Ecliptae herba (EH) has been widely used in HCC patients in China, but its hepatoprotective mechanism is still unclear.  The number of hepatocellular carcinoma (HCC) cases worldwide has increased significantly. As a traditional Chinese medicine (TCM) with a long history,  In this study, we applied a network pharmacology-based strategy and experimental verification to systematically unravel the underlying mechanisms of EH against HCC. First, six active ingredients of EH were screened from the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP) by the ADME method. Subsequently, 52 potential targets of 6 active ingredients acting on HCC were screened from various databases, including TCMSP, DGIdb, SwissTargetPrediction, CTD, and GeneCards. Then, by constructing protein-protein interaction (PPI) network from STRING, we displayed the intricate connections among these 52 targets through Cytoscape software. We also applied enrichment analysis, including Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses, to provide an outline and set of concepts for describing gene functions and the advanced functions of biological systems of these 52 targets from genomic and molecular level information. Finally, molecular docking and biological experiments were used to reconfirm these results.  We hypothesized that EH might exert anti-HCC activity by acting on hub genes, including RELA, MMP9, PTGS2, ESR1, EGFR, AR, AKT1, HIF1A, AHR, CYP3A4, ABCG2, and MMP2. Moreover, based on GO and KEGG analysis, we speculated that EH may exert hepatoprotective effects on HCC through the following mechanisms: regulation of the PI3K-AKT signaling pathway to promote apoptosis and inhibit the abnormal proliferation of HCC, downregulation of HIF-1A expression by activating the HIF-1 signaling pathway, prevention of HCC by regulating lipid metabolism, and inhibition of nonalcoholic fatty liver disease (NAFLD) by the cytochrome P450 subfamily. Subsequent biological experiments verified that EH inhibits the PI3K-AKT signaling pathway through its active ingredients, quercetin, and wedelolactone, thereby inhibiting the proliferation of HCC cells and promoting the apoptosis of HCC cells.  The network pharmacological strategy provides an efficient method to systematically explore the pharmacological mechanism of EH in HCC. Our study demonstrated that the anti-HCC proliferation activity of EH is mainly exerted by two active ingredients (quercetin and wedelolactone), which inhibit the proliferation of HCC cells (HepG2 and Huh-7) by inhibiting PI3K-AKT signaling. Cancer statistics show that liver cancer is the fourth most common cause of cancer-related death globally, with the World Health Organization estimating that more than 1 million people will die from liver cancer by 2030 . There aEcliptae herba (EH), known as “Mo-Han-Lian” in China, has been used as a “liver-nourishing” treatment in traditional Chinese medicine (TCM) for several thousand years [in vitro and in vivo experiments have shown that EH has hepatoprotective and antihepatotoxic effects in recent years, the detailed therapeutic targets and molecular mechanisms of EH against HCC have not been systematically elucidated.Traditional Chinese medicine, which originated in China, has been practiced by Chinese people for thousands of years because of its satisfactory therapeutic effect and few side effects –10. It hnd years . EH has nd years –17. Ethand years , 19. Itsnd years . AlthougNetwork pharmacology is an innovative method to investigate the interrelationship between drugs and diseases by integrating multiple sources of information which is an efficient method for understanding the effects of EH in HCC treatment. The strategy is based on the concept of “Medicine-Target-Gene-Disease,” which can illuminate intricate drug-disease interactions, and is considered to be very suitable for studying the molecular mechanisms of drugs and drug discovery –22.In this study, we performed an integrated strategy, including network pharmacology-based analysis, molecular docking, and biological experimental verification, to identify the potential therapeutic targets and the associated molecular mechanisms of EH against HCC.https://tcmspw.com/tcmsp.php). In this database, the Chinese characters of “Mo-Han-Lian” were input to identify related ingredients and their pharmacokinetic property data.We collected the ingredients of EH from the Traditional Chinese Medicine System Pharmacology Database , and the SwissTargetPrediction web server (http://www.swisstargetprediction.ch/).We collected relevant targets of EH active ingredients by screening different databases. These databases include the TCMSP, the Drug Gene Interaction Database  and the GeneCards web server (https://www.genecards.org/). We searched for the keyword “hepatocellular carcinoma” on these web servers, and the species was restricted to “Homo sapiens.” Then, we used an open web tool (http://bioinformatics.psb.ugent.be/webtools/Venn/) to filter the common targets of different databases to improve the reliability of the data obtained. We used this web tool to match the predicted target of EH active ingredients with the relevant target of HCC to obtain the overlapping targets. These overlapping targets are the targets that EH may play a role in the treatment of HCC.Related targets of HCC were collected through the Comparative Toxicogenomics Database  web server. On this database, the species option was “Homo sapiens,” protein interactions had a confidence score >0.4, and the disconnected nodes were hidden in this network. The resulting data were imported into the visualization software Cytoscape to establish protein-protein interaction networks of EH against HCC. Based on this E-H network, the hub gene network was obtained through the CytoHubba plugin in Cytoscape software. In addition, we constructed the C-T-P network from the results of the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis. For each network, we selected five parameters to evaluate its topological characteristics, which were calculated by the Cytoscape plugin, Network Analyzer. These topological parameters were “Degree,” “Betweenness Centrality,” “Closeness Centrality,” “Clustering Coefficient,” and “Topological Coefficient.”In this process, a compound-target-disease network (C-T-D network), EH target-HCC target network (E-H network), compound-target-pathway network (C-T-P network), and hub gene network were constructed to reveal the pharmacological mechanism and therapeutic target of EH in the treatment of HCC. The graphical interactions in the C-T-D network were visualized by Cytoscape software (version 3.7.2). The E-H network was obtained by uploading the overlapping targets to the STRING . The organism of interest was “Homo sapiens,” the method of interest was “Over-Representation Analysis,” and the functional database was “Gene Ontology.” Then, the overlapping gene list was uploaded to the database to obtain GO enrichment analysis results, which included three parts, biological process (BP), cellular component (CC), and molecular function (MF). Then, we constructed a network of enriched GO terms through WebGestalt analysis of the overlapping genes and TCGA RNASeq liver hepatocellular carcinoma (LIHC) database, with the option of method of interest selected as “Network Topology-based Analysis,” and the option of functional database selected as “TCGA RNASeq LIHC.”In this study, we performed Gene Ontology (GO) analysis of the overlapping targets through the WEB-based Gene SeT AnaLysis Toolkit  data obtained from WebGestalt, with the option of method of interest being “Over-Representation Analysis,” and the functional database being “pathway.”In addition, we calculated and evaluated significant pathways using KEGG (https://www.rcsb.org/). AutoDock Vina and PyMOL 1.8 software were utilized to perform docking studies for EH and selected genes. First, the 2D chemical structures of the active ingredients of EH were downloaded from PubChem (https://pubchem.ncbi.nlm.nih.gov/) with the format for SDF and converted to a .pdb format by PyMOL 1.8. For protein preparation, the protein was input into the PyMOL 1.8 to remove the water molecules and heteroatoms and saved in a .pdb format. Second, the .pdb format of the protein and active ingredients was converted to the .pdbqt format via AutoDockTools (version 1.5.6) and used to perform a series of operations, including adding hydrogen atoms and calculating and adding Gasteiger charges. Third, we defined the specific pocket for active ingredients of EH binding with the hub genes using the grid box function of AutoDockTools. We used the command prompt for molecule docking analysis and the output results were displayed by PyMOL.In this study, we performed molecular docking to further reveal the binding pattern of EH and hub genes. The X-ray crystal structures of hub genes were obtained from the Protein Data Bank  medium containing 1% penicillin-streptomycin  and 10% fetal bovine serum  under 5% CO2 at 37°C.Hepatocellular carcinoma cell lines (HepG2 and Huh-7) were obtained from South Medical University Affiliated Maternal & Child Health Hospital of Foshan . HepG2 cells were cultured with DMEM  containing 1% penicillin-streptomycin  and 10% fetal bovine serum  under 5% COμ membrane, and then stored at −20°C.Wedelolactone was purchased from MedChemExpress , and quercetin was purchased from Shanghai Fushen Biotechnology Co., Ltd. . Raw EH was purchased from Foshan Zhongtian Chinese Medicine Pieces Co., Ltd. . The raw EH was authenticated by Deng Dongmei from the Department of TCM of the South Medical University Affiliated Maternal and Child Health Hospital of Foshan. The extraction step of EH was carried out using a previously reported protocol . The finHepG2 or Huh-7 cells  were transplanted into each well of a 96-well plate and treated with different concentrations of EH, wedelolactone, and quercetin for 48 hours. Then, the proliferation of HCC cells was measured by CCK-8 assays . The cell viability of the sample was calculated by the OD value.μg/L EH, 8 μg/mL wedelolactone, and 8 μg/mL quercetin for 48 hours, according to the Annexin FITC/propidium iodide (PI) cell apoptosis kit  instructions for the experimental procedures. FACSCanto II  was used for the analysis, and the results were analyzed by FlowJo V10 software.HepG2 or Huh-7 cells were treated with 150 μg/mL EH, 8 μg/mL wedelolactone, and 8 μg/mL quercetin for 48 hours, western blotting was performed using previously reported protocol standards [After treatment of HepG2 or Huh-7, cells were treated with 150 tandards . The antP value < 0.05.Each experiment was repeated three times independently. Data are presented as the mean ± standard error of the mean (SEM) and analyzed using GraphPad Prism 7 . One-way analysis of variance (ANOVA) was used, and the difference was considered significant when P), hydrogen-bond donors (Hdon), and hydrogen-bond acceptors (Hacc). According to the ADME thresholds of DL > 0.18, OB > 30%, Caco-2 > 0, BBB permeability < −0.3, MW < 500 Da, Log P < 3, Hdon < 5, and Hacc < 10, 6 active ingredients were selected out [A total of 48 chemical ingredients of EH were obtained from TCMSP , and maximal clique centrality (MCC). As shown in P value < 0.05 were considered significant pathways. The top 10 biological processes were mainly involved in proliferation, cell death, cell communication, and cell apoptosis. The biological processes included the apoptotic process (GO:0006915), regulation of the apoptotic process (GO:0042981), regulation of cell death (GO:0010941), regulation of programmed cell death (GO:0043067), positive regulation of cell communication (GO:0010647), and cell proliferation (GO:0008283). These results suggest that EH may induce anti-HCC activity by regulating cell proliferation, death, and apoptosis. In molecular function analysis, EH exerted anti-HCC activity mainly through the following processes: transition metal ion binding (GO:0046914), identical protein-binding (GO:0042802), protein dimerization activity (GO:0046983), oxidoreductase activity (GO:0016491), heme binding (GO:0020037), and transcription factor binding (GO:0004879). In addition, the cellular components of the 52 targets of EH in the treatment of HCC were mainly the nuclear chromosome (GO:0000228), transcription factor complex (GO:0005667), nuclear chromosome part (GO:0044454), chromosome (GO:0005694), and chromosomal part (GO:0044427).After uploading 52 therapeutic targets of EH in the treatment of HCC in WebGestalt, we obtained the results of the top 10 GO enrichment analyses, which were divided into biological processes, molecular functions, and cellular components Figures . For thiTo further reveal the mechanism of action of EH in the treatment of HCC, we built a GO network in the TCGA RNASeq LIHC database through the Network Topology-based Analysis (NTA) method on the WebGestalt web server  and 8(e), treatment of HepG2 and Huh-7 cells with 150 μg/mL wedelolactone and 8 μg/mL quercetin significantly inhibited the expression of p-PI3K p85 (Tyr467), p-PI3K p55 (Tyr199), and p-Akt (Ser473) in HepG2  and cell apoptosis . We concluded that the regulation of cell proliferation and apoptosis plays a vital role in the development of HCC. The effect of EH on HCC through cell proliferation and apoptotic processes involves 29 targets and 31 targets in these biological processes, respectively. These targets include AKT1, EGFR, PI3K, GSK3B, and MET. Additionally, KEGG results showed that 19 of the top 20 KEGG pathways were involved in the PI3K-AKT signaling pathway, including “hepatocellular carcinoma (hsa05225)” . This fiThe liver plays an important role in lipid metabolism, and excessive lipid accumulation in hepatocytes can lead to nonalcoholic fatty liver disease, which is a chronic liver disease that has emerged as a major cause of liver cancer in Western countries in recent years , 39. CytIn addition, we simulated the interaction pattern of these 6 active ingredients of EH with the hub genes by molecular docking technology, and the results showed that these molecules effectively bind to the binding pocket of these genes by forming several hydrogen bonds. However, this phenomenon does not allow them to selectively interact with specific proteins. Therefore, it is necessary to improve the selectivity of these compounds through subsequent structural modification.Although network pharmacology has revealed the potential mechanism by which EH inhibits HCC, validation experiments are still needed to verify the above results. CCK-8 and cell apoptosis experiments confirmed that EH and its main active ingredients, quercetin, and wedelolactone, can significantly inhibit the proliferation of HCC and promote apoptosis, which is consistent with the GO analysis. To further clarify the molecular mechanism by which EH inhibits HCC, we confirmed through western blot assay that the active components of EH, quercetin, and wedelolactone can inhibit the PI3K-AKT signaling pathway in the HCC cell line. These results suggest that the pharmacological effects of EH on the inhibition of the abnormal proliferation of HCC and the promotion of HCC cell apoptosis are achieved by regulating the PI3K-AKT signaling pathway.Overall, this study explored the underlying mechanism of EH against HCC based on the strategies of network pharmacology and experimental verification. These mechanisms of network pharmacology prediction mainly involve the following aspects: regulation of the PI3K-AKT signaling pathway promotes apoptosis and inhibits abnormal proliferation of HCC cells, downregulation of HIF-1A expression is mediated by activating the HIF-1 signaling pathway, and prevention of HCC is induced by regulating lipid metabolism and inhibiting NAFLD by the cytochrome P450 subfamily. Moreover, we verified through experiments that EH inhibits the PI3K-AKT signaling pathway through its active ingredients, quercetin, and wedelolactone, thereby inhibiting the proliferation of HCC cells and promoting apoptosis of HCC cells. Although the network pharmacology method can be a good solution to this issue, further experimental verification is necessary.


These results indicate that the proposed model with good accuracy in screening the CGFC and inferring potential mechanisms in the formula of TCM, which provides reference for the optimization and mechanism analysis of the formula in TCM.Traditional Chinese medicine (TCM) usually plays therapeutic roles on complex diseases in the form of formulas. However, the multicomponent and multitarget characteristics of formulas bring great challenges to the mechanism analysis and secondary development of TCM in treating complex diseases. Modern bioinformatics provides a new opportunity for the optimization of TCM formulas. In this report, a new bioinformatics analysis of a computational network pharmacology model was designed, which takes Chai-Hu-Shu-Gan-San (CHSGS) treatment of depression as the case. In this model, effective intervention space was constructed to depict the core network of the intervention effect transferred from component targets to pathogenic genes based on a novel node importance calculation method. The intervention-response proteins were selected from the effective intervention space, and the core group of functional components (CGFC) was selected based on these intervention-response proteins. Results show that the enriched pathways and GO terms of intervention-response proteins in effective intervention space could cover 95.3 and 95.7% of the common pathways and GO terms that respond to the major functional therapeutic effects. Additionally, 71 components from 1,012 components were predicted as CGFC, the targets of CGFC enriched in 174 pathways which cover the 86.19% enriched pathways of pathogenic genes. Based on the CGFC, two major mechanism chains were inferred and validated. Finally, the core components in CGFC were evaluated by  Depression belongs to the mental health disorder, which is an emotional disorder that causes persistent sadness and loss of interest, and is the leading cause of worldwide disability . PreviouBupleurum scorzonerifolium Willd.  (6 g), Citrus reticulata Blanco  (6 g), Ligusticum striatum DC.  (4.5 g), Cyperus rotundus L.  (4.5 g), Citrus × aurantium L.  (4.5 g), Paeonia lactiflora Pall.  (4.5 g), and Glycyrrhiza uralensis Fisch. ex DC.  (1.5 g). CHSGS has been widely applied in treating depression and has achieved remarkable results (Chai-Hu-Shu-Gan-San (CHSGS) is comprised of seven botanical drugs and were extracted with water solution:  results . Previou results . It has  results . In addi results . These eTCM usually plays therapeutic roles in the form of formulas in treating complex diseases. The formula has a multicomponent and multitarget mode of action during the therapy process, and these components and targets constitute the all-to-all effect network of TCM formulas in treating diseases. In the treatment procedure, some components in the effect network have auxiliary function, while others have major therapeutic actions, which were defined as the core group of functional components (CGFC). It refers to the components with suitable pharmacological features and closely associated with the effectual response to diseases. Detecting the CGFC that takes fundamental function in treating complex diseases is a big challenge due to the incomplete comprehending of the complex mechanism of multicomponents and multitargets in TCM. Optimizing formulas and obtaining CGFC are the key steps to reduce components with side effects or without activity and analyze the treatment mechanism of Chinese botanical drug formulas. Several network pharmacology–based formula optimization models have been proposed. However, these models mainly focus on the analysis of the component–target network and ignore the construction of the effect propagation space which links the drug targets to the pathogenic genes . StudiesCurrently, a new system pharmacology strategy was developed to capture the CGFC and clarify the molecular mechanisms of CHSGS in treating depression. The potential pathogenic genes of depression were extracted by analyzing the literature reports and published databases. All components of CHSGS were obtained from the database and literature and were further screened to obtain the potential active components. Three prediction tools were utilized to predict the targets of these active components. And then, the potential pathogenic genes and active component–target networks were utilized to establish effective intervention space to identify the intervention-response proteins. The intervention-response proteins selected from the effective intervention space were utilized to screen the CGFC by using the cumulative contribution coefficient (CCC) module. The CGFC was utilized to speculate the mechanisms of CHSGS in the therapy of depression.Genes related to depression reported in DisGeNET , GeneCarhttp://lsp.nwsuaf.edu.cn/tcmsp.php), TCM@Taiwan (http://tcm.cmu.edu.tw/zh-tw), TCMID (http://www.megabionet.org/tcmid/), SymMap (https://www.symmap.org/), and ETCM (http://www.nrc.ac.cn:9090/ETCM/index.php/Home/Index/index.html). The concentration of chemical components of the botanical drugs in CHSGS was extracted from the published reports , TCMID   , HitPickThe component–target (C-T) networks of CHSGS were established by utilizing Cytoscape software (Version 3.7.0) . NetworkConstructing effect intervention space from complex networks acted by TCM can retain highly correlated small molecular targets and pathogenic genes to the greatest extent. We map the component–target network to the PPI network which integrated from Biogrid, String, DIP, HPRD, INTACT, and MINT, and then, map the pathogenic genes with the literature support to the PPI network to establish the component–target–pathogenic gene–disease network. The importance of nodes in the network is the main basis for analyzing the key components of the network.N means nodes which represent components, targets, pathogenic genes, and disease. E means edges which represent component–target–pathogenic gene–disease interactions:i in the network; The j and k. j to node k and through node i. i; n means the total number of nodes in the network. EIS represent the effective intervention space. The nodes in the effective intervention space were identified as intervention-response proteins. And then, we performed functional pathway analysis using intervention-response proteins and depression pathogenic genes to evaluate whether the intervention-response proteins could cover the pathogenic genes of depression at the functional level.There are many reported methods for calculating node importance, such as degree, closeness centrality, betweenness centrality, clustering coefficient, neighborhood connectivity, and average shortest path length. Here, we design a new method to characterize the importance of nodes; we defined i in the effective intervention space asn components, so that the cumulative contribution rate of targeted to pathogenic genes is the largest. The specific calculation formula is as follows:The CGFC is hidden in the components of the effective intervention space. We define the network coverage of each component Therapeutic Mechanisms of CHSGS for Depressiony. From the optimal substructure properties, the recursive formula for calculating Set the subproblems of the given question as:p-values of GO and KEGG analyses were set at 0.05 as the cut-off criterion. The ggplot2 package was used to create graphs in the R statistical programming language (version 3.4.2). The above results were annotated by Pathview (https://www.bioconductor.org/).The clusterProfiler  package Pathview  in the RWe defined i, respectively. Vanillic acid (≧98% purity by HPLC) was obtained from Jingzhu Biotechnology Co., Ltd. , and corticosterone (purity ≧98%) was purchased from TCI Shanghai . RPMI-1640 medium, fetal bovine serum (FBS), 0.25% trypsin, and 3--2,5-diphenyltetrazolium bromide (MTT) were purchased from Shanghai Sangon Biotechnology Co. Ltd. . Poly-L-polylysine (PLL) was purchased from Sigma-Aldrich Co. . The commercial kit for measuring the lactate dehydrogenase (LDH) was purchased from Nanjing Jiancheng Bioengineering Co., Ltd. .The differentiated PC12 cells were purchased from the Cell Bank of the Chinese Academy of Sciences . Cells were cultured in an incubator at 37°C with an RPMI-1640 medium containing 10% FBS. When the cells reached 80% confluency, the cells were treated with vanillic acid for 3 h; then, the cells were treated with corticosterone (400 μM).4 per/well) were seeded in 96-well plates, which were coated with PLL (0.01%). After 24 h incubation, PC12 cells were treated with 0.1, 1, 10, 25, 50, and 100 μM vanillic acid and corticosterone. MTT was superinduced to a 96-well plate for 4 h; then, the culture supernatant was removed. Finally, DMSO was utilized to dissolve the purple crystals. The plate reader was utilized to detect the absorbance at 570 nm.PC12 cells , Citrus reticulata Blanco (CP), Ligusticum striatum DC. (CX), Cyperus rotundus L. (XF), Citrus × aurantium L. (ZQ), Paeonia lactiflora Pall. (SY), and Glycyrrhiza uralensis Fisch. ex DC. (GC) have 55, 7, 22, 21, 4, 36, and 75 specific active components, respectively. These results show that different components in CHSGS act as a synergistic mode; in this process, the specific components play a leading role.Except the shared components, most of the botanical drugs play therapeutic roles through their specific ingredients. We used three tools to predict the target genes of the active components and got 1,286 predicted target genes. To probe the therapeutic mechanism of CHSGS in treating depression, 249 active components and 1,286 targets  were utiMoreover, we previously described that the common components shared by two or more botanical drugs and specific components of certain botanical drugs, kaempferol (CHSGS20), vanillin (CHSGS26), naringenin (CHSGS137), limetin (CHSGS158), L-Menthone (CHSGS35), and patchouli alcohol (CHSGS29) also have higher targets number. The degrees of these components are 78, 95, 36, 61, 16, and 16, respectively. These results suggested that the pivotal roles of these components in treating depression and further confirmed that CHSGS has the effect of multi-targets treatment of depression.In the component–target network, the mean degree of targets for different components is 7.63. Interestingly, majority of these targets are confirmed related to the pathogenesis of depression and that may indicate potential therapeutic mechanisms of CHSGS on depression. These results indicated that CHSGS act synergistically to treat depression in a multi-component manner.i in the T-P network, if the important score of a node is more than the median important score of all nodes in the network, such node is believed to play a pivotal role in the network structure and can be treated as a hub node (p < 0.05) and 1905 (p < 0.05). The intervention-response proteins enriched pathways and GO terms were found to cover 95.3 and 95.7% of the 150 common pathways and 1991 common GO terms  network. This complex network contains 2,548 nodes and 41,378 edges. In this huge network, the correlation and transmission of target genes to pathogenic genes is the fundamental part. Thus, we take targets–pathogenic genes (T-P) subnetwork in the CTPD network for further analysis. Importance of nodes in a network is the key topological property that can be used to evaluate the roles of nodes in the network. For each node hub node . FollowiGO terms . These rThere are three categories of intervention-response proteins in effective intervention space. The first category is the direct interactions between the component targets and pathogenic genes. We defined this category as the essential common targets. The second category is the interactions of disease-specific targets. The third category is the interactions of component-specific targets. In order to test whether the effective intervention space can be replaced by essential common targets, disease-specific targets or component-specific targets for further optimization. We performed pathway analysis on essential common targets, disease-specific targets, component-specific targets, respectively. Results show that the coverage proportion of enriched pathways of three categories compared with the enrichment pathways of pathogenic genes is 90.7, 82, and 84.7%, respectively, . The covThe CCC module was established to optimize effective components and get the CGFC, which would be used to clarify the molecular mechanism of CHSGS in the therapy of depression. According to the contribution accumulation results, the top eight components including vanillic acid (CHSGS4), DTR (CHSGS163), apocynin (CHSGS109), isovanillic acid (CHSGS79), phenylacetic acid , Karenzu DK2 (CHSGS177), benzoic acid , and quercetin (CHSGS2) contribute to 53.23% target coverage of effective proteins. For further analysis, 61 components can contribute to 90.08% target coverage of effective proteins, while the target coverage of effective proteins quickly increased to 95.12% after the 71 components were taken in the CCC model. Thus, we selected the 71 components as the CGFC . Higher p < 0.05), and the number of pathogenic gene–enriched pathways was 181 (p < 0.05). The CGFC target–enriched pathways were found to cover 86.19% of the pathogenic gene–enriched pathways (p < 0.05), compared to the pathogenic genes, the coverage of enriched pathway on CGFC targets and full targets was 83.7 and 81.5%, respectively. This result indicates that the CGFC selection model could effectively select key targets and remove noise. These major targets of CGFC were frequently involved in the PI3K-Akt signaling pathway (hsa04151), MAPK signaling pathway (hsa04010), cAMP signaling pathway (hsa04024), Rap1 signaling pathway (hsa04015), calcium signaling pathway (hsa04020), oxytocin signaling pathway (hsa04921), phospholipase D signaling pathway (hsa04072), sphingolipid signaling pathway (hsa04071), relaxin signaling pathway (hsa04926), thyroid hormone signaling pathway (hsa04919), ErbB signaling pathway (hsa04012), and VEGF signaling pathway (hsa04370), etc. , etc. . Among tIn order to further clarify the potential mechanism of CGFC-mediated CHSGS in treating depression, we compared and analyzed the pathways enriched by CGFC targets and pathogenic genes and found that 17 of the top 30 pathways were overlapped , and amoIn this comprehensive pathway, we use the maximum targeting weight (MTW) model to calculate the cascade pathways, which indicate drug enter cells through extracellular receptors to cause a cascade effect of downstream genes. 13 Cascade pathways with scores greater than 0.7 were retained for further analysis .These cascade pathways are integrated, two main cascade targeting modules can be obtained . The firTo validate the accuracy and reliability of our model, the important CGFC predicted were experimentally validated in PC12 cells. The effect of vanillic acid was evaluated in PC12 cells. The results showed that 0.1, 1, 10, 25, and 50 μM vanillic acid had almost no effects to PC12 cells . MTT resPrevious pharmacological studies have shown that vanillic acid treatment could block oxidative damage in PC12 cells and exert the effect of neuroprotective . It has TCM plays therapeutic roles in treating complex diseases with the characteristics of multi components and multi targets. These components and targets form a complex intervention network. How to find the most effective intervention relationship in this intervention network and find CGFC is the key to understand the material basis and molecular mechanism of TCM and is also the basis for the secondary development of TCM. At present, the main purpose of formulas optimizing based on network pharmacology is to improve the curative effect of the formula and reduce the nonpharmacological factors. According to the principle of compatibility of TCM, each formula consists of several botanical drugs, each of which contains hundreds of chemical components. Whether botanical drugs or ingredients in the formula are necessary, especially when treating a specific disease, still needs analysis and verification. Through compound optimization, these botanical drugs and components with a better intervention effect can be screened out, while those botanical drugs and components with the antagonistic effect and even side effects are removed, making the compound simpler and more effective .In order to better optimize the classical formulas with clinical efficacy, network pharmacology methods and bioinformatics algorithms are used to screen the key corresponding relationships from component targets to pathogenic genes. In this process, we established a new node importance calculation method. Based on this method, we constructed the effective intervention space and traced the CGFC from the effective intervention space, then inferred the possible mechanism using the maximum target weight model based on the CGFC. It provides methodological reference for the secondary development of TCM and the development of new drugs.At present, how to optimize and obtain the CGFC and analyze their mechanism of action is the basis for quantification of TCM. TCM emphasizes a holistic and systematic view and regards the integrated treatment of different botanical drugs and ingredients as a coordinated whole. Network pharmacology has the characteristics of systematisms and integrity and conforms to the core theory of TCM. Network pharmacology emphasizes multitarget regulation of signal pathways to improve drug efficacy and reduce toxic and side effects. Network pharmacology is widely used to speculate the potential mechanism in treating complex diseases in TCM . For exaIn the process of analyzing the therapeutic mechanism, network pharmacology formed a fixed analysis rule. In this rule, the first step is to collect the components of botanical drugs, do ADME/T screening for selecting the active components, then predict targets and infer the molecular mechanism. The flow chart really solves the molecular mechanism of some formulas for treating complex diseases in TCM, such as decoding the synergistic mechanism of zhi-zhu wan for functional dyspepsia , analyziWe used a dynamic programming algorithm to infer the CGFC from the genes in the effective intervention space and made functional analysis and verification. It was found that the target genes of the CGFC were enriched to 174 pathways, with 156 pathways coinciding with the enriched pathways of pathogenic genes, accounting for 86.19%, while the enriched pathways of the whole CHSGS target genes only have 150 pathways coinciding with the pathogenic genes, accounting for 82.87% of the pathogenic gene enrichment pathways. This result indicates that after optimization, invalid or weak effect relationships were removed, and the functional coverage rate was improved. It also shows the reliability of our effective intervention space and the strategy of selection of CGFC.via phosphorylation, a number of cellular substrates, including transcription factors, ion channels, transporters, exchangers, intracellular Ca2+ -handling proteins, and the contractile machinery . The receptor mediates various biological actions of adenylate cyclase-activated polypeptide 1 (ADCYAP1). ADCYAP1 is the main regulator of central and peripheral stress responses needed to restore and maintain internal balance . It can achinery . Dopamin neurons .A network pharmacology model–based bioinformatics algorithm was established to extract the core components group and decode the mechanisms of CHSGS in the treatment of depression. Compared with other published work, the effective intervention space construction strategy based on the novel node importance calculation method, CGFC prediction and validation strategy, and maximum targeting weight model for mechanism speculation were reported. Our research is a computational mining work based on pharmacological basic data, which provides a feasible scheme to reduce the verification scale for the experiment, provides methodological reference for the optimization of the core components group and interpretation of the molecular mechanism in the treatment of complex diseases using TCM.However, there are some limitations in this study. Firstly, more components from the core group of functional components should be selected for validating the reliability of our approach. Secondly, the precise mechanisms were speculated by maximum targeting the weight model warrant further validation. Finally, the undirected network was utilized in our algorithm, which ignores the activation or inhibition effects of the targets.In the era of big data and artificial intelligence, network pharmacology is helpful to study the TCM formula systematically. There are some suggestions for future research in network pharmacology. The dose-effect relationship of TCM components should be considered. Metabolites of TCM after entering the body may also be the material basis for exerting therapeutic effects, and the metabolic process of TCM in the body also needs to be considered in the network pharmacology study. In summary, there is still a long way to go in the quantification and digitization of TCM.


Wumei pill (WMP) has a long history of colitis treatment in China, but the protective mechanisms have not been elucidated. To uncover the potential mechanisms of WMP against ulcerative colitis (UC), the network pharmacology approach was utilized in this study.  Public databases were utilized to identify the potential targets of WMP and genes related to UC. Based on the identified overlapping common targets, drug-ingredient-target gene network, Gene Ontology (GO) analysis, Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis, and protein-protein interaction (PPI) analysis were conducted. Molecular docking was carried out to verify the selected key active ingredients and core targets.  129 active ingredients and 622 target genes were obtained. The drug-ingredient-target gene network revealed 52 active ingredients of WMP acting on 73 targets related to UC. GO analysis revealed that biological processes were mainly associated with oxidative stress, such as, reactive oxygen species metabolic processes, response to oxidative stress, cellular response to oxidative stress, response to reactive oxygen species, and regulation of reactive oxygen species metabolic processes. KEGG analysis revealed that the immune- and inflammation-related pathways, tumor-related signaling pathways, and microbial infection-related signaling pathways were the most significant. PPI network identified 13 core target genes. The molecular docking results indicated the formation of stable bonds between the active ingredients and core target genes.  The approach of network pharmacology reveals the key ingredients, potential core targets, and biological process of WMP in the treatment of UC. The mechanisms of action of WMP involve anti-inflammation, antioxidation, and modulation of immunity, which provides evidence for the therapeutic role of WMP in UC. Ulcerative colitis (UC) is an inflammatory bowel disease characterized by relapsing and remitting mucosal inflammation . GloballChinese herbal medicine (CHM) has been widely used in colitis treatment in China, with potential benefits including high efficacy, safety, and relatively low economic costs . Since CNetwork pharmacology is a priori analytical approach that combines system network analysis with pharmacology and can efficiently elucidate the relationship between drugs, compounds, diseases, and targets , 9. Therhttps://tcmspw.com/tcmsp.php) [A systematic search was conducted in Traditional Chinese Medicine Systems Pharmacology (TCMSP) database (msp.php)  to obtaimsp.php) .https://www.uniprot.org/) [Target genes corresponding to WMP that obtained from TCMSP were imported into UniProt (ot.org/)  to obtaihttps://www.genecards.org/), PharmGKB database (https://www.pharmgkb.org/), OMIM database (https://omim.org/), TTD database (http://bidd.nus.edu.sg/group/cjttd/), and DrugBank database (https://www.drugbank.ca/). Targets from GeneCards with the relevance score ≥ 5 were screened out [Targets related to UC were retrieved from the follow public databases: GeneCards database  [PPI analysis was carried out in STRING database (db.org/)  and visudb.org/)  by calcuhttps://pubchem.ncbi.nlm.nih.gov/) [http://www.rcsb.org/) [Molecular docking was performed to validate the compound-target associations. Structures of compounds were downloaded from the PubChem (ih.gov/) , and thesb.org/) . Moleculsb.org/) . The valsb.org/) , and thesb.org/) .From the TCMSP database, a total of 129 active ingredients were identified, including 8 ingredients of Wumei, 8 of Xixin, 7 of Guizhi, 14 of Huanglian, 37 of Huangbai, 2 of Dangui, 22 of Rensen, 5 of Huajiao, 5 of Ganjiang, and 21 of Fuzi. Moreover, 1927 target genes were obtained and subsequently uploaded to UniProt to obtain standard gene symbols. After eliminating the redundancy, 1710 target genes were identified finally. Details of the 129 active ingredients and the 1710 target can be found in supplementary In total, 5273 target genes were identified, including 549 from the GeneCards, 3 from OMIM, 15 from PharmGKB, 15 from TTD, and 189 from DrugBank. After eliminating the redundancy, 622 target genes were obtained finally. Details of the 622 target genes can be found in supplementary A Venn diagram  identifiThe results of GO and KEGG pathway analyses were visualized in A PPI network with 73 nodes and 2150 edges was obtained in STRING. Through Cytoscape plugin cytoHubba, the results of the first screening found 32 nodes and 440 edges. For the second screening, a dense region network with 13 nodes and 78 edges was obtained. The 13 core target genes included CCL2, HIF1A, JUN, NFKBIA, MMP9, CXCL8, IL1B, TP53, AKT1, IFNG, PPARG, PTGS2, and ICAM1. Details are presented in With molecular docking, the results showed that all values of the Vina score less than “−5,” suggesting that the key active ingredients have good binding ability with the core target genes , Table 1UC is an urgent global public health concern. Although conventional drugs such as 5-aminosalicylates, hormones, and immunosuppressive agents have played a good role in the treatment of UC, they still failed to achieve ideal effects and were often accompanied by side effects . HoweverFor the targets of WMP on UC, GO analysis showed that the enriched biological processes were mainly focused on oxidative stress. Oxidative stress stems from the altered balance between reactive oxygen species production and the ability to rapidly detoxify reactive intermediates , and excα and lipocalin-2 mRNA and enhance the expression of Slip protein, which in turn inhibits inflammation in UC organoids [κB inhibitor family, which plays a key role in inflammation, oxidative stress, and immunity [γ agonists may be a new target for UC treatment [PPI network was constructed to explore the key active ingredients and core targets. The results revealed that quercetin, kaempferol, ginsenoside rh2, frutinone A, and dianthramine were the key active ingredients of WMP in the treatment of UC. Previous studies have shown that these key active ingredients have antioxidant and anti-inflammatory effects and can modulate the immune system at the molecular level . It is rrganoids . Kanagolrganoids . Similarrganoids . Moreoverganoids . HIF1A arganoids . For JUNrganoids . NFKBIA immunity . Expressimmunity . IL-1B iimmunity . TP53 isimmunity . Similarimmunity . IFNG enimmunity . PPARG ireatment . PTGS isreatment . Similarreatment .Furthermore, the molecular docking validation results showed that the key active ingredients had good binding ability to the core target genes, indicating that the potential mechanism of WMP for UC treatment revealed by the network pharmacology approach is reasonable.Limitations must be acknowledged. First, the use of network pharmacology to reveal the mechanism of WMP in the treatment of UC is an in silico prediction method; thus, further in vivo experimental validation is required to support the reliability of the prediction results. Furthermore, the application of network pharmacology in CHM research is only in its infancy; further integration of multiple disciplines such as bioinformatics, computer science, and pharmacology needs to be promoted to improve the scientificity of research methods.The approach of network pharmacology reveals the key ingredients, potential core targets, and biological process of WMP in the treatment of UC. The mechanisms of action of WMP involve anti-inflammation, antioxidation, and modulation of immunity, which provides evidence for the therapeutic role of WMP in UC.


Staphylococcus aureus infection. The PPI network analysis showed that 9 common DEGs were found in GSE100927 and GSE16561, and a cluster with 6 nodes and 12 edges was also identified by PPI network analysis. In conclusion, this study suggested that FCGR3A and MAPK pathways were connected with ACI.Atherosclerotic cerebral infarction (ACI) seriously threatens the health of the senile patients, and the strategies are urgent for the diagnosis and treatment of ACI. This study investigated the mRNA profiling of the patients with ischemic stroke and atherosclerosis via excavating the datasets in the GEO database and attempted to reveal the biomarkers and molecular mechanism of ACI. In this study, GES16561 and GES100927 were obtained from Gene Expression Omnibus (GEO) database, and the related differentially expressed genes (DEGs) were analyzed with R language. Furthermore, the DEGs were analyzed with Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis. Besides, the protein-protein interaction (PPI) network of DEGs was analyzed by STRING database and Cytoscape. The results showed that 133 downregulated DEGs and 234 upregulated DEGs were found in GES16561, 25 downregulated DEGs and 104 upregulated DEGs were found in GSE100927, and 6 common genes were found in GES16561 and GES100927. GO enrichment analysis showed that the functional models of the common genes were involved in neutrophil activation, neutrophil degranulation, neutrophil activation, and immune response. KEGG enrichment analysis showed that the DEGs in both GSE100927 and GSE16561 were connected with the pathways including Cell adhesion molecules (CAMs), Cytokine-cytokine receptor interaction, Phagosome, Antigen processing and presentation, and  Cerebral infarction is the second fatal disease in modern society, which is a global issue threatening the health of humans. Statistically, more than 70% cerebral infarction is induced by ischemia, and ACI serves as a major type in it , 2. AtheHigh-throughput screen is characterized with high efficiency and accuracy in gene sequencing, which has been increasingly applied in clinical research . MicroarTherefore, this study attempted to identify the differentially expressed genes (DEGs) of the patients and normal persons and investigate the pathological mechanism of ischemic cerebral infarction induced by atherosclerosis by delving the microarray datasets in Gene Expression Omnibus (GEO) database.https://www.ncbi.nlm.nih.gov/geo/). The samples of 39 patients with stroke and 24 normal individuals in GES16561 tested on GPL6883 were selected for subsequent analysis, and the samples of 29 patients with carotid atherosclerosis and 24 normal persons in GSE100927 were selected for subsequent analysis.The microarray datasets including GES16561 and GES100927 were obtained from National Center Biotechnology Information Gene Expression Omnibus  > 2 were selected as DEGs.https://david.ncifcrf.gov/) were analyzed to reveal the functions of the DEGs. After that, the results were visualized by topGO package. The Kyoto Encyclopedia of Genes and Genomes (KEGG) were performed on DAVID and then the results were visualized with ggplot2 package.The Gene Ontology analysis was performed to identify the functions of the DEGs in GSE16561 and GSE100927. In brief, the packages including clusterProfiler and topGO were preloaded in R studio, and the ENTREZIDs of the DEGs obtained from DAVID(http://string-db.org) to obtain the results of PPI network, and then the results were further analyzed with Cytoscape (version 3.7.1).The interaction of the DEGs was performed by protein-protein interaction network (PPI) analysis. In brief, the DEGs of datasets were uploaded on STRING database (To reveal the profiling difference of genes in the patients and normal persons, the clinical microarray datasets including GES16561 and GES100927 were analyzed by limma package. The results showed that 134 downregulated DEGs and 234 upregulated DEGs were found in GES16561 and 25 downregulated DEGs and 104 upregulated DEGs were found in GSE100927 Figures .To delve the molecular functions of DEGs, the GO enrichment analysis was performed. The results showed that the DEGs in GSE16561 were enriched into 100 related molecular functions, and the DEGS in GSE100927 were enriched into 268 molecular functions. Moreover, for GSE16561, the DEGs were involved in neutrophil activation, neutrophil degranulation, neutrophil activation involved in immune response, neutrophil-mediated immunity, and so on . For GSEStaphylococcus aureus infection, Osteoclast differentiation, Leishmaniasis, Chemokine signaling pathway, Phagosome, and so on. For GSE16561, the DEGs were involved in Primary immunodeficiency, Hematopoietic cell lineage, T cell receptor signaling pathway, Staphylococcus aureus infection, Tuberculosis, and so on (Staphylococcus aureus infection (To explore the regulation mechanism of DEGs, the DEGs of GSE16561 and GSE100927 were analyzed by KEGG enrichment analysis. The result have indicated that 68 DEGs in GSE100927 were enriched in 18 pathways and 163 DEGs in GSE16561 were enriched in 16 pathways. For GSE100927, the DEGs were involved in nd so on . Moreovenfection .To further reveal the molecular mechanism of the genes in the progression of ischemic stroke induced by atherosclerosis, the interactions of DEGs were analyzed by STRING database and Cytoscape. According to the MCODE analysis, the clusters of the top three scores of GSE100927 and GSE16561 were selected for visualization. For GSE16561, cluster 1 involved 23 nodes and 348 edges, cluster 2 involved 19 nodes and 158 edges, and cluster 3 involved 8 nodes and 40 edges Figures . For GSEIncreasing studies have indicated that the development of cerebral infarction induced by atherosclerosis is one of the major types of ischemic stroke . AlthougAtherosclerosis has been thought as the major reason of ischemic stroke . Plaque κB pathway [The progression of ACI is related to the abnormal expression of multiple genes. In this study, the DEGs of GSE16561 and GSE100927 were analyzed by GO enrichment analysis. It was found that the DEGs in GSE16561 and GSE100927 were also related to neutrophil activation, neutrophil degranulation, and neutrophil activation involved in immune response, which suggests that both atherosclerosis and ischemic stroke are connected with the abnormally activation of the neutrophils. Neutrophils involve the various inflammatory reactions, and the inflammation induced by neutrophils has been confirmed to play the major roles in the formation of atherosclerosis [ pathway . Denorme pathway . Moreove pathway . The stu pathway . Besides pathway , 22. ThuThis study confirmed that the DEGs in GSE16561 were enriched in Herpes simplex infection, Viral carcinogenesis, Cytokine-cytokine receptor interaction, MAPK signaling pathway, and HTLV-I infection pathways, while the DEGs in GSE100927 were enriched in Phagosome, Transcriptional misregulation in cancer, Tuberculosis, Chemokine signaling pathway, and Cytokine-cytokine receptor interaction pathways. However, it was also found that both GSE16561 and GSE100927 were enriched in MAPK, Cytokine-cytokine receptor interaction, antigen processing and presentation, and cGMP-PKG signaling pathways. MAPK pathway is implicated in various cellular activities including cell apoptosis and inflammation , 24. AbnPPI network analysis has been confirmed as an effective strategy for tracking genes and investigating molecular mechanism of diseases . In this


Paediatric common infection pathways have been developed in collaboration between the BSAC and national paediatric groups, addressing the management of cellulitis, lymphadenitis/lymph node abscess, pneumonia/pleural empyema, pyelonephritis, tonsillitis/peritonsillar abscess, otitis media/mastoiditis, pre-septal/post-septal  cellulitis, and meningitis. Guidance for the management of a child presenting with a petechial/purpuric rash and the infant under 3 months of age with fever is also provided. The aim of these pathways is to support the delivery of high-quality infection management in children presenting to a hospital. The pathways focus on diagnostic approaches, including the recognition of red flags suggesting complex or severe infection requiring urgent intervention, approaches to antimicrobial stewardship (AMS) principles and guidance on safe and timely ambulation aligned with good practice of outpatient parenteral antimicrobial therapy (OPAT). Antimicrobial resistance (AMR) is an urgent and increasing threat, especially in children.Most patients in the UK (adults and children) are seen in district general hospitals, not teaching hospitals/tertiary centres. Whilst most district general hospitals offer adult infection services , almost no district general hospitals have specialists in paediatric infectious diseases or paediatricians with a specific interest in infection. Currently, most paediatricians refer to their local antibiotic guidelines, where they exist, but management can differ between clinicians in the same team, within teams in hospitals, and between local hospitals (authors’ experience). This may lead to confusion within teams and ultimately patient risk.,There is an increasing evidence base supporting the use of shorter parenteral antimicrobial courses for numerous pathologies in adults. Evidence from the paediatric literature indicates that for children with severe infections who initially received IV antibiotics, earlier IV to oral switches and shorter total durations of antibiotic therapy are associated with equivalent outcomes, assuming the child can tolerate/absorb oral antimicrobials and adherence to oral treatment regimens without regular oversight is not a concern.,Embedding paediatric antimicrobial stewardship principles within general paediatric services has been shown to safely reduce the duration of IV antimicrobials through earlier cessation of antimicrobial therapy or switching from IV to oral agents, compared with children being managed outside of services with paediatric AMS oversight.The aim of this collaboration was to develop pathways for the management of children (<18 years old) presenting to district general hospitals with common community-acquired infections, including cellulitis, lymphadenitis/lymph node abscess, pneumonia/pleural empyema, pyelonephritis, tonsillitis/peritonsillar abscess, otitis media/mastoiditis, pre-septal/post-septal  cellulitis, and meningitis. Pathways for the management of children presenting to hospital with petechial/purpuric rashes and for infants under 3 months of age presenting with fever were also developed.The aim of this article is to inform how the pathways were created. We present the rationale behind the development of the pathways, including details of the robust methodology applied to inform each pathway, collaboration with specialist groups, and consultation process with wider stakeholders. Plans for widespread implementation of the pathways are also included.The rationale for these pathways was to support the delivery of high-quality infection management in children presenting to hospital, focusing on diagnostic approaches including the recognition of red flags suggesting complex or severe infection requiring urgent intervention, approaches to management adhering to the principles of AMS and guidance on safe and timely ambulation aligned with the principles of outpatient parenteral antimicrobial therapy (OPAT). Review of the current evidence on antimicrobial prescribing in children and areas for improvement were used to derive a list of questions to be answered within each pathway .13–15Although the aim of the pathways was to provide guidance aligned with the principles of AMS, recommendations were not made on the choice of antimicrobial agent(s) within these pathways. This approach was taken because deciding on choice of antimicrobial agent requires local discussions with key stakeholders along with review of local antimicrobial resistance rates; the Pathway Working Party were concerned that offering guidance on choice of antimicrobial agent may be challenged by local clinicians, leading to unnecessary variability in care and potentially missed opportunities for optimal care delivery. The pathways signpost users to the recently developed National Institute for Health and Care Excellence (NICE) guidelines for common infections, where applicable.The pathways are aimed at paediatricians, emergency department clinicians, advanced paediatric nurse practitioners, microbiologists, and pharmacists.et al. (2016)JAC-AMR Online). The same databases were searched using criteria associated with paediatric OPAT (pOPAT) , CINAHL, EMBASE and MEDLINE databases were comprehensively searched from 1 January 2014 to 16 October 2019 using the search criteria of McMullan et al.n = 690) (see n = 483) was completed by two individuals by screening titles and abstracts. There were 233 references selected for further appraisal and the full articles available in English were reviewed. References from the literature search considered to be of relevance (n = 109) were categorized according to pathway  was the host organization. Working Party membership comprised consultants in paediatric medicine, paediatric emergency medicine, ambulatory care, paediatric infectious diseases, and medical microbiology, an antimicrobial pharmacist, and a clinical nurse specialist in paediatric infectious diseases. Pathways were drafted in partnership with national professional groups .www.bsac.org.uk), and a formal 2 week consultation process was completed (19/10/20–02/11/20). The pathways were revised in light of the 151 comments received  and each pathway was revised before wider consultation. To gather feedback and improve the quality of the content of the pathways, the draft pathways were sent to a comprehensive list of stakeholders , uploadereceived .https://bsac.org.uk/paediatricpathways/.Ten pathways were created. Pathways for the management of children presenting to hospital with common community-acquired infections including cellulitis, lymphadenitis/lymph node abscess, pneumonia/pleural empyema, pyelonephritis, tonsillitis/peritonsillar abscess, otitis media/mastoiditis, pre-septal/post-septal  cellulitis, meningitis, management of children presenting to hospital with petechial/purpuric rashes, and infants under 3 months of age presenting with fever, are freely available online at: Each pathway comprises sections on diagnosis, including differential diagnoses, necessary investigations, and guidance on how to assess severity (e.g. red flags) that may indicate severe/complex infection and a need for urgent intervention. Management options for mild, moderate, and severe infection are presented, including when antibiotics  are required. Although the specific choice of antimicrobial agent was not included, criteria for IV-to-oral switch and total duration of antibiotic course were provided, as was guidance for patient admission or ambulation. Indications for source control/surgical management were included, as was the need for ongoing monitoring, follow-up, and further investigations. Links to national guidance and safety-netting advice were provided.The impact of AMR on children is being increasingly recognized, and local systems, processes and pathways need to be introduced to reduce the unnecessary variability of antimicrobial prescribing in children. This piece of work is based on the large body of evidence supporting the role of empirical antimicrobial guidelines and clinical pathways in improving the quality of antibiotic prescribing and antimicrobial stewardship.However, although NICE provides guidance on the choice of antibiotic for managing common infections in children,https://www.uk-pas.co.uk/).The pathways presented here provide a practical approach to antimicrobial management in children presenting to hospital, including clear guidance on when children can be safety ambulated and the clinical governance systems required to facilitate this. The strength of these pathways lies in the robust scientific process adopted to identify relevant literature, in addition to collaboration with relevant national bodies to develop each pathway. In order to avoid overlap with NICE guidance, the pathways signpost readers to guidance on choice of antibiotic where it is available, such as guidance provided by the UK Paediatric Antimicrobial Stewardship (UK PAS) Network (Data support the availability of online access to improve dissemination of clinical pathways.In the short term, it is hoped that successful implementation of these pathways will result in reduced variability in care and ultimately improve the clinical management of children presenting to district general hospitals, which in many cases will result in shortened duration of antibiotic courses, earlier transition from IV to oral therapy and timely ambulation from hospital. We hope that in the longer term, these pathways will contribute to a reduction of the development of antimicrobial resistance in children.dlab029_Supplementary_dataClick here for additional data file.


Isorhamnetin reduced adenosine triphosphate levels (ATP) in platelets stimulated by collagen and TRAP-6. We also evidenced that isorhamnetin’s antiplatelet activity was related to the inhibition of mitochondrial function without effect on reactive oxygen species (ROS) levels. Additionally, we investigated isorhamnetin’s effect on thrombus formation in vitro under flow conditions on the damaged vessel wall. In this context, we demonstrate that isorhamnetin at 20 µM induced a significant inhibition on platelet deposition, confirming its antithrombotic effect. Our findings corroborate the antiplatelet and antithrombotic potential of isorhamnetin present in many foods of daily consumption.With the diet, we ingest nutrients capable of modulating platelet function, which plays a crucial role in developing cardiovascular events, one of the leading causes of mortality worldwide. Studies that demonstrate the antiplatelet and antithrombotic potential of bioactive compounds are vital to maintaining good cardiovascular health. In this work, we evaluate the flavonol isorhamnetin’s antiplatelet effect on human platelets, using collagen, thrombin receptor activator peptide 6 (TRAP-6), and phorbol myristate acetate (PMA) as agonists. Isorhamnetin induced a significant inhibition on collagen- and TRAP-6-induced platelet aggregation, with half-maximum inhibitory concentration (IC The number of deaths from diet-related cardiovascular events increased between 2010 and 2016 . A balan16H12O7) is a methylated flavonol present in leaves, flowers, and fruits of many plants [Phaseolus vulgaris L., belonging to the group of legume plants consumed in the five continents as an essential component of the diet [Isorhamnetin  and arachidonic acid. The antiplatelet mechanism was related to activation of protein kinase B (AKT), which decreases the activation of platelets [Epidemiological and clinical studies have shown that beans’ consumption is inversely related to coronary artery diseases and the risk of cardiovascular events ,13. Varilatelets .Isorhamnetin has been used in traditional medicine to prevent and treat various diseases ,15 due tIn the context of cardioprotective potential, isorhamnetin protects against cardiac hypertrophy by blocking the PI3K-AKT pathway ,15. The A causal factor in cardiovascular disorders is platelet activation. Platelets play a fundamental role in thrombus formation, atherogenesis, and atherosclerotic lesions progression . The inhThrombin 6 receptor activating peptide (TRAP-6), collagen, and phorbol myristate acetate (PMA), were obtained from Sigma-Aldrich . Antimycin (AA), citrate-dextrose solution, mepacrine, dihydroethidium (DHE), intracellular calcium fluorescence indicator (Fluo-3-AM), and trifluoromethoxyphenylhydrazone (FCCP) were also obtained from Sigma-Aldrich . FITC annexin V apoptosis, PE mouse IgG1 isotype control, and FITC mouse anti-human CD61 were obtained from BD Biosciences . Isorhamnetin was obtained from Cayman Chemical, USA. All assays incorporated as vehicle control dimethyl sulfoxide (DMSO) 0.2%.v/v and then centrifuged at room temperature for 10 min at 240× g to obtain platelet-rich plasma (PRP). PRP was centrifuged (800× g) at room temperature for 8 min [v/v). The platelets were washed again by centrifugation at 800× g for 8 min [Platelets were obtained from six healthy volunteers (men and women) who did not consume medication for two weeks. Donors signed the informed consent according to the protocol approved by the Scientific Ethics Committee of the University of Talca (protocol no. 19/2018), following the Declaration of Helsinki . Blood wor 8 min . Then plor 8 min . The plaor 8 min . Flow cy8 platelets/mL) were incubated with isorhamnetin  for 10 min at 37 °C. Subsequently, platelets were centrifuged (800× g) for 8 min, and the supernatant was analyzed with the lactate dehydrogenase (LDH) cytotoxicity assay kit . A microplate reader  was used to measure the reaction’s absorbance at 490 nm. The positive control was Triton X-100 (10%) [Washed platelets (3 × 1000 (10%) .8 platelets/mL) were incubated for 5 min with CaCl2 (2 mM) plus isorhamnetin  or vehicle . Similar concentrations in vitro have been evaluated in other studies [50) was obtained from isorhamnetin’s concentration curves .Inhibition of platelet aggregation was evaluated by a turbidimetric method using a lumi-aggregometer  ,26. Wash studies . Platele studies . The pla studies . The con8 platelets/mL) were incubated for 5 min with CaCl2 (2 mM) and subsequently with isorhamnetin  for 10 min at 37 °C. In addition, PS was induced in activated conditions by collagen (1 mg/mL)/TRAP-6 (5 µM). The samples were then diluted with annexin V-binding buffer and pre-incubated in the dark with annexin V-FITC and anti-CD61-PE for 30 min. Samples were examined on an Accuri C6 flow cytometer .Externalization of phosphatidylserine (PS) in platelets was determined by flow cytometry, following the methodology described by Mendez et al. 2020, with slight modifications ,29. Wash8 platelets/mL) were incubated for 5 min with CaCl2 (2 mM) and isorhamnetin . The luciferin (Chrono-Lume) reagent and the platelet agonists (TRAP-6 5 µM and collagen 1 µg/mL) were added consecutively. ATP secretion (100%) was obtained with the vehicle (DMSO 0.2%) plus the platelet agonists. Adenosine (10 µM) was used as a positive control of inhibition.Extracellular adenosine triphosphate (ATP) was determined using Chrono-lume  . Washed 8 platelets/mL) were incubated with Fluo-3-AM (0.4 µM) at room temperature for 30 min as previously described by Mendez et al. 2020 [7 platelets/mL) with Tyrode’s buffer without calcium, and the samples were incubated with the negative control, FCCP (1 µM) or isorhamnetin  for 10 min at 37 °C. The effect of isorhamnetin on the intracellular calcium levels was calculated relative to the vehicle (DMSO 0.2%) using the Accuri C6 flow cytometer .Washed platelets  with DHE (10 µM) in the presence of isorhamnetin  and CaCl2 (2 mM) for 30 min at 37 °C. Antimycin 10 µM was a positive control. ROS formation was analyzed by Accuri C6 flow cytometer  [Reactive oxygen species production (ROS) was determined in washed platelets  ,30.2 (2 mM) was added to the washed platelets (5 × 107 platelets/mL). Then, TMRM (100 nM) was added and incubated with DMSO 0.2%, isorhamnetin , or FCCP 1 µM at 37 °C for 30 min. The samples were studied in the Accuri C6 flow cytometer  [Mitochondrial membrane potential (Δψm) was assessed with the permeating cellular dye tetramethylrhodamine methyl ester perchlorate (TMRM) as before described . CaCl2  .y-axis. The data were linearized, and regression analysis was calculated .The effect of isorhamnetin (20 µM) on platelet adhesion and thrombus formation was evaluated under controlled blood flow conditions in the Badimon Perfusion Chamber ,33. The 50 was calculated from the dose-response curves of isorhamnetin. Results were examined by analysis of variance (ANOVA) and Tukey’s post hoc test to determine the significant differences between samples [t-test. p values < 0.05 were considered statistically significant.Data were represented as the mean ± standard error of the mean (SEM) of the experiments and were analyzed using Prism 6.0 software . The IC samples . DiffereLDH is released from the cell because of damage or lysis. In this context, isorhamnetin’s cytotoxic effects were evaluated at 10, 50, and 100 µM. It was observed that isorhamnetin at none of the concentrations evaluated induced cytotoxic activity A. The PS50 of 8.1 ± 2.6 µM) had a powerful concentration-dependent antiplatelet effect when platelet aggregation was induced by collagen. When TRAP-6 was used as an agonist, the compound inhibited platelet aggregation with IC50 of 16.1 ± 11.1 µM. While PMA-induced platelet aggregation had a lower activity (IC50 > 100 µM) and at 100 µM inhibited PMA-induced platelet aggregation by about 30 ± 6%. Based on these results, we decided to continue the antiplatelet studies with isorhamnetin 1, 10, and 20 µM.The efficacy of isorhamnetin  to inhibit platelet aggregation was evaluated by turbidimetry stimulated by collagen (1 µg/mL), TRAP-6 (5 µM), and PMA (100 nM) A–C. The Platelet activation is stimulated by platelet secretion products, such as ATP. About the control, isorhamnetin at 20 µM lowers ATP levels to 0.28 ± 0.03 and 0.28 ± 0.05 (folds of control) in collagen- and TRAP-6-stimulated platelet, respectively A. MeanwhThe association between developing CVD and mitochondrial damage is well-known. We study if the isorhamnetin has an antiplatelet activity through the inhibition of mitochondrial bioenergetics. In this context, we observed that isorhamnetin at 1, 10, and 20 µM significantly decreases the membrane potential compared to the control B.Platelet activation is regulated by the endogenous generation of ROS induced by a wide variety of stimuli, for example, at the mitochondrial level by antimycin A. In our study, isorhamnetin was shown not to affect ROS C.2+ levels relative to baseline  and at high shear rate . Isorhamnetin at 20 µM induced a significant reduction in platelet deposition versus an untreated blood sample  at both Platelets are the smallest blood cells, and their activation at sites of the vascular lesion is essential for thrombus formation . The treIsorhamnetin has broad pharmacological effects, such as anti-osteoporosis , anti-inLDH activity can be used as an indicator of cell membrane integrity and, therefore, a measure of cytotoxicity . IsorhamPlatelet agonists activate different pathways in signal transductions. TRAP-6 acts as a PAR1 agonist in platelet activation ,47; glycMitochondria via ATP production are involved as central drivers in platelet activation. In this context, inhibition of mitochondrial function has been reported as an antiplatelet target via a decrease of ATP levels ,54. We dIt has been described that atherothrombosis is initiated by collagen exposure when endothelial damage occurs, and this triggering platelet activation/adhesion ,55. In tAlthough this study has some limitations since the effects of isorhamnetin on platelet aggregation are more complex in vivo conditions, our results support scientific evidence that isorhamnetin helps reduce platelet activation and thereby thrombotic and cardiovascular risk.Diet and nutrition influence the progression and/or treatment of CVD. Isorhamnetin may be a promising scaffold compound to develop new antiplatelet agents with specific action on thrombotic diseases. Isorhamnetin did not induce cytotoxic effects. Its antiplatelet potential was significant in collagen-stimulated platelet aggregation. Consecutively, it induced a fall in membrane potential and a decrease in ATP levels. The evidence presented in this work suggests that this flavonoid may play an important role in health maintenance and possibly protect against cardiovascular disease associated with the inhibition of platelet function and a possible reduction in the risk of thrombosis. Certainly, future studies, such as absorption and metabolism, are needed to translate the antiplatelet activity of in vitro dose–response to in vivo concentrations (human).


To study the molecular targets and mechanisms of Dayuan Decoction in the treatment of fever.  The traditional Chinese medicine system pharmacological database and analysis platform (TCMSP) was used to search for the main active components and action targets of Dayuan Decoction. The GeneCards database was used to retrieve fever-related gene targets. The common gene targets were obtained by selecting the intersection of the action targets of the main active components of Dayuan Decoction and the fever-related gene targets using a Wayne diagram. Cytoscape 3.7.2 software was used to construct the drug active component action target network diagram. The protein-protein interaction PPI network of coacting targets was constructed using the STRING database. David data were used for Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses of common gene targets.  A total of 89 main active components and 832 acting gene targets of Dayuan Decoction were screened, of which 50 were common gene targets of Dayuan Decoction and fever, including apoptosis regulatory factor Bel (BCL2L1), interleukin 6 (IL-6), interleukin 10 (IL-10), and tumor necrosis protein 53 (TP53). A total of 47 GO and 59 KEGG enrichment entries were obtained using GO and KEGG enrichment analyses, involving the cancer pathway, hypoxia-inducible factor 1 (HIF-1) signaling pathway, and mitogen-activated protein kinase (MAPK) signaling pathway.  Dayuan Decoction has multitarget regulation characteristics and a multifaceted role in the treatment of fever. Mycoplasma pneumoniae, Rickettsia sp., and spirochetes), fungi, and parasites), and noninfectious fever are both prevalent. The typical normal human body temperature is between 36 and 37°C (armpit). Low heat is 37.3–38°C, medium heat is 38.1–39°C, high heat is 39.1–41°C, and ultrahigh heat is >41°C.The heat generation and dissipation processes of the human body maintain a dynamic equilibrium under the supervision of the temperature regulation center . When thAnemarrhena asphodeloides (3 g), peony (3 g), Scutellaria baicalensis (3 g), and licorice (1.5 g)” are the ingredients in this treatment [Wu Youke's essay on epidemic disorders during the Ming Dynasty inspired Dayuan Decoction. Betel nut 6 g), Magnolia officinalis 3 g), grass nut 1.5 g), reatment . Clinica g, grassreatment . It is p g, reatm g, Magnoreatment .Dayuan Decoction exhibits a clear antifever effect; however, its mechanism of action remains unknown and a systematic and complete investigation is required . With thhttp://tcmspw.com/index.ph) is a systematic pharmacological database of Chinese herbal medicines that can be used to explore the relationship between drugs, targets, and diseases. The TCMSP was used, with the keywords betel nut, Magnolia officinalis, grass nut, Anemarrhena aspedoloides, peony, Scutellaria baicalensis, and licorice as the main seven traditional Chinese Medicine Boards of Dayuan Decoction. The screening criteria were oral bioavailability (OB) ≥ 30%, drug likeness (DL) ≥ 0.18, and half‐life (HF) ≥ 4 to screen the main active components of Dayuan decoction.The TCMSP (https://www.uniprot.org). The database corrects and standardizes the target information of the active components to obtain the corresponding gene targets. Using the GeneCards (https://www.genecards.org/) database with “fever” as the search term, the fever-related gene targets were retrieved. Dayuan Decoction gene targets of the main active ingredients and disease-related gene targets were obtained by selecting the intersection of the Wayne diagram to obtain the common gene target, the gene target of Dayuan Decoction for the treatment of fever, loading the search results, and saving them in Excel format. This completed the collection of all human fever-related gene data.The related target function is one of the functions of the TCMSP, which can be used to predict the action target information of the main active components of the Dayuan decision, using UniProt . In the software, if there are more connections between a target and other targets, the target is important and is a key node. The common gene target obtained from the Wayne diagram was imported into STRING , the species was set as “Homo sapiens,” and the protein phase of common gene targets was constructed through a protein-protein interaction (PPI) network.Betel nut, https://david.ncifcrf.gov/), the species was set as “Homo sapiens,” and GO and KEGG pathway enrichment analyses were conducted. P < 0.01 and enriched gene counts > 5 was the cut-off value standard to further determine the molecular signaling pathway of blue Dayuan Decoction in the treatment of fever. GO analysis is composed of three aspects, namely, the biological process (BP), cellular component (CC), and molecular function (MF), to explain the essence of genes. KEGG pathway enrichment analysis is helpful to understand the signaling pathway process and function of a gene or protein in the process of the signaling pathway.The database for annotation, visualization, and integrated Disco (David) is commonly used for the annotation of gene functions. Using David , drug response (14), and lipopolysaccharide-mediated signaling pathway (13). The items and number of genes involved in the process of cell composition were cytoplasm (22), cytosol (20), and nucleus (19). The items and the number of genes involved in molecular function processes were protein binding (30), similar protein binding (13), and protein homopolymerization activity (12).A total of 192 GO entries were obtained through screening GO enrichment analysis from the David database, including 16 cell composition, 157 biological process, and 19 molecular function entries. The results were visually analyzed using R software to obtain a bubble diagram of the GO enrichment analysis Figures –5. The aP < 0.01), as shown in Amoeba protozooses (10).A total of 35 enrichment entries were obtained through KEGG pathway enrichment screening (As shown in The obtained active compound was predicted to the target through the TCMSP database, and then, the target information was queried through DAVID, STRING, and other databases, and the drug target-metabolic pathway-active ingredient network  was consFever is defined as an increase in body temperature over the usual range as a result of pyrogen activity or other causes . ExogenoWhen compared to 40 instances treated with conventional medication, the curative efficacy of Dayuan Decoction was superior, and there were no negative effects reported. Wang et al. used Dayuan Decoction in the treatment of malignant fever associated with moist heat buildup, which had a positive clinical impact as well as being gentle and steady in nature, with few negative effects and excellent tolerance . The netThe nonspecific disease-related route includes the nervous system signaling pathway hsa04722: neurotrophin signaling pathway, which is also known as the neurotrophin signaling pathway. Neurotrophins are involved in the differentiation of nerve cells . The neuFinally, the mechanism of action of Dayuan Decoction in the treatment of fever includes a range of metabolic-centered pathways, which may be activated by the simultaneous responses of multiple target complicated pathways. This research expands the body of evidence supporting the therapeutic impact of Dayuan Decoction on malignant fever and suggests that Dayuan Decoction may have the potential to cure neurodegenerative disorders in the future. The intricacy of the chemical components of traditional Chinese medicine necessitates that this research be presented only from the standpoint of facts, and several shortcomings still exist. This requires the continued enhancement of the detection strength of the LC-MS technique and metabolomics and further investigation and validation of these findings through experimental studies.


Background: Cyclophosphamide is a common tumor chemotherapy drug used to treat various cancers. However, the resulting immunosuppression leads to leukopenia, which is a serious limiting factor in clinical application. Therefore, the introduction of immunomodulators as adjuvant therapy may help to reduce the hematological side effects of cyclophosphamide. Lvjiaobuxue granule has been widely used in the clinical treatment of gynecological diseases such as anemia and irregular menstruation. Recently, it has been found to increase the function of white blood cells, but its mechanism of action is still unclear. We aimed to reveal the mechanisms of Lvjiaobuxue granule against acute leukopenia by an integrated strategy combining metabolomics with network pharmacology.Methods: Subcutaneously inoculated 4T1 breast cancer cells to prepare tumor-bearing mice, intraperitoneal injection of cyclophosphamide to establish a 4T1 tumor-bearing mice leukopenia animal model, using pharmacodynamic indicators, metabolomics, network pharmacology and molecular biology and other technical methods. To comprehensively and systematically elucidate the effect and mechanism of Lvjiaobuxue granule in improving cyclophosphamide-induced leukopenia in 4T1 tumor-bearing mice.Results: Lvjiaobuxue granule can improve the blood routine parameters and organ index levels of the leukopenia model of 4T1 tumor-bearing mice. Metabolomics studies revealed that 15 endogenous metabolites in the spleen of mice were considered as potential biomarkers of Lvjiaobuxue granule for their protective effect. Metabonomics and network pharmacology integrated analysis indicated that Lvjiaobuxue granule exerted the leukocyte elevation activity by inhibiting the branched-chain amino acids (BCAAs) degradation pathway and increasing the levels of valine, leucine and isoleucine. The results of molecular biology also showed that Lvjiaobuxue granule can significantly regulate the key enzymes in the catabolism of BCAAs, which further illustrates the importance of BCAAs in improving leukopenia.Conclusion: Lvjiaobuxue granule exerts obvious pharmacological effects on the leukopenia model of 4T1 tumor-bearing mice induced by cyclophosphamide, which could be mediated by regulating the branched-chain amino acid degradation pathway and the levels of valine, leucine and isoleucine. Cyclophosphamide is the most common cancer chemotherapy agent and used in anticancer for various types of cancer, especially breast cancer . UnfortuAfter constant efforts, the treatment against the side effects of chemotherapy still leaves much to be desired. In recent years, prescriptions are composed of a combination of multiple drugs, which are considered to be a promising treatment strategy to improve the anti-tumor effect and reduce the side effects of chemotherapy drugs . TraditiColla corii asini, Angelica sinensis (Oliv.) Diels and other compound preparations in the treatment of deficiency of Qi and blood. It is known as the “Holy Medicine in Blood” and widely used in the clinical treatment of gynecological diseases such as anemia, irregular menstruation and so on. Besides, a large number of clinical studies have found that it has the effect of increasing white blood cells , Astragalus mongholicus Bunge , Codonopsis pilosula (Franch.) Nannf , Rehjnannia glutinosa (Gaertn.) DC , Atractylodes macrocephala Koidz  and Angelica sinensis (Oliv.) Diels  at a ratio of 36:30:30:20:15:10.Lvjiaobuxue granule is a new kind of good medicine for the treatment of Qi and blood deficiency and fatigue. It fills the blank of od cells . HoweverColla corii asini  in cyclophosphamide-induced mice  and Angelica sinensis (Oliv.) Diels  , Biological Magnetic Resonance Data Bank [BMRB (http://www.bmrb.wisc.edu/)] and published literature. In total, 32 endogenous metabolites were identified, belonging to amino acids, organic acids, sugars, lipids, etc. the details of the metabolites were shown in The representative To explore the metabolites levels in 4T1 tumor-bearing mice with leukopenia induced by cyclophosphamide, the supervised multivariate methods PLS-DA and OPLS-DA were used for processing . The PLSR2 and Q2 values were lower than the original ones   . Further= 0.649) . The corα-glucose, leucine, and phenylalanine in the spleen of the model group were observed. The changes of these endogenous metabolites were considered to be a direct result of the leukopenia. Meaningfully, the levels of metabolites were regulated by LBG treatment   . The corThe metabolic networks involved in a series of enzymes and genes were constructed by using the Metscape plug-in running on Cytoscape 3.7.1, that the internal correlation of the differential metabolites based on enzyme or gene levels could be better understood . As a reAstragalus mongholicus Bunge , Codonopsis pilosula (Franch.) Nannf , Rehjnannia glutinosa (Gaertn.) DC , Atractylodes macrocephala Koidz  and Angelica sinensis (Oliv.) Diels  respectively. The ingredients were retrieved from these ingredients via the ADME parameters and literature confirmation. Consequently, a total of 32 active ingredients of Lvjiaobuxue granule were filtered out for further analysis , among which the valine, leucine and isoleucine degradation were the most closely related  method was used to evaluate the binding potential between selected valine, leucine and isoleucine degradation pathway targets and the chemical components with top 7° (Degree ≥ 5) (https://www.rcsb.org/)  . The docsb.org/) . The resp < 0.01) in the model group (p < 0.05 or p < 0.01). The BCKDHA and ACADS enzyme levels in the model group were significantly increased in the catabolism pathway of BCAAs. Abnormal expression of BCAAs degrading enzymes cause BCAAs to overly decomposed in the model group. Lvjiaobuxue granule can reverse the levels of two enzymes, thereby improving the abnormal metabolism of BCAAs. In the leukopenia model group, the abnormal expression of BCAAs degrading enzyme lead to the excessive decomposition of BCAAs.To further confirm the results of the biological targets network, we verified the BCAAs catabolism pathway. Two key and irreversible reactions of BCAAs catabolism require the participation of branched-chain keto acid dehydrogenase (BCKDH) complex and acyl-CoA dehydrogenase (ACAD). The levels of BCKDHA and ACADS were significantly increased  single medicine, which can stimulate bone marrow hematopoiesis, promote the proliferation and differentiation of hematopoietic stem progenitor cells, increase the number of blood cells produced, and effectively improve the white blood cell level of leukopenia. Radiotherapy and chemotherapy can cause bone marrow suppression in cancer patients, and their hematopoietic microenvironment will also be destroyed. In addition, DST can prevent the DNA damage of bone marrow hematopoietic cells caused by radiotherapy and chemotherapy, and have the effect of protecting the hematopoietic microenvironment . At the metabolite level, this study only used 1H-NMR metabolomics technology to reveal the changes in the level of branched-chain amino acids, and only detected the upstream metabolites leucine, isoleucine and valine in the catabolism of BCAAs. Unable to detect its downstream substances. 2). At the genetic level, although this study uses the method of network prediction to find out the genes that affect the changes of metabolites and the genes regulated by Lvjiaobuxue granule, it is still necessary to further determine the accuracy of the prediction results. 3). At the level of enzymes and proteins, this study only used ELISA to determine the content of the key metabolic enzymes ACADS and BCKDHA in the BCAAs catabolic pathway, but it is not known whether the enzyme activity changes when the BCAAs catabolic pathway changes. 4). At the pathway level, this study conducted a systematic study on the regulation of BCAAs from the metabolite to the molecular level. However, both KEGG pathway analysis and metabolomics results show that Lvjiaobuxue granule have effects on other pathways  and metabolites  also have a certain effect. In view of the overall regulatory effect of TCM, it is not comprehensive to study only the decomposition pathway of BCAAs.In summary, we first established a model of leukopenia after chemotherapy, which can be used to study the effect of drugs on leukopenia. Determine the method and dosage of cyclophosphamide. This model can reflect the pathophysiological nature of clinical patients. Based on metabolomics and network pharmacology, a new comprehensive strategy was developed to construct an “active ingredients-targets-differential metabolites” network, which involved 32 active ingredients, 28 potential targets, and 11 differential metabolites. KEGG results showed that Lvjiaobuxue granule can improve leukopenia in mice by regulating the decomposition pathway of valine, leucine and isoleucine (BCAAs). The molecular docking results showed that the active ingredients of Lvjiaobuxue granule and the six targets on the decomposition pathway of BCAAs had a high binding force, indicating the reliability of the biological target network analysis results. Molecular biology verification results showed that Lvjiaobuxue granule can significantly reduce the key metabolic enzymes ACADS and BCKDHA in the catabolism of BCAAs, thereby playing a role in the treatment of leukopenia. This research provided data and theoretical support for in-depth study of its mechanism, and lay a foundation for clinical application.