Type 2 diabetes mellitus is a high-risk factor for acute cardiovascular events. Some reports show that Empagliflozin has a protective effect on cardiovascular events and diabetes mellitus, and Empagliflozin can act on the CaMKII pathway. However, the specific gene of action is not precise. Therefore, this study investigated the target genes of Empagliflozin by integrated gene analysis and molecular docking method to provide a theoretical basis for further elucidating the mechanism of action of Empagliflozin.In this study, we obtained 12 datasets from GEO, divided into experimental and validation groups, with a total of 376 samples. We then integrated CaMKII pathway-related genes from OMIM, NCBI, and genecards databases. We then intersected them with the differential genes we obtained to obtain 5 common genes and performed functional enrichment analysis. We then performed group comparisons in the validation set, and we obtained 2 clinically significant genes. Then we performed group comparison in the validation set, and we obtained 2 clinically significant genes, followed by molecular docking analysis with pymol, autodock software. We obtained molecular docking models for the 2 genes.CaMK2G and PPP1CA, genes associated with the CaMKII pathway and type 2 diabetes and acute cardiovascular events, by integrative gene analysis and validated their expression in the relevant dataset. We also derived that Empagliflozin acts on amino acid TRP-125 of CaMK2G gene and GLN-249 ASP-210 ASP-208 of PPP1CA through CaMKII pathway, thus acting on type 2 diabetes and acute cardiovascular events by molecular docking technique.In this study, we obtained  Patients with type 2 diabetes are at increased risk of developing cardiovascular disease . Empaglihttps://www.ncbi.nlm.nih.gov/geo/) [In this study, 12 datasets with a total of 376 samples(The specific information such as the origin of the tissue is shown in ov/geo/) , and difExclusion criteria were as follows: 1. The data retrieved had drug treatment or other confounding factors in the experimental group. 2. The data retrieved did not have genetic samples. 3. The data obtained from the survey did not have grouping information. 4. The data obtained by probing did not have grouping information. 5. The source of the samples was not Homo sapiens.We screened out the experimental group for differentially expressed gene analysis and the validation group for critical genes. The specific information is as follows .Quality control was performed on the datasets obtained from GEO. Subsequently, the R package "limma" was used to analyze variance between samples. P-values of genes and adj. p-values after hypothesis testing were obtained after correction. The meaning of the corrected P-value is the P-value obtained after hypothesis test calibration , 6. Log2To avoid the effect of differences in gene expression in different tissues on the analysis results, we used the original grouping method in the dataset. We performed the analysis of differential gene expression within the original dataset. For example, in the dataset GSE48060, the raw data were obtained from blood and grouped into MI and control groups. We analyzed the raw matrix data into the R language for differential gene expression comparison.Heatmaps and volcano maps of differential genes were obtained using the heatmap package in R software.Metascape is an online functional enrichment analysis website. It mainly includes interactive enrichment and more . WebGestIn order to obtain the maximum number of differential genes and avoid drug-induced gene count reduction as a confounding factor, our original dataset was not selected for patients using Empagliflozin or not using Empagliflozin. Instead, we selected samples of patients with the disease and normal controls.The CaMKII Pathway was selected as a screening condition because it has been demonstrated that the CaMKII Pathway may be a pathway for the pharmacological effects of Empagliflozin in type 2 diabetes and acute myocardial infarction. To test the possibility of this hypothesis, we conducted the present study. There are no relevant experiments to illustrate the pharmacological pathway of Empagliflozin in type 2 diabetes mellitus and acute myocardial infarction. Therefore, this study sought to demonstrate the possibility of CaMKII Pathway as a pharmacological pathway of Empagliflozin in type 2 diabetes and acute myocardial infarction.http://omim.org) is an online database of genetic information used by clinicians, molecular biologists, and genomic scientists [www.genecards.org) is an integrated database of gene information. It brings together genetic data from several considerable public resources, such as HGNC [OMIM  is an online database of genetic information. It includes the retrieval and storage of genomic data. It provides free downloadable data to a wide range of researchers . We analhttp://starbase.sysu.edu.cn/index.php) [To observe the main biological functions and significant pathways of the five essential genes, we entered these five gene names as keywords into the search box of the Metascape website and webgestalt website. The search was performed by:1. We entered differential genes as keywords into the search box.2, limiting their species to homo sapiens.3. the Method of Interest in the webgestalt website was limited to over-representation analysis. In addition, we obtained upstream miRNAs for five essential genes by using the mRNA-miRNA module of the online miRNA database StarBase  .We used the dataset GSE60993 GSE61144 as the validation group, extracted the expression of five essential genes, and compared them in groups according to the clinical stage of their disease, using t-tests with p-values as a measure of test effectiveness.https://www.kegg.jp/) is a comprehensive database, of which the PATHWAY database is widely used for gene pathway studies [We performed a KEGG pathway analysis to observe the pathways enriched by the five essential genes. KEGG  and prediction accuracy of autodock software are relatively high , 21. We http://pubchem.ncbi.nlm.nih.gov, a public database of active ingredients of small molecules and RNAi reagents is available) to retrieve its active molecule as a ligand [http://sparql.uniprot.org/, a database for storing protein information) with "CaMK2G" and "PPP1CA" as keywords to obtain the protein files of 1.7A and 2.25A as receptors, respectively [For this purpose, we entered Empagliflozin as a keyword in the PubChem website  is a comprehensive database, of which the PATHWAY database is widely used for gene pathway studies. kegg mapper is a visualization tool for the KEGG database, which allows visualization of gene enrichment pathways. We entered the names of five critical genes as keywords into the search box of KEGG Mapper and limited the search mode to hsa. Among them, the Diabetic cardiomyopathy pathway and the cAMP signalling pathway should be of most interest to us. Notably, in the CaMP pathway, CNGC forms CaMK and participates in the CaMP pathway, which transfers calcium ions to cells. In the pathway of regulation of TRP channels by inflammatory mediators.CaMKII is involved in regulating TRP channels by inflammatory mediators by interacting with TRPV1 protein.PPP1 is involved in regulating TRP channels by inflammatory mediators through interaction with TRPM8 protein. In the Diabetic cardiomyopathy pathway, CaMKII is involved in myocardial contraction by interacting with RyR2 protein and causing contractile dysfunction. PPP1 is involved in Cardiomyocyte death , a serine/threonine phosphatase. Increased PP1 activity is associated with the development of heart failure [PPP1CA, suggesting that Empagliflozin may indeed interact with the CaMKII pathway-related PPP1CA gene and its transcribed protein to produce its drug effect.Study shows that calcium is closely associated with vascular smooth muscle cells via the PP1 signalling pathway . PPP1CA  failure , 30. Stu failure . Mouse s failure . These aCaMK2G PPP1CA screened by bioinformatics analysis had low activation energy with the active ingredient of Empagliflozin, suggesting that the active ingredient of Empagliflozin is likely to modify the activity of the CaMKII pathway by binding to CaMK2G PPP1CA and thus exert its effect on type 2 diabetes and acute cardiovascular events.In our study, the CaMKII pathway-associated CaMK2G and PPP1CA, a gene associated with the CaMKII pathway and type 2 diabetes and acute cardiovascular events, by integrative gene analysis. We verified their expression in the relevant dataset, and we concluded that CaMK2G PPP1CA could bind to the active ingredient of Empagliflozin at low activation energy by molecular docking technique. We concluded that the clinical effects of Empagliflozin are associated with CaMK2G and PPP1CA in CaMKII pathway by the above analysis.The present study obtained S1 File(RAR)Click here for additional data file.


As a frequent cause of death in cancer patients, liver cancer usually occurs in hepatitis B and cirrhosis. In China, Chinese people have been using traditional Chinese medicine (TCM) in treating various chronic liver diseases, which could effectively improve the symptoms and slow down the progression of liver diseases. However, due to the complexity rules of TCM prescription, their action mechanisms are still not clearly understood, which may affect the popularization of effective prescriptions. This study aims to identify the core TCM herbs in the treatment of hepatitis B, liver cirrhosis, and liver cancer so as to clarify the mechanism of action of the core herb networks.  There were 1,673 prescriptions for chronic liver diseases collected in this study, of which 854 were hepatic B prescriptions, 530 were for liver cirrhosis, and 289 were for liver cancer. The basic characteristics of herbal medicine were firstly explained via descriptive analysis, then the core prescriptions of herbal medicine were analyzed through association rule, and finally, the mechanism of core prescriptions was explored with the help of systematic network pharmacology and by applying such databases as TCMIP, HERB, OMIM, GeneCards, KEGG, and software like RStudio and Cytoscape. Oldenlandia (BaiHuaSheSheCao), Bearded Scutellaria (BanZhiLian), Curcumae Rhizome (EZhu), and Cardamom (DouKou) were most frequently prescribed to eliminate cancer toxin, invigorate the spleen, and activate blood. These core herbs mainly act through signal transduction and immune system pathways, in which the PI3K-Akt pathway plays a key role. The core prescription for liver cirrhosis regulated more endocrine system pathways than the hepatitis B prescription, and liver cancer prescription regulated more nervous system-related pathways.  The rule of the core prescriptions in these cases was characterized by the application of herbs with both cold and warm properties, in which bitter herbs with cold property took priority. Tonifying deficiency, clearing heat, and activating blood circulations to remove stasis were common treatment principles for the three liver diseases. Turmeric Root Tuber (YuJin), White Peony Root (BaiShao), Bupleurum (ChaiHu), Salvia miltiorrhiza (DanShen), and Astragali Radix (HuangQi) were prescribed the most in hepatitis B treatment to invigorate the spleen and soothe the liver. Astragali Radix (HuangQi), Tuckahoe (FuLing), Atractylodis Macrocephalae Rhizoma (BaiZhu), Fructus Polygoni Orientalis (ShuiHongHuaZi), and Curcumae Rhizome (EZhu) were most frequently applied in liver cirrhosis treatment to replenish qi and activate blood.  Three core prescriptions for hepatitis B, liver cirrhosis, and liver cancer treatment were identified, which acted mainly through signal transduction and immune system pathways to regulate immunity and cell growth and participate in inflammation inhibition, in which liver cancer prescription regulated more pathways, especially more nervous system-related pathways than the other two. The global morbidity and mortality rate of liver cancer has been increasing in the past years, which has become the third most common cause of death from tumors . Asia anWith the development of complementary and alternative medicine around the world, the role of traditional Chinese medicine (TCM) in tumor prevention and treatment has drawn more and more attention. Over 60% of the antitumor medicines were developed from natural herbal products these years . In ChinBy analyzing the application characteristics, the prescription rules of the herbs, and constructing diagrams, data mining could help to discover the potential correlation between herbs, which might provide support for doctors in developing treatment protocols . AnalysiThe “multicomponent and multitarget network” in systematic pharmacology happened to coincide with the holism concept of TCM prescription, which might effectively bridge the gap between modern medicine and TCM and provide new research methods for TCM theories and would greatly promote the synergistic development between TCM and modern medicine . By analThis study proposed a comprehensive method based on data mining and systematic pharmacology, analyzed the rules of TCM prescriptions, and revealed their mechanisms. Clinical data of patients with liver diseases from the two centers were collected, and the prescriptions of these cases were analyzed by descriptive statistics and association rules to explore the prescription rules and core prescriptions in the cases. The important targets were retrieved from the database, and Metascape and KEGG were used to further analyze the biomarker pathways. Workflow is shown in In this study, 1,673 prescriptions from the outpatient departments of the Affiliated Hospital of Shandong University of Traditional Chinese Medicine by Professor Changjian Yin and the Affiliated Hospital of Capital Medical University by Professor Ying Qian were analyzed retrospectively from January 2020 to June 2021, among which, 854 cases were diagnosed as chronic hepatitis B, 530 were cirrhosis, and 289 were liver cancer.Hepatitis B diagnostic criteria referred to the guidelines for the prevention and treatment of chronic hepatitis B . DiagnosThe diagnostic criteria of decompensated liver cirrhosis referred to the Chinese guidelines on the management of liver cirrhosis . DiagnosThe diagnostic criteria of primary liver cancer referred to the diagnostic criteria of the guidelines for diagnosis and treatment of primary liver cancer . DiagnosThe general clinical information and the information on Chinese herbal medicine prescriptions of these cases were collected, including the patients' age and gender and the formulae prescribed to them. The data were entered into Microsoft Office Excel, and the database was subsequently established. The herbs in the database were further standardized, and their properties, flavors, and efficacies of the herbs were completed according to the National Pharmacopoeia Commission and Chinese Materia Medica. Two researchers completed the data entry separately.This study was reviewed by the Ethics Committee of Beijing University of Chinese Medicine, with ethical batch number 2019BZHYLL0204, which was strictly implemented in accordance with the ethical system.Microsoft Office Excel software was used to statistically analyze the distribution of patients' visits, age, and gender, and GraphPad Prism was used for visual display. Firstly, a general descriptive analysis was performed on the properties, flavors, and efficacies of the herbs in the prescriptions. Then the properties, flavors, and other aspects of the high-frequency herbs were analyzed and visually displayed so as to comprehensively show the therapeutic ideas of the prescriptions. Finally, association rules were used to screen the core herbs with strong association.Descriptive analysis was performed on the characteristics  of the herbs. The siqi (four properties) included five items: cold, heat, warm, cool and mild, and the wuwei (five flavors) included seven items: sour, bitter, sweet, pungent, salty, astringent, and light. The herbs were divided into 21 categories according to their efficacies, which are deficiency-tonifying herbs, heat-clearing herbs, blood-activating and stasis-removing herbs, damp-draining herbs, qi-regulating herbs, exterior-relieving herbs, aromatic damp-resolving herbs, phlegm-resolving, cough-suppressing and panting-calming herbs, digestant herbs, blood-stanching herbs, wind-damp-dispelling herbs, tranquilizing herbs, liver-wind-calming herbs, astringent herbs, interior-warming herbs, purgative herbs, detoxification, parasiticide and itching-relieving herbs, worm-expelling herbs, resuscitative herbs, emetic herbs, and suppuration and granulation-promoting herbs. The top 10 core herbs in terms of frequency used in the three diseases were chosen for visual analysis of their properties, flavors, and proportion of use in all cases. RStudio 3.6.1 software and Mul-chart software were applied for analysis and visual display.Apriori algorithm was a frequent itemset algorithm that formed the association rules, which was used to analyze the clear rules in TCM treatment and obtain the core herbs in this study. Each herb was treated as an itemset, each prescription was treated as a transaction, and they were used to find out the frequent itemset, to mine the association rules between the herbs in the prescriptions, and to discover the core prescriptions. RStudio 4.0.2 software was applied for analysis and visual display.https://www.TCMIP.cn/TCMIP/index.php/Home/Login/login.html) [https://herb.ac.cn/) [In this study, the integrative medicine pharmacology research platform TCMIP v2.0 (in.html)  and the .ac.cn/)  were usehttps://www.genecards.org/) and Online Mendelian Inheritance In Man (OMIM) database (https://www.omim.org/). A score ≥10 was set in the GeneCards database to select the targets of high correlation with the disease, and the obtained targets in the two databases were merged with each other to screen out the disease-related targets.Relevant targets for the three diseases were collected via the integration of the GeneCards database (https://string-db.org/), and the species was selected as “Homo sapiens” to derive the data of protein-protein interaction (PPI). The resulting PPI data were imported into Cytoscape 3.8 (https://www.Cytoscape.org/) for processing the PPI network diagram.The overlapped targets were selected as the core herbs of the relevant ones for the treatment of hepatitis B, cirrhosis, and liver cancer. The target was then processed with String (https://metascape.org/gp/index.html#/main/step1) was applied for KEGG pathway analysis. The data were uploaded as a multiple gene list, the species were selected as “Homo sapiens,” and the mode was set as custom analysis, P value <0.01, min overlap >3. After removing the disease-related pathways, each of the top 15 important pathways was selected, and the Microbiology Information website (https://www.bioinformatics.com.cn) was applied to plot the KEGG pathway bubble map.Common targets were acquired after overlapping the obtained herb targets, and Metascape  online tool Mapper-Search and Color Pathway. For better presentation, the intermediate genes were hidden.As shown in There were a total of 1,673 prescriptions, including 854 prescriptions for hepatitis B, 530 prescriptions for cirrhosis, and 289 prescriptions for liver cancer. The classification of herbs is shown in Oldenlandia (BaiHuaSheSheCao), 227 times.In this study, the top 10 herbs in frequency used in three liver diseases were further extracted, with a total of 21 herbs, and their properties, flavors, and frequency of use are shown in Oldenlandia (BaiHuaSheSheCao), Curcumae Rhizome (EZhu), Paris Rhizome (ChongLou), Common Pleione Pseudobulb (ShanCiGu), and so on were frequently used in treating liver cancer.Oldenlandia (BaiHuaSheSheCao), Bearded Scutellaria (BanZhiLian), Curcumae Rhizome (EZhu), and Cardamom (DouKou) have the strongest association with liver cancer and are all at the core, also called the core prescriptions of the disease.Apriori algorithm was used in analyzing the association rules of the herbs in each of the three diseases. The data were firstly transformed by transactions, and the top 10 frequent items for each disease are listed in https://bioinformatics.psb.ugent.be/cgi-bin/liste/Venn) ; after the free targets were filtered, there were 155 targets for hepatitis B, 58 targets for cirrhosis, and 126 targets for liver cancer. The data of the PPI network were then obtained, the file with “tsv” format was downloaded, and the PPI network diagram shown in  was obtaP value cut-off <0.01). The diseases-related pathways in the KEGG pathway were eliminated in order to present the pathways of the core herbs on the three chronic liver diseases directly, and a total of 116 pathways were obtained with 64 pathways for hepatitis B, 68 pathways for cirrhosis, and 96 pathways for liver cancer  may promote the proliferation and migration of liver cancer cells through the PI3K/AKT signaling pathway [Oldenlandia (BaiHuaSheSheCao) and Bearded Scutellaria (BanZhiLian) were important herbs in the core prescription of liver cancer. They significantly induced apoptosis of cancer cells by regulating PI3K/Akt and effectively inhibited tumor growth in vitro and in vivo [As an important factor in liver disease progression, chronic inflammation and cell growth and apoptosis are involved in the whole process of hepatitis B, liver cirrhosis, and liver cancer. The pathways related to signal transduction and the immune system were mostly intervened in the core prescriptions of the three diseases, which intervened jointly in the PI3K-Akt signaling pathway, TNF signaling pathway, HIF-1 signaling pathway, Th17 cell differentiation, and IL-17 signaling pathways, and were involved in immune regulation, inflammation, cell cycle and other important roles –43. It cma cells . The hep pathway . When th pathway . Turmeri pathway . Astraga pathway . Oldenla in vivo .Apart from regulating immunity and inhibiting inflammation, the prescription for hepatitis B had a regulatory effect on the endocrine system as well, which might be an important mechanism for preventing the progression and degeneration of hepatitis B. Compared with hepatitis B prescription, liver cirrhosis prescription regulated more endocrine system pathways. Studies have found that growth hormone resistance and low insulin-like growth factor-1, thyroid dysfunction, and other problems are common in patients with liver cirrhosis, which will affect negatively its prognosis . It has β, and TNFα, as well as the anti-inflammatory cytokine IL-6 and its upstream factor NF-κB, were important targets that were regulated by the three prescriptions [κB could be activated in almost all chronic liver diseases [It was found from the subnetworks  compiledriptions . NF-κB cdiseases –63, whicdiseases . Comparediseases . Intervediseases , 67. As diseases .This research applied a combined method of data mining and integrated network pharmacology and explored the prescription rules and mechanism of action of TCM in the treatment of various chronic liver diseases. To the best of our knowledge, this study is the first of its kind in TCM to analyze various related diseases jointly, which has created a new paradigm in methodology for the analysis of related diseases.With the application of this method, the core prescriptions and the herbs involved for the three liver diseases were identified, which mainly intervened in the process of chronic hepatitis B, cirrhosis, and liver cancer through the intervention of signal transduction and immune system-related pathways and regulated the immune and cell growth processes, in which the PI3K-Akt was an important signaling pathway of it. Hepatitis B prescription was helpful for angiogenesis regulation, which might be the reason why it prevented the disease progression. The prescriptions for liver cirrhosis and liver cancer interfered more with the endocrine system pathways, and the liver cancer prescription had a broader regulating effect on the nervous system. The findings may cast some light on the treatment of chronic liver diseases as well as prevention of liver cancer using TCM. The prescriptions in this study came from two professors in outpatient clinics; even though there was a lack of evaluation on the efficacy of prescriptions and the results of the analysis may be biased, the two professors are prestigious Chinese medicine experts who boast leading positions in the field of liver disease in China. Their prescriptions have been an important source of learning for doctors in the hepatology department of traditional Chinese medicine, and thus, it is still of great significance to analyze their prescriptions. In addition, the source of the cases would be expanded in future studies, and further experimental studies are required for the evaluation and validation of the action mechanisms of the core prescriptions.Three core prescriptions for chronic liver disease treatment were identified in this study, and it was found that they mainly functioned through regulating signal transduction and immune system-related pathways. Compared with the other two liver diseases, the core prescription for liver cancer regulated more neural system-related pathways.


R. The gene–pathway network of FFXST against IS was created to obtain the key target genes. Molecular docking was used to validate the core targets using AutoDock Vina 1.1.2. A total of 455 candidate compounds of FFXST and 18,544 disease-related differential genes were screened. Among them, FFXST targets for IS treatment had 67 active compounds and 10 targets in the PPI network related to STAT1, STAT3, and HIF1A. The biological processes of GO analysis included the regulation of reactive oxygen species metabolic process, cellular response to chemical stress, regulation of angiogenesis, regulation of vasculature development, positive regulation of cytokine production, and response to oxidative stress. The KEGG enrichment analysis showed that Kaposi sarcoma-associated herpesvirus infection, microRNAs in the cancer signaling pathway, Th17 cell differentiation, and HIF-1 signaling pathway were significantly enriched. The network pharmacology outcomes were further verified by molecular docking. We demonstrated that FFXST protection against IS may relate to the regulation of oxidative stress, immune inflammatory response, and angiogenesis through the relevant signaling pathways. Our study systematically illustrated the application of network pharmacology and molecular docking in evaluating characteristics of multi-component, multi-target, and multi-pathway of FFXST for IS.Fufang Xueshuantong (FFXST) capsule is a traditional Chinese medicine (TCM) preparation used to activate blood circulation, resolve stasis, benefit qi, and nourish yin in clinical practice. However, its potential mechanism and molecular targets after ischemic stroke (IS) have not been investigated. The aim of this research was to investigate the molecular mechanisms of FFXST in the treatment of IS based on network pharmacology and molecular docking. We used the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP) to collect candidate compounds of four herbs in FFXST; disease-related differential genes were screened using the Gene Expression Omnibus (GEO) database, and a compound–disease network was created using Cytoscape 3.8.2 software. The topological analysis of the protein–protein interaction (PPI) network was then created to determine the candidate targets of FFXST against IS. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were conducted using the clusterProfiler package in  As estimated by the latest WTO report, stroke is the second leading cause of death in the world, and approximately 87% of stroke-related deaths belong to ischemic stroke (IS) . Current2+ overload, oxidative stress, energy metabolism disorders, and apoptosis  as an important way to treat ischemic stroke . Fufang culation  in the bculation . Huangqiculation . Danshenculation . XuansheOwing to its multi-component, multi-target, and multi-pathway synergistic characteristics, TCM compounds can treat various diseases through potential ingredient–target interactions. Network pharmacology is a bioinformatics network construction and network topology analysis strategy, which observes the complex network relationship of “drug–target–disease” from the overall level and then guides the basic and clinical research of TCM, which is systematic and consistent with the overall view of TCM . In the http://lsp.nwu.edu.cn/tcmsp.php), with oral bioavailability (OB) ≥ 30% and drug-like properties (DL) ≥ 0.18 as the filter conditions, the names of FFXST single-flavor Chinese medicines  were used as keywords to search (https://www.uniprot.org/) was used to convert the obtained target names into standard gene names to obtain the gene targets of FFXST.Through the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform . The statistical significance of differential expression was set at p-value filter < 0.05 and logFC filter > 0.5. The differentially expressed genes presented in the form of a volcano map were depicted using the R package, and the top 20 differential genes were selected to render the heat map.The differentially expressed genes in IS patients were derived from the GEO database , Biological General Repository for Interaction Datasets (BioGRID), Human Protein Reference Database (HPRD), IntAct Molecular Interaction Database (IntAct), Molecular INTeraction (MINT) database, and Biomolecular Interaction Network Database (BIND)  . VisualiThe targets with the greatest correlation (core targets) are obtained using Cytoscape CytoNCA plugin according to betweenness centrality (BC), closeness centrality (CC), degree centrality (DC), eigenvector centrality (EC), local average connectivity (LAC), and network centrality (NC) .R, the GO and KEGG enrichment analyses were carried out. Enrichment analysis of biological process (BP), cellular component (CC), and molecular function (MF) of potential targets was performed separately. First, the Bioconductor in the R package “org. Hs. eg.db” was installed and run, converting key target genes from each component for the treatment of ischemic stroke into Entrez IDs. Then, the clusterProfiler in the R package was installed, the species as a human source was set according to the converted Entrez ID, p < 0.05 was selected for the threshold of the key target gene GO and KEGG analyses, and the top 10 enrichment results were output in the form of histograms and bubble charts.Using the clusterProfiler package in https://pubchem.ncbi.nlm.nih.gov/) was used to download small molecule ligand files of chemical composition, and they were imported into Chem3D software for spatial structure conversion and energy optimization. The gene ID of the core target was retrieved from the UniProt database, and the corresponding PDB format file was downloaded from the PDB database (http://www1.rcsb.org/). After water molecule removal and ligand isolation using PyMOL software, the macromolecular receptor file of the obtained core target was imported into AutoDockTools 1.5.7 software for hydrotreating. The docking core target and its corresponding chemical composition were performed using AutoDock Vina 1.1.2. The binding energy was revealed as an evaluation index for molecular docking.The top three core targets in the PPI network were screened for molecular docking with their corresponding chemical compositions. The PubChem database , cellular component (CC), and molecular function (MF). The top 20 terms are shown in via AutoDock Vina 1.1.2 software  were selected from the component–target–pathway network. These core targets docking with kaempferol, cryptotanshinone, and quercetin bioactive components were verified experimentally software . The STAtware  in FFXST play important roles in the treatment of IS and are related to a variety of molecular targets and signaling pathways, indicating that these medicinal ingredients have potent research value. Quercetin, kaempferol, and crytotanshinone were identified as the active ingredients involved in most targets, and the molecular docking outcomes also validated that they exhibit strong binding efficacy with STAT1, STAT3, and HIF1A. Quercetin is a promising natural dietary compound with preventive and therapeutic effects on a variety of diseases , and itsm stress . Kaempfem stress . Kaempfem stress . Cryptotregation . Cryptoton cells .The topological analysis of the PPI network was performed for 33 intersection genes, revealing 33 strongly related proteins, among which 10 proteins  are the predicted targets. Consistent with the results of the gene–pathway network, STAT3, STAT1, and HIF1A are the main targets in the PPI network with median values, suggesting that they may be the core targets of FFXST in the treatment of IS. STATs are a transcription factor family mediating cell proliferation, apoptosis, and other cellular events . They arin vivo is mainly produced through pathways such as succinate dehydrogenase (SDH) and NADPH oxidase in the cellular mitochondrial succinate  are epithelial cells, endothelial cells, B cells, and macrophages. KSHV infection of cells leads to increased synthesis and secretion of cytokines, which promotes cell proliferation and differentiation, and its infection is accompanied by pathological changes in the nervous system . MicroRNgulation . Th17 cegulation . Among tgulation . Therefogulation . The stugulation , which wThe potential mechanism and targets of FFXST for IS were investigated using network pharmacology and molecular docking in this study. Quercetin, kaempferol, and crytotanshinone were identified as the active ingredients associated with most targets. FFXST may exert anti-IS function and the regulation of pathways including Kaposi sarcoma-associated herpesvirus infection, microRNAs in the cancer signaling pathway, Th17 cell differentiation, and HIF-1 signaling pathway. STAT1, STAT3, and HIF1A were the important targets of FFXST in the treatment of IS. This research initially explored the active components, core targets, and signaling pathways of FFXST in the treatment of IS, and the active components and core targets were further verified by means of molecular docking. This study systematically illustrated the characteristics of multi-component, multi-target, and multi-pathway of FFXST for IS. We demonstrated that FFXST protection against IS may relate to the regulation of oxidative stress, immune inflammatory response, and angiogenesis through the relevant signaling pathways. However, due to the complexity of TCM ingredients, the preliminary exploration of FFXST based on network pharmacology is still insufficient. Aiming at the molecular targets and signaling pathway of FFXST for IS in the prediction results, further experimental verification will be conducted in the later stage, in order to provide a theoretical basis and reference for the pharmacological study of the pharmacodynamic components of FFXST in the treatment of IS and provide a basis for strengthening the optimization of experimental design and further discussion in the later stage.


Liuwei Dihuang Pill is widely used to treat tinnitus in China. However, the underlying mechanism of Liuwei Dihuang Pill in treating tinnitus still remains unclear.To explore the potential pharmacological mechanism of Liuwei Dihuang Pill in the treatment of tinnitus based on network pharmacology and molecular docking.The active components of the Liuwei Dihuang Pill were obtained from the traditional Chinese medicine systems pharmacology database and analysis platform (TCMSP) database. Cytoscape software was used to draw the active component-target network diagram of Liuwei Dihuang Pill, and obtain the core components. Then the corresponding targets were also obtained from the TCMSP database. Targets related to tinnitus were obtained from the GeneCards, DisGeNET, TTD and DrugBank databases. The String database was used to construct protein–protein interaction (PPI) network of common targets of drugs and diseases, then the core targets were screened out. The Annotation, Visualization and Integrated Discovery (DAVID) database was used for gene ontology (GO) enrichment and Kyoto encyclopedia of genes and genomes (KEGG) pathway analysis of common targets. Finally, the molecular docking between the core component and the core target was carried out by AutoDock.The core components of Liuwei Dihuang Pill in the treatment of tinnitus including quercetin, stigmasterol, kaempferol, β-sitosterol, tetrahydroalstonine, which may act on core targets such as STAT3, transcription factor AP-1 (JUN), tumor necrosis factor (TNF), interleukin-6 and MAPK3. HIF-1 signaling pathway, Influenza A, P53 signaling pathway, and Toll-like receptor signaling pathway play a role in anti-inflammatory, improving microcirculation in the blood-labyrinth barrier, increasing cochlear blood flow, and preventing hair cell damage. The molecular docking results showed that the affinity between core components and core targets was good.The potential mechanism of Liuwei Dihuang Pill in the treatment of tinnitus was preliminarily discussed in this study, which may provide a theoretical basis and evidence for further experimental research. The prevalence of tinnitus ranged from 4.3% to 51.33%, but varied with age and sex. The highest increase in prevalence from the previous decade in age occurs during the fifth and sixth decades, and the highest prevalence occurred in the seventh decade at 34.27%. Until now, the pathological mechanisms of tinnitus have not been clearly elucidated, but the main pathogenesis of tinnitus is now associated with abnormal neuronal firing, mechanical dysfunction of the cochlea, abnormal micromechanical activity of the cochlea, mechanical feedback of the cochlea, and abnormal vibration of outer hair cells.,5Obviously, the deeper mechanism of tinnitus needs to be studied from various aspects and from a broad perspective, rather than being limited to lesions in the auditory conduction pathway. While there is currently no golden standard treatment for tinnitus, counseling, psychotherapy, pharmacological approaches, masking devices, individualized sound stimulation, and cognitive behavioral therapy are the most widely used strategies. Its mechanism of action may be related to the following factors, including enhances cochlear blood flow，protects against ototoxic stimuli and restrains apoptosis，impedes age-associated degenerative processes in the inner ear，affects neurotransmitter actions，and so on. While some of these treatment modalities are effective in reducing awareness of tinnitus or related distress, they can be time-consuming, expensive, and not covered by insurance, so are only available to a limited population.Tinnitus is the absence of a corresponding external sound source or electrical stimulation, but subjectively there is a sound in the ear or brain. Liuwei Dihuang Pill is often used to treat tinnitus. Besides, Liuwei Dihuang pill can also be used to treat a variety of other diseases, including type 2 diabetes, postmenopausal osteoporosis, triple negative breast cancer, and Primary nephrotic syndrome. Modern scientific research had confirmed the function of Liuwei Dihuang pill manifested in anti-inflammation, Inhibition of apoptosis, and so on. Numerous papers have shown that Liuwei Dihuang pill has a good clinical effect on tinnitus.,17 However, the effects and mechanism of Liuwei Dihuang Pill of tinnitus were not clear.Liuwei Dihuang Pill originated in Key to Therapeutics of Children’s Disease which was written by Qian Yi in Song Dynasty. Liuwei Dihuang pill includes 6 traditional Chinese medicines, including Radix Rehmanniae Preparata, Rhizoma Dioscoreae, Fructus Corni, Poria, Rhizoma Alismatis and Cortex Moutan, which has the function of nourishing kidney Yin. There includes complementary reinforcing and reducing methods in the whole prescription, which play a role in nourishing kidney yin and improving ear function. Molecular docking is based on the receptors and ligands with known structures, in accordance with the 3 complementary principles of geometry, energy, and chemical environment, to identify the interaction between molecules, and to predict the best binding mode between molecules. It has important value and potential advantages in the exploration of the potential target and action mechanism of active ingredients in traditional Chinese medicine, and the study of the pharmacological mechanism of prescription of Chinese herbal compound. This study aims to explore the potential mechanism of Liuwei Dihuang Pill in the treatment of tinnitus using traditional Chinese medicine network pharmacology and molecular docking methods, to provide a theoretical basis for further research. The graphical abstract was shown as follow , Uniprot database (http://www.uniprot.org), GeneCards database (https://www.genecards.org), DisGeNET database (https://www.disgenet.org), TTD database (http://bidd.nus.edu.sg/group/cjttd), DrugBank database (https://www.drugbank.ca), String database , the Annotation, Visualization and Integrated Discovery (DAVID) database (https://david.ncifcrf.gov), PDB database (https://://www.rcsb.org). Online drawing tools: Venny , Bioinformatics (http://www.bioinformatics.com.cn). Software: Cytoscape software (version 3.8), AutoDock software (version 4.2.6), AutoDock Tools software (version 1.5.6), PyMOL software (version 2.4.0).Databases: Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform , meeting the 2 conditions of oral bioavailability (OB) ≥ 30 % and drug-like (DL) ≥ 0.18. After the active ingredients of each drug were obtained, the corresponding targets were also searched in the TCMSP database, and the target name was standardized by the Uniprot database. Cytoscape software was used to draw the active components-targets network diagram of Liuwei Dihuang Pill, and the topological analysis was carried out through the network analyzer function of the software. The core components of Liuwei Dihuang Pill were obtained according to the degree value.Tinnitus-related targets were retrieved from the GeneCards, DisGeNET, TTD, and DrugBank databases using “tinnitus” (Mesh) as the key search term. After merging the targets obtained from the above database, as well as removing duplicates, the tinnitus-related targets were obtained.The interaction targets of Liuwei Dihuang pill and tinnitus were obtained through Venny, and then the Venn diagram was drawn. The PPI network between interaction targets was built using the String database. The mode was “Multiple Protenin,” and the organism was “Homo sapiens.” The minimum required interaction score was set to “Highest confidence (0.900),” and the disconnected nodes in the network were hidden. Other parameters remained unchanged. The obtained node1, node2, and combined score were imported into Cytoscape software for visual analysis. The network analyzer function of the software was used for topological analysis, and the core targets of Liuwei Dihuang Pill in the treatment of tinnitus was obtained according to the degree value.P value. For each GO enrichment and KEGG pathway, 20 items with the lowest P value were selected to draw the advanced bubble diagram by Bioinformatics.Gene Ontology (GO) enrichment and KEGG pathway analysis were performed on the interaction targets of Liuwei Dihuang Pill and tinnitus using DAVID database. The Select Identifier was set to “OFFICIAL GENE SYMBOL” and the species and background were set to “Homo sapiens.” Then the cell component (CC), molecular function (MF), biological process (BP), and KEGG pathway analysis were performed. The data was exported and sorted according to the P value in the KEGG pathways were imported into Cytoscape software, and the interaction targets between the compounds and disease targets included in the above KEGG pathways were also imported into the software, then the targets-pathways interaction network was constructed.The 20 items with the lowest AutoDock molecular docking was performed between the selected core components and the core targets. The mol2 formats of the core components were downloaded from the TCMSP database, and transformed into pdb formats by using PyMOL, then saved as pdbqt formats by using AutoDock Tools. The pdb formats of the core targets was downloaded from the PDB database (the protein complex with ligand and resolution of < 3A was selected), and the water removal, hydrogenation, and removal of the original ligand were performed by PyMOL. The atomic type was set as Assign AD4 type, and then imported into AutoDock Tools to save as pdbqt formats. The spatial position of the original ligand in the protein complex was defined as the active pocket, and the Lamarckian genetic algorithm was selected to run molecular docking by AutoDock. The binding free energy was used to screen the best docking results. Finally, the results were visualized by PyMOL.The components and targets of Liuwei Dihuang Pill were obtained from the TCMSP database, and OB ≥ 30% and DL ≥ 0.18 were used as the included criteria. After eliminating the non-target components and removing the duplication, 42 active components were obtained. Among them, there are no unique ingredients of Radix Rehmanniae Preparata, 11 unique ingredients of Rhizoma Dioscoreae, 12 unique ingredients of Fructus Corni, 6 unique ingredients of Poria, 6 unique ingredients of Rhizoma Alismatis, and 5 unique ingredients of Cortex Moutan. Besides, there was 1 common ingredient of Radix Rehmanniae Preparata, Rhizoma Dioscoreae, and Fructus Corni. There was 1 common ingredient of Radix Rehmanniae Preparata, Rhizoma Dioscoreae, Rhizoma Alismatis, Fructus Corni, and Cortex Moutan. The 42 active components were arranged according to the OB value from large to small, and the top 20 components were shown , 82 molecular functions (MF), 456 biological processes (BP), and 108 KEGG pathways were obtained. The data was exported and sorted according to the The top 20 enrichment results of GO-CC were: extracellular space, cytosol, extracellular region, membrane raft, nucleoplasm, caveola, neuron projection, nucleus, protein complex, mitochondrion, apical plasma membrane, external side of plasma membrane, mitochondrial outer membrane, lysosome, PML body, plasma membrane, cell surface, nuclear chromosome, cytoplasm, receptor complex Fig. A.The top 20 enrichment results of GO-MF were: enzyme binding, protein binding, identical protein binding, steroid binding, protein homodimerization activity, transcription factor binding, steroid hormone receptor activity, chromatin binding, sequence-specific DNA binding, heme binding, protein heterodimerization activity, protein kinase binding, transcriptional activator activity, RNA polymerase II core promoter proximal region sequence-specific binding, cytokine activity, core promoter sequence-specific DNA binding, serine-type endopeptidase activity, BH3 domain binding, protein kinase activity, ubiquitin protein ligase binding, protease binding , Toxoplasmosis, Colorectal cancer, Proteoglycans in cancer, Prostate cancer, HIF-1 signaling pathway, Malaria, Influenza A, p53 signaling pathway, Tuberculosis, Toll-like receptor signaling pathway, Leishmaniasis, Pertussis, African trypanosomiasis, Pancreatic cancer, Small cell lung cancer Fig. .The targets-pathways interaction network was constructed by Cytoscape software Fig. . In general, when the binding free energy is less than 0, it indicates that the ligand and receptor can bind spontaneously. The binding energy results obtained by molecular docking in this study were shown  and the core targets . The lower the binding free energy between the ligand and the receptor, the more stable the conformation is, which indicating the stronger the affinity of the 2.wn Table . Y. Wang studied the prescription of Chinese medicine for tinnitus and found that Rhizoma Dioscoreae, Poria, Rhizoma Alismatis and Cortex Moutan are high-frequency medicines for tinnitus, especially suitable for tinnitus with deficiency of kidney essence.In traditional Chinese medicine, there is a close relationship between the ear and the kidney, so nourishing the kidney and filling the essence is a common treatment for tinnitus, and Liuwei Dihuang pill can be selected by classics. Radix Rehmanniae Preparata has been used in traditional Oriental Medicine for the treatment of inner ear diseases, such as tinnitus and hearing loss. Fructus Corni also have been used for treatment of tinnitus. In the study of H. H. Yu, we investigated the protective effect of C. officinalis on hydrogen peroxide-induced cytotoxicity in auditory cells HEI-OC1. Y. Hirose et al experimentally investigated whether quercetin could prevent hair cell loss in the zebrafish lateral line and guinea pig cochlea, which found that quercetin reduced antioxidant activity and protected the cochlea from noise exposure in rodent. T. Yang found that quercetin could significantly prevent gentamicin-induced hair cell damage by reducing ROS and NO formation. Stigmasterol has anti-inflammatory, anti-oxidant, anti-cancer, and cholesterol-lowering effects, and can be extracted from bell grass and other active ingredients to treat deafness and tinnitus. J. Li screened active ingredients for the prevention and treatment of deafness based on cell and zebrafish injury models, and found that stigmasterol had a preventive effect on cisplatin-induced zebrafish hair cell damage. Kaempferol has antioxidant and antiapoptotic properties, protecting cells from cisplatin-induced apoptosis in a dose-dependent manner in HEI-OC1 cells. β-Sitosterol has antioxidant, cholesterol-lowering, anti-inflammatory, immunomodulatory, and anti-tumor effects. R. Paniagua-Pérez et al found a mean inhibition of inflammatory inhibition of 75% by β-sitosterol in the mouse ear edema test.In this study, the core components of Liuwei Dihuang Pill in the treatment of tinnitus were preliminarily screened by the methods of traditional Chinese medicine network pharmacology and molecular docking, which were quercetin, stigmasterol, kaempferol, β-sitosterol, tetrahydroalstonine and so on. Quercetin is a flavonoids compound with antioxidant, anti-inflammatory, antitussive, antiasthmatic, and antiallergic effects. Hearing loss usually gives rise to subjective tinnitus, a phantom ringing sensation that occurs in the absence of external sounds, resulting from a compensatory increase in gain or neural amplification in the central auditory system to compensate for the loss of sensory input from the cochlea. The inner ear is highly susceptible to ischemia and oxidative damage, Current therapeutic strategies have proven that the goal of the traditional oriental medicine principle of increase humoral principles is a relevant approach for reducing the development of hearing loss by improving microcirculation in the blood-labyrinth barrier and increasing cochlear blood flow. The potential benefits of traditional oriental medicine drugs extend to a multi-target approach to different auditory structures of the inner ear that are associated with increased cochlear blood flow, antioxidant, anti-inflammatory, anti-apoptotic and neuroprotective activities. Studies have shown that about 25 herbs and 40 active compounds in traditional oriental medicine are used in the treatment of hearing loss and tinnitus, which include quercetin. Both in vitro and in vivo experiments have shown beneficial effects on acquired sensorineural hearing loss caused by external noise, aging, ototoxic drugs, or diabetes. J. Zhai explored the effects of serum inflammatory factors and neurotrophic factors of the cochlear tissue of presbycusis in rats and found that presbycusis is closely related to TNF-α and other serum inflammatory factors in cochlear tissue, and its abnormal expression in the cochlea of aged rats is of great significance for the diagnosis, treatment and prognosis of presbycusis. Studies have shown that tumor necrosis factor alpha (TNF-α) expression is elevated by loud noise exposure in the auditory periphery and cochlear nucleus and is associated with tinnitus, and pharmacological blockade of TNF-α expression prevents noise-induced neuroinflammation and tinnitus. Conversely, infusion of TNF-α into primary auditory cortex resulted in behavioral signs of tinnitus in wild-type and TNF-α knockout mice with normal hearing.In this study, the core targets TP53, JUN, MAPK1, AKT1 and TNF were obtained according to the PPI network. Hearing loss and tinnitus induced by aspirin and sodium salicylate are considered to be completely reversible problems that disappear within a few days after discontinuation use, and the 3 genes with the greatest increase in apoptotic gene expression all belong to the TNF family or its corresponding receptor genes, the tumor suppressor gene P53, was also significantly up-regulated (2.00-fold). Hypoxia-inducible factor (HIF) is a key mediator of adaptation to hypoxia, enhancing transcription of various target genes, reducing oxygen-dependent pathways by regulating metabolism. Transcription factor HIF-1 (hypoxia-inducible factor-1) regulates the expression of genes involved in glucose supply, growth, metabolism, redox reactions and blood supply. Hypoxia and ischemia play an important role in the pathogenesis of tinnitus and hearing loss. B. Mazurek investigated the possible cellular and molecular biological causes of peripheral hearing loss and tinnitus, discussed the role of hypoxia and ischemia in the cochlea and in the etiology of the neurosensory types of tinnitus, and found that HIF-1 may act as a key transcription factor play important roles in cellular adaptation to hypoxia. TNF plays a regulatory role in reducing, maintaining and improving inflammatory responses, and Riva C et al also explored auditory function in cd/1 mice at 4, 12, and 24 weeks of age and correlated it with the presence of oxidative damage in the cochlea and the regulation of cochlear HIF-1 responsive target genes, associated with multiple inflammatory pathways such as TNF-α, or cell death with p53 protein, Bax protein and surviving factors with insulin-like growth factor-1. Furthermore, hypoxia modulates molecular processes in both acute and chronic tinnitus. While under hypoxic/ischemic conditions, HIF-1 induces changes in gene expression that may contribute to the remodeling of specific structures within the cochlea. p53 is a key component of signaling networks that protect cells from various stresses. Studies have shown that the Mdm2/p53 interaction has an important role in ensuring the survival of proliferative progenitor cells and differentiated cells of the auditory organ. H. Xiong show that activation of miR-34a/SIRT1/p53 signaling contributes to cochlear hair cell apoptosis, and has implications for age-related hearing loss. Changes in the upregulated modulator of apoptosis p53 directly or within the apoptosis-related cascade may play an important role during development, especially in the outer ear, where TP53 mRNA expression was significantly downregulated in patients with ear atresia compared to controls. Tlr4 regulates multiple aspects of the immune response in the cochlea and contributes to cochlear pathogenesis after acoustic damage. C. Yang showed that higher expression of the TLR2 gene was found in patients with profound hearing loss compared to those with milder hearing loss (P < .05). G. T. Van Well also found that the TLR system appears to play an important role in the immune response to the BM and subsequent neuronal damage and cochlear inflammation. In addition, the foreign literature reports that the hepatitis B vaccine and Toxoplasma gondii can cause acute tinnitus， sudden deafness, and other diseases.,53 Epidemiological studies have found many common respiratory diseases such as influenza A in the middle ear effusion of children with acute otitis media.The HIF-1 signaling pathway, Influenza A, p53 signaling pathway, Toll-like receptor signaling pathway, Hepatitis B, TNF signaling pathway, Toxoplasmosis, etc. The results obtained by enrichment analysis are all closely related to tinnitus, mainly related to anti-inflammatory, improving the microcirculation of the blood labyrinth barrier, increasing cochlear blood flow, reducing hair cell damage, and so on. Mechanisms associated with the development of tinnitus and acquired hearing impairment include hair cell loss, signal transduction disturbances in the region of the outer and inner hair cells and the spiral ganglion, impaired cochlear blood flow, mechanical impairment, and hypoxia and ischemia.Based on network pharmacological analysis, this study demonstrated that Liuwei Dihuang Pill treated tinnitus through multi-compounds, multi-targets, and multi-pathways, and preliminarily clarified the related potential mechanism of Liuwei Dihuang Pill in the treatment of tinnitus. Through KEGG pathway enrichment analysis, it was found that Liuwei Dihuang Pill played an important role in the treatment of tinnitus, including HIF-1 signaling pathway, Influenza A, P53 signaling pathway, and Toll-like receptor signaling pathway, which might play a role in anti-inflammatory, improving microcirculation in the blood-labyrinth barrier, increasing cochlear blood flow, and preventing hair cell damage. Although subsequent validations are needed to determine the exact mechanism of Liuwei Dihuang Pill, our present study provide promising directions for future research.The authors would like to thank Jiangxi Hospital of Integrated Traditional Chinese and Western Medicine, Affiliated Hospital of Jiangxi University of Traditional Chinese Medicine, Jiangxi University of Traditional Chinese Medicine, and Pinghu Hospital of Traditional Chinese Medicine. This paper is supported by State Administration of Traditional Chinese Medicine , and Jiangxi Provincial Health and Family Planning Commission . The authors are grateful to Mr. Yulong Ji for his help with the preparation of this paper. We would like to thank the editors and reviewers for their helpful remarks that improved this paper.Conceptualization: Zhongbiao Wu, Zhongyan Zhu.Data curation: Zhongbiao Wu, Zhongyan Zhu, Xinmei Huang.Formal analysis: Zhongbiao Wu, Zhongyan Zhu, Weikun Wu.Funding acquisition: Zhongbiao Wu, Chengcheng Deng, Qiang Xie.Investigation: Jian Cao, Chengkun You.Methodology: Jian Cao, Weikun Wu, Shiping Hu, Chengkun You.Project administration: Zhongbiao Wu, Zhongyan Zhu.Resources: Jian Cao, Weikun Wu, Xinmei Huang.Software: Zhongyan Zhu, Shiping Hu.Supervision: Zhongbiao Wu, Zhongyan Zhu.Validation: Shiping Hu, Chengcheng Deng, Qiang Xie.Visualization: Chengcheng Deng, Qiang Xie.Writing – original draft: Chengcheng Deng, Qiang Xie, Xinmei Huang, Chengkun You.Writing – review & editing: Xinmei Huang, Chengkun You.


ALB, IL6, IL1B, VEGFA, STAT3, MMP9, TLR4, CCL2, CXCL8, and IL10. GO functional enrichment analysis yielded 92 entries for biological process (BP), 47 entries for cellular component (CC), 31 entries for molecular function (MF), and 20 signaling pathways, which were obtained from KEGG pathway enrichment screening. Based on these findings, the FoxO signaling pathway is critical in the treatment of CHD by safflower. The in vitro results showed that safflower had an ameliorating effect on ox-LDL-induced apoptosis and mitochondrial membrane potential. The western blot results showed that safflower decreased Bax expression and acetylation of FoxO1 proteins while increasing the expression of Bcl-2 and SIRT1 proteins. Safflower can be used in multiple pathways during CHD treatment and can exert anti-apoptotic effects by regulating the expression of Bax, Bcl-2, and SIRT1/FoxO1 signaling pathway-related proteins.Safflower has long been used to treat coronary heart disease (CHD). However, the underlying mechanism remains unclear. The goal of this study was to predict the therapeutic effect of safflower against CHD using a network pharmacology and to explore the underlying pharmacological mechanisms. Firstly, we obtained relative compounds of safflower based on the TCMSP database. The TCMSP and PubChem databases were used to predict targets of these active compounds. Then, we built CHD-related targets by the DisGeNET database. The protein-protein interaction (PPI) network graph of overlapping genes was obtained after supplying the common targets of safflower and CHD into the STRING database. The PPI network was then used to determine the top ten most significant hub genes. Furthermore, the DAVID database was utilized for the enrichment analysis on Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG). To validate these results, a cell model of CHD was established in EAhy926 cells using oxidized low-density lipoprotein (ox-LDL). Safflower was determined to have 189 active compounds. The TCMSP and PubChem databases were used to predict 573 targets of these active compounds. The DisGeNET database was used to identify 1576 genes involved in the progression of CHD. The top ten hub genes were  According to the American Heart Association's Heart Disease and Stroke Statistics (2020 edition), cardiovascular disease is a growing public health concern worldwide . AccordiCarthamus tinctorius L.), which belongs to the subclass Dicotyledoneae and family Compositae, are commonly used in clinics to treat CHD, hypertension, and cerebrovascular diseases, as well as to enhance blood flow and remove blood blockage [κB pathway, which prevents lipopolysaccharide (LPS)-induced TNF signaling activation and cell death in H9c2 cardiomyoblast cells [Traditional Chinese medicine (TCM) is multitargeted and multidirectional and is therefore used in the treatment of CHD. Dehydrated flowers of the safflower plant  and module networks, which were then analyzed for Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment. We aimed at investigating the potential therapeutic targets of safflower in the treatment of CHD, which would serve as a foundation for further research into the pharmacological mechanism of safflower. Subsequently, we used in vitro experiments to validate the predicted molecular mechanism of safflower in coronary heart disease therapy as determined by network pharmacology. The results of these experiments provide a foundation for the clinical application of safflower in CHD patients. The research process is illustrated in http://lsp.nwu.edu.cn/tcmsp.php). TCMSP is an open database that contains the oral bioavailability (OB) of nearly 500 Chinese herbal medicines and their drug similarity (DL), blood-brain barrier (BBB), intestinal epithelial pass permeability (Caco-2), and other reference values. The compounds contained in safflower were screened using the TCMSP database, with oral bioavailability set to OB ≥ 30% and drug likeness set to DL ≥ 0.18.The effective compounds of safflower were obtained by screening the Traditional Chinese Medicine Systems Pharmacology Platform (TCMSP) database (https://pubchem.ncbi.nlm.nih.gov/). The top 100 target genes corresponding to each compound were searched from the chemical-gene co-occurrences in the literature module of the PubChem database. Subsequently, genes corresponding to the targets of the safflower compounds were determined using the STRING database (https://www.string-db.org/).Typically, medicinal components interact with relevant biological pathways via their targets. To estimate the targets of compounds found in safflower, details on the molecular structure of the bioactive constituents in safflower were obtained from the TCMSP and PubChem databases (https://www.disgenet.org/). DisGeNET is an exploratory platform containing genes associated with human diseases and their comorbidities. According to Score gda> 0.1, targets related to the pathogenesis of CHD were selected.Enter the keyword “coronary artery disease” in the DisGeNet database to search for the corresponding target. The intersecting genes were discovered by comparing safflower target genes to CHD-related pathogenesis targets.The Cytoscape software (version 3.6.0) was used to construct the disease-compound-target-pathway network, followed by network topology analysis. Cytoscape is a visualization software that can integrate biomolecular interaction networks, data on high-throughput gene expression, and other molecular details.To explain the relationship between projected targets and other human proteins, a PPI network was created, which has been shown to be a viable technique for drug development. The protein-protein interaction network of potential therapeutic targets of safflower for CHD was built using the STRING database. In the designated setting, protein associations with a confidence score greater than 0.4 were selected after duplicates were removed.The PPI network was imported into Cytoscape using the CytoHubba plugin, and the MCC algorithm was used. The top ten hub genes targeted by safflower to treat CHD were identified.https://david.ncifcrf.gov/) database for GO bioprocess analysis and KEGG pathway analysis. GO enrichment analysis comprises the following three aspects: biological process (BP), molecular function (MF), and cellular component (CC). The GO enrichment analysis results were selected based on P-values, and the top 10 pathways were selected. KEGG can be used to evaluate the target signaling pathways of medicines and identify those with the highest correlation, which is critical for determining the potential mechanism of safflower in CHD therapies. Finally, the threshold value of P < 0.05 was determined. Major pathways were identified, and bioinformatics software was used to map the signaling pathways. The top 20 pathways were selected based on the outcomes of KEGG pathway enrichment analysis.The screened targets were analyzed using the DAVID . Cells were commonly cultivated in Dulbecco's modified Eagle's medium (DMEM), 10% fetal bovine serum (FBS), and 1% penicillin/streptomycin. The cells were randomly separated into three groups during the logarithmic growth phase: the control group, oxidized low-density lipoprotein (ox-LDL) group , and safflower group. Each group had at least three replicates, and cell counting was performed to ensure that the number of cells in each group was the same. Except for the control group, the cells were cocultured with 100 µg/ml ox-LDL for 24 h and then switched to a normal medium or a safflower drug-containing medium for another 24 h, and then collected for further analysis.n = 5) and blank serum (n = 5). The typical human dose of safflower (10 g) was converted to an appropriate drug dosage for the SD male rats according to the body surface area. SD rats were given a safflower solution (1 g/kg) intragastrically via gavage for 7 days. The normal control group were given an equal volume of normal saline (NS). Blood samples were taken from the aorta one hour after the last gavage. The supernatant was extracted, filtered, and inactivated in a water bath at 56°C for 30 min, and the sera from the same group were mixed and stored at −20°C.Ten male Sprague-Dawley (SD) rats  were provided by Changsha Tianqin Biotechnology Co. Ltd. . All SD rats were housed under conventional conditions  and provided with equal access to standard rodent feed and water. All experiments were conducted in accordance with China's National Guidelines for Experimental Animal Care and Use. The study was approved by the Ethical Committee of Hainan Medical University(No. HYLL-2021-140). SD rats were categorized into two groups for the preparation of safflower drug-containing serum (μl/100 μl medium) was added to the media for 2 h at 37°C in a 96-well plate. A microplate reader  was used to measure the absorbance of the media at 450 nm.A Cell Counting Kit-8 was used to check the viability of cells . When cells reached 80% confluence, they were treated with ox-LDL at 0, 25, 50, 100, or 200 µg/ml for 24 h. Subsequently, CCK8 solution . In each group, apoptotic cells were counted in three random fields using a fluorescent microscope.A JC-1 fluorescent probe was used to measure mitochondrial membrane potential according to the manufacturer's instructions . EAhy926 cells were incubated with 1 ml JC-1 at 37°C for 20 min, washed twice with PBS, and then observed and photographed using a fluorescent microscope.β-actin (Abcam). After three washes, the luminescence developed, and the gel imager was exposed for imaging. The strips were analyzed for grayscale using ImageJ software  and processed for semi-quantitative analysis, with three replicates each.Cells were collected and lysed on ice in 6-well plates. BCA protein assay kits were used to measure the concentrations . SDS-PAGE was used to separate the protein samples, which were then transferred to polyvinylidene fluoride (PVDF) membranes. After blocking for 2 h at room temperature with 5% nonfat milk in Tris-buffered solution containing 0.1% Tween 20 (TBST), membranes were incubated overnight at 4°C with primary antibodies (1 : 1000) against Bax (Abcam), Bcl-2(Abcam), Sirt1 (Thermo Fisher Scientific), acetylated FoxO1 (Thermo Fisher Scientific), and P < 0.05.All experimental data are presented as mean ± SD. Prism software was used for data processing and analysis, and one-way analysis of variance was used for comparison between groups. Significance was set to We obtained 189 safflower compounds from the TCMSP database. Using the screening conditions of OB ≥ 30% and DL ≥ 0.18, 34 active compounds that met the criteria were obtained, including 6-hydroxykaempferol, baicalein, qt_carthamone, quercetagetin, beta-carotene, baicalin, beta-sitosterol, stigmasterol, kaempferol, and quercetin. Precise information on the main bioactive components is shown in The TCMSP and PubChem databases were used to retrieve 2527 targets from the 34 candidate components. After expelling the overlapping targets, a total of 573 targets of safflower compounds were acquired. The DisGeNET data comprise 1576 genes associated with CHD.Using the web tool Venny 2.1, the intersection of the targets corresponding to coronary heart disease and safflower active compounds was determined using a Venn diagram. According to the findings, a total of 239 putative targets related to both CHD and safflower were identified .The STRING database was used to create a PPI network to further elucidate the biological mechanisms underlying the pharmacological effects of safflower on CHD. The nodes and edges in the network indicate proteins and protein-protein interactions, respectively. According to the results, the network had 239 nodes and 5819 interactions . The aveThe hub gene was screened in the interaction network using CytoHubba, a Cytoscape plugin. The top 10 hub genes for saffron treatment of CHD were identified using the MCC algorithm , and a hThe BP, CC, and MF annotations of the 239 selected proteins were determined using the DAVID database. GO analyses revealed 92 enriched GO terms in the category of the biological process (BP), including cholesterol homeostasis, lipoprotein metabolic process, high-density lipoprotein particle remodeling, cholesterol efflux, and reverse cholesterol transport. GO analyses indicated 47 enriched GO terms in the category of cellular component (CC), including extracellular space, extracellular region, cell surface, and receptor complex. GO analyses reflected 31 enriched GO terms in the molecular function (MF) category, including heme binding, cholesterol transporter activity, transcription factor binding, RNA polymerase II transcription factor activity, and ligand-activated sequence-specific DNA binding .P-values, along with the HIF-1 signaling pathway, PI3K-Akt signaling pathway, Toll-like receptor signaling pathway, FoxO signaling pathway, and TNF signaling pathway. Based on these findings, the FoxO signaling pathway was defined as one of the most valuable signaling pathways during the treatment of CHD with safflower. http://www.kegg.jp/kegg/mapper.html).The 239 putative targets were registered into the DAVID database for KEGG pathway enrichment analysis to explain the crucial signaling pathways targeted by safflower in CHD therapeutic approaches. To more specifically define the method of action of safflower in the treatment of CHD, a network of disease-compound-target-pathways was constructed. The network, shown in μg/ml) were applied to EAhy926 cells for 24 h. The survival rates of each group of cells are shown in μg/ml for 24 h, cell viability decreased, as shown in μg/ml, ox-LDL reduced cell viability by approximately 50%. According to the experimental results, ox-LDL 100 μg/ml applied for 24 h could inhibit EAhy926 cell viability more effectively, which was used as a modeling condition for subsequent experiments.Different concentrations of ox-LDL  in EAhy926 cells to determine how safflower affected ox-LDL-induced cardiac damage. Compared to control cells, the protein levels of SIRT1 were considerably reduced in ox-LDL-treated cells, and the protein levels of Ac-FoxO1 were significantly increased in ox-LDL-treated cells, as shown in As demonstrated in β-sitosterol, kaempferol, quercetin, baicalein, flavoxanthin, lignan, precarthamin, carthamone, quercetagetin, and 3-methoxydaidzein that have the potential to treat CHD. β-Sitosterol protects the heart by modulating antioxidant enzymes via the estrogen/phosphatidylinositol 3-kinase pathway [κB and AP-1 signaling pathways [κB signaling [CHD is a severe hazard to human health because it is a complex cardiovascular disease. It is characterized by ischemia, hypoxia, or necrosis of the myocardium caused by narrowing or blockage of the heart vessels due to coronary artery sclerosis or spasm. CHD is caused by a variety of factors. Single target drugs are typically ineffective for the treatment of this multifactorial disease. Furthermore, repeated or continuous high-dose administration of a single specific drug can result in drug resistance. As a result, the development of new multitarget drugs is critical. TCM has multicomponent and multitarget qualities, meaning it can target multiple biological systems to manage symptoms and treat basic problems. Safflower has been used to treat CHD for over a thousand years –7, but i pathway . In vitrpathways . Kaempfepathways . The pospathways . Based oignaling . Baicaliignaling . Accordiignaling .VEGFA, STAT3, IL-6, IL-1B, MMP9, CCL2, CXCL8, TLR4, IL-10, and ALB were chosen as candidate core genes. VEGFA is a direct target gene of miR-451 according to TargetScan and dual-luciferase reporter gene assay data and could be a new biomarker for CHD [Because of the high degree of node and closeness centrality in this network,  for CHD . In a PD for CHD . In endo for CHD . In the  for CHD . Based o for CHD –26. The  for CHD . Atheros for CHD . MMP9 is for CHD . When AS for CHD . CCL2 wa for CHD . Interle for CHD . TLR4 is for CHD ,34. IL-1 for CHD . One stu for CHD . The rel for CHD .α, miR-27a, and miR-23a expression levels were shown to be substantially linked to the severity of stenosis in the PBMCs of patients with CAD. Compared to healthy controls, the expression of SIRT1 was shown to be significantly lower in CAD patients [These targets are mostly connected with signal transduction pathways such as TNF, HIF-1, FoxO, Toll-like receptor, and PI3K-Akt signaling, according to our KEGG pathway analysis. As a pro-atherogenic cytokine, TNF increases the expression of cytokines and adhesion molecules, as well as the migration and mitogenesis of VSMCs and endothelial cells . HIF-1 ipatients . Macrophpatients . FOXO ispatients . SIRT1 ipatients . The SIRpatients . FoxO1 ipatients . FoxO1 mpatients . SIRT1 dpatients . SIRT1/Fpatients . In thisNumerous studies have found that apoptosis is important for the pathogenesis of CHD . After oThe mitochondrial apoptotic pathway in endothelial cells is regulated by several signaling pathways, among which the SIRT1/FoxO1 pathway can sense energy changes and regulate the expression of mitochondrial pathway apoptotic genes such as Bcl-2 and Bax. SIRT1 is deacetylated and is inhibited under physiological conditions; however, when cells are stimulated by pathological factors such as ischemia and hypoxia and LPS, SIRT1 is activated, resulting in increased expression and deacetylation activity. Activated SIRT1 can deacetylate FoxO1, and the deacetylated FoxO1 enters the cytoplasm from the nucleus ,55; FoxOβ-sitosterol, kaempferol, quercetin, baicalein, and 3'-methoxydaidzein. Possible targets include VEGFA, STAT3, IL-6, IL-1B, MMP9, CCL2, CXCL8, TLR4, IL-10, ALB, and putative pharmacological activities of other bioactive components linked to antioxidative stress, anti-apoptosis, and anti-inflammation. In addition, coronary artery disease can lead to the activation of the SIRT1/FoxO1 signaling pathway and activation of the apoptotic signaling pathway, and safflower can reverse this result. According to the findings, network pharmacology predictions could be useful in illustrating the molecular mechanisms of the Chinese herbal drug safflower in the treatment of CHD.Using network pharmacology, the targets, mechanism of action, and associated signaling pathways of safflower in the therapy of CHD were assessed in this study. It was demonstrated that the effective compounds of safflower in the treatment of CHD may be dipalmitin, flavoxanthin, lignan, precarthamin, carthamone, quercetagetin,


After pathway analyses, we found indoximod had effects on nonalcoholic fatty liver disease through regulated TNFa, AP-1, AKT, PI3K, etc. Furthermore, we established the NAFLD cell model with LO2 cells induced using PA; ELISA showed that the levels of TG, ALT, and AST were significantly improved by indoximod. In summary, our study offers optimal therapeutic drugs, which may provide novel insight into the precise treatment of NAFLD and promote researches.Nonalcoholic fatty liver disease (NAFLD) has become one of the problems affecting the health of the population worldwide. The progressive disease includes nonalcoholic steatohepatitis (NASH) and fibrosis, which with no approved therapy, system identification of effective drugs remains challenging. In this work, we applicated drug perturbation gene set enrichment analysis to screen drugs for the development of NAFLD. A total 15490 small-molecule compounds were analyzed in our study; based on the  Nonalcoholic fatty liver disease (NAFLD) is a common multifactorial liver disease that becomes a common public health problem seriously affecting people's lives and health . NAFLD wThe pathogenesis of NASH was very complicated. After NASH was defined as a new disease in 1980, Powell et al. found that obesity and diabetes were the main risk factors, and NASH might be a metabolic-related disease . Animal NAFLD was contributed by multiple factors in long-term cooperation . MonotheIn order to screen the potential therapeutic drugs for liver diseases, in the present test, we firstly get the disease high-throughput molecular profiles from the Gene Expression Omnibus (GEO) database, then analyzed the biological functions and pathways of differentially expressed genes, and used drug perturbation gene set enrichment analysis to screen the potential therapeutic drug. Our research will provide strategies for the treatment of NAFLD.https://www.ncbi.nlm.nih.gov/geo/) database (GSE130970), including 5 normal, 23 nonalcoholic steatohepatitis, and 25 hepatic fibrosis. T-test  was set as the cutoff criteria to evaluate differentially expressed genes (DEGs). The heat maps were plotted based on the pheatmap package of R.We get the expression profiling of liver biopsies from the course of nonalcoholic fatty liver disease from the GEO (Gene Expression Omnibus https://pubchem.ncbi.nlm.nih.gov/) is a public repository for archiving biological tests of small molecules generated through high-throughput screening experiments, medicinal chemistry studies, chemical biology research, and drug discovery programs. The drug expression profile data is from the Library of Integrated Network-Based Cellular Signatures (LINCS) L1000 dataset that measured changes in genes before and after treatment of human cells with over 20000 small-molecule compounds including most of the FDA-approved drugs.PubChem  annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway of the DEGs were analyzed and visualized by clusterProfiler package of R. dpGSEA is an approach for drug screening, which used the drug perturbation gene set as a background and disease-related characteristic genes for enrichment analysis. In this way, drugs related to characteristic polar shadows can be detected or potential treatments can be discovered .2. Palmitic acid (PA) was added at a 0.3 mmol/liter concentration for 24 h to establish a cellular model of NAFLD. Cells were divided into control, PA, and PA+indoximod groups. Indoximod (200 μM) was added to cell cultures for 12 h, followed by coincubation with PA for another 24 h. Levels of TG, alanine aminotransferase (ALT), and aspartate aminotransferase (AST) were measured by commercial kits according to the manufacturers' instructions. ELISA kits for TG (SBJ-H0240), ALT (SBJ-H0658), and AST (SBJ-H0027) were purchased from Nanjing Sen Bei Jia Biotechnology .Based on the previous methods of established cellular model of NAFLD , normal Firstly, we identified DEGs in different stages of nonalcoholic fatty liver. As shown in Figures Next, we performed GO and KEGG analysis of DEGs. The results had shown that DEGs in NASH are mainly involved in metabolism-related processes, such as purine-containing compound metabolic process, glycerophospholipid metabolic process, and glucose metabolic process which is related to liver function. KEGG analysis results had shown that DEGs mainly participate in regulation of the chemokine signaling pathway and FoxO signaling pathway . Meanwhip value of enrichment score (p < 0.05), we have individually identified candidate drugs for each pathological state. Finally, we found 7 drugs (or compound) have potential therapeutic effects on NASH and fibrosis  inhibitors suppress the progression of nonalcoholic fatty liver disease. In addition, the other six drugs were all reported to be related to hepatic disease. Acifran has the effect of lowering blood lipids; sirolimus, bisindolylmaleimide, and abexinostat have been reported to have the effect of treating liver cancer; and progesterone and indoximod have the effect of reducing liver damage. This result also suggested that these six drugs may have potential therapeutic effects on NAFLD. Through the pathway analysis, we found that indoximod could influence nonalcoholic fatty liver. Finally, we verified the effectiveness of indoximod through cell experiments. There are certain limitations to our study. We will investigate the mechanism of indoximod in treatment of NAFLD.Taken together, our experiment data provided a potential therapeutic agent for the development of NAFLD; meanwhile, it also provided ideas for screening new drugs for complex diseases.