeNET database. The common target network of drug-diseases was constructed using Cytoscape for visualization analysis. Potential mechanisms were identified through enrichment analysis of gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG). Three key targets  were selected from the key networks with high correlation scores in PPI for molecular docking, through molecular docking, the interaction between target and protein is visualized.59 active components and 648 targets of TMDZ capsules and 2286 targets of IS were obtained through database mining. Compound-target network is constructed with 117 nodes and 1185 edges. GO and KEGG suggest that lipids and atherosclerosis, fluid shear forces and atherosclerosis, neurodegenerative pathways – multiple diseases and blood circulation play important roles in the treatment of IS.This study reveals the molecular mechanism of TMDZ capsules in the treatment of IS by integrating molecular docking with a network pharmacological strategy, which not only confirmed the clinical efficacy of TMDZ capsule, but also laid the foundation for further experimental research. Its 2 major categories are ischemic stroke (IS) and hemorrhagic stroke. About 18 million people suffered from IS in 2020, with two-thirds of IS occurring in people over 65 years of age,,5 and South Asians are higher risk of developing IS particularly, accounting for 40% of global IS deaths, it imposes an enormous burden on health care systems worldwide. IS accounts for 70% to 80% of stroke, and the research on effective treatment of IS is one of the hot spots of stroke at present.,7Stroke is the most common cardiovascular and cerebrovascular disease in clinic With the clinical gradual attention of traditional Chinese medicine (TCM) treatment of diseases and Chinese medicine has also made positive progress in the treatment of cardiovascular and cerebrovascular diseases, so we should actively adopt the method of integrated traditional Chinese and western medicine for clinical treatment.,9 The common pathogenic factors of IS are arterial thrombosis and venous thrombosis, which are distinguished by embolism of attached blood vessels, arterial thrombosis is associated with a high risk of IS. In view of the behavioral disorders caused by IS, TCM put forward the concept of “qi deficiency and blood stasis.”,10,11 From the TCM for the treatment of IS, it was found that the Qiangli Tianma Duzhong Capsule (TMDZ capsule) contained part of the TCM for the treatment of IS, According to the investigation, the TMDZ capsule with TCM ingredients has obvious clinical efficacy in the treatment of cardiovascular and cerebrovascular diseases.IS is characterized by early absence of obvious symptoms and high recurrence rate, it is easy to develop into cerebral infarction with the gradual aggravation of the disease. TMDZ capsule is a multi-target drug with good efficacy in the treatment of IS, the network pharmacology method can explain the characteristics of “multi-component and multi-target” of TMDZ capsule, which is consistent with the complex etiology and multi target treatment concept of IS.,14 In this study, network pharmacology was adopted to predict the active ingredients, targets and pathways of TMDZ capsules, the specific steps are shown in Figure At present, TMDZ capsule has been registered in the national medical insurance, in clinical practice, it is mainly used for the symptoms of meridian pain, limb numbness, inconvenient walking, waist and leg pain, headache and dizziness caused by stroke. TMDZ capsules can improve microcirculation, slow heart rate, prolong ejection time, increase cerebral blood flow, reduce myocardial oxygen consumption, have good sedative and hypnotic effects, and reduce sympathetic nerve excitability, these effects have certain therapeutic on related symptoms caused by IS.http://www.tcmip.cn/ETCM/index.php/Home/Index/) and Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform  can provide comprehensive information, which is ingredients, targets and pharmacokinetic characteristics of commonly used TCM herbs and herbal formulas. In this study, we accessed the ETCM and TCMSP to predict the ADME-related properties of TMDZ capsules by searching for the chemical name “Gastrodia elata, Eucommia ulmoides , Radix Aconiti Kusnezoffii, Aconitum carmichaelii Debx, Radix Angelicae Pubescentis, Ligusticum, scrophularia ningpoensis, Angelica sinensis, rehmannia, Cyathula officinalis, Viscum coloratum and Notopterygium incisum,” which can collect components, targets and other relevant properties of TMDZ capsules.The Encyclopedia of Traditional Chinese Medicine , Online Mendelian Inheritance in Man database (http://www.omim.org), DrugBank database (https://www.drugbank.ca/) and the therapeutic target database (http://db.idrblab.net/ttd/), the results of the 5 databases was summarized, duplicates were removed, finally, IS-related targets were obtained.IS targets are collected from different databases in preparation for subsequent analysis. Keywords “ischemic stroke” for Genecard database (All targets were standardized using the Uniprot database and the species should be selected as “Homo snpiens” when using the Uniprot database for target and Gene name conversion.https://bioinfogp.cnb.csic.es/tools/venny_old/).The core targets of TMDZ capsules in the treatment of IS were obtained by intersecting the targets of the active chemical components in TMDZ capsules with the genes related to IS disease and the Venn diagram was prepared, which requires the use of the Venny online platform  for PPI, which can explain the therapeutic mechanism of action of TMDZ capsule more comprehensively. In order to meet the accuracy and quantity requirements of PPI, the confidence score was selected to be greater than 0.4, the species was limited to Homo sapiens, then PPI data was obtained. The results are saved in tsv format. PPI results are imported into Cytoscape3.8.2 software to draw the interactive network, then core targets were selected by double degree value.Protein-protein interaction (PPI) network diagrams are constructed to illustrate the interactions of proteins and interactions between proteins and other molecules, which can predict intracellular effects. The core target of TMDZ capsule treatment IS entered into the String online platform (http://metascape.prg/gp/index.html), results were obtained with human was defined as the species. After getting the results, these results are visualized as GO term figure and bubble chart based on bio-informatics platform (www.bio-informatics.com.cn). metascape platform is a powerful gene functional database, which can provide comprehensive functional annotations of pathways, including biological processes, cellular component and molecular functions.To further clarify relevant functions and pathways, gene ontology (GO) functional enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis were carried out by the metascape platform (https://www.rcsb.org/), the chemical structure of the 3 components was drawn by ChemDraw18.1. The prepared protein structure and molecular structure were imported into PyMOL to remove water molecules and small molecule ligands, the AutoDockTools software was used to convert the protein and drug components into pdqt format files and identify active pockets, finally, molecular docking was performed by vina software. The greater the absolute value of binding energy, the stronger the binding ability of the compound and the protein target.Degree of nodes is a key indicator to describe network nodes. The top 3 core targets of degree values  were selected from PPI networks. The corresponding chemical components of p-hydroxybenzaldehyde, carotenoside and palmitic acid were found, then Molecular docking of the target and the corresponding chemical components was performed. The protein structure of the key target was obtained from the PDB database , 99 active compounds were obtained after removing the duplicate values. A total of 648 genes were identified from the ETCM and TCMSP databases, 345 targets from Gastrodia elata, 248 targets from Eucommia ulmoides, 8 targets from Radix Aconiti Kusnezoffii, 17 targets from Aconitum carmichaelii Debx, 35 targets from Radix Angelicae Pubescentis, 4 targets from Ligusticum, 23 targets from scrophularia ningpoensis, 62 targets from Angelica sinensis, 93 targets from Cyathula officinalis, 61 targets from Viscum coloratum and 38 targets from Notopterygium incisum, excluding 326 common genes, finally, 322 genes were identified.A total of 2334 targets were identified, 2144 targets were identified in Genecard database, 129 targets were identified in OMIM database, 61 targets were identified in DrugBank database and 0 targets were identified in the therapeutic target database database. 2286 targets were defined after removing 48 duplicate targets.Through Venny online platform, 130 common targets of drugs and diseases were obtained, as shown in Figure Protein interaction relationship data and protein interaction network diagram were obtained, as shown in Figure In order to further explore the mechanism of action of TMDZ capsule treatment of IS, potential therapeutic targets were analyzed for GO and KEGG enrichment (the top 10 results of each analysis are shown in Table Through GO functional enrichment analysis and KEGG pathway analysis, ion channel complex, lipid binding, blood circulation, lipids and atherosclerosis, fluid shear stress and atherosclerosis, pathways of neurodegeneration multiple diseases and other processes were related to the treatment of IS with TMDZ capsule. Through the above process, we can speculate the mechanism of action of TMDZ capsule in the treatment of IS.Molecular docking simulates the interaction between small molecular ligands and bio-macromolecule receptors, which can predict the binding mode and affinity between them and selects the lead drug of the target, it could aid drug development. In this study, the top 3 targets with the highest degree value were selected for molecular docking, the results were shown in Figure –27 In recent years, TMDZ capsules have been widely used for the treatment of IS. However, the underlying mechanism of action is still poorly understood when TMDZ capsules are used to treat IS. Network pharmacology approach was applied to explore the underlying pharmacological mechanism of actions of TMDZ capsules and a total of 130 potential targets related to the treatment of IS were identified in this study. Through these targets, we can identify the main ingredients for the treatment of IS including sitosterol, vanillin, palmitic acid, carotene, gastrodin, p-hydroxybenzaldehyde, citric acid, kaempferol, curcumin, ferulic acid, epicatechin and other ingredients. At present, it has been reported that sitosterol, vanillin, carotene, gastrodin, p-hydroxybenzaldehyde and other components can reduce blood cholesterol, inhibit the formation of cerebral arteriosclerosis, inhibit the excessive accumulation of cholesterol in nerve cells, anti-apoptosis of nerve cells, maintain the homeostasis of neurogliocyte and protect the integrity of blood-brain barrier.–32 In the treatment of IS, these components can inhibit the accumulation of cholesterol in nerve cells, regulate the cholesterol transport function of cholesterol transporter NPC1L1 on the cell membrane, reduce the expression levels of Cathepsin B and Caspase-3 and inhibit the apoptosis pathway of Cathepsin B-caspase. kaempferol, curcumin, ferulic acid, epicatechin and other components regulate the phosphorylation level of Akt and Src proteins, prolong the reaction time of PT and APTT, increase the activity of plasma t-PA, decrease the activity of PAI-1, increase the content of intracellular CGMP, antagonistic endothelin, blocking calcium channels, dilating blood vessels, reducing blood viscosity, reducing platelet aggregation, restoring damaged endothelial tissue, enhancing the activity of plasma anticoagulant system, enhancing the activity of fibrinolytic system, improving blood perfusion, thrombolysis, inhibiting thrombosis, activating platelets, reducing the release of platelet inflammatory factors, reducing the density and coverage of platelet accumulation in cerebral thrombosis and other ways to anti-oxidation, anti-inflammation, anti-tumor and neuroprotection and other pharmacological effects. These pharmacological effects can well protect cranial nerve function, reduce brain damage, reduce cerebral infarction volume and neurological score in IS. These components play a good role in the treatment of IS.IS is a common cardiovascular and cerebrovascular disease, which is one of the main causes of death and disability, it is a global public health problem. IS has a great impact on the life and health of patients and has caused a large economic burden to the country.GO analysis showed that TMDZ capsule played a therapeutic role in the treatment of IS mainly through ion channels, enzyme reactions and hormone reactions. In the early stage and recovery stage of IS, TMDZ capsule can prevent and treat IS by regulating the levels of ion channels, enzyme levels and oxidoreductase activity. KEGG pathway enrichment analysis showed that TMDZ capsule played a role in the treatment of IS mainly through lipid and atherosclerosis, fluid shear stress and atherosclerosis, pathways of neurodegeneration multiple diseases and other pathways. promoting endothelial cells to release neurotrophic factors VEGF-A and BDNF, enhancing nerve cell to resistance damage, promoting the proliferation of neural stem cells and regulating cholesterol levels.,52 This role is mainly related to lipids, ion channels, atherosclerosis pathways and neurodegeneration pathways. The results showed that TMDZ capsules had the effects of anti-oxidation, reducing blood cholesterol, inhibiting cerebral arteriosclerosis, inhibiting thrombosis, activating platelets and reducing the release of platelet inflammatory factors. Furthermore, the mechanism of action of TMDZ capsules was further explained in the prevention and treatment of IS.Through molecular docking, it can be seen that p-hydroxybenzaldehyde, carotoside and palmitic acid molecules are closely connected with the targets of ALB, TNF and INS, which can be inferred that the main drugs in the TMDZ capsule are gastrodia and eumoides. Its main components play a role in the prevention and treatment of IS by reducing the level of EAA neurotransmitter Glu,This study systematically explored the active ingredients of TMDZ capsules and the potential targets and signaling pathways of TMDZ capsules in the treatment of IS. The results showed that the TMDZ capsule was composed of 12 drugs, a total of 59 active compounds and 648 targets. There were 2286 targets for IS, and 130 targets for the common action of TMDZ capsules and IS. TMDZ capsule mainly acts through vanillin, p-hydroxybenzaldehyde, palmitic acid, carotene, quercetin, β-carotene and other components in the treatment of IS. The corresponding targets of these components are ALB, TNF, INS, and so on. It mainly plays a role through lipid and atherosclerosis, fluid shear stress and atherosclerosis, neurodegenerative pathway-multiple diseases and other pathways. In addition, molecular docking successfully verified the tight connection between the active compounds and the IS targets. This study explored the mechanism of action of TMDZ capsules in the treatment of IS by network pharmacology, which provided some reference for the clinical use of TMDZ capsules in the treatment of IS.Data curation: Fengjiao Yang.Formal analysis: Fengjiao Yang.Methodology: Ya Yan.Project administration: Guangming Wang.Resources: Yun Gu.Validation: Fengjiao Yang.Visualization: Ya Yan, Yun Gu.Writing – original draft: Fengjiao Yang.Writing – review & editing: Guangming Wang.


Curcuma longa L. (CL) has been reported as a promising therapeutic. We aimed to uncover the core components and mechanisms of CL against hepatic fibrosis via a network pharmacology approach.Hepatic fibrosis is a great concern in public health. While effective drugs for its treatment are lacking, The main components of CL were obtained and screened. While targets of components and disease were respectively collected using SwissTargetPrediction and online databases, common targets were assessed. A protein–protein interaction (PPI) network was constructed, and core targets were identified. GO and KEGG pathway enrichment analyses were performed, and molecular docking was conducted to validate the binding of core components in CL on predicted core targets.Nine main components from CL based on high-performance liquid chromatography (HPLC) and 63 anti-fibrosis targets were identified, and a PPI network and a component target-disease target network were constructed. Apigenin, quercetin, demethoxycurcumin, and curcumin are likely to become key phenolic-based components and curcuminoids for the treatment of hepatic fibrosis, respectively. KEGG pathway enrichment analysis revealed that the HIF-1 signaling pathway (hsa04066) was most significantly enriched. Considering core targets of the PPI network and a network of the common targets and pathways enriched, AKT1, MAPK1, EGFR, MTOR, and SRC may be the core potential targets of CL against hepatic fibrosis. Molecular docking was carried out to verify the binding of above core components to core targets.The therapeutic effect of CL on hepatic fibrosis may be attributed to multi-components, multi-targets, and multi-pathways. Efforts to explore medicines facilitating fibrosis resolution will reduce the incidence of end stage liver diseases. Studies have revealed key mechanisms leading to liver fibrosis, which include chronic damage to hepatocytes and the epithelial or endothelial barrier, release of inflammatory cytokines, recruitment of inflammatory cells, activation of hepatic myofibroblasts, excessive production of ECM and dysregulation of ECM degradation.,3 However, over the last decade, despite a variety of experimental and clinical studies targeting single mechanism, there is still no effective drug to treat hepatic fibrosis in the clinic. Etiology-focused therapies are the main treatment for patients, which cannot directly contribute to fibrosis resolution and liver repair. Given that multiple pathologies are associated with hepatic fibrosis, the development of novel therapeutics targeting multiple pathogenic pathways is desirable.Hepatic fibrosis is a reversible wound-healing response characterized by the accumulation of extracellular matrix (ECM) following liver injury by, for example, hepatitis B virus and/or hepatitis C virus infections, alcohol, or metabolic syndrome inducing nonalcoholic steatohepatitis. Perpetuation of hepatic fibrosis results in cirrhosis, which is the 16th leading cause of death and severely affects the quality of life.,5Curcuma longa L. (CL), known as “Jianghuang” in Chinese, is widely used as a food spice and herbal medicine in Asia. Classical TCM documents, such as Tang Materia Medica and Rihuazi Materia Medica, demonstrated that CL can promote blood circulation and remove stasis, and treat abdominal mass, etc. Clinically, formulae containing CL, such as Shengjiang Powder, Zhongman Fenxiao Pill, and Jianghuang Powder, have also been widely used to treat chronic liver diseases. Researches have demonstrated the therapeutic effects of CL on carbon tetrachloride induced liver injury, liver fibrosis, and cirrhosis, suggesting that CL has the potential as a treatment option for hepatic fibrosis.,7 The effects of CL are largely attributed to its predominant active components. Previous studies showed that CL consists of different components with following percentage; curcuminoid compounds 2% to 5%, carbohydrates nearly 40% to 70%, proteins 6% to 8%, oils 5% to 8%, and minerals and other elements 3% to 5%. Curcuminoids and essential oils are 2 major components present in CL.–10 Salama et al showed Hepatoprotective effect of ethanolic extract of CL on thioacetamide induced liver cirrhosis in rats. Lin et al found that curcumin, the main compound of CL, exerted anti-fibrogenic effects and induction of apoptosis in rat hepatic stellate cells (HSCs). Remarkable hepatoprotective, anti-inflammatory, and anti-fibrotic effects of analogs of curcumin and non-curcuminoid constituents have also been reported.,14 Despite a series of studies on the biological activities of active components in CL, the potential targets and underlying mechanisms of CL in hepatic fibrosis remain largely unclear. Due to the effects of CL and its bioactives on hepatic fibrosis in researches and a variety of pathological processes involved, we therefore hypothesized that CL possibly exert a treatment effect by acting on multiple pathological processes.Traditional Chinese medicine (TCM) has been used to treat hepatic fibrosis for thousands of years, and both bioactive components and formulae have been proved to be effective. which increased the quality of this study significantly. Our study may offer new insights into the mechanisms of CL and provides a more specific and effective treatment for hepatic fibrosis.As an approach for drug discovery, network pharmacology combines network biology and multi-pharmacology. This study aimed to identify effective target proteins of main components in CL and uncover the mechanism of the anti-fibrosis effect through a network pharmacology approach and molecular docking. A scheme of the study protocol is shown in Figure  the CAS numbers of the relevant molecules were obtained. The chemical structures were found through the PubChem website and saved as sdf files, which then were used to predict the oral availability and drug-like properties of the components on the SwissADME website . The screening condition was “GIabsorption” as “High,” and “Druglikeness”  met 3 of the 5 rules. This finally determined the main active components of CL. To obtain the targets of the main active components, SwissTargetPrediction  was employed.The main components in CL were obtained from a recent study based on HPLC.https://auth.lifemapsc.com/now ), DrugBank database , therapeutic target Database , and DisgeNET database  using “hepatic fibrosis,” “liver fibrosis,” or “cirrhosis” as a key phrase, and duplicate targets were removed using Microsoft Excel software. The intersection of CL-related targets and disease targets was assessed by Venny 2.1 . Common targets represent the target of main active components in CL against hepatic fibrosis.Disease targets were collected from the GeneCards database . The organism was set to Homo sapiens, and only the minimum required interaction score > 0.4 was chosen as significant. PPI networks consist of nodes, which represent a target protein, and edges, which represent PPI. The thickness of an edge represents the combined score. Degree refers to the number of other nodes directly connected to a node. The higher the degree is, the more important the node is. Core targets were identified through network analysis using Cytoscape software (v.3.9.1) and its plugin . In the present study, the top 10 proteins ranked by degree were selected and identified as core targets.A PPI network was constructed by using the STRING database version 11.5 https://metascape.org/gp ) is a comprehensive tool for gene annotation and enrichment analysis. GO and KEGG pathway enrichment analyses were performed using Metascape. The enriched terms with P < .01, a minimum count of 3, and an enrichment factor >1.5 were considered significant. The top 20 enriched terms were visualized using an online tool . A target-enriched KEGG pathway network for the main components against hepatic fibrosis was also constructed by Cytoscape software. Red nodes represent enriched KEGG pathways, and yellow nodes represent target proteins.Metascape . Molecular docking calculations were performed using the SwissDock web service .To validate the binding of core components in CL on predicted core targets, the 3D molecular structure of compounds was retrieved from the PubChem database and the structure files of target proteins were acquired from the RCSB Protein Data Bank  , which may be the most relevant to AD treatment. The details are provided in Figure 63 common targets and 9 main active components from CL were used to construct a main component target-disease target network Fig. . All comP < .01. There were 902, 59, and 80 GO terms related to biological processes (BP), cell components, and molecular functions (MF), respectively. The primary enriched BP terms were positive regulation of cell migration (GO:0030335), positive regulation of cell motility (GO:2000147), positive regulation of cellular component movement (GO:0051272), etc , plays an important role in the adaptation to hypoxic conditions and is closely related to vascularization, metabolic regulation, cell multiplication and survival , Phospholipase D signaling pathway (hsa04072), Relaxin signaling pathway (hsa04926), Endocrine resistance hsa01522), and PI3K-Akt signaling pathway (hsa04151) . Studies showed that apigenin can alleviate hepatic fibrosis by inhibiting HSC activation and autophagy via TGF-β1/Smad3 and p38/PPARα pathways, and regulating VEGF-mediated FAK phosphorylation through the MAPKs, PI3K/Akt, HIF-1, ROS, and eNOS pathways.,37In GO analysis, main components of CL were involved in positive regulation of cell migration, positive regulation of cell motility, and positive regulation of cellular component movement. All of these BP are closely associated with ECM remodeling and recruitment of inflammatory cells, which is of great importance in the progression of hepatic fibrosis. Main involved cellular components included ECM, external encapsulating structure, and vesicle lumen, and MF concerned included carbonate dehydratase activity, phosphotransferase activity, and kinase activity. KEGG pathway enrichment analysis revealed that HIF-1 signaling pathway, Phospholipase D signaling pathway, Relaxin signaling pathway, Endocrine resistance and PI3K/Akt signaling pathway were most enriched pathways associated with hepatic fibrosis. As the most significantly enriched pathway, HIF-1 signaling pathway mediates the body responses to hypoxic microenvironment, induces the angiogenesis, migration and proliferation of fibroblasts and keratinocytes, anaerobic metabolic transformation, and systemically increases the number of red blood cells. The role of HIF-1 in the development of hepatic fibrosis has been clearly identified by experiments, which closely interacts with VEGF, PI3K/Akt, MAPK, TGF-β and NF-kB signaling pathways, and plays an important role in HSC activation and ECM synthetization.A network of the common targets and pathways enriched showed that AKT1, MAPK1, PIK3CB, PIK3CA, RELA, EGFR, MTOR, SRC, CCND1, and BCL2 possibly participated in the above KEGG pathways. Considering the main targets showed by PPI network, we speculate that AKT1, MAPK1, EGFR, MTOR, and SRC may be the core potential targets of CL against hepatic fibrosis, and molecular docking was further performed. As mentioned above, both curcumin and demethoxycurcumin were strongly bound to MAPK1, EGFR, and SRC, which indicated that curcumin and demethoxycurcumin might be the core components against hepatic fibrosis, and MAPK1, EGFR, and SRC might be the core targets. MAPK cascade usually consists of at least 3 core kinases, defined as MAP3K, MAPKK, and MAPK. Once activated, the signal is propagated through sequential phosphorylation and activation of sequential kinases, which, in turn, leads to the phosphorylation of hundreds of target regulatory proteins identified in the cytoplasm, mitochondria, endoplasmic reticulum, and Golgi apparatus, as well as in the nucleus. Previous studies showed that, during the progression of hepatic fibrosis, MAPK1 played a crucial role in the transduction of proliferative stimuli into HSC, activation of type I collagen and Connective tissue growth factor synthesis, migration in response to chemoattractants and ROS, etc.,40,41 Up to now, anti-fibrogenic drugs and strategies, including curcumin, have been designed to directly affect MAPK1 or to affect the pathways upstream to MAPK cascade, but few studies have reported some benefit in hepatic fibrosis patients.–45 Based on our results, MAPK1 plays an important role in 19 of 20 most enriched pathways in KEGG analysis and strongly binds to core components of CL. It worthy to be further studied as a potential core target of CL against hepatic fibrosis.MAPK1, also known as extracellular signal-regulated kinase 2, acts as an essential component of the MAPK/ERK cascade, which mediates intracellular signaling triggered by extracellular stimuli such as growth factors and cytokines as well as by intracellular stimuli such as oxidative and endoplasmic reticulum stress and gives rise to various cellular responses including proliferation, migration, differentiation, survival or apoptosis, autophagy, and inflammatory reactions. EGFR can bind ligands of the EGF family and activating several signaling cascades, such as the ras/raf/MEK/MAPK pathway, p38-MAPK, phospholipase C/protein kinase C pathway, the PI3K/Akt–mTOR pathway and the STAT pathway, to convert extracellular cues into appropriate cellular responses. Important evidence has accumulated on the central role of the EGFR signaling system in conveying strong reparative and regenerative signals to hepatocytes upon liver injury and inflammation.,48 Studies also showed that different EGFR ligands exert anti-fibrogenic or pro-fibrogenic functions by activating different EGFR downstream signaling pathways.,50 Drugs and natural components have been reported to inhibit the the phosphorylation of EGFR and its downstream pathways and prevent the progression of cirrhosis and regressed fibrosis in different animal models.,52 EGFR was involved in 10 of 20 most enriched pathways and had effective free energy against key components in molecular docking. Further verification and exploration needs to be done in future studies.EGFR, also known as ErbB1 or HER-1, is a transmembrane protein receptor endowed with tyrosine kinase activity.,54 and results for SRC targeting have also been reported in the treatment of hepatic fibrosis.,56SRC consists of 4 SRC homology domains, and is usually activated following engagement of different classes of cellular receptors, including immune response receptors, integrins and other adhesion receptors, receptor protein tyrosine kinases, G protein-coupled receptors as well as cytokine receptors. SRC participates in signaling pathways that control a diverse spectrum of biological activities including gene transcription, immune response, cell adhesion, cell cycle progression, apoptosis, migration, and transformation. Members of the SRC family kinases have been broadly investigated in cancer due to their pro-oncogenic characteristics,Taken together, the potential core targets showed in our study, like MAPK1, EGFR, SRC, etc, have been reported to play an important role in some significant enriched pathways, like HIF-1 signaling pathway and PI3K/Akt pathway. Potential core active components including apigenin and curcumin, have also been reported to exert an anti-fibrotic function via targets and pathways mentioned above. Our molecular docking results further verified that these components could bind closely to potential core targets. Therefore, the therapeutic effect of CL on hepatic fibrosis may be attributed to these potential core components, core targets and signaling pathways.Our study was not without limitations. First, since the network pharmacology research was conducted based on existing database, which might be incomplete, the results were deductions based on previous experimental results and computer simulation predictions, and the potential core targets and mechanisms obtained need experimental verification in vivo or vitro. Furthermore, the binding affinity of potential core active components with potential targets awaits further verification.In conclusion, these findings implicated that CL mainly acted into multi-component, multi-target, multi-pathway in treating hepatic fibrosis. Potential core components, core targets and signaling pathways were screened by network pharmacology analysis. Further pharmacological experiments are needed to validate the above therapeutic mechanisms of CL. Our study combined bioinformatics analysis, network pharmacology, and molecular docking to reveal the potential core components, targets and mechanisms of CL, and might provide a theoretical basis for the use of CL against hepatic fibrosis.Conceptualization: Qiang Han, Peng Zhang.Data curation: Qiang Han, Jiahui Zhu.Formal analysis: Qiang Han, Jiahui Zhu.Funding acquisition: Peng Zhang.Methodology: Peng Zhang.Software: Qiang Han, Jiahui Zhu.Writing – original draft: Qiang Han, Jiahui Zhu.Writing – review & editing: Peng Zhang.


Western blotting, RT-qPCR and ELISA analysis showed that BXD inhibited not only the expression of IL-1β, TNF-α and NO, but also NF-κB, MMP9 and PI3K/AKT signaling pathways. This study applied network pharmacology and in vivo experiments to explore the possible mechanisms of BXD against neuroinflammation, providing insight into the treatment of neuroinflammation.Neuroinflammation plays an important role in the pathogenesis of neurological disorders, and despite intensive research, treatment of neuroinflammation remains limited. BaiXiangDan capsule (BXD) is widely used in clinical practice. However, systematic studies on the direct role and mechanisms of BXD in neuroinflammation are still lacking. We systematically evaluated the potential pharmacological mechanisms of BXD on neuroinflammation using network pharmacological analysis combined with experimental validation. Multiple databases are used to mine potential targets for bioactive ingredients, drug targets and neuroinflammation. GO and KEGG pathway analysis was also performed. Interactions between active ingredients and pivotal targets were confirmed by molecular docking. An experimental model of neuroinflammation was used to evaluate possible therapeutic mechanisms for BXD. Network pharmacological analysis revealed that Chrysoeriol, Kaempferol and Luteolin in BXD exerted their anti-neuroinflammatory effects mainly by acting on targets such as NCOA2, PIK3CA and PTGS2. Molecular docking results showed that their average affinity was less than −5 kcal/mol, with an average affinity of −8.286 kcal/mol. Pathways in cancer was found to be a potentially important pathway, with involvement of PI3K/AKT signaling pathways. In addition,  Neuroinflammation is an inflammatory response within the brain or spinal cord, a complex immune response to various injurious stimuli to the nervous system. This inflammation is mainly mediated by cytokines, chemokines, second messengers and reactive oxygen species (ROS) produced by resident glial cells (microglia and astrocytes) upon activation , 2. NormCurrently, treatment of neuroinflammation includes acupuncture interventions and medication. Although acupuncture therapy is gaining recognition as a natural treatment with a long history of simplicity, effectiveness, and no side effects, it is not always feasible because the mechanism of action of acupuncture for neuroinflammation remains unclear.Therefore, a variety of related drugs such as NSAIDs and steroidal anti-inflammatory drugs are already available on the market. Minocycline appears to inhibit the pro-inflammatory phenotype of microglia without affecting the beneficial regulatory functions of microglia. Statins have many effects on the central nervous system, including affecting microglia activation, preventing ROS formation, inhibiting matrix metalloproteinases (MMPs) and reducing excitotoxicity, and are able to reduce pro-inflammatory immune responses while enhancing regulatory immune responses . DespiteIn this context, herbal medicine is attracting more and more attention from researchers as a source of novel compounds. A variety of herbal extracts and their monomeric compounds block microglia activation, and herbs or components that follow the efficacy of “tonifying deficiency” , “cleariIn order to explore the potential mechanisms of action of BXD, a network analysis approach based on “drug-target-disease” interactions was used. In 2007, HOPKINS  first inhttps://tcmsp-e.com/tcmsp.php) for screening of active ingredients [The constituent herbs of BXD were uploaded to TCMSP  [https://sea.bkslab.org/) [https://db.idrblab.net/ttd/) [https://go.drugbank.com/ut/med-search-210910) [https://www.disgenet.org/) [https://www.genecards.org/) and OMIM database (https://omim.org/) [https://www.uniprot.org/) and all protein targets obtained by retrieval were corrected to Uniprot ID [http://www.bioinformatics.com.cn/).Potential targets for BXD were obtained from the TCMSP, SwissTargetPrediction Tools (ion.ch/)  and SEA ab.org/)  and wereet/ttd/) , drugban-210910) , DisGeNEet.org/) , GeneCarim.org/)  to obtaiiprot ID . Potentihttps://string-db.org/) of the intersection target to build a PPI network. The protein interaction relationship network and data were obtained and saved in TSV format. The network was imported into Cytoscape 3.8.2 for topological analysis and core therapeutic targets were selected based on parameters such as degree, betweenness centrality (BC) and closeness centrality (CC). PPI networks were visualized and key genes were filtered out by CytoHubba plugin [Upload String  with a threshold P < 0.05 [The anti-neuroinflammatory targets of BXD described above were imported into the David database  [https://cadd.labshare.cn/cb-dock2/php/index.php) [The structures of the individual compounds screened above were downloaded from the PubChem database. Also, enter the entry number in the RCSB PDB database (on: 1 Å)  in orderdex.php) –50 to peLipopolysaccharide (LPS) ; Nitric Oxide (NO) assay kit ; Mouse IL-1β ELISA kit ; BCA Protein Assay Kit ; SDS-PAGE Gel Rapid Preparation Kit ; anti-PI3K rabbit antibody , anti-Phospho-AKT1/2/3 rabbit antibody , anti-AKT1 rabbit antibody (AF0836. Affinity), anti-NF-kB p65 rabbit antibody , anti-MMP9 rabbit antibody , anti-TNF alpha rabbit antibody  and anti-β-actin rabbit antibody ; HRP-conjugated goat anti-rabbit IgG ; Nissl staining solution ; Trizol ; Oligo (dT)18 Primer , PrimeScript II RTase  and Recombinant RNase Inhibitor .Male C57BL6 mice, body mass 18–22 g, purchased from Jinan Penyue Laboratory Animal Breeding Co., Ltd. All mice were housed in environmentally controlled, SPF-rated animal houses. All care and experimental procedures were carried out according to the requirements of the National Institutes of Health Guide for the Care and approved by the Animal Experiment Ethics Committee of Shandong University of Traditional Chinese Medicine (No. SDUTCM20200312008).LPS, a characteristic endotoxin of the outer membrane of Gram-negative bacteria, and acts as a potent immune system stimulant, activating and releasing inflammatory mediators , which pThe presence and disappearance of Nissl body is an important indicator of whether nerve cells are damaged, and when encephalitis, cerebral ischemia, and axonal reactions occur, Nissl body can lyse or even disappear. Mice were anesthetized with isoflurane 24 h after the onset of neuroinflammation, fixed by infusion of paraformaldehyde and brains were rapidly harvested. Brains were serially coronally sectioned and immerse in Nissl staining solution for staining. And analyzed using Image J.Brain tissue from the hippocampus was rapidly extracted 24 hours after the onset of neuroinflammation. The cytokines were detected according to the ELISA kit instructions and the secretion levels were calculated.Brain tissue was rapidly extracted from the hippocampus 24 h after the onset of neuroinflammation. After extraction of total hippocampal protein from the 3 groups, their protein concentrations were assayed with the BCA kit. Blots were transferred to PVDF membranes  after SDS-PAGE electrophoresis. Primary antibodies (1:1000) using anti-PI3K, phospho-AKT1/2/3, AKT1, NF-kB p65, MMP9, TNF alpha and β-actin rabbit antibody overnight at 4°C. The secondary antibody was incubated for 1 hour at room temperature and the protein bands were recorded on the gel imaging instrument .RNA was extracted from hippocampal tissue by adding Trizol  and the RNA concentration was determined using an ultra-micro spectrophotometer . Total RNA was reverse transcribed into cDNA and amplified for quantitative analysis using specific primers on a fluorescent quantitative PCR instrument. See Measures that conformed to a normal distribution were expressed as mean ± SEM and one-way ANOVA was used for comparisons between multiple groups. Statistical analysis of the data was carried out using Graphpad Prism 8.0 software. A visible graphical summary demonstrating the technical routes is provided .sunpeng@sdutcm.edu.cn.The data used to support the findings of this study are available from the corresponding author upon request. Lead contact, further information and requests for resources and reagents should be directed to and will be fulfilled by Peng Sun, The active ingredients of each component of BXD were obtained from the TCMSP database, and a total of 130 active ingredients were obtained after de-duplication .The 130 active ingredients obtained were searched in TCMSP and 713 drug targets of action were obtained after screening and de-weighting. After removing duplicate targets, a total of 11,145 neuroinflammation-related disease targets were screened using TTD, DisGeNET, Genecards, OMIM and DrugBank disease databases, and the target genes corresponding to BXD were intersected to obtain 575 common gene targets as key targets for the treatment of neuroinflammation in BXD, see A total of 1807 GO-enriched entries were obtained as a result of the experiment. The top ten BP terms  were resA total of 199 signalling pathways were enriched by KEGG pathway analysis. The BXD anti-neuroinflammation is mainly involved in the following pathways : PathwayAmong the ten major signaling pathways, Pathways in cancer  exerted A compound-target network for BXD was constructed using the screened compounds and their targets, see P = 0.0079, P = 0.0110, P = 0.0157, P = 0.0052).Microscopically: Neuroinflammation is a relatively common disease worldwide. Most anti-neuroinflammatory drugs have a single target and are of limited effectiveness. Some traditional Chinese medicines have been shown to have anti-neuroinflammatory effects, however there are fewer studies on the anti-neuroinflammatory effects of BXD. This study uses a network pharmacology approach to initially explore the mechanism of action of BXD in the treatment of neuroinflammation.in vivo [The active ingredient-target network shows that BXD is multi-component and multi-target for the treatment of neuroinflammation. The results show that chrysoeriol, isorhamnetin, kaempferol, luteolin, and quercetin are the key active ingredients. Mapping the active ingredients to the common targets of neuroinflammation, 154 common targets of drug-disease action were obtained. Jadaun et al.  demonstrin vivo .Pathways in cancer encompass many inflammatory factors that mediate immune responses by a variety of mechanisms, and there are multiple targets in this pathway that are relevant to the pathogenesis of neuroinflammation, such as the Potassium channel AKT1 (AKT1), Nitric oxide synthase, inducible (NOS2), Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit alpha isoform (PIK3CA), Matrix metalloproteinase-9 (MMP9), Nuclear factor NF-kappa-B p105 subunit (NFKB1), Mitogen-activated protein kinase 1 (MAPK1), and Prostaglandin G/H synthase 2 (PTGS2). This study identified multiple enrichment of all potential target species on pathways in cancer, which may be key pathways for BXD to regulate neuroinflammation.in vivo anti-inflammatory activity results, indicating that the network pharmacology prediction results have a high degree of accuracy. Zhang et al. [Activation of AKT in the PI3K/AKT signaling pathway occurs when protein growth factors bind to receptors on the cell surface, inducing phosphorylation of the tyrosine portion of the receptor in the cytoplasm, leading to the aggregation and activation of PI3K . Previoug et al.  demonstrg et al.  later foNeuroinflammation is an inflammatory response that occurs in the CNS. Excessive production of pro-inflammatory cytokines , chemokines  and ROS cause neuronal and glial cell dysfunction in the form of axonal degeneration, myelin loss and cell death . As an iin vivo experiments to identify relevant targets and related pathways of BXD formulations for the treatment of neuroinflammation, and initially reveals the modern value of targeted multi-target therapy of BXD at the molecular level in the study of neuroinflammation.In summary, BXD may exert its anti-inflammatory effects by modulating NF-κB, MMP9, TNF-α, IL-1β, NO and other components of the PI3K/AKT signaling pathway through chrysoeriol, kaempferol and luteolin. This study combines bioinformatics and There are also some shortcomings in this study. It is difficult to fully reflect the overall situation due to limitations such as the lack of comprehensive data in the database and the lack of precision of the action targets obtained. In addition, for complex chemical compounds and multi-targets of BXD, the further study is still needed. In the future, more studies also suggest comparing the changes in PI3K-AKT pathway in the same animals before and after BXD intervention, and the hippocampus of each group of mice could be sampled by microdialysis to further confirm that BXD ameliorates neuroinflammation by altering the PI3K-AKT pathway.