Shenzhuo formula (SZF) is a traditional Chinese medicine (TCM) prescription which has significant therapeutic effects on diabetic kidney disease (DKD). However, its mechanism remains unknown. Therefore, this study aimed to explore the underlying anti-DKD mechanism of SZF.  The active ingredients and targets of SZF were obtained by searching TCMSP, TCMID, SwissTargetPrediction, HIT, and literature. The DKD target was identified from TTD, DrugBank, and DisGeNet. The potential targets were obtained and PPI network were built after mapping SZF targets and DKD targets. The key targets were screened out by network topology and the “SZF-key targets-DKD” network was constructed by Cytoscape. GO analysis and KEGG pathway enrichment analysis were performed by using DAVID, and the results were visualized by Omicshare Tools.  We obtained 182 potential targets and 30 key targets. Furthermore, a “SZF-key targets-DKD” network topological analysis showed that active ingredients like M51, M21, M5, M71, and M28 and targets like EGFR, MMP9, MAPK8, PIK3CA, and STAT3 might play important roles in the process of SZF treating in DKD. GO analysis results showed that targets were mainly involved in positive regulation of transcription from RNA polymerase II promoter, inflammatory response, lipopolysaccharide-mediated signaling pathway, and other biological processes. KEGG showed that DKD-related pathways like TNF signaling pathway and PI3K-Akt signaling pathway were at the top of the list.  This research reveals the potential pharmacological targets of SZF in the treatment of DKD through network pharmacology and lays a foundation for further studies. Diabetic kidney disease (DKD) is one of the most common chronic microvascular complications of diabetes. It may be caused and shaped by the interaction of many factors such as endoplasmic reticulum dysfunction, high sugar-mediated generation of terminal advanced glycation endproducts (AGE), increased activation of the renin angiotensin aldosterone system, increased generation of reactive oxygen species (ROS), and activation of extracellular matrix (ECM) and protein kinase C , 2. It iin vivo processes, the systematic mechanism research of SZF has been hindered.Shenzhuo formula (SZF) as a traditional Chinese medicine (TCM) prescription has certain advantages in the treatment of DKD .It is crRecently, network pharmacology has been developed rapidly with the use of multiomics, high-throughput screening, network visualization and analysis, or other techniques –9. It cahttp://lsp.nwu.edu.cn/tcmsp.php) [http://www.megabionet.org/tcmid/) [http://www.swisstargetprediction.ch/) [http:lifecenter.biosino.org/hit/) [http://bidd.nus.edu.sg/group/cjttd/) [https://www.drugbank.ca/) [http://www.disgenet.org/) [http://string-db.org) [http://www.cytoscape.org/) [http://david.ncifcrf.Gov) [https://www.Omicshare.com/) were used for visual analysis of GO and KEGG results.The Chinese Traditional Medicine System Pharmacological Database Analysis Platform  , Traditi/tcmid/) , SwissTaion.ch/) , and HITrg/hit/)  were use/cjttd/) , DrugBanank.ca/) , and Diset.org/)  were use-db.org) . Composipe.org/)  was usedcrf.Gov)  was usedWe relied on TCMSP, TCMID database, and literatures mining to search for the chemical constituents of SZF .As we all know, TCM drugs enter human body and then take effect through absorption, distribution, metabolism, and excretion (ADME) processes. Among them, oral bioavailability (OB) and drug similarity (DL), the key parameters of ADME components, were used as the screening criteria for active ingredients in this study. In this section, we used TCMSP to collect active compounds and their ADME properties. And then the active compounds that meet “OB ≥ 30%, DL ≥ 0.18” were selected as potential active ingredients.SwissTargetPrediction and HIT databases were used to collect the drug targets. In addition, TTD, DrugBank and DisGeNet databases were used to search for DKD targets by entering the key words of “diabetic kidney disease” and “diabetic nephropathy.” Further, we matched SZF targets with DKD targets to obtain common targets.PPI network of common targets was obtained using STRING. Furthermore, the PPI network topology analysis was carried out using Cytoscape 3.2.1 software and then key targets were obtained. To further explore the interactions between the active ingredients and their related targets at a system level, a “SZF-key targets-DKD” network was constructed by Cytoscape3.2.1.GO analysis is widely used for gene function classification and mainly includes the molecular function (MF), biological processes (BP), and cellular components (CC) . In thisThrough TCMSP and TCMID database, a total of 87 active compounds of Hedysarum Multijugum Maxim, 210 of Radix Salviae, 35 of Hirudo, and 92 of Radix Rhei Et Rhizome were obtained. Then by ADME  screening, a total of 101 active compounds were selected, including 20 active compounds of Hedysarum Multijugum Maxim, 65 of Radix Salviae, and 16 of Radix Rhei Et Rhizome . In addition, through literature mining, another 4 active compounds were collected, including 2 active compounds of Hedysarum Multijugum Maxim , 23, 1 oAfter matching SZF targets with DKD genes, a total of 182 common targets of SZF were obtained. We only show 50 of them in The PPI network of the 182 common targets was obtained using STRING . Then, wThe DAVID was used to carry out GO analysis. And the GO terms were visualized by the Omicshare Tools . The GO KEGG pathway enrichment analysis showed that a total of 104 pathways were obtained. The top 20 pathways are displayed in Previous studies have suggested that SZF has a therapeutic effect on DKD ,6. HowevThrough the SZF-key targets-DKD network, we could know that most active ingredients were linked with no less than one target, which indicated the character of multi-target of TCM active ingredients. In the meanwhile, different active compounds from different herbs acted on the same targets, which demonstrated that SZF had a synergistic effect in treating DKD. In addition, there were 8 active ingredients whose degrees were greater than 2 times of average in SZF-key targets-DKD network topology analysis. Interestingly, 3 of them had been proven to have kidney protection effect by experiments. For example, quercetin liposomes had renal protective effects of reducing oxidative stress, attenuating AGE expression, and delaying the progression of DKD . LuteoliMoreover, the results of the SZF-key targets-DKD topology analysis also showed that there were 5 targets whose degrees were greater than 2 times of the average. Particularly, 3 of these had been proven to be closely related with DKD. For instance, EGFR activation had a significant role in activating pathways that mediate podocyte injury and loss in diabetic nephropathy . DownregAs shown in GO analysis, the potential targets of SZF acting on DKD were mainly associated with various biological processes, such as lipopolysaccharide-mediated signaling pathway, inflammatory response, positive regulation of cyclase activity, protein kinase B signaling, positive regulation of MAP kinase activity, and response to estradiol, which had a strongly direct correlation with the pathogenesis of DKD –38.Similarly, KEGG pathway enrichment analysis showed that SZF took an anti-DKD effect by multiple pathways. Through further research, we found that some pathways had been already verified to exert anti-DKD potential by experiments, such as TNF signaling pathway , HIF-1 sin vitro or in vivo studies.In conclusion, this study based on the network pharmacology had preliminarily explained the anti-DKD mechanism of SZF from the perspective of multi-active ingredients, multi-targets, and multi-pathway. In the future, we will further investigate its mechanism by molecular docking, using


To predict the main active ingredients, potential targets, and key pathways of Jiawei Chaiqin Wendan decoction treatment in vestibular migraine and explore possible mechanisms by network pharmacology and molecular docking technology.  The active ingredients and related targets of Jiawei Chaiqin Wendan decoction were obtained from the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP). The corresponding genes of the target were queried by UniProt database, and the “drug-compound-target-disease” network was constructed by Cytoscape 3.7.2 software. GO functional enrichment analysis and KEGG pathway enrichment analysis were carried out by R software and Bioconductor, and column chart and bubble chart were drawn by Prism software and OmicShare database for visualization. Finally, the mechanism and potential targets of Jiawei Chaiqin Wendan decoction in the treatment of vestibular migraine were predicted. P < 0.05). KEGG pathway enrichment analysis screened 156 signal pathways (P < 0.05), involving PI3K-Akt signal pathway, AGE-RAGE signal pathway in diabetes complications, MAPK signal pathway, HIF-1 signal pathway, IL-17 signal pathway, etc. Molecular docking results showed that quercetin, luteolin, kaempferol, tanshinone IIa, wogonin, naringenin, nobiletin, dihydrotanshinlactone, beta-sitosterol, and salviolone have good affinity with core target proteins IL6, PTGS2, MAPK1, MAPK3, and CGRP1.  The “drug-compound-target-disease” network contains 154 active ingredients and 85 intersection targets. The key targets include AKT1, IL6, MAPK3, VEGFA, EGFR, CASP3, EGF, MAPK1, PTGS2, and ESR1. A total of 1939 items were obtained by GO functional enrichment analysis ( The active ingredients in Jiawei Chaiqin Wendan decoction may regulate the levels of inflammatory factors and neurotransmitters by acting on multiple targets such as IL6, MAPK3, MAPK1, and PTGS2, so as to play a therapeutic role in vestibular migraine. Vestibular migraine (VM) is a chronic and disabling disease that is common and has a genetic tendency; the main symptoms are recurrent dizziness/vertigo, which may be accompanied by nausea and vomiting with or without headache . StudiesThe diagnosis of VM is mainly based on the characteristics of the patient's clinical onset and lacks specific signs and objective diagnostic basis. In the past few decades, uncertainty about the diagnosis of VM has limited the progress of treatment . There iChaiqin Wendan decoction is made of Wendan decoction from Yao Sengyuan in the Southern and Northern dynasties. The modified Chaiqin Wendan decoction (JCWD) is an effective empirical prescription for the treatment of vertigo in clinic. This prescription is composed of Radix Bupleuri (Chaihu), Radix Scutellariae (Huangqin), Pinellia tuber (Banxia), Dried tangerine Peel (Chenpi), Fructus Aurantii Immaturus (Zhishi), Poria cocos (Fuling), Radix Paeoniae Alba (Baishao), Angelica sinensis (Danggui), Salvia miltiorrhiza (Danshen), Gastrodia elata (Tianma), Uncariae Ramulus Cumuncis (Gouteng), and Shi Cassia (Shijueming). At present, clinical observation indicates that Chaiqin Wendan decoction has a significant effect on the treatment of patients with dizziness and headache caused by Triple Burner Congestion –11. Therhttp://tcmspw.com/tcmsp.php) [http://www.uniprot.org/), and gene annotation was performed.The TCMSP (msp.php)  databasehttps://www.genecards.org/) and OMIM database (https://omim.org/) by retrieving “vestibular migraine” look up vestibular migraine related genes and remove duplicates. Through Venny2.1 software, the target of active ingredients of traditional Chinese medicine obtained above was intersected with disease-related genes.Using GeneCards , and the species was selected as “Homo sapiens,” with a medium confidence of 0.400 to predict the associated PPI network. Download protein-protein interaction TSV file, import Cytoscape3.7.2 software, and use CytoHubba plug-in to screen core target proteins.Multiple proteins were introduced into String11.0 (http://www.bioconductor.org) to GO functional annotations and KEGG pathway enrichment analysis. P < 0.05 was selected for functional annotation clustering. In order to study the biological effects of VM targets of drug therapy by regulating specific pathways, the top 10 items were screened according to the enrichment degree and a bar chart was drawn. Similarly, P < 0.05 was selected as the significant pathway for cluster analysis, and the top 20 items were screened according to the enrichment degree to draw a bubble plot.There is the core target gene ID through the R software and Bioconductor (https://www.ncbi.nlm.nih.gov/pubmed). The 3D structures of ligands were generated by Chem3D software, and their energy was minimized and exported to mol2 format. All compounds were saved as ligand parameter files in pdbqt format by AutodockTools-1.5.6 software. The 3D structure of the core target protein was downloaded from PDB database (https://www.rcsb.org/) and saved in PDB format. The solvent and water molecules in the target protein receptor molecules were removed by pymol software. Then, the protein receptor molecules were hydrogenated and calculated by AutodockTools software and saved in pdbqt format. After the ligand and protein receptor molecular preprocessing is completed, the appropriate box center and box lattice parameters are set by AutodockTools-1.5.6 software, the active pocket sites that small molecular ligands may bind to are included, and the parameter text file is set, and the other parameters remain default. Molecular docking was carried out by Autodock Vina, and 3D diagrams were drawn by PyMol software for the docking results with the strongest binding activity of each target protein to show the hydrogen bond and hydrophobic effect formed by it.The 2D structures of key compounds were downloaded in PubMed  and OMIM database (https://omim.org/) by retrieving “vestibular migraine” search related genes, a total of 826 were extracted after duplication. Through Venny2.1 software, the intersection of traditional Chinese medicine target and disease genes was obtained, and 85 genes related to JCWD treatment of VM were obtained, as shown in In GeneCards  and 915 edges, as shown in Eighty-five JCWD gene targets for VM treatment were introduced into String11.0 to construct a Protein-protein interaction network (PPI), and isolated nodes were removed, as shown in P < 0.05）, among which 1731 items of biological processes (BP) were closely related to the reactions of metal ions, blood circulation, reactive oxygen species, and oxidative stress, lipopolysaccharide, steroids, and bacterial origin molecules. The 141 items of molecular function (MF) are mainly related to protein phosphatase binding, nuclear receptor activity, and steroid receptor activity, transcription factor activity, cytokine activity, and receptor binding, NO synthase regulator activity, etc. 67 results of cell composition (CC) are mainly related to membrane microdomain, receptor complex, components, and inherent components of synaptic membrane and vesicle cavity, as shown in GO function enrichment analysis and KEGG pathway enrichment analysis were carried out by R software and Bioconductor database installation packages such as clusterProfiler and enrichplot. 1939 GO enrichment results were obtained  pathway, MAPK in the activator protein-1 pathway, and cytokine signal inhibitors in the signal transduction and transcriptional activator 3 pathway, regulating inflammatory mediators and playing an anti-inflammatory role. Luteolin can inhibit all proinflammatory cytokines, such as interleukin-1β, IL-2, IL-6, IL-8, IL-12, IL-17, TNF-α, interferon-β(IFN-β), and granulocyte-macrophage colony stimulating factor and upregulate anti-inflammatory cytokine IL-10 [κB pathway, inhibit the expression of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), TNF-α, IL-1β and IL-6, and reduce the inflammatory response of spinal dorsal root neurons induced by lipopolysaccharide [κB-α and the activation of NF-κB induced by LPS by inhibiting the activation of NIK-IKK and MAPKs  signal pathways, so as to exert its anti-inflammatory activity [From the point of view of the active components of the drug, quercetin can exert its anti-inflammatory effect by inhibiting the production of IL-6, TNF-and VEGF . In addipathways . Kaempfepathways . Luteoline IL-10 . Wogoninccharide ,25. Tansactivity .α, IL-2, IL-6, and other proinflammatory factors [Protein-protein interaction (PPI) network was used to screen out 10 core genes by Matthews correlation coefficient (MCC) algorithm. They are VEGFA , MAPK3 (mitogen-activated protein kinase 3), MAPK1 (mitogen-activated protein kinase 1), EGF , EGFR , IL-6 (interleukin-6), PTGS2 (prostaglandin endoperoxide synthase 2), STAT3 , MMP9 , and MMP2 . Vascular endothelial growth factor (VEGF) has heparin active growth factor, which can play a role in vascular endothelial cells to cause the division and proliferation of vascular endothelial cells, thus forming new blood vessels and improving vascular permeability. The main causes of migraine include vasodilation and protein exudation. In the onset stage of migraine, patients' serum VEGF level is abnormally elevated, blood vessels dilate, and their permeability increases, eventually leading to extravasation of plasma protein . Epiderm factors .Through the analysis of KEGG pathway, it is found that MAPK signal pathway, HIF-1 signal pathway, and IL-17 signal pathway are the main treatment pathways, which play a synergistic anti-inflammatory effect. MAPK signaling cascades include three different regulatory pathways: extracellular signal-regulated kinase (ERK1/2), c-jun N-terminal kinase (JNK) and p38 protein (P38), which are responsible for mitosis, proliferation, differentiation, and survival of cells during development , as wellIn this study, the active components of JCWD and the potential targets and key pathways acting on vestibular migraine were predicted by the method of network pharmacology, which once again confirmed the process of multicomponent, multitarget, and multipathway treatment of diseases with traditional Chinese medicine compound prescription. The active components such as quercetin, kaempferol, luteolin, wogonin, and tanshinone II as in Jiawei Chaiqin Wendan decoction may regulate the levels of inflammatory factors and neurotransmitters (such as CGRP) by acting on IL6, MAPK3, MAPK1, PTGS2, and other targets, thus playing a role in the treatment of vestibular migraine. Due to the limitations of the database and the software itself, the above results need to be further verified by experiments.


The 31 main components of Lianhua Qingwen (LHQW) were obtained through a literature and database search; the components included glycyrrhizic acid, emodin, chlorogenic acid, isophoroside A, forsythia, menthol, luteolin, quercetin, and rutin. Sixty-eight common targets for the treatment of novel coronavirus pneumonia (NCP) and influenza virus pneumonia (IVP) were also obtained. A “component-target-disease” network was constructed with Cytoscape 3.2.1 software, and 20 key targets, such as cyclooxygenase2 (COX2), interleukin-6 (IL-6), mitogen-activated protein kinase14 (Mapk14), and tumor necrosis factor (TNF), were screened from the network. The David database was used to perform a Kyoto Encyclopedia of Genes and Genomes (KEGG) signal pathway enrichment analysis and gene ontology (GO) biological process enrichment. Results showed that the key targets of LHQW in the treatment of NCP and IVP mainly involved biological processes, such as immune system process intervention, cell proliferation, apoptosis and invasion, toxic metabolism, cytokine activity, and regulation of the synthesis process. KEGG enrichment analysis revealed that 115 signalling pathways were related to the treatment of LHQW. Amongst them, IL-17, T cell receptor, Th17 cell differentiation, TNF, toll-like receptor, MAPK, apoptosis, and seven other signalling pathways were closely related to the occurrence and development of NCP and IVP. Molecular docking showed that each component had different degrees of binding with six targets, namely, 3C-like protease (3CL), angiotensin-converting enzyme 2 (ACE2), COX2, hemagglutinin (HA), IL-6, and neuraminidase (NA). Rutin, isoforsythiaside A, hesperidin and isochlorogenic acid B were the best components for docking with the six core targets. The first five components with the best docking results were isoforsythiaside, hesperidin, isochlorogenic acid B, forsythin E, and quercetin. In conclusion, LHQW has many components, targets, and pathways. The findings of this work can provide an important theoretical basis for determining the mechanism of LHQW in treating NCP and IVP. Lianhua Qingwen (LHQW) is composed of 13 traditional Chinese medicines (TCMs), namely, Forsythiae Fructus , Lonicerae Japonicae Flos , Ephedare Herba , Armeniacae Semen Amarum , Gypsum Fibrosum , Isatdis Radix , Dryopteridis Crassirhizomatis Rhizoma , Houttuyniae Herba , Pogostemonis Herba , Rhei Radix et Rhizoma , Rhodiolae Crenulatae Radix et Rhizoma , l-Menthol , and Glycyrrhizae Radix et Rhizoma . According to the pathological theory of TCM, LHQW is good medicine for dredging collaterals, dispersing the lung, opening with pungent and bitter drugs, and clearing away heat and detoxification . LHQW haGiven the complexity of the composition and mechanism of traditional Chinese medicine (TCM), systematically explaining the mechanism of TCM in the treatment of diseases is difficult. Network pharmacology is a new technology that integrates system biology, multidirectional pharmacology, and computer analysis. It can systematically link drugs with diseases and observe the intervention and influence of drugs on disease networks –9. The sIn recent years, network pharmacology has been widely used to systematically explain the mechanism of action of TCM and TCM compound prescription in the treatment of diseases –12, and https://pubchem.ncbi.nlm.nih.gov/), the target protein of the compound was obtained from the Traditional Chinese Medicine Systems Pharmacology (TCMSP) database (http://tcmspw.com/tcmsp.php), National Center for Biotechnology Information (NCBI) database (https://pubchem.ncbi.nlm.nih.gov/), and https://dx.doi.org/10.1093/nar/gkz382 (SWISS) database (http://www.swisstargetprediction.ch/). At the same time, the protein database UniProt (http://www.uniprot.org/uploadlists/) was used to convert the targets to a unified gene name.The main components of LHQW can be identified and quantified through literature retrieval. Through verification and conversion of the PubChem database (https://www.genecards.org/) is a platform that provides all known human genes in genome, proteome, transcription, heredity, and function [GeneCards database (function . NCP- anhttps://string-db.org/) to construct a protein-protein interaction  network map. The database parameter was set to “Homo sapiens,” and the protein type was set to the rating criterion (confidence level >0.9). Then, the PPI network file was downloaded. The active components, their corresponding LHQW targets, and PPI were input into Cytoscape 3.2.1 to construct the “native component-target” network of LHQW. The topology parameters of the network were analysed using the network analyser function of Cytoscape and derived. The related topological data, such as degree (DG) and betweenness centrality (BC), were examined. DG and BC are important topological parameters for evaluating the specific gravity of a node in the network. DG can reflect the number of links between one node and other nodes in the network, and BC reflects the ratio of the number of paths passing through the node to the total number of shortest paths in all the shortest paths in the network. The larger the topology parameters of the node are, the more critical the target in the network is [To determine the interaction between viral pneumonia potential therapeutic targets and targets of LHQW, this study introduced the selected targets into the STRING network platform . GO enrichment can be used to understand the main action process of the target. The KEGG pathway can be adopted to understand the role of this target in metabolism, signal transduction and other pathways by observing the distribution of targets in the pathway. The main signalling pathways and biological processes involved in the efficacy of LHQW were obtained by screening the target genes with P < 0.05. The P value is the statistical significance of target enrichment/annotation, and P < 0.05 indicates that the pathway has analytical significance. The smaller the P value is, the more significant the enrichment result is.The core targets of LHQW in the treatment of viral pneumonia were subjected to gene ontology (GO) biological process and Kyoto Encyclopedia of Genes and Genomes (KEGG) signal pathway enrichment analyses by using the David database (https://www.rcsb.org/), and the target protein and component were pretreated using AutoDock 1.5.6 and Discovery Studio 2.5 software. Then, the target and component were docked using AutoDock Vina. Generally, negative values of binding energy are associated with the stable conformation of ligand binding to the receptor. If the binding energy is less than −5 kcal/mol, then the ligand and receptor can bind spontaneously; binding energy ≤−7 kcal/mol denotes good binding [The protein structure file was downloaded from the Protein Data Bank (PDB) website . Main ingredients of LHQW (red triangle) and medicinal composition of LHQW (green diamond).To further explore the mechanism of LHQW, 68 target proteins of LHQW against viral pneumonia were input into the STRING database, and the PPI network file was obtained . The medP < 0.05 were screened out. In general, the GO terms were classified into three parts: biological process (BP), molecular function (MF), and cellular component (CC). The visualisation results of BP, CC, and MF enrichment analyses were obtained using OmicShare, as shown in Twenty key targets were annotated with GO terms, and 91 terms with P < 0.05 were selected. The visualisation results of the top 20 KEGG pathways with the most significant P value were obtained using OmicShare, as shown in The enrichment analysis of KEGG signalling pathways was carried out on 20 key targets, and 115 signal pathways with The occurrence and development of a new coronavirus and influenza virus exhibit similarities and differences after infection. The former mainly involves two targets (3CL and ACE2) –30, wherThe top five components for combination with each target are shown in Dimax = (Yi − Ymin)/(Ymax − Ymin), and OD = (D1D2... DK)k1/ (k is the index number) [To further analyse the results of each component combined with the targets, this study performed a multi-index comprehensive analysis and multi-index data processing by referring to the general evaluation normalisation method reported in literature , 37. In  number) , 39. Theα, IL1, etc.), and chemokines , and upregulates the expression of COX2 and NOS2, which induce the synthesis of inflammatory mediators, such as PGE2 and NO [κB, as regulatory factors of the inflammatory response, bind to the IL-6 response element in the promoter of the acute phase protein gene [α is an important member of inflammatory cytokines [α can regulate immune cells, and as an endogenous heat source, it can cause fever and interfere with apoptosis. As an early cytokine of viral pneumonia, the expression of TNF-α can regulate other proinflammatory factors downstream. An increase in other cytokines stimulates the production of TNF-α and triggers the cytokines that are dependent on TNF-α. IL-6 and TNF-α are the key cytokines [α, as an early cytokine, can regulate other proinflammatory factors downstream. An increase in other cytokines stimulates the production of both and initiates the TNF-α-dependent cytokine cascade reaction, thus forming a vicious spiral that can cause a cytokine storm and aggravate the patient's condition. The activation and inhibition of these targets not only aggravate the disease but also promote each other, resulting in a vicious cycle that is closely related to the inflammatory injury of the lung; it is also the key target of this study. Therefore, an in-depth study of these targets can help reveal the scientific nature and connotation of the treatment of IVP and NCP.In this study, 31 main components of LHQW were qualitatively and quantitatively obtained by compiling literature, and 712 component targets were obtained from three databases. A total of 68 common targets for the treatment of viral pneumonia were obtained. The expression of COX2 is abnormal in IVP and NCP –43 and m2 and NO , 45. The2 and NO , 47, suc2 and NO , 49. NOSein gene , 51. Wheytokines , 53 thatytokines , 55 for α, IL-2, IL-4, IL-15, and IL-17, can be inhibited relatively. IL-17 signalling pathways regulate IL-17 family expression (first discovered by Th17 cells), and by binding to different receptors, they activate downstream signalling pathways JAK-STAT and NFKB to regulate the secretion of other inflammatory factors; IL-17 signalling pathways are also involved in the occurrence and development of lung injury [β and IL-6, which can activate signal transduction and the transcription protein pathway by inducing the expression of transcription factors. Th17 mediates inflammatory responses mainly by secreting IL-17, IL-21, IL-2, IL-4, TNF-α, and other cytokines [κB is located at the hub of the downstream signalling pathway in TLR [α, IL-1, IL-6, IL-8, and other inflammatory factors; they can also upregulate the expression of COX2 and NOS2, which can induce the synthesis of inflammatory mediators (PGE2 and NO). The accumulation of these cytokines can directly reflect the degree of inflammation. A moderate inflammatory response is a kind of defence reaction of the body, and it is conducive to the clearance of pathogens and the recovery of the body. However, an excessive reaction can cause cascading inflammatory reactions that are difficult to control. TLR and NF-κB may form a complex cycle network with the participation of certain cytokines, which can regulate each other and form a vicious cycle. Therefore, the 20 intervening key targets and their pathways are key to the treatment of viral pneumonia with LHQW.According to the KEGG pathway enrichment analysis in this study, the IL-17 pathway is the most important signal pathway. IL-17 signal pathways are important to the study of multiple coronaviruses. For example, SARS viruses induce the activation of pattern recognition receptors and IL-17 pathways . The Midg injury , 58. In g injury . Severalg injury , 61, andytokines , 63, butytokines . NF-κB iy in TLR . It can κB, TLR, MAPK, and other signalling pathways and downregulation of the gene expression of COX2 and iNOS. As a result, the production of proinflammatory factors, such as IL-6, TNF-α, and chemokines , is reduced to intervene in acute lung injury. Luteolin [κB signalling pathway and the expression of apoptosis-related factors  to reduce the immune damage on the host cells caused by the virus. It also regulates cell apoptosis and plays an antiviral role. Patchouli alcohol [α and IL-6. Forsythia A [κB, and Th17 signalling pathways. Phillyrin [α, IL-6, IL-1β, CXCL10, etc.) induced by SARS-CoV-2 by regulating the activity of the NF-κB signalling pathway. Therefore, forsythin can be used as a new strategy for coronavirus disease. Research has confirmed that glycyrrhizic acid [In addition, the results of molecular docking showed that the 31 main components contained in LHQW key targets had different degrees of combination. In particular, flavonoids and their glycosides and phenylpropanoids had the best binding result. The varied components contained in LHQW could affect multiple pathways by acting on multiple targets to achieve overall intervention in the development of viral pneumonia and overall treatment. Previous studies have found that chlorogenic acid , cryptocLuteolin , 76 has  alcohol  has anti alcohol , 79, cansythia A  can inhihillyrin  can signzic acid  is similκB and MAPK signalling pathways in activated T cells and inhibiting the continuous burst of cytokines. Most of the components reported in these studies are the main components of honeysuckle and forsythia suspense, which belong to the monarch medicine of the LHQW recipe and are the most important components in the treatment of diseases. They clear away heat, detoxify the body, and disperse wind heat. The results of molecular pairing in this study also showed that the main components of honeysuckle and forsythia account for the largest number and are the key to the efficacy of LHQW. The ingredients reported in the above-mentioned studies are basically consistent with the results of the current study, suggesting that the material basis analysis results of this study conform to the law of the monarch, the subject, and TCM to a certain extent. Presently, most studies focus on nonvolatile components, and the few reports on antiviral, anti-inflammatory, and immune regulation of the volatile components of LHQW focus on cooling, expectorant, and antibacterial effects and nasal congestion reduction [Several studies  have foueduction . The resAn abnormal immune system is the main common point in the occurrence and development of IVP and NCP. The pathway enrichment analysis and biological function annotation in this study showed that the mechanism of LHQW in the treatment of the two diseases is closely related to immune, inflammatory, and viral signalling pathways. Therefore, the mechanism of LHQW treating IVP and NCP could focus on restoring the physiological function of the immune system, reducing the persistent injury of inflammation to the body, inhibiting the invasion and proliferation of the virus, and promoting the apoptosis of the virus.LHQW contains 31 main components. Isochlorogenic acid B, isoforsythiaside, forsythoside E, rutin, quercitrin, hesperidin, and other components are the key to the simultaneous treatment of NCP and IVP by LHQW. These components can interfere with signalling pathways, such as IL-17, T cell receptor, Th17 cell differentiation, TNF, toll-like receptor, MAPK, and apoptosis by acting on targets, including 3CL, ACE2, COX2, HA, IL-6, and NA, to realise the anti-inflammatory, antivirus, and immune regulation comprehensive efficacy of LHQW. Although the mechanism of treating IVP and NCP differs and the different contents are mainly reflected in the manner of virus infection and the material basis of antivirus, the main links are closely related to regulating immunity, reducing inflammation, and combating the virus. The process embodies the characteristics of seeking common ground whilst reserving differences in the treatment process of IVP and NCP. It also shows the multicomponent, multitarget, and multichannel advantages of LHQW and provides a research method for the mechanism and substance foundation of the treatment of IVP and NCP.


Recurrent spontaneous abortion (RSA) is intractable infertility and can be ameliorated with the use of traditional Chinese medicine preparation, the Wenjing decoction. This study aimed to identify the therapeutic mechanism of Wenjing decoction on specific target proteins involved in RSA. n = 3). The interaction between DEGs and the predicted target proteins was identified by the Venn diagram. Using the Cytoscape software and clusterProfiler package, enrichment analysis was conducted for the intersected target proteins. Additionally, the protein-protein interaction (PPI) network and pharmacological network were generated using the Cytoscape software.  Wenjing decoction contains Wuzhuyu, Danggui, Chuanxiong, Guizhi, Shengjiang, Banxia, Gancao, Ejiao, Mudanpi, Chishao, Dangshen, and Maidong. Using TCMSP and BATMAN databases, we queried for active ingredients and predicted their target proteins by BATMAN. Using the edgeR package, we analyzed the differentially expressed genes (DEGs) in the GSE121950 database between control samples and RSA (α (tumor necrosis factor-α), IL-10 (interleukin-10), TLR4 (Toll-like receptor 4), JUN (Jun proto-oncogene), IL-1B (interleukin-1-beta), CYBB (cytochrome b558 heavy chain gene), PTGS2 (prostaglandin-endoperoxide synthase 2), APOE (apolipoprotein E), SPI1 , and MPO (myeloperoxidase) which showed higher degrees.  In total, 31, 2, 7, 7, 5, 13, 93, 11, 29, and 21 active ingredients were identified from Wuzhuyu, Danggui, Chuanxiong, Guizhi, Shengjiang, Banxia, Gancao, Mudanpi, Chishao, and Dangshen, respectively. Additionally, 100 intersected target proteins were revealed by the Venn diagram. Moreover, 98 functional terms and 24 pathways  were enriched. In the PPI network, 10 proteins involved in these five pathways were identified, namely, TNF- The abovementioned genes and pathways might be involved in the pharmacological activity of Wenjing decoction in RSA. Abortion involves pregnancy termination before 28 weeks, with fetal weight below 1000 g . Nearly Some traditional Chinese medicines (TCMs) have shown promise in women with recurrent miscarriage by improving embryo development and live birth rate . The TCMTCM typically has multiple components and multiple targets. The active ingredients and candidate targets can be explored by network pharmacology , 13. In Tetradium ruticarpum) 15 g, Danggui (Angelica sinensis) 10 g, Chuanxiong (Ligusticum chuanxiong hort) 10 g, Guizhi (Cinnamomum cassia Presl) 10 g, Shengjiang  10 g, Banxia (Pinellia ternata) 20 g, Gancao  10 g, Ejiao (Colla corii asini) 10 g, Mudanpi (Paeonia suffruticosa) 10 g, Chishao (Radix Paeoniae Rubra) 10 g, Dangshen (Codonopsis pilosula) 10 g, and Maidong (Ophiopogon japonicus) 30 g. Based on TCM names, the medicinal ingredient information (including molecular weight (MW), molecule name, lipid-water partition coefficient (AlogP), the number of constituents, the number of hydrogen bond donors/acceptors (Hdon/Hacc), blood-brain barrier (BBB), intestinal epithelial permeability (Caco-2), drug-likeness (DL), oral bioavailability (OB), and drug half-life (HL)) was extracted from the Traditional Chinese Medicine Systems Pharmacology  database [The TCM preparation of the Wenjing decoction contained Wuzhuyu (database .The active ingredients were identified using the (ADME) parameters  from thehttp://bionet.ncpsb.org/batman-tcm/) [Using the BATMAN (an-tcm/)  and TCMSan-tcm/)  databasehttp://www.ncbi.nlm.nih.gov/geo/) database. This dataset was deposited by Huang et al. [Q ≤ 20) bases exceeding 40% of the total bases were deleted, and the reads with length <35 were omitted. Using the Tophat Software  [http://subread.sourceforge.net) [https://www.gencodegenes.org/) [The raw data from the GSE121950 was downloaded from the Gene Expression Omnibus  , the clehttp://www.bioconductor.org/packages/release/bioc/html/edgeR.html) [p values and log fold change (FC) values. The mRNAs/lncRNAs with p values of <0.05 and |logFC| > 1 were considered as differentially expressed genes (DEGs). The target protein intersection of the active ingredients and DEGs was analyzed subsequently.Using the edgeR package  , the rawhttp://www.cytoscape.org) [p value ≤0.01 was the threshold for GO_BP enrichment analysis. In addition, the R package clusterProfiler [p value ≤0.01 was set as the threshold.The Gene Ontology (GO)_biological process (BP) enrichment analysis of the intersected target proteins was conducted using the ClueGO + CluePedia plug-in Cytoscape software (ape.org) . The adjProfiler  was usedhttp://string-db.org/) [The interactions among the intersected target proteins were predicted by the STRING database (db.org/) , and thedb.org/) . The reqdb.org/) .The TCM data in the TCMSP database were queried with the keywords to obtain the chemical composition and other relevant information for each TCM. The data showed 176, 125, 189, 220, 265, 116, 280, 55, 119, 134, 55, and 3 constituents in Wuzhuyu, Danggui, Chuanxiong, Guizhi, Shengjiang, Banxia, Gancao, Mudanpi, Chishao, Dangshen, Maidong, and Ejiao, respectively. The constituents were screened further to identify ingredients with the best ADME parameters. There were 31, 2, 7, 7, 5, 13, 93, 11, 29, and 21 active ingredients in Wuzhuyu, Danggui, Chuanxiong, Guizhi, Shengjiang, Banxia, Gancao, Mudanpi, Chishao, and Dangshen, respectively .The BATMAN online tool was used to predict the target proteins for the active ingredients in the TCMSP database, and the target records for 10 herbs, including Wuzhuyu, Danggui, Chuanxiong, Guizhi, Shengjiang, Banxia, Gancao, Mudanpi, Chishao, and Dangshen, were obtained. In total, we identified 933 target proteins of 49 active ingredients from the 10 TCMs after screening the targets with a score larger than 20 in BATMAN.From the raw data of GSE121950, a total of 17163 mRNAs were annotated. Differential expression analysis showed that there were 1371 DEGs (including 453 upregulated and 918 downregulated genes) between RSA and the control samples . The VenFor the 100 intersected target proteins, 98 GO_BP terms were enriched significantly. The pie diagram showed the involvement of 23 different functions, such as “cellular chemotaxis” (13.27%), “reactive oxygen species biosynthetic process” (7.55%), “regulation of nitric oxide biosynthesis” (7.35%), “positive regulation of the vasculature development” (7.35%), “regulation of inflammatory response” (6.12%), and others . In addiα (TNF-α), interleukin-10 (IL-10), Toll-like receptor 4 (TLR4), c-Jun (JUN), interleukin-1β (IL-1B), cytochrome b558 heavy chain gene (CYBB), prostaglandin-endoperoxide synthase 2 (PTGS2), apolipoprotein E (APOE), Salmonella pathogenicity island 1 (SPI1), and myeloperoxidase (MPO), from the PPI network were screened  in RSA development was shown before, and the serum TLR4 and CCL2 levels in pregnant women could serve as RSA indicators [TLR4 and increased TLR2 induced by ligand treatment of spermatozoa influenced unexplained RSA [TNF-α, IL-10, and TLR4 might be targets for Wenjing decoction.The PPI network of the intersected target proteins, TNF-tibility . TNF-α o for RSA , 26. Thegnancies . The invdicators . Reducedined RSA . TherefoIL-1B (C-511T) polymorphism may promote the development of recurrent miscarriages, while the IL-6 (G-634C) polymorphism may reduce its risk [PTGS2, might play a pharmacological role in treating blood stasis syndrome [MPO, are higher in women with RPL exhibiting metabolic syndrome, and the oxidative stress, subclinical inflammatory state, and metabolic syndrome contribute to pregnancy outcome prediction in women with RPL [IL-1B, PTGS2, APOE, and MPO might also be targeted by Wenjing decoction.The its risk . Dangguisyndrome . Apo E3/syndrome . The levwith RPL , thus suα, IL-10, TLR4, JUN, IL-1B, CYBB, PTGS2, APOE, SPI1, and MPO were involved in the pharmacological network pathways and might be responsible for the pharmacological activity of Wenjing decoction.Enrichment analysis of the 100 intersected target proteins showed enrichment of “AGE-RAGE signaling pathway in diabetic complications,” “TNF signaling pathway,” “osteoclast differentiation,” “C-type lectin receptor signaling pathway,” “chemokine signaling pathway,” “leukocyte transendothelial migration,” and “MAPK signaling pathway.” A previous study showed the contribution of the C-type lectin receptors in tailoring the adaptive responses and innate recognition, and several C-type lectin receptors function in protective antifungal immunity . Systemiin vitro or in vivo studies are still warranted to investigate the effects of Wenjing decoction on RSA.This study is a preliminary research to screen the primary targets of Wenjing decoction. RSA is a serious disease involving fetus/baby and mother. Misinformation about RSA may lead to more RSA. Therefore, the results of this study should be interpreted cautiously. Further clinical studies as well as TNF-α, IL-10, TLR4, JUN, IL-1B, CYBB, PTGS2, APOE, SPI1, and MPO pathways might be targeted by the Wenjing decoction. Furthermore, “C-type lectin receptor signaling pathway,” “chemokine signaling pathway,” “leukocyte transendothelial migration,” “TNF signaling pathway,” “MAPK signaling pathway,” and “AGE-RAGE signaling pathway in diabetic complications” might correlate with the pharmacological activity of Wenjing decoction. However, the genes and pathways involved and their significance in women with RSA treated by Wenjing decoction should be explored further.We selected 100 intersected target proteins of the active ingredients by cross-validation. During RSA, the


Proliferative diabetic retinopathy (PDR) is a sight-threatening diabetic complication in urgent need of new therapies. In this study we identify potential molecular mechanisms and target candidates in the pathogenesis of PDR fibrovascular tissue formation. We performed mRNA sequencing of RNA isolated from eleven excised fibrovascular membranes of type 1 diabetic PDR patients and two non-diabetic patients with rhegmatogenous retinal detachment with proliferative vitreoretinopathy. We determined differentially expressed genes between these groups and performed pathway and gene ontology term enrichment analyses to identify potential underlying mechanisms, pathways, and regulators. Multiple pro-angiogenic processes, including VEGFA-dependent and -independent pathways, as well as processes related to lymphatic development, epithelial to mesenchymal transition (EMT), wound healing, inflammation, fibrosis, and extracellular matrix (ECM) composition, were overrepresented in PDR. Overrepresentation of different angiogenic processes may help to explain the transient nature of the benefits that many patients receive from current intravitreal anti-angiogenic therapies, highlighting the importance of combinatorial treatments. Enrichment of genes and pathways related to lymphatic development indicates that targeting lymphatic involvement in PDR progression could have therapeutic relevance. Together with overrepresentation of EMT and fibrosis as well as differential ECM composition, these findings demonstrate the complexity of PDR fibrovascular tissue formation and provide avenues for the development of novel treatments. Proliferative DR (PDR) is the end-stage disease, with hypoxia-induced pathological neovascularization at the vitreoretinal interface, often accompanied by vitreous haemorrhage (VH), tractional retinal detachment (TRD) and diabetic macular oedema (DME). PDR progression involves a complex interplay of biochemical, immunological and inflammatory factors, but the exact mechanisms remain unclear2.Diabetic retinopathy (DR) is a sight-threatening microvascular complication of diabetes, developing in nearly all patients with type 1 diabetes (T1D), and in majority of patients with T2D3, but global RNA expression of PDR tissues by sequencing has not been investigated.Panretinal photocoagulation (PRP) and intravitreal anti-VEGFA injections are used to treat DR complications, but often to poor or transient effects. Vitrectomy is performed in cases of prolonged VH and/or TRD, and of combined tractional and rhegmatogenous retinal detachment. Microarray analysis has previously revealed gene expression differences in PDR tissues compared to normal human retina4, abnormal wound healing where proliferative and contractile membranes form on the retina upon ischemia, cell death and blood-retinal barrier breakdown due to RRD. RPE cells and glial cells are major contributors to these membranes, but exact pathological mechanisms remain unclear5. RPE cells undergo epithelial-to-mesenchymal transition (EMT), though other cell types like hyalocytes and glial cells also take part in extracellular matrix (ECM) deposition and fibrosis6.Rhegmatogenous retinal detachment (RRD), detachment of the neurosensory retina from the retinal pigment epithelium (RPE), occurs when fluid enters and accumulates in the subretinal space after e.g. retinal tears upon trauma. 5–11% of RRD cases develop proliferative vitreoretinopathy (PVR)We here performed bulk RNA sequencing to determine gene expression patterns in PDR fibrovascular tissues and avascular fibrous RRD-PVR tissues in order to better understand the mechanisms of these sight-threatening diseases and identify potential therapeutic targets.To analyse gene expression in PDR, we sequenced mRNA isolated from fibrovascular membranes of PDR patients n = 11) and mRNA from avascular fibrotic tissues of non-diabetic RRD-PVR patients (n = 2) and compared their transcriptomes . Of these, 910 were upregulated and 537 downregulated in the PDR cohort  analysis, the PDR patients segregated from the RRD-PVR patients along PC1 which explained 52.6% of the variation Fig. , indicatLymph vessel development, Lymphangiogenesis, Lymph vessel morphogenesis and Lymphatic endothelial cell differentiation were also significant , 53 cellular components (CCs) and 37 molecular functions (MFs) were significant in GOEA Fig. A-C, as want Fig. A. Enrichant Fig. B-C. KEGGant Fig. D.Figure VEGF Family Ligand-Receptor Interactions and Nitric Oxide Signalling in the Cardiovascular System, pathways involved in inflammation and wound healing, such as Leukocyte Extravasation Signalling, IL-8 Signalling and STAT3 Pathway, and EMT-related pathways such as Regulation of the EMT in Development Pathway. In addition to pro-angiogenic pathways, Inhibition of Angiogenesis by Thrombospondin 1 (TSP1) was predicted to be activated  of all DEGs identified 40 significantly activated IPA canonical pathways Fig. A. These To investigate potential factors regulating the observed gene expression changes, we performed IPA upstream regulator analysis (URA) and identified 21 activated upstream regulators (URs)  signalling pathways were also predicted to be activated  promotes blood vessel maturation and stability via signalling through tyrosine kinase with immunoglobulin and epidermal growth factor homology domains (TIE)-2 receptor8. ANGPT2/TIE2-signalling induces vessel destabilization. Both ANGPT1 and ANGPT2 were upregulated in PDR, as were TIE1 and TIE2 /Notch-1 signalling by inducing Wnt/β-catenin pathwayted Fig. A-B. Notc8). The hepatocyte growth factor (HGF)/cMet pathway has been suggested as an anti-VEGFA therapy escape mechanism9. HGF was upregulated in PDR. Though the MET gene was downregulated, within this group its expression was highest in the anti-VEGFA-treated patient , receptor found on proliferating ECs and involved in TGF-β–mediated neovascularization, was upregulated in PDR  was upregulated, as was TBX1, which can activate VEGFR3 transcription in ECs . The poorly studied hypoxia-inducible transmembrane protein 204 (TMEM204), which can interact with VEGFR2 and -3 to regulate lymphatic vessel development12, was also upregulated in PDR . Cell movement of lymphatic system cells, Migration of lymphatic system cells, Lymphangiogenesis and Proliferation of lymphatic endothelial cells were significantly increased functions in IPA downstream effects analysis  precursors, as are SOX18 and NR2F2. NR2F2 and SOX18 induce PROX1 expression in LEC specificationPDR Fig. A. NucleaPDR Fig. B. NR2F2 sis Fig. A-B.15. Though not a DEG, MAFB was predicted an activated UR , Prox-1 and Klf4 expression UR Fig. . Variabi5. GO-terms Chemotaxis, Leukocyte migration, Response to wounding, Immune system development, Inflammatory response, Coagulation and Platelet activation, as well as KEGG pathways Platelet activation and Leukocyte transendothelial migration were overrepresented based on DEGs upregulated in PDR , TRPM1 and OTX2 (bipolar cells21), TMEM119 (microglia22), SNCG , and GNGT1 (photoreceptor cells23).Since RPE cells are known to contribute to RRD-PVR pathogenesis24. DEGs were present in all categories, and included both upregulated and downregulated genes, suggesting diverse fibrotic mechanisms in both diseases 36, recently approved in the US for treatment of DME and wet age-related macular degeneration (AMD)37. ALK-1 involvement in angiogenesis is not fully understood, but combined targeting of VEGFR and ALK-1 has been suggested for inhibition of anti-VEGFA-resistant angiogenesis in cancer8.As expected, multiple angiogenic pathways were overrepresented in PDR. Rather than low absolute expression in PDR tissues, the observed downregulation of VEGFA in PDR is likely to reflect prominent VEGFA level in RRD-PVR tissues, as VEGFA is involved in PVR pathogenesis16, we identified overrepresentation of lymphatic development-related GO-terms and pathways. Our results also show upregulation of VEGFR3, TBX1, SOX18 and NR2F2 in PDR, consistent with LEC programming. PROX1 and LYVE1 were not differentially expressed, but had variable expression among PDR patients, consistent with our previous findings16. Lymphatic marker podoplanin is not expressed in endothelium in PDR tissues16, and is also expressed in RPE cells38. Therefore, the upregulation of podoplanin in RRD-PVR likely reflects abundance of RPE cells in RRD-PVR tissues.Consistent with our previous results29. Myofibroblasts that originate from endothelial-to-mesenchymal transition (EndMT) are suggested to contribute to fibrosis in PDR fibrovascular membrane formation, and factors that regulate EMT can also regulate EndMT39. Therefore, the observed overrepresentation of EMT processes in PDR may result from the presence of vasculature. Mesenchymal cell differentiation was also implicated in RRD-PVR, consistent with fibrotic tissue formation via EMT from RPE cells6. Fibrosis also involves ECM deposition, and we identified differential matrisome gene expression between PDR and RRD-PVR. In the future, in-depth analysis of the ECM composition can help define the complex cell-ECM interactions and the physical microenvironment within fibrotic tissue formation.Inflammation, fibrosis, and wound healing are relevant to both RRD-PVR and PDR40 and found GO-terms related to inflammation, immune response, wound healing, and coagulation significantly enriched in PDR. This is consistent with the enriched GO-terms and transcriptional pathways found here in the PDR tissues. As the proteome of a tissue may exhibit variability not detectable in its transcriptome, comparative validation of both the proteome and transcriptome of the pathological PDR tissues remains of future interest.Our current study is restricted to analyses of mRNA expression. Previously, we compared the proteomes of PDR and DR vitreousIn absence of healthy control tissue, the avascular RRD-PVR tissues enable us to explore the PDR transcriptome, especially with regards to the PDR vascular aspects. Expression patterns related to inflammation, fibrosis, and wound healing found in our data instead reflect the specific differences between these mechanisms central to both RRD-PVR and PDR. Nevertheless, this comparison can serve as a basis for future individual investigations of the complex fibrotic/fibrovascular and inflammatory mechanisms behind the pathological tissue formation in these two sight-threatening vitreoretinal eye diseases.Bulk RNA sequencing provides tissue-level gene expression data. Approaches such as single-cell RNA sequencing and spatial transcriptomics will be instrumental to define the tissue heterogeneity and cell-specific expression patterns in fibrovascular PDR formation and avascular RRD-PVR progression.The small cohort size is another limitation in our current study. For instance, the cohort included only one anti-VEGFA (Ranibizumab) treated patient, ruling out statistical analyses related to this treatment. This patient showed, however, an interesting expression profile suggestive of putative therapy escape mechanisms and further studies will be of interest to investigate the impact of anti-VEGFA and other treatments, e.g. statins, in larger cohorts.In conclusion, our findings on fibrotic, fibrovascular and inflammatory changes and potential therapy escape mechanisms open new avenues for the study of PDR and RRD-PVR progression and for the development of novel therapies. Further investigation of these processes may also help to understand other devastating posterior-segment vitreoretinal diseases where similar pathological mechanisms are involved, such as wet-AMD and retinopathy of prematurity.Patients were enrolled in the tertiary care ophthalmology clinic, unit of vitreoretinal diseases, at Helsinki University Hospital (HUH) Eye Clinic in 2010–2019. Signed informed consent was obtained from each patient. The study was approved by the Institutional Review Board and Ethical committee of HUH and conducted according to the Declaration of Helsinki.10. The indications for vitrectomy were active PDR, with or without TRD threatening or involving the macula, or severe non-clearing VH. For our bulk RNA cohort, 11 PDR membranes were excised from 11 vitrectomized PDR eyes of 11 patients using segmentation and delamination, cut when needed with microscissors, and removed from the vitreous cavity with intraocular end-gripping microforceps (MaxGrip Alcon Laboratories). The PDR patients had type 1 DM. Of the 11 PDR patients, one received pre-operative intravitreal VEGFA and all except one received previous photocoagulation. Summary baseline characteristics of patients can be found in Supplementary Table 1, and individual characteristics in Supplementary Table 2.Transconjunctival microincision vitreoretinal surgery by 23- or 20-gauge three-port pars plana vitrectomy was performed as previously described16.For bulk RNA sequencing, excised tissues were immersed in RNA stabilization reagent  and stored at − 80 °C until processing. Tissue samples utilized for immunohistochemistry were obtained and processed as previously described10.The RNeasy Fibrous Tissue Mini kit (Qiagen) was utilized for total RNA extraction, using ceramic beads  and TissueLyser-II for homogenization of tissue. Preparation of total RNA, washes and elution were performed according to kit protocol. The Agilent Bioanalyzer RNA-nano chip (Agilent) was used for evaluation of RNA integrity, and Qubit RNA kit (Life Technologies) for RNA quantification. Extracted RNA was stored at − 80 °c until library preparation, evaluation and sequencing, which were performed as previously describedMouse monoclonal antibodies against CD31 , Notch-1 , SOX18  and CD45  and rabbit polyclonal antibodies against CD68  were used.10. Heat-induced antigen retrieval  was used. Images were taken using 3DHISTECH Pannoramic 250 FLASH II digital slide scanner.Immunohistochemistry (IHC) was performed as previously described41. Gene ontology (GO)-term and Kyoto Encyclopaedia of Genes and Genomes (KEGG) enrichment analysis42 was performed with The Database for Annotation, Visualization and Integrated Discovery (DAVID) Bioinformatics Resources 6.844, and R Studio 41. P values were adjusted using FDR with a 0.05 cut-off. Pathway analysis was generated through the use of Ingenuity Pathway Analysis  on the DEGs.Differential expression analysis was performed using HiSeq 2000 platform (Illumina), Lexogen Quantseq 2.2.3 FWD pipeline on BlueBee genomics cloud-based analysis platform. Hierarchical clustering was performed based on the differentially expressed genes  < 0.05), and principal component (PC) analysis on the 500 most variable genes, using R Studio Supplementary Information 1.Supplementary Information 2.Supplementary Information 3.Supplementary Information 4.Supplementary Information 5.Supplementary Information 6.Supplementary Information 7.Supplementary Information 8.


Glycine max) productivity. Breeding soybean for tolerance to high salt conditions is therefore critical for increasing yield. To explore the molecular mechanism of soybean responses to salt stress, we performed a comparative transcriptome time-series analysis of root samples collected from two soybean cultivars with contrasting salt sensitivity.Soil salinity is a primary factor limiting soybean  showed more differential expression of genes than the salt-sensitive cultivar ‘Dong Nong No.50’ (DN50). We identified 17,477 genes responsive to salt stress, of which 6644 exhibited distinct expression differences between the two soybean cultivars. We constructed the corresponding co-expression network and performed Gene Ontology term and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis. The results suggested that phytohormone signaling, oxidoreduction, phenylpropanoid biosynthesis, the mitogen-activated protein kinase pathway and ribosome metabolism may play crucial roles in response to salt stress.Our comparative analysis offers a comprehensive understanding of the genes involved in responding to salt stress and maintaining cell homeostasis in soybean. The regulatory gene networks constructed here also provide valuable molecular resources for future functional studies and breeding of soybean with improved tolerance to salinity.The online version contains supplementary material available at 10.1186/s12870-022-03541-9. Of the current 230 million hectares (ha) of irrigated land, 45 million ha (19.5%) are estimated to be affected by high salt; similarly, 32 million ha (2.1%) of the 1500 million ha used for dryland agriculture are affected by varying degrees of salt stress ). For GO functional enrichment, we focused on Biological Process. For both GO and KEGG enrichment analysis, the minimum number of genes was set to five for a GO term to be significantly enriched, and groups with > 50% overlap were merged. GO term/pathway network connectivity (kappa score) was set to Medium (0.5). DEGs were classified into two groups: one group containing DEGs annotated as salt-responsive shared by both QH34 and DN50, and the other containing time point-specific genes for QH34. For both groups, GO function and KEGG pathway enrichment analysis were independently performed for each gene network cluster.To construct gene co-expression networks and study the coordination of gene expression under salt stress, a raw gene count table generated from FeatureCounts was normalized by median normalization using the EBSeq R package , then Loe igraph . The vise igraph   synthesis kit . qPCR was performed on a Step One Plus™ Real-Time PCR system  using ChamQ Universal SYBR qPCR Master Mix . Three independents biological replicates were analyzed. Samples used for RNA extraction were collected from plant roots treated as mentioned above.https://crm.vazyme.com/cetool/singlefragment.html) and the coding sequences (Williams 82) were amplified. The resulting PCR products were ligated into the 35S-pBI121 (kanamycin resistance) vector and transformed into Escherichia coli DH5α by freeze-thaw method. Plasmids with the correct insert and free of mutations were introduced into Agrobacterium (Agrobacterium rhizogenes) strain K599 (streptomycin-resistant) using the freeze-thaw method. K599 colonies with the target construct were used to inoculate 10 mL YEP liquid medium and incubated at 28 °C for 10 h with shaking (220 rpm). After 10 h, 100 μL bacterial culture was spread onto a YEP solid plate and cultured in an incubator (28 °C) for 2 d for subsequent hairy root experiments.The seven genes were filtered for RT-qPCR validation based on their functional annotations, expression patterns, respective pathways and the data from literature reports , 61–64. OE) and evaluated their salt sensitivity by growth in half-strength Hoagland’s solution with or without added NaCl, side by side with control hairy roots transformed with empty vector (EV). The salt-sensitive cultivar DN50 was used for functional validation by overexpressing the candidate genes. DN50 was sown in soil . Six days after sowing, the tip of a 1-mL medical syringe was used to puncture four wounds 0.5–1 cm below the cotyledons in a cross pattern. Then, cultures from the relevant Agrobacterium colonies were smeared onto the four wounds with pearl wool. The seedlings were covered with a clear plastic lid to maintain high humidity. Six days later, the plastic cover was removed and the infected area was covered with vermiculite to maintain humidity. Then, 14 d later, the taproot was cut off when the hairy root was ~ 5–10 cm in length and cultured in half-strength Hoagland’s solution (pH 6.0) for 3 d to adapt environment, and consistent seedlings were selected for salinity treatment of 3 d with 100 mM NaCl; controls received no NaCl. The root length before and after salt treatment was measured and the root relative elongation was calculated to evaluate salt tolerance. In addition, about 35 d after Agrobacterium infection, overexpressing hairy root plants and the control were transplanted into mixed soil. After 3 d, the hairy root plants were irrigated with half-strength Hoagland’s solution containing 200 mM NaCl, or half-strength Hoagland’s solution without NaCl as the control group. Each plant was irrigated with 50 mL solution every 2 d. After 12 d of salt treatment, the above-ground phenotypes were determined.We generated transgenic hairy roots overexpressing the target gene (Additional file 1: Fig. S1. The measurement of salt tolerance in 22 soybean accessions.Additional file 2: Fig. S2. Evaluation of RNA-seq data from the two soybean cultivars DN50 and QH34.Additional file 3: Fig. S3. Heatmap of negative regulation abscisic acid activity signaling pathway.Additional file 4: Fig. S4. Heatmap of oxidoreductase activity signaling pathway.Additional file 5: Fig. S5. Heatmap of negative regulation of proteolysis signaling pathway.Additional file 6: Fig. S6. GO enrichment analysis for uDEGs.Additional file 7: Fig. S7. KEGG pathway analysis of uDEGs.Additional file 8: Fig. S8. Pathway analysis of gene network clusters for sDEGs.Additional file 9: Fig. S9. Heatmap of hormone signaling pathways.Additional file 10: Fig. S10. Heatmap of MAPK signaling pathway.Additional file 11: Fig. S11. Heatmap of phenylpropanoid biosynthesis signaling pathway.Additional file 12: Fig. S12. Heatmap of ribosome metabolism biosynthesis signaling pathway.Additional file 13: Table S1. The source information of 22 soybean germplasms.


Major Depression Disorder (MDD) is a common mental disease that has become one of the world's major medical diseases. Currently, the Fructus Aurantii (FA) has been widely used to treat depression. However, the active substance ingredients and potential mechanisms of the shell antidepression have not yet been clarified.  First, we used ultraperformance liquid chromatography-quadrupole/time-of-flight tandem mass (UPLC-QTOF-MS/MS) technology to identify the chemical composition of the FA. Then, it is predicted for active ingredients, pharmaceutical disease target screening by DiscoveryStudio 2016 (DS), Metascape, and other databases, PPI network diagram, and FC core pathway. Finally, the system network pharmacology results are verified by molecular contact verification.  Forty-six compounds in FA were identified, and twelve active ingredients were determined. Various database information, PPI network analysis of 41 intersections, and 20 core targets including DRD2, MTOR, FASP3, and PIK3P1 were integrated. Finally, the MDD treatment is indicated by molecular docking, and the most relevant potential signal pathway is the PI3K-Akt signaling pathway. Major Depression Disorder (MDD) is the most common chronic mental disease nowadays, mainly manifested as depression, loss of pleasure, cognitive impairment, panic, lack of initiative, anorexia, insomnia, suicide injury, and other behaviors. Therefore, it is difficult to treat this disease due to its complexity, heterogeneity, and high recurrence –4. Many Therefore, in this work, the multicomponent approach used by the traditional Chinese medicine was compared to the traditional treatment of MDD in consideration of the shortcomings of western medicine.Citrus Aurantium L. It often used as a Chinese medicine for regulating qi and used in clinical practice and is one of the components of the most common Chinese medicine prescriptions, including Chaihu Shugan San and Weichang Anwan [Fructus Aurantii (FA) is the dry immature fruit of the ng Anwan , 9. The ng Anwan , lower tng Anwan , have anng Anwan , promoteng Anwan , and havng Anwan . HoweverThe FA is mainly produced in the province of Jiangxi, and it is of the highest quality. Therefore, this experiment used FA as raw material, and the chemical composition was determined by UPLC-QTOF-MS/MS. In addition, the active ingredient, disease target, and the action pathway were investigated to obtain the underlined mechanism used by FA on MDD, which was further verified by molecular docking –17.FA was purchased in Yicheng Town, Zhangshu City, Jiangxi Province .Citrus aurantium L. Twelve pure compounds (purity >98%) such as eriodictyol, 5-demethylnobiletin, naringenin, nobiletin, 3′, 4′, 3, 5, 6, 7, 8-heptamethoxyflavone, auraptene, ferulic acid, umbelliferone, hesperidin, naringin, neohesperidin, and limonin were selected according to the 2015 “Chinese Pharmacopoeia” method to process FA and purchased from Sichuan Vicky Biotechnology Co. Ltd. . Methanol was purchased from Xilong Scientific Co, Ltd. . Acetonitrile , formic acid (ACS), and ethanol were purchased from the National Medicine Group Chemical Reagent Co., Ltd. . The water used in this work was ultrapure, obtained by the Milli-QB system.Professor Geifei, who is the head of the Chinese Medicine Resources Discipline Group, at the Jiangxi University of Traditional Chinese Medicine characterized the dry unripe fruit of The impurities were removed from FA, then it was washed, moisturized, and cut into thin slices, and the broken core was sifted out after drying . FA crudμm microporous membrane to obtain the standard solutions.Twelve milligrams of each reference compound  was weighed, transferred to 10 ml volumetric flasks, and diluted with methanol to reach the volumetric mark. The solutions were filtered through a 0.22 μm) was used to perform the chromatographic separation with a flow rate of 0.3 ml/min at 40°C. A linear gradient program with a mobile phase system including solvent A  and solvent B  was performed as follows: solvent A at 5% ∼20% for 0.01∼2 min, 20%∼30% for 2∼10 min, 30∼55% for 10∼25 min, 55%∼100% for 25∼30 min, 100% A for 30∼32 min, and 100%∼5% for 0.5 min, with isocratic elution performed at 5% for 2.5 min.Chemical analysis was conducted on a UPLC-  hybrid triple quadrupole-time-of-flight mass spectrometer  connected with an electrospray ionization source (ESI). Acquity UPLC BEH C18 column  and gas 2 (GS2) were both set to 60 kPa, curtain gas (CUR) was set to 35 kPa, ion spray voltage floating (ISVF) was set to 5500 V, ion source temperature (TEM) was set at 500°C, collision energy (CE) was set at 45 eV, collision energy spread (CES) was set at 45 ± 10 eV, the declustering potential (DP) was set at 100 V, and nitrogen was used as a nebulizer and auxiliary gas. Samples were analyzed in positive ionization modes with a sFAnnin mass-to-charge (m/z) range from 50 to 1,000. Data were collected in information-dependent acquisition (IDA) mode and analyzed by PeakView®1.2 software (AB Sciex).The chemical composition of FA was obtained from existing databases and documents, including TCMSP, SCHINDER, TCMIP, China Knowledge Network, and Geen Medical. The data were sorted out and the FA composition database was established. (+) MS data was imported into PeakView®1.2, “XIC Manager” was used to analyze the 70% ethanol extract of FA, and each chemical composition in the retention time and its corresponding primary and secondary mass spectrum data were obtained. It combined the base peak chart with the reference substance and compared it with the data reported in the relevant references, and the chemical composition was confirmed.https://www.swisstargetprediction.ch/) and Pharmmapper (https://www.lilab-ecust.cn/pharmmapper/).In this study, the chemical compounds were imported into Discovery Studio 2016 (DS), through “ADMET Descriptors” to screen the active ingredients. The related targets of FA components were obtained from SwissTargetPrediction (https://www.geneFArds.org/) and Online Mendelian Inheritance in Man  to identify disease targets associated with MDD.The keyword “depression” was used in the Genecards database (https://bioinfogp.cnb.csic.es/tools/venny/index.html) was used to obtain the overlapping targets between FA-related targets and MDD-related targets. Overlapping targets were imported into the Retrieval of Interacting Genes/Proteins (STRING) 11.0 (https://www.string-db.org/), and the interaction results were saved. The interaction resulting file was added to Cytoscape v3.9.0 software, and the Protein-Protein Interaction (PPI) network was obtained.VENNY2.1 software , “Homo sapiens” was selected as “Organism of interest,” and “Pathway” and “KEGG” were selected as “Functional Database.” Finally, “genome” was selected as the “Select reference set,” and the KEGG pathway analysis was performed.Overlapping targets were imported into Metascape . The protein structure was used to simulate dehydration, hydrogenation, and removal of proteins using DiscoveryStudio2016 (DS) software, and then molecular docking was performed.The computational software was used to simulate the interaction between active compounds and core targets and to explore the binding ability and binding mode between compounds and targets. Therefore, 12 drug components were selected for the molecular docking with 14 core targets. The PDB format of the target protein was downloaded from the structural bioinformatics protein database  is an important structure in the human body to maintain a stable brain environment; thus, lipophilic and water-soluble drugs do not cross easily the BBB, limiting the effect of 95% of drugs on the central nervous system . DepressChina Pharmacopoeia.” In addition, according to the literature, meranzin hydrate and nobiletin can reduce ROS, MDA, IL-6, and TNF-α levels in MDD rats; these compounds can cause anti-inflammatory and antioxidative stress [Forty-six compounds were screened by DS software, and the screening standard used was “ADMET_BBB ≥ −0.3” . Seven ae stress . Naringie stress . Hesperie stress . AlthougAll the targets of the compounds in FA were collected from the SwissTargetPrediction software and Pharmmapper software. A total of 827 FA-related targets were obtained after the removal of the repeated targets.The screening standard used was “Relevance score>10” , which aThe disease targets and composition targets were combined and imported into Venny2.1 software, and 41 overlapping targets were obtained by the Venny diagram  and PPI WebGestalt and Metascape were used to perform Go function and KEGG pathway enrichment analysis on 20 core targets. The results showed the involvement of 12 Biological Processes (BP), 16 Cellular Components (CC), and 13 Molecular Functions (MF). BP was related to cell communication, response to stimulus, and biological regulation, CC was related to membrane, cell projection, and endomembrane system, and MF was related to protein binding, molecular transducer activity, and ion binding .There is a close relationship between the core target and the signaling pathway. The screening results with “value of enrichment ratio” resulted in 10 pathways, including EGFR tyrosine kinase inhibitor resistance (hsa04080), ErbB signaling pathway (hsa04072), thyroid hormone signaling pathway (hsa04919), PI3K-Akt signaling pathway (hsa04151), and neuroactive ligand-receptor interaction (hsa04722). Some antidepressants exert a therapeutic effect through the ErbB signaling pathway and often induce the downregulation of NRG1/ErbB4; thus, our hypothesis was that antidepressants could play a role through this channel . TherefoCytoscape v3.9.0 software was used to build an “ingredients-disease-gene symbols” network, as shown in Cytoscape software analysis revealed that the values of C4 , C1 , C5 , C2 , and C7  were at the top of the list, meaning that these compounds could be the core components of FA effective in the treatment of MDD.The binding ability of the receptor to the ligand is closely related to the molecular docking score. The higher the score, the stronger the binding ability of the receptor. The docking scoring results showed that most of the receptors had good binding ability to the ligands except ABL1 and FASP3 genes, which had low docking scores . TherefoBased on these data, we found that the interaction between each component and the core target is the basis of biological activity. Therefore, FA in the treatment of MDD is a complex process among a series of multitargets and multicomponents.MDD is a common mental disease that has become one of the world's major diseases, resulting in the change of the quality of life and work ability. The number of patients increases year by year. At present, most of the patients suffering from clinical depression are treated by the western medicine, which commonly use selective serotonin reuptake inhibitors (fluoxetine and paroxetine) and serotonin and norepinephrine reuptake inhibitors (venlafaxine and duloxetine). However, many problems are associated with the use of the western medicine, such as high recurrence rate, poor tolerance, and adverse reactions. The treatment using traditional Chinese medicine is gradual, and the existing research results show that the curative effect of the Chinese medicine does not differ significantly from that of the western medicine. Nevertheless, the Chinese medicine has a faster effect, less adverse reactions, and more benefits. Therefore, the research on MDD of traditional Chinese medicine has been more intense and fruitful in recent years.FA is one of the commonly used medicinal compounds, which is widely used to cure gastrointestinal diseases. Previous studies showed that MDD often leads to gastrointestinal dysfunction, and the FA extract can regulate the HPA axis  and neurUPLC-Q-TOF-MS/MS technology was used to identify FA chemical components. Then, the combination of the literature with the related software resulted in a selection of 12 main active ingredients. Finally, the overlapping target was used to perform the PPI network analysis. DRD2, DRD4, OPRM1, and mTOR were the most relevant targets for the treatment of MDD by FA. DRD2 is divided into two subtypes, D2S and D2L , it is aMetascap was used to perform the pathway analysis on 20 core targets. The results showed that FA mainly activates and regulates PI3K-Akt signaling pathway (hsa04151). EGFR tyrosine kinase inhibitor resistance (hsa04080) and ErbB signaling pathway (hsa04072) exert pharmacological effects. The feature to pay attention to is PI3K-Akt signaling pathway, Akt is upstream of the mTOR signaling pathway, and the phosphorylated form can activate the mTOR signaling pathway. Key targets for PI3K/Akt signaling pathway influence the glutamic acid system function, hippocampal neuron apoptosis, and mitochondrial function involved in MDD. Many studies show the main role of TCM as an antidepressant by acting on the PI3K/Akt/mTOR signaling pathway. Caihujialong Tang increases the expression and activity of PI3K in the rat hippocampus to protect neurons, thus exerting an antidepressant effect . JiaotaiFA in China is often used as a hydraulic medicine, commonly used to cure gastrointestinal diseases, but the literature does not give any explanation regarding its therapeutic effects on MDD, such as the mitigation of diarrhea caused by MDD. Therefore, our results by UPLC-Q-TOF-MS/MS technology and system pharmacology revealed a potential mechanism of FA to treat MDD, providing a reference for the development and use of antidepressants.


This study aimed to investigate the possible mechanism of the Zhishi and Baizhu herb pair in the treatment of gastric cancer by means of network pharmacology and molecular docking and to provide a theoretical basis for experiments and clinical application of traditional Chinese medicine for treating gastric cancer.  The main active chemical components of Zhishi and Baizhu were screened through Traditional Chinese Medicine Systems Pharmacology (TCMSP) database and selected by using the thresholds of oral bioavailability ≥30% and drug-likeness ≥18%. The targets of Zhishi and Baizhu were obtained from TCMSP, Therapeutic Targets Database (TTD), and the DrugBank database. The corresponding genes of the targets were retrieved from the UniProt database, and the gastric cancer targets were obtained from the GeneCards database and TTD. Subsequently, the networks were built between the main drug components, drug targets, and gastric cancer targets. Then, the enrichment analyses of GO and KEGG were applied to predict the potential roles of gastric cancer pathogenesis via the R package clusterProfiler. Finally, molecular docking was used to determine the affinity between the targets and components.  Twenty-seven main active components were predicted from the Zhishi-Baizhu herb pair, and a total of 120 intersection genes were screened from 303 potential medicine genes and 1,839 disease genes. The enrichment included the PI3K-Akt and IL-17 signaling pathways, and the network analysis showed that the Zhishi-Baizhu herb pair acted on seven key targets, namely, AKT1, MMP9, IL-6, CCND1, BCL2, MTOR, and MDM2 (where they played a role in treating gastric cancer). Molecular docking showed that luteolin and naringenin could stably bind to the targets.  The possible mechanisms of the components of the Zhishi-Baizhu herb pair in treating gastric cancer might be related to luteolin and naringenin, which intervened with the targets AKT1, MMP9, IL-6, CCND1, BCL2, MTOR, and MDM2, and are linked with the PI3K-Akt and IL-17 signaling pathways. This knowledge will lay a solid foundation for further experimental and clinical studies. Gastric cancer (GC) is a common malignant tumour in the world today and ranks third in fatalities and fifth in prevalence among malignant tumours ; it is tCurrently, surgical resection, perioperative adjuvant or neoadjuvant radiotherapy, immunotherapy, molecular targeted agents, and chemotherapy are the most effective treatments for this disease –5. HowevIn recent years, the position of Chinese medicine in treating GC has also become increasingly crucial. As reported in previous studies, Chinese medicine can effectively inhibit the invasion and metastasis of GC cells and greatly improve the prognosis of GC patients, subsequently improving the quality of the lives of patients , 9.In traditional Chinese medicine, GC belongs to the category of gastralgia, dysphagia, stomach reflux, abdominal mass, and others . Its maiAtractylodes macrocephala Koidz.) has been frequently used for treating digestive diseases. This pairing was found by a famous Chinese physician named Zhang Zhongjing in the Eastern Han Dynasty [The herbal pairing of Zhishi (Aurantii Fructus Immaturus) and Baizhu  and a bitter, pungent, and acidic flavour, with a meridian tropism in the stomach and spleen. It is used for treating abdominal mass, dissipating phlegm, resolving mass issues, and settling stomach-Qi , 16. As Baizhu was first discovered in Shennong's Classic of Materia Medica as an upper grade drug; it has a warm nature and a bitter, sweet taste, with a meridian tropism in the stomach and spleen. It can invigorate the spleen and replenish Qi, induce diuresis, stop perspiration, dry dampness of the body, and calm a foetus . The detThe compatibility of Zhishi and Baizhu is a classical prescription and the pairing had long been used to cure internal-injured spleen-stomach diseases , and theNetwork pharmacology and molecular docking are new technologies based on systems biology and database molecular correlation analysis for the exploration of new drugs and prediction of their related targets . Networkhttps://tcmspw.com/tcmsp.php) database [https://bidd.nus.edu.sg/group/ttd/ttd.asp) [https://www.drugbank.ca) [https://www.uniprot.org/) [Homo sapiens”), and the GC-related human genes were downloaded from the GeneCards database (https://www.genecards.org/) [https://bioinfogp.cnb.csic.es/tools/venny/index.html) [The main chemical compounds of the Zhishi (ZS) and Baizhu (BZ) herb pair were acquired from the Traditional Chinese Medicine Systems Pharmacology   and combbank.ca) . Subsequot.org/)  to screeds.org/)  and TTD ex.html) .https://string-db.org) [Homo sapiens” from species, and the compound-target-disease network was built and displayed using Cytoscape 3.6.1 software (https://cytoscape.org/) [We inputted these overlapped targets into STRING tools (-db.org)  to constpe.org/) . We usedpe.org/) .Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses of potential targets were performed using the R package.P value < 0.05 as the condition to visualise the results. Three selected analysis items were selected for GO enrichment analysis: molecular functions, cell components, and biological processes. Thus, we obtained the important terms of enriched GO terms and KEGG pathways to study the specific mechanism of the ZS-BZ herb pair against GC.ClusterProfiler was used to search the related functions and pathways ; we set https://www.rcsb.org/) [Homo sapiens” and refined at resolutions of 2.0–3.0 Å [The 3D structures of the target proteins were downloaded from the RCSB PDB database (sb.org/) ; the sou.0–3.0 Å , and the.0–3.0 Å , 40, add.0–3.0 Å , and theHomo sapiens”; a total of 303 human genes related to these medicine targets were screened out. In addition, a total of 1,839 GC-related targets were screened from the GeneCards database. All of the suitable herb targets are also shown in Excluding no known targets of compounds, a total of 27 main chemical compounds from the ZS (20) and BZ (7) herb pair were obtained from the TCMSP database, and 743 targets were retrieved from the TCMSP database, TTD, and DrugBank using the names of all the selected compounds above. The main compounds were luteolin, naringenin, 12-senecioyl-2E,8E,10E-atractylentriol, nobiletin, and tetramethoxyluteolin. The selected compounds are shown in The STRING tool was applied to construct the PPI network of the herb compound-disease-target relationships for the 120 overlapping targets. We selected a score >0.7 from the analysis. The network of PPI contained 120 nodes and 671 edges, the average node of degree was 11.2, and the average local clustering coefficient was 0.533 . The results of GO analysis were separated into three parts, as follows: (1) biological processes: response to lipopolysaccharide, cellular response to drugs, regulation of apoptotic signaling pathway, response to nutrient levels, and regulation of DNA-binding transcription factor activity  celactivity ; and (3)activity .P < 0.05, KEGG pathway enrichment of the top 20 pathways was identified . They also relieve oppression and masses, eliminate pathogenic factors without damaging healthy qi, and strengthen the spleen and stomach .luteolin and naringenin as the results of the molecular docking method.Network pharmacology and molecular docking methods were used to explore the possible pharmacological mechanisms of the ZS-BZ herb pair related to gastric cancer in this study, which indicated that 27 compounds and 120 target genes were related to gastric cancer. According to the results of GO and KEGG pathway enrichment analyses, we found that the effects of the ZS-BZ herb pair against gastric cancer may be due to the active compounds of the ZS-BZ herb pair, especially for luteolin and naringenin which could influence the regulation of the PI3K-Akt and IL-17 signaling pathway. They may also be related to the apoptotic signaling pathway, cyclin-dependent protein kinase holoenzyme complex, and receptor complex and transcription factor complex. Based on the results of PPI analysis and Cytoscape Hub, AKT1, MMP9, IL-6, CCND1, BCL2, MTOR, and MDM2 were selected as key targets, and they reflect a strong affinity with Gastric mucosal carcinogenesis is a progressive process. The most recognized mode of intestinal gastric cancer is atrophic gastritis intestinal metaplasia dysplasia intramucosal cancer proposed by Correa  in 1988.The PI3K-Akt signaling pathway is significant in the formation and progression of many malignancies, and it has become a hot spot as a therapeutic target for cancer. It involves the regulation of tumour cell of gastric cancer survival, proliferation, invasion, and migration , 50. VarAKT1 and MTOR genes in gastric mucosa of gastric cancer patients and showed that they play vital roles in cell growth, proliferation, metabolism, angiogenesis, and survival in mediating cellular functions [The experiments showed the overexpression of unctions . Furtherunctions .It has been reported that IL-6 is highly upregulated in many cancers and is one of the most important cytokine families in tumorigenesis and metastasis  and is hSome scientific tests have established that luteolin is an active component of ZS, which is a potential chemotherapeutic agent against gastric cancer. It can suppress cell cycle arrest, proliferation, colony formation, and accelerate apoptosis in gastric cancer cells in vitro and in vivo by regulating the PI3K and AKT signaling pathways . In addiNaringenin induces apoptosis of gastric cancer SGC7901 cells by decreasing the expression of MMP9 by downregulating the AKT pathway . It can In summary, the results of our study showed that the ZS-BZ herb pair may play a therapeutic role in treating gastric cancer based on the active compounds luteolin and naringenin. These two compounds mainly act on seven targets, namely, AKT1, MMP9, IL-6, CCND1, BCL2, MTOR, and MDM2; they may be related to the regulation of the PI3K-Akt signaling pathway and IL-17 signaling pathway. Our efforts have provided evidence and we have obtained a good understanding of the multitarget, multicompound, and multipathway synergy of traditional Chinese medicine in treating gastric cancer. However, in vivo and in vitro experiments and clinical studies should be conducted to verify the mechanism of the ZS-BZ herb pair against gastric cancer.