Visualization is a key recurring requirement for effective analysis of relational data. Biology is no exception. It is imperative to annotate and render biological models in standard, widely accepted formats. Finding graph-theoretical properties of pathways as well as identifying certain paths or subgraphs of interest in a pathway are also essential for effective analysis of pathway data. Given the size of available biological pathway data nowadays, automatic layout is crucial in understanding the graphical representations of such data. Even though there are many available software tools that support graphical display of biological pathways in various formats, there is none available as a service for on-demand or batch processing of biological pathways for automatic layout, customized rendering and mining paths or subgraphs of interest. In addition, there are many tools with fine rendering capabilities lacking decent automatic layout support.Systems Biology Layout and Rendering Service) for automatic layout of biological data in various standard formats as well as construction of customized images in both raster image and scalable vector formats of these maps. Some of the supported standards are more generic such as GraphML and JSON, whereas others are specialized to biology such as SBGNML  and SBML (The Systems Biology Markup Language). In addition, SyBLaRS supports calculation and highlighting of a number of well-known graph-theoretical properties as well as some novel graph algorithms turning a specified set of objects of interest to a minimal pathway of interest.To fill this void, we developed a web service named SyBLaRS (We demonstrate that SyBLaRS can be used both as an offline layout and rendering service to construct customized and annotated pictures of pathway models and as an online service to provide layout and rendering capabilities for systems biology software tools.GitHub and freely distributed under the MIT license. In addition, a sample deployment is available here for public consumption.SyBLaRS is open source and publicly available on  PLOS Computational Biology Software paper.This is a With help from widely accepted standard file formats such as SBGNML  and SBMLVisualization of relational data via maps/pathways is compelling in all domains including biology as it is believed to bring out patterns, broad relationships and emerging trends in data, for an insightful analysis . In visuMany web-based  and stanA similar past effort providing such a layout service  works onSystems Biology Layout & Rendering Service (SyBLaRS) with the aim to fill the void for programmatically generating graphical representations of pathways while optionally highlighting paths or sub-pathways of interest and automatically laying them out  in popular standard formats,Lay out the provided map in specified layout style chosen from many available ones and return the map with layout information,Both lay out the provided map in specified layout style and create an image of it as raster image or scalable vector image formats, andOptionally highlight a path or a sub-pathway  based on the results of a graph-based query.SyBLaRS accommodates a number of novel methods as well as widely known and used ones on automatic layout of pathways , 17, calEven though SyBLaRS supports a wide range of automatic layout algorithms written as Cytoscape.js extensions, of particular interest is fCoSE  that supGitHub repository for a complete set of options.All available layout algorithms are customizable for one’s specific needs  through the associated API. Similarly, all are suitable for interactive use as they complete in at most a few seconds for pathways of up to several hundred biological entities and similar number of interaction edges. Please refer to the associated One can predict the behavior of biological networks using measured graph-theoretical or structural properties of those systems as well as the local rules governing individual objects of the networks . Hence, Degree centrality,Closeness centrality,Betweenness centrality, andPage rank.Such properties are displayed as part of node labels and optionally emphasized through node highlight as exemplified in Oftentimes one is interested in discovering and analyzing certain types of paths or sub-pathways in a given biological pathway model, especially in large ones. In order to facilitate such queries, the algorithms in  were adaSyBLaRS exposes the shortest paths algorithm of Dijkstra as provided by Cytoscape.js library, where the user specifies the source and target nodes and a single path of a shortest length between these nodes are highlighted with the specified color.The remaining graph mining queries were originally designed in  with theEven though these algorithms seem like straightforward implementations of well-known graph algorithms such as Dijkstra’s shortest paths , they diThey support compound structures through modified DFS and BFS traversals, where upon reaching a particular compound node or a node within a compound node, we also visit its children or parent/sibling nodes, so as to seamlessly continue the traversal. The user has the option to choose the traversal direction .all such paths between a group of source and target nodes. Besides, some of these algorithms allow one to define additional distance. For instance, if the actual shortest path between one source and one target node is 3 and the additional distance is 2, then these algorithms will find all paths of length between 3, 4 or 5.Dijkstra’s algorithm finds one of many potentially available shortest paths from a single dedicated node to another dedicated one, whereas algorithms such as Paths-between and Paths-from-to find This is a simple neighborhood query where one can specify a group of nodes, from all of which a parallel compound BFS is started. Any node discovered during these traversals gets added to the result (see This query aims to find those nodes that are common to the up (common regulator), down (common target) or both streams of a specified group of entities. One often wants to know if there is a gene in the upstream of some genes in a pathway, which can provide a causal explanation for the co-regulation  . Or two With the latest high-throughput sequencing technologies, one can scan for alterations in large quantities of samples . A problk defines the maximum length of these links. Paths-between query finds a “maximal” pathway comprising all the nodes of interest complemented by the “missing links” among these nodes. Its parameter all shortest paths from a source node set S to a target one T. The query could be constrained with a maximum length k of such paths. Furthermore, another parameter d is provided for relaxing the shortest requirement.Finding shortest paths between a single or all pairs of vertices in a graph is a well known and commonly used algorithm . This quEven though traversal based queries discussed above might be quite useful in mining sub-pathways of interest, they should be used with caution in large pathways due to their exponential nature in number of such paths of interest.GitHub repository for a complete set of options.Optionally, an image of the provided map is created in the specified format . Construction can be tailored in certain ways such as the image dimensions and the color scheme to be used. With some file formats, a single color is specified and SyBLaRS uses different shades of that color for rendering graph elements of varying types, whereas in some others, there are specific color schemes  to choose from . The usecytoscape-sbgn-stylesheet [cytoscape-graph-algos extension. SyBLaRS’s architecture is sketched in sbgnml-to-cytoscape [libsbmljs [cytoscape-graphml extension to convert SBGNML, SBML and GraphML files, respectively, into JSON format as accepted by Cytoscape.js. Cytosnap, which is a package to render graphs on the server side with Cytoscape.js, is used to apply layout and/or generate images of pathway graphs.SyBLaRS uses a package named ylesheet  that proytoscape  and libsibsbmljs  packagesour sample public deployment, or programmatically in the background without a user interface, say to produce an image of a newly curated pathway as it becomes available. Supported input file formats are SBGNML [JSON.Once a service is set up, requests can be made through a web page, like the one used by e SBGNML , SBML [2e SBGNML , and JSOBelow is an example request to our sample deployment via curl:curl -X POST -H “Content-Type: text/plain” --data“request_body” syblars.cs.bilkent.edu.tr/file_formatfile_format is one of sbgnml, sbml, graphml and json, and the request_body includes the map content and any layout, image and query options as detailed in the GitHub repository README.where, the All three operations, performing a layout on the provided map, running a query on it and creating an image of it, are optional. If preferred, the service will return the layout information in JSON format. The format of the optional output of an image of the map is JPG, PNG or SVG.SyBLaRS has multiple use cases as a service including, but not limited to the following.One can use SyBLaRS to quickly generate images for pathway models, where an optional automatic layout can also be performed. In fact, this could be automated in such a way that, in pathway databases where new models emerge periodically, a tool using a SyBLaRS service could construct such images on demand.GitHub. See We tested this use case for a hundred BioModels pathways  that areSyBLaRS may also be used as a layout service in pathway analysis applications, where the tool has rendering capabilities of its own but not a proper automatic layout facility.This use case has been tested with Newt , a web bThe publicly available deployment of SyBLaRS may also be used by individuals who would like to highlight certain paths of interest in their pathways or simply construct images of their pathways for inclusion in a document such as a scientific article. With SyBLaRS’s user-friendly and simple graphical user interface, one can easily generate such images without having to install software or learn sophisticated pathway visualization tools.SyBLaRS can be used both as an offline layout and rendering service to construct customized and annotated pictures of pathway models and as an online service to provide layout and rendering capabilities for systems biology software tools. In addition, it may be used for merely constructing automatically laid out static images of your pathway models with optional support to highlight paths or sub-pathways of your interest.GitHub and freely distributed under the MIT license. In addition, a sample deployment is available here for public consumption. The service in this deployment may also be used programmatically from your own application.SyBLaRS is open source and publicly available on As future work, we would like to embed newly calculated layout information in the provided file using the same format as the input, as opposed to returning it separately as JSON. We would also like to extend our query and layout  algorithms.S1 VideoThis video demonstrates how the public SyBLaRS deployment can be used to perform automatic layout and querying features of the service as well as construction of an image of the provided pathway model.(MP4)Click here for additional data file.


Meibomian gland dysfunction is one of the main causes of dry eye disease and has limited therapeutic options. In this study, we investigated the biological function of the beta 2-adrenergic receptor (ADRB2)/protein kinase A (PKA) pathway in lipid synthesis and its underlying mechanisms in human meibomian gland epithelial cells (HMGECs). HMGECs were cultured in differentiation media with or without forskolin (an activator of adenylate cyclase), salbutamol (an ADRB2 agonist), or timolol (an ADRB2 antagonist) for up to 4 days. The phosphorylation of the cAMP-response element-binding protein (CREB) and the expression of peroxisome proliferator activator receptor (PPAR)γ and sterol regulatory element-binding protein (SREBP)-1 were measured by immunoblotting and quantitative PCR. Lipid synthesis was examined by LipidTOX immunostaining, AdipoRed assay, and Oil Red O staining. PKA pathway activation enhanced PPARγ expression and lipid synthesis in differentiated HMGECs. When treated with agonists of ADBR2 , PPARγ expression and lipid synthesis were enhanced in HMGECs. The ADRB2 antagonist timolol showed the opposite effect. The activation of the ADRB2/PKA signaling pathway enhances lipid synthesis in HMGECs. These results provide a potential mechanism and therapeutic target for meibomian gland dysfunction, particularly in cases induced by beta-blocker glaucoma drugs. Meibomian gland dysfunction (MGD) is a major cause of evaporative dry eye disease and is characterized by terminal duct obstruction and/or qualitative/quantitative changes in glandular secretion . The meiAcinar cells of meibomian glands (meibocytes) mainly produce meibum and maturate from proliferating, non-lipid-producing basal cells to non-proliferating, lipid-accumulating differentiated cells. Lipid biosynthesis and accumulation are hallmarks of meibocyte maturation. It has been reported that various signal pathways are involved in this maturation process. Notch signaling enhances human meibomian gland epithelial cell (HMGECs) differentiation and lipid production . The losPKA, a tetrameric enzyme consisting of two catalytic subunits, each bound to a regulatory subunit dimer , is knowIn addition, it is clinically important to study the PKA pathway because eye drops that modulate the PKA pathway are widely used in glaucoma patients. Beta blockers are one of the most widely used anti-glaucoma eye drops and are known to cause damage to the ocular surface, including MGD ,20,21. HWe hypothesized that the PKA signaling pathway plays a role in the differentiation and lipid accumulation of meibocytes, similar to adipocytes, and we examined the role of the PKA pathway in the differentiation and lipid accumulation of meibocytes using HMGECs. We further investigated the function of adrenergic receptors, which could affect the PKA pathway and meibocyte maturation.CREB phosphorylation was assessed to evaluate the PKA pathway in HMGECs. HMGECs were treated with forskolin, which activates adenylyl cyclase, thereby increasing the intracellular concentration of cAMP and activating the PKA pathway. The phosphorylation of CREB at the ser133 residue was assessed by immunoblotting at 0, 15, 30, 60, and 120 min. The phosphorylation of CREB in HMGECs increased after 15 min and gradually decreased after 120 min. We then treated HMGECs with escalating doses of forskolin to HMGECs and the level of phosphorylated CREB (pCREB) was examined at 30 min. As shown in It is generally accepted that cAMP signaling, followed by PKA activation, plays an important role in lipogenesis in white adipose tissue . To deteDiverse hormones and extracellular signals utilize cAMP-dependent signal transduction to exert their effects; sympathetic activation of beta-adrenergic receptors by catecholamines is a classic example . TherefoTo investigate the influence of ADRB2 signal cues on lipid synthesis and PPARγ expression levels, we first evaluated the effect of salbutamol, a selective beta 2-adrenergic receptor agonist, on the PKA signaling pathway in differentiated HMGECs. Salbutamol was added to differentiated HMGECs and the phosphorylation of CREB was assessed at a series of time points, then at 10 min each with escalating doses. The finding that the ADRB2 receptor agonist facilitated the lipogenesis of differentiated HMGECs led us to examine the effect of the ADRB2 receptor antagonist on HMGECs. Timolol, a beta-adrenergic receptor blocking agent and one of the representative anti-glaucoma therapeutics, was chosen and used to differentiate HMGECs. It is well-known that long-term use of topical antiglaucoma eye drops, including timolol, is associated with meibomian gland dysfunction ,20. ZhanWe treated HMGECs with physiological doses of timolol  on diffeThe lipogenesis of differentiated HMGECs was compared at day 4, and timolol-treated HMGECs showed a significantly reduced lipid droplet accumulation, as evaluated by the AdipoRed assay and LipidTOX immunostaining C,D.Finally, lipid synthesis was confirmed by Oil Red O staining at day 4 after forskolin. HMGECs with differentiation media were treated with salbutamol or timolol at day 0 and 2. Consistent with previous results, HMGECs treated with forskolin and salbutamol demonstrated increased lipid accumulation. The HMGECs treated with timolol showed a significant reduction in lipid accumulation .In this study, we demonstrated that the PKA pathway plays an important role in HMGEC differentiation and lipid accumulation. Forskolin, a PKA pathway activator, enhances both differentiation and lipid synthesis in HMGECs. We then evaluated the function of ADRB2, which is a G-protein-coupled membrane receptor and an upstream signal transducer of the PKA pathway, in the differentiation of HMGECs. Salbutamol, an ADRB2 agonist, successfully induced the activation of the PKA pathway and subsequent PPARγ expression and lipid synthesis, whereas timolol showed the opposite effect. These results suggest that the PKA pathway might be related to the pathophysiology of MGD and that drugs affecting the PKA signaling pathway could have an effect on MGD.PKA includes two regulatory subunits and two catalytic subunits . When cAThe role of the PKA pathway in meibomian gland dysfunction and dry eye disease is not well-understood. Ru et al. reported the protective effects of α-melanocyte-stimulating hormone against dry eye using a scopolamine-induced dry eye rat model, and that these protective effects were mediated by the PKA-CREB pathway . HoweverThe PKA pathway is known to play an important role not only in lipid synthesis but also in lipolysis of adipocytes, in connection with cAMP signals . cAMP isOne of the most representative signals activating the PKA pathway is an increase in cAMP levels via beta-adrenergic receptors . As variIn conclusion, we demonstrated that the activation of the PKA pathway enhanced PPARγ expression and lipid synthesis in HMGECs. The physiological relevance of ADRB2 to the PKA pathway was also evaluated. The ADRB2 agonist salbutamol enhanced the PKA pathway and subsequent PPARγ expression and lipid synthesis, whereas the ADRB2 antagonist timolol had the opposite effect. These results show that the ADRB2-PKA pathway could be implicated in the pathogenesis of MGD and provide an understanding of how drugs, such as timolol, could aggravate MGD in patients. In addition, the ADRB2-PKA pathway may be a potential target for MGD treatment.v/v) fetal bovine serum and 10 ng/mL epidermal growth factor , with or without drug compounds, according to each experiment. To assess the phosphorylation of CREB, chemicals were treated with fresh differentiation media after 2 h of incubation. To evaluate the PPARγ pathway and lipid accumulation, differentiation media were added on days 0 and 2. Forskolin  was added to the differentiation medium at a final concentration of 1 µM. Salbutamol (Sigma) and timolol maleate (Sigma) were supplemented at final concentrations of 1 µM and 0.0002%, respectively.The immortalized HMGEC—kindly provided by Dr. Sullivan (Schepens Eye Research Institute)—were cultured in keratinocyte serum-free medium  as previously described . HMGECs qPCR was performed as previously described . Total RImmunoblotting was performed as previously described . The celOil Red O staining and AdipoRed assays were performed as previously described, with some modifications . The celFor the AdipoRed assay, the cells were grown in a 48-well plate in triplicate for each cycle. The cells were stained with AdipoRed (Lonza) according to the manufacturer’s instructions. After 10 min, the stained cells were placed in a fluorimeter  and the fluorescence was measured at excitation and emission wavelengths of 485 and 572 nm, respectively.The differentiated cells, grown in the Nunc™ Lab-Tek™ II Chamber Slide™ system (Thermo Fisher Scientific), were fixed with 4% paraformaldehyde (Biosesang) for 30 min at room temperature and then washed twice with phosphate-buffered saline. The cells were stained by incubation with LipidTOX (Invitrogen) for 30 min at room temperature and mounted with gel/mount containing DAPI . Fluorescence images were obtained using a Zeiss LSM 700 confocal microscope .p < 0.05 was considered significant. All experiments were repeated at least three times. The results of the cell culture experiments were compared using Student’s t-test using GraphPad Prism 8 .The results of multiple experiments are presented as the mean ± standard error of the mean, and


A transcriptome analysis proved 2680 differently expressed genes (DEGs) between the model and didymin groups and suggested that the PI3K/Akt and metabolic pathways might be the most relevant targets. Meanwhile, the metabolome analysis revealed 67 differently expressed metabolites (DEMs) between the didymin and model groups that were mainly clustered into the glycerophospholipid metabolism, which was consistent with the transcriptome study. Importantly, a comprehensive analysis of both the omics indicated a strong correlation between the DEGs and DEMs, and an in-depth study demonstrated that didymin alleviated metabolic disorder and hepatocyte injury likely by inhibiting the glycerophospholipid metabolism pathway through the regulation of PLA2G4B, LPCAT3, and CEPT1 expression. In conclusion, this study demonstrates that didymin can ameliorate LPS/D-Gal-induced ALI by inhibiting the glycerophospholipid metabolism and PI3K/Akt and TLR4/NF-κB pathways.Based on the multiomics analysis, this study is aimed at investigating the underlying mechanism of didymin against acute liver injury (ALI). The mice were administrated with didymin for 2 weeks, followed by injection with lipopolysaccharide (LPS) plus D-galactosamine  to induce ALI. The pathological examination revealed that didymin significantly ameliorated LPS/D-Gal-induced hepatic damage. Also, it markedly reduced proinflammatory cytokines release by inhibiting the TLR4/NF- Acute liver injury can be induced by various risk factors, such as alcohol, lipopolysaccharide (LPS), ionizing radiation, chemotherapy, and acetaminophen. LPS-induced liver injury is defined as a class of syndrome with liver inflammatory responses . As an iRecently, multiomics analysis has been considered a promising strategy to elucidate the complicated pathogenesis of multiple diseases , 7. TranOriganum vulgare L. [Didymin, also known as isosakuranetin-7-O-rutinoside, is a common flavonoid found in many kinds of herbs, such as lgare L. . Didyminlgare L. . In our lgare L. , suggestMale C57BL/6J mice  were obtained from Changsha Tianqin Biotechnology Co., LTD . The animal study was approved by the Experimental Animal Center of Guangxi Medical University .n = 10): normal control group, didymin control group (0.8 mg/kg didymin), model group, bifendate-treated group  [μg/kg) and D-Gal (800 mg/kg) intraperitoneally to induce ALI [The schedule of animal experiments is shown in duce ALI . After 4Fresh liver tissues were fixed in 4% paraformaldehyde, embedded in paraffin, and sliced into sections . Liver hActivities of serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), total protein (TP), albumin (ALB), globulin (GLB), and A/G (ALB/GLB) were determined using commercial kits by an Automatic Blood Biochemical Analyzer .The enzymes in the supernatants, including glutathione reductase (GSH-Rd), superoxide dismutase (SOD), myeloperoxidase (MPO), malondialdehyde (MDA), and inducible nitric oxide synthase (iNOS), were detected using the commercially available kits  according to the manufacturer's protocols. Hepatic reactive oxygen species (ROS) were detected using an ELISA kit .β), and tumor necrosis factor-α (TNF-α), were determined using the commercial ELISA kits  according to the manufacturer's instructions.Inflammatory cytokines in serum, including interleukin-6 (IL-6), interleukin-1 beta  at 4°C overnight, followed by incubation with the peroxidase-coupled secondary antibody for 30 min at room temperature. Immunoreactivity was visualized with 3, 3'-diaminobenzidine tetrachloride (DAB). Additionally, sections were counterstained with hematoxylin and observed under the microscope .Tissue sections were dewaxed and rehydrated with freshly distilled water. Endogenous peroxidase was blocked by incubating the samples with 3% HThe total protein from the liver tissues was prepared using RIPA buffer  with 1% Halt protease inhibitor cocktail and 1% Halt phosphatase inhibitor cocktails . The concentration of extracted protein was measured using BCA Protein Assay Kit . Protein samples were subjected to a 7.5%-12.5% SDS polyacrylamide electrophoresis and then electrophoretically transferred to a nitrocellulose membrane. The membrane was incubated with the primary antibodies  and further reverse-transcribed into cDNA using HiScript™ RT SuperMix  with Veriti Thermal Cycler . The mRNA levels were determined using the 7500 Fast Real-Time PCR System SDS software version 2.0.5 (Applied Biosystems) according to the 2t method . The priμm, Waters Corp.). Mobile phase gradient elution using (a) HPLC-grade water and (b) acetonitrile (0.1% formic acid) was performed at a flow rate of 0.4 mL/min. The gradient program for solvent B was set as follows: 5-20% B from 0 to 1 min, 20-40% B from 1 to 2.5 min, 40-90% B from 2.5 to 9 min, 90-100% B from 9 to 10 min, 100% B from 10 to 14 min, and 5% B from 14 to 15 min. The autosampler was maintained at 4°C. A sample of 10 μl was injected for each run. Mass spectrum (MS) was performed on a Xevo G2-S Q-TOF instrument . The conditions of MS were defined as previously described [https://www.metaboanalyst.ca/MetaboAnalyst/).The liver samples and quality control (QC) were prepared as described in our previous study . ChromatP value <0.05 using the R packages.Correlation analysis of transcriptome and metabolome data was performed to explore the correlation between differentially expressed genes and metabolites using RStudio with the various packages. The correlation coefficient cluster heat map, the Spearman correlation coefficient (SCC), and the cojoint KEGG pathway analysis were also analyzed using the R packages. Besides, a nine-quadrant graph was used to show the log2 (Fold change (FC)) of genes and metabolites with SCC > 0.8 and P value <0.05 was considered statistically significant.Statistical analysis was performed using the software of SPSS (Ver. 17.0) for Windows. Differences between the groups were assessed using a one-way analysis of variance (ANOVA) with a Tukey's test for post hoc multiple comparisons. The data were presented as means ± standard deviation (SD). A The pathological changes were assessed by H&E staining. As shown in Liver function was assessed by detecting the serum indexes, including AST, ALT, ALB, GLB, A/G, and TP. As shown in Figures β, TNF-α and IL-6 content, TNF-α mRNA level, and IL-6 protein expression  revealed that the gene expression among the normal, model, and didymin-treated (didymin 0.8 mg/kg) groups was distinct. And the heat map showed that the trend of the model group was opposed to the normal control; didymin could reverse the abnormal trend to some extent, suggesting that didymin could restore the LPS/D-Gal-induced abnormal transcriptome profile Figures . RNA-seqThe transcriptome analysis above predicted that the PI3K/Akt signaling pathway might be the target of didymin. Therefore, the effect of didymin on this pathway was assessed. As shown in Figures κB signaling pathway is involved in inflammation and ALI. Thus, the expressions of the TLR4/NF-κB pathway-related indicators were detected in the present experiment. As shown in κB was inhibited by bifendate or didymin. Moreover, the western blot analysis showed that didymin significantly reduced the expression levels of TLR4, MyD88, and NF-κB and the phosphorylations of p-IKKα/β and p-IκBα as well  analysis indicated that the Ultra-High Liquid Chromatograph-Mass Spectrum (UPLC-MS) experimental condition was high repeatability and stable, as evidenced by the similar total ion chromatogram (TIC) plot among the QC samples and a relatively tight clustering in the Principal Component Analysis (PCA) Score Plot and Loading Plot  (As shown in ct >0.1) .r| > 0.80) were divided into 1-9 quadrants: the 1 and 9 quadrants indicated the negative correlation between the genes and metabolites; while the quadrants 3 and 7 suggested that they were positively correlated; the 5 quadrant meant no significance, and the rest of quadrants suggested a partial correlation. These results from the heat map, and the nine-quadrant diagrams indicated that many genes might play direct or indirect regulatory roles in the alterations in the corresponding metabolites. In addition, the cojoint KEGG pathway enrichment analysis of transcriptome and metabolome was performed as previously described [P < 0.05).To further elucidate the correlation between transcriptome and metabolome, the correlation coefficient was calculated using R packages with the Spearman method. As shown in escribed . As showhttps://www.metaboanalyst.ca/MetaboAnalyst/). As shown in To understand more detailed information on how didymin regulated the glycerophospholipid metabolism, the integrative analysis of transcriptome and metabolome was further conducted by the MetaboAnalyst 5.0 database . IKKα can phosphorylate IκBα and IκBβ, which results in their ubiquitination and the activation of TLR4/NF-κB signaling [κB and exert anti-inflammatory actions, preventing hyperglycemia-induced human umbilical endothelial cell dysfunction and death [κB, and the phosphorylation of IKKα/β and IκBα, which was consistent with the previous research [κB pathway and, consequently, reduce inflammation.Oxidative stress has been regarded as a crux influence cause of hepatic injury. The imbalance of the oxidative and antioxidant systems easily leads to oxidative damage to the hepatocytes, playing an important role in liver injury. In this study, the activities of SOD and GSH-Rd were drastically decreased in the LPS/D-Gal group, while the level of MDA was strongly elevated; however, these abnormal changes were largely reversed by didymin treatment, suggesting that didymin could recruit the antioxidative defense system and, as a result, decrease oxidative damage. In addition, excessive inflammation is also one of the risk factors that induces and accelerates liver injury. The current study showed that didymin significantly reduced the levels of iNOS, TNF-ignaling , ultimatnd death . In thisresearch . Our datNext, the underlying mechanisms of didymin against liver injury were further investigated by transcriptome analysis. Transcriptome is the collection of all transcripts within a cell, including mRNA, rRNA, tRNA, and other noncoding RNAs. Unlike conventional PCR experiments, transcriptome sequencing can comprehensively obtain almost all transcripts of a specific organ or tissue in a certain state and reflect their expression levels, predicting the pathogenesis of a disease or the therapeutic mechanism of drugs as a whole. In the current study, the RNA-seq revealed more than two thousand differently expressed genes (DEGs) between the model and didymin-treated groups, and these DEGs were enriched into many pathways. Interestingly, the metabolic and PI3K/Akt pathways likely were the most relevant targets, as indicated by the KEGG pathways analysis.Since the transcriptomics analysis suggested the key role of the PI3K/Akt pathway in didymin treating liver injury, the effect of didymin on this signaling pathway was then assessed. Numerous studies have demonstrated that the PI3K/Akt pathway is involved in the onset and development of liver injury . Akt recP < 0.05 and impact >0.1).Besides, the transcriptome analysis also predicted that the metabolic pathways might be involved in the protective effects of didymin on ALI. Thus, an untargeted metabolomics approach was employed to define the changed metabolic pathways and find clues to uncover the potential mechanisms. The mass spectrum showed that LPS/D-Gal administration led to a significant change in the global liver metabolic profiles. Interestingly, didymin treatment could restore the abnormal change nearly to the normal level. Further analysis revealed 67 differently expressed metabolites (DEMs) between the model and didymin groups, which were enriched into seven metabolism-related pathways. Among the significantly changed pathways, the glycerophospholipid metabolism pathway might be the most important /24 : 0) and PC ) by regulating PLA2G4B, LPCAT3, and CEPT1 expression, ultimately inhibiting the glycerophospholipid metabolism pathway. It has been confirmed that phosphoethanolamines (PEs) and phosphocholines (PCs) are the key phospholipids of cell membranes [To understand how didymin regulated metabolism, we conducted an in-depth analysis of the transcriptome and metabolome. We found that the DEGs and DEMs were closely related; many genes might play direct or indirect regulatory roles in the alterations in the corresponding metabolites. And the evidence also indicated that didymin regulated the metabolism of many substances embranes , which aκB signaling pathways and the glycerophospholipid metabolism pathway (In conclusion, didymin can meliorate LPS/D-Gal-induced ALI in C57BL/6J mice by regulating the PI3K/Akt and TLR4/NF- pathway . Our stu


Circular RNA (circRNA) plays an important role in the regulation of gene expression and the occurrence of human diseases. However, studies on the role of circRNA in acute myocardial infarction (AMI) are limited. This study was performed to explore novel circRNA-related regulatory networks in AMI, aiming to better understand the molecular mechanism of circRNAs involvement in AMI and provide basis for further scientific research and clinical decision-making.The AMI-related microarray datasets GSE160717 (circRNA), GSE31568 (miRNA), GSE61741 (miRNA), and GSE24519 (mRNA) were obtained from the Gene Expression Omnibus (GEO) database. After differential expression analysis, the regulatory relationships between these DERNAs were identified by online databases circBank, circInteractome, miRDB, miRWalk, Targetscan, and then two circRNA-miRNA-mRNA regulatory networks were constructed. Differentially expressed genes (DEGs) in this network were selected followed by enrichment analysis and protein–protein interaction (PPI) analysis. Hub genes were identified using Cytohubba plug-in of Cytoscape software. Hub genes and hub gene-related miRNAs were used for receiver operating characteristic curve (ROC) analysis to identify potential biomarkers. The relative expression levels of these biomarkers were further assessed by GSE31568 (miRNA) and GSE66360 (mRNA). Finally, on the basis of the above analysis, myocardial hypoxia model was constructed to verify the expression of Hub genes and related circRNAs.A total of 83 DEcircRNAs, 109 CoDEmiRNAs and 1204 DEGs were significantly differentially expressed in these datasets. The up-regulated circRNAs and down-regulated circRNAs were used to construct a circRNA-miRNA-mRNA regulatory network respectively. These circRNA-related DEGs were mainly enriched in the terms of “FOXO signaling pathway,” “T cell receptor signaling pathway,” “MAPK signaling pathway,” “Insulin resistance,” “cAMP signaling pathway,” and “mTOR signaling pathway.” The top 10 hub genes ATP2B2, KCNA1, GRIN2A, SCN2B, GPM6A, CACNA1E, HDAC2, SRSF1, ANK2, and HNRNPA2B1 were identified from the PPI network. Hub genes GPM6A, SRSF1, ANK2 and hub gene-related circRNAs hsa_circ_0023461, hsa_circ_0004561, hsa_circ_0001147, hsa_circ_0004771, hsa_circ_0061276, and hsa_circ_0045519 were identified as potential biomarkers in AMI.In this study, the potential circRNAs associated with AMI were identified and two circRNA-miRNA-mRNA regulatory networks were constructed. This study explored the mechanism of circRNA involvement in AMI and provided new clues for the selection of new diagnostic markers and therapeutic targets for AMI. Cardiovascular disease is a serious threat to human health. Ischemic heart disease remains one of the leading causes of death and disability worldwide . The higAcute myocardial infarction is the result of myocardial ischemic necrosis caused by acute interruption of myocardial blood flow, often characterized by acute thrombosis superimposed on atherosclerotic plaque rupture . Early sCircular RNAs, characterized by a covalently closed continuous loop, is a type of circular single-stranded RNA different from other linear RNAs . They arThis study was performed to construct the circRNA-miRNA-mRNA regulatory Network and identify novel potential biomarkers in AMI. Datasets GSE160717, GSE31568, GSE61741, GSE24519, and GSE66360 were downloaded and then analyzed using Bioinformatics analysis. Our study would provide new insights of molecular mechanisms of circRNAs involved AMI, aiming to understand the development and progression of AMI and find targets for its diagnosis and treatment. The workflow of the specific analysis is shown in 1 GSE160717 is based on the GPL21825 platform (074301 Arraystar Human CircRNA microarray V2). GSE31568 and GSE61741 are based on the GPL9040 platform (febit Homo Sapiens miRBase 13.0). GSE24519 is based on the GPL2895 platform . GSE66360 is based on the GPL2895 platform ([HG-U133_Plus_2] Affymetrix Human Genome U133 Plus 2.0 Array). These datasets satisfy the following characteristics: (1) Organism: Homo sapiens; (2) Sample source: Blood; and (3) Grouping: AMI group and healthy controls. The microarray datasets GSE160717 (circRNA), GSE31568 (miRNA), GSE61741 (miRNA), GSE24519 (mRNA), and GSE66360 (mRNA) were obtained from the Gene Expression Omnibus (GEO) database.2 Benjamini-hochberg method was used to correct adj. P for potential false positive results. FDR < 0.05 and |logFC| > 1.0 was set as the cut-off criteria of DERNAs. CoDEmiRNAs in GSE31568 and GSE61741were identified using Venn diagram web tool.3 Based on relative expression information in raw sequencing data, the heatmap of DEcircRNAs were produced using the package gg plots of R software (version: x64 3.2.1) (Raw sequencing data of GSE160717 (circRNA), GSE31568 (miRNA), GSE61741 (miRNA), and GSE24519 (mRNA) were downloaded from the abovementioned platform and preliminary differential expression analysis between AMI samples and control was performed using GEO2R.4 3.2.1) .4 the circRNA name in GSE160717 containing six digits was converted into circBase ID containing seven digits. circRNAs that do not match in the annotation file were further blatted to complete the ID conversion. The miRDB5 database was used to convert the miRNA ID in GSE31568 and GSE61741 from the previous version to the latest version.Using circBase database,6 database is the first comprehensive cancer-specific circRNA database which can predict the microRNA response element (MRE) sites, RNA binding protein (RBP) sites, and potential open reading frames (ORF)  . Structu7 and circInteractome8 (9 Targetscan (version7.210), and miRWalk11  was used to perform Gene Ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of DEGs in these two circRNA-miRNA-mRNA regulatory networks, respectively  was used to predict the interaction between proteins, and a PPI network was established by Cytoscape software (version 3.7.1) subsequently.In order to better understand the molecular mechanism of circRNA’s involvement in transcriptional regulation, a protein–protein interaction network (PPI network) of DEGs in the circRNA-miRNA-mRNA network was constructed. The String database (version10.014) database.The scores of Maximal Clique Centrality (MCC) algorithm was set as the criteria , 31, andBased on hub genes identified in PPI network, corresponding miRNAs and circRNAs were selected from circRNA-miRNA-mRNA regulatory networks mentioned above. R software ggplot2, ggalluvial, and dplyr package was used to draw sankey diagrams . The heaP < 0.05 were screened as potential biomarkers. Their relative expression levels in these datasets were further evaluated by box plots. Graphpad Prism 9 software was used for analysis and mapping to compare the control and AMI groups. Comparison between the two groups was performed by independent sample T test. P < 0.05 was considered statistically significant.Hub gene and hub gene-related miRNAs in the sankey diagram were considered as candidate biomarkers for AMI. Based on their raw expression data in datasets GSE31568 (miRNA), GSE61741 (miRNA), GSE24519 (mRNA), and GSE66360 (mRNA), Graphpad Prism (version:8.0) software was used to plot the receiver operator characteristic curves and calculate the area under the curve (AUC) respectively to evaluate the predictive ability of these biomarkers for AMI. Meanwhile, hub genes and miRNAs with AUC ≥ 7.0 and 2.Human cardiomyocyte AC16 cell line were purchased from BeNa Culture Collection . The cells were cultured in Dulbecco’s modified Eagle’s medium  with 10% fetal bovine serum  and 1% penicillin-streptomycin  at 37°C with 5% CO2, 5% CO2, and 94% N2) in DMEM to generate a hypoxia model. AC16 cells under normoxia condition all the time were regarded as the control group.The AC16 cardiomyocytes were exposed to 24 h of hypoxia , and the mRNA and circRNA cDNAs were synthesized with Hifair® II 1st Strand cDNA Synthesis Kit (gDNA digester plus) . The qRT-PCR reactions were carried out in the Bio-Rad CFX96 Real-time PCR Detection System, using Hieff UNICON® Universal Blue qPCR SYBR Green Master Mix . GAPDH was used as the reference genes and the relative fold change was evaluated by the method of 2–ΔΔCt. All primers were presented in P < 0.05 was considered statistically significant.According to the manufacturer’s protocol, total RNA was isolated using FastPureThe microarray datasets GSE160717 (circRNA), GSE31568 (miRNA), GSE61741 (miRNA), and GSE24519 (mRNA) were obtained from the Gene Expression Omnibus (GEO) database. Based on GEO2R software analysis and screening criteria mentioned above, DERNAs in each dataset were identified. There were 83 DEcircRNAs, including 50 upregulated and 33 downregulated, in GSE160717. There were 249 DEmiRNAs, including 101 upregulated and 148 downregulated, in GSE31568. There were 143 DEmiRNAs, including 88 upregulated and 55 downregulated, in GSE61741. Finally, there were 1204 DEGs, including 766 upregulated and 438 downregulated, in GSE24519. The DEcircRNA’s name in GSE160717 containing six digits was converted into circBase ID containing seven digits . Figure In order to better understand the molecular mechanism of circRNAs involved in the process of AMI. The up-regulated circRNAs and down-regulated circRNAs were used to construct a circRNA-miRNA-mRNA regulatory network respectively. Gene Ontology knowledgebase and KEGG database were used to characterize the functional roles of the DEGs in the circRNA-miRNA-mRNA Regulatory Network mentioned above. The DEGs obtained from the circRNA-miRNA-mRNA Regulatory Network were introduced into the online database String. After removing the isolated genes without interaction, a PPI network with 105 nodes and 203 edges was established . The 74 The KOBAS online tool was used for pathway enrichment analysis. The results indicated that “the DEGs in module 1” and “hub genes” are significantly enriched in the pathway of Calcium signaling pathway. The DEGs of module 2 are enriched in the pathway of RNA polymerase. In addition, the DEGs of module 3 are significantly enriched in the pathway of Peroxisome. P < 0.05 in two datasets were considered as potential biomarkers. Finally, hsa-miR-31-3p, hsa-miR-1291, hsa-miR-646, SRSF1 and HNRNPA2B1 were identified as potential markers of AMI. Receiver operating characteristic curve (ROC) analysis was performed on 10 miRNAs and 10 hub genes in the Sankey diagram and AUC values were calculated. Meanwhile, molecules with AUC ≥ 7 and Hub genes  and Hub gene related circRNAs were further verified in AC16 hypoxia modle. The results showed that 4 Hub genes and 8 Hub gene related circRNAs were differentially expressed. They were hsa_circ_0023461(up), hsa_circ_0030368 (down), hsa_circ_0004561 (up), hsa_circ_0001147 (down), hsa_circ_0004771(down), hsa_circ_0061276 (down), hsa_circ_0045519(up), hsa_circ_0075503(down), ATP2B2(down), GPM6A (down), SRSF1(down), and ANK2(up) . The expAcute myocardial infarction has a high incidence and mortality in the middle-aged and elderly worldwide. In the AMI-related research field, circRNA, as a key molecule for gene expression regulation, has attracted more and more attention from scientists in recent years. However, the available circRNA information of AMI is still very limited, and the selection of AMI-related key circRNAs is also a challenge. We use bioinformatics method to conduct data mining on AMI-related datasets, extract relevant biological information from high-dimensional data, and construct the corresponding circRNA-miRNA-mRNA regulatory Network, aiming to explore the mechanism of circRNA involvement in acute myocardial infarction. These identified circRNA and circRNA-related molecules may be used as biomarkers for AMI, providing basis for further scientific research and individualized diagnosis and treatment of clinical patients.In the present study, we screened 83 DEcircRNAs, 109 CoDEmiRNAs and 1204 DEGs in AMI-related datasets. Subsequently, the endogenous competition regulatory networks corresponding to the up-regulated and down-regulated circRNAs were constructed, respectively. GO and KEGG pathway enrichment analysis showed that DEGs in these two networks were significantly enriched in pathways related to cell cycle, apoptosis, autophagy and immune. These circRNA-related DEGs are involved in “FOXO signaling pathway,” “T cell receptor signaling pathway,” “MAPK signaling pathway,” “Insulin resistance,” “cAMP signaling pathway,” and “mTOR signaling pathway.” These discovered pathways may be the key molecular mechanism for circRNAs participating in AMI. Based on PPI analysis, 10 hub genes, ATP2B2, KCNA1, GRIN2A, SCN2B, GPM6A, CACNA1E, HDAC2, SRSF1, ANK2, and HNRNPA2B1 were identified. These genes are enriched in the pathway of Calcium signaling pathway. This indicated that circRNA may also participate in the regulation of calcium signaling pathway. In addition, 10 miRNAs and 13 circRNAs related to these 10 hub genes were screened out and hsa-miR-31-3p, hsa-miR-1291, hsa-miR-646, SRSF1 and HNRNPA2B1 were identified as potential markers of AMI.CircRNA is stable and highly conserved among species, so it may be a key marker or therapeutic target for disease , 34. A din vivo can improve cardiac insufficiency and prevent the development of poor remodeling  can be found in the article/JW, CL, and ZL performed the data analysis and drafted the manuscript. HC, YH, and YZ prepared the figures and contributed toward the study design. TZ and HZ optimized the analysis protocol and participated in the data analysis. XH and JC revised the figures and designed and supervised the study. All authors have read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Scutellaria baicalensis, which has anti-inflammatory, anti-angiogenesis, and anti-fibrosis effects. Nevertheless, the effect of wogonin on pulmonary hypertension (PH) still lacks systematic research. This study aims to elucidate the potential mechanism of wogonin against PH through network pharmacology and further verify it through biological experiments in pulmonary arterial smooth muscle cells (PASMCs). The potential targets and pathways of wogonin against PH were predicted and analyzed by network pharmacology methods and molecular docking technology. Subsequently, the proliferation of PASMCs was induced by platelet-derived growth factor-BB (PDGF-BB). Cell viability and migration ability were examined. The method of Western blot was adopted to analyze the changes in related signaling pathways. Forty potential targets related to the effect of wogonin against PH were obtained. Based on the protein–protein interaction (PPI) network, gene-ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG) enrichment, and molecular docking, it was shown that the effect of wogonin against PH is closely related to the proliferation of PASMCs and the hypoxia-inducible factor-1α (HIF-1α) pathway. A variety of results from biological experiments verified that wogonin can effectively inhibit the proliferation, migration, and phenotypic transformation of PDGF-BB-mediated PASMCs. In addition, the anti-proliferation effect of wogonin may be achieved by regulating HIF-1/ NADPH oxidase 4 (NOX4) pathway. Wogonin is one of the main active components of  Pulmonary hypertension (PH) is a progressive disease characterized by increased vasoconstrictor tone at the early stage, leading to small pulmonary artery remodeling, elevated pulmonary vascular resistance, and right heart failure . The exaThe process of pulmonary vascular remodeling involves abnormal proliferation of pulmonary arterial smooth muscle cells (PASMCs), the injury of pulmonary arterial endothelial cells (PAECs), and deposition of extravascular collagen . The mecScutellaria baicalensis Georgi, is one of the major active components of the traditional Chinese medicine Scutellaria baicalensis. Recent studies have indicated that wogonin has great potential in the treatment of cardiovascular diseases including diabetic cardiomyopathy, cardiac hypertrophy, and dilating blood vessels [Natural products are one of the major sources of modern innovative medicines. With the continuous progress of modern pharmacology and molecular biology techniques, natural products have attracted more attention because of their advantages of low side effects and multiple targets. A variety of compounds from multiple natural sources, including tanshinone IIA and ligustrazine, are being tried and applied in the treatment of PH at preclinical and clinical stages ,7,8. Wog vessels ,10,11. NBased on the above status, in the present study, the approaches of network pharmacology and molecular docking were employed to analyze the key targets and signaling pathways of wogonin in the treatment of PH. Based on the results, experiments in vitro were performed to explore the effects of wogonin on PASMC proliferation, and the previously predicted potential mechanism of wogonin was experimentally verified at the molecular level .Wogonin, bosentan, and PDGF-BB were purchased from MedChemExpress (Israel Shekel). High-glucose Dulbecco’s Modified Eagle’s Medium (DMEM) and fetal bovine serum (FBS) were purchased from Gibco . Phalloidin and Hochest 33342 were purchased from Abcam . Primary antibodies specific for vascular cell adhesion molecule 1 (VCAM-1), intercellular adhesion molecule 1 (ICAM-1), and HIF-1α were procured from ProteinTech . Primary antibodies against NOX4, α-smooth muscle actin (α-SMA), and calponin 1 were purchased from Abcam . The Cell Counting Kit-8 (CCK-8) assay was purchased from Dojindo Laboratories . Other reagents and chemicals were all analytical grade.http://www.lilab-ecust.cn/pharmmapper/ (accessed on 18 August 2021), fit score >3) [http://stitch.embl.de/ (accessed on 18 August 2021)) [http://ctdbase.org/) [http://www.swisstargetprediction.ch (accessed on 18 August 2021), score > 0.1) [http://herb.ac.cn/ (accessed on 18 August 2021)) [To collect a comprehensive list of targets of wogonin, four databases were searched: PharmMapper Server (core >3) , STITCH t 2021)) , Comparase.org/) , SwissTae > 0.1) , and HERt 2021)) . https://www.genecards.org/ (accessed on 19 August 2021), score > 10) [http://www.disgenet.org/ (accessed on 19 August 2021)) [http://ci.smu.edu.cn/ (accessed on 19 August 2021), hit score > 5) [The PH-related genes were identified using the Genecards database (re > 10) , the Dist 2021)) , and GenThe intersection of targets of wogonin and PH-related proteins was considered potential therapeutic targets of wogonin against PH.cn.string-db.org) database to construct the PPI network. The species is set as “Homo sapiens”. The obtained PPI network is imported into Cytoscape 3.8.2 software for visualization. Using the NetworkAnalyzer plugin, the topological parameters of the network were analyzed. The larger the degree value of a node in a network, the higher its core properties in the network are represented.The gene list of therapeutic targets of wogonin against PH was uploaded to the STRING (https://david.abcc.ncifcrf.gov/ (accessed on 1 September 2021)) was employed for enrichment analysis of therapeutic targets of wogonin against PH [https://www.omicshare.com/ (accessed on 1 September 2021)) for visualization.The Database for Annotation, Visualization and Integrated Discovery (DAVID) v6.8 (http://www.pdb.org/ (accessed on 25 September 2021)). The Discovery Studio was used to embellish by removing the ligands, adding hydrogen, and removing water. Then, wogonin was docked with PDGFR-β and HIF-1α using the -CDOCKER method to evaluate their interaction energy and binding mode [The 3D structure of wogonin (ID: 5281703) was downloaded from the PubChem database. The crystal structure of the PDGF receptor-β  and hyping mode ,24.2 in air at 37 °C. Before treatments, HPASMCs were cultured in 0.5% FBS medium overnight for starvation. When the experiments were conducted, cells were pretreated with wogonin , bosentan (10 μmol/L) or vehicle (DMSO), followed by stimulation by PDGF-BB (40 ng/mL) for a further 48 h [Human pulmonary arterial smooth muscle cells (HPASMCs) were purchased from Cell Bioscience . HPASMCs were cultured in high-glucose DMEM containing 10% FBS and penicillin/streptomycin (1:100) and maintained in a humidified atmosphere of 5% COher 48 h ,26,27.A CCK8 cell proliferation assay kit was used to measure cell viability according to the manufacturer’s instructions. CCK-8 (10 μL/well) was added to the medium for 3 h at 37 °C. The absorbance for each sample was assessed at 450 nm using a microplate reader. Proliferating cell nuclear antigen (PCNA) is a typical indicator of cell proliferation . VasculaMigration was evaluated by a wound-healing assay . PASMCs 2EDTA, 1 mM EGTA, 1% NP-40, 1% sodium deoxycholate, 2.5 mM sodium pyrophosphate, 1 mM beta-glycerophosphate, 1 mM Na3VO4, 1 µg/mL leupeptin, phosphatase inhibitor, and protease inhibitor. After lysis on ice for 30 min add the loading buffer, mix thoroughly and boil for 10 min in boiling water. Protein samples were separated by SDS-PAGE (8–10% polyacrylamide gels) and transferred to nitrocellulose membranes  [The HPASMCs were washed with ice-cold PBS and harvested by scraping in a lysis buffer containing 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM Naica, MA) . The prip-values < 0.05.The mean ± SEM was used to describe the data. For statistical analysis, the data were analyzed using GraphPad Prism 6 . Statistical comparisons were made using one-way ANOVA followed by Dunnett’s post hoc test. Significance was defined as After removing the repeated targets from various databases, 113 targets of wogonin were collected. The 235 PH-related proteins and genes were identified using the Genecards database, the DisGeNET platform, and GenCLiP online tool. The 40 intersection genes of wogonin and PH-related proteins were considered to be potential therapeutic targets of wogonin against PH . The PPIDAVID was used for the enrichment analysis of 40 potential targets of wogonin against PH. The BP and MF terms involved in wogonin were explored. The biological processes that wogonin may regulate include “negative regulation of apoptotic process”, “response to hypoxia”, “positive regulation of nitric oxide biosynthetic process”, “positive regulation of smooth muscle cell proliferation”, “positive regulation of cell proliferation”, and “cellular response to interleukin-1” A. The moThe KEGG database was used for enrichment analysis. The top 20 pathways were screened. The results showed that the potential genes of wogonin against PH were mainly enriched in “HIF-1 signaling pathway”, “TNF signaling pathway”, and “PI3K-Akt signaling pathway” et al. .The structure of PDGFR and HIF-1α were taken from the PDB database. The binding mode of wogonin in the active site of PDGFR is represented in In order to investigate the anti-proliferation effect of wogonin, the PASMCs were treated with PDGF-BB for 48 h with different concentrations of wogonin . The results of cell viability are shown in The effect of wogonin on PASMCs migration was detected by the wound healing experiment. The experimental result showed that wogonin (1 μM) significantly inhibited PDGF-BB-induced PASMCs migration after scratches were detected at 12 h and 24 h. To further verify the inhibitory effect of wogonin on PASMCs migration, the immunofluorescence in PASMCs was carried out to detect their protein levels of ICAM and VCAM. The results in The transformation of PASMCs from contractile/differentiated phenotype to synthetic/dedifferentiated phenotype can lead to vascular remodeling and promote the development of PH. Smooth muscle cells with contractile phenotype highly express α-SMA and calponin. Western blotting was used to detect the effects of wogonin on α-SMA and calponin expression. Compared with the control group, the expression of α-SMA and calponin in the model group was significantly decreased, while the expression of α-SMA was upregulated by wogonin (10 μM). The results suggest that PDGF-BB can promote the transformation of PASMCs from contractile to synthetic phenotype, while wogonin (10 μM) has the potential effect to inhibit this transformation B,C. We aHypoxia can promote the binding of HIF-1 to the hypoxia response element (HRE) in response to hypoxia status, while prolonged HIF-1 activation can lead to changes in pulmonary vascular structure. In addition to hypoxia stimulation, PDGF-BB can also induce HIF-1α in smooth muscle cells. The results confirmed that PDGF-BB can promote the expression of HIF-1α in smooth muscle cells, and the level of HIF-1α in the wogonin (3 μM) group was significantly decreased, indicating that wogonin can inhibit the expression of HIF-1α in PDGF-BB-induced PASMCs A. HIF-1αScutellaria baicalensis, has attracted increasing scientific attention in recent years because of its potent activity on the cardiovascular system. The phenomenon of pulmonary vascular remodeling is often preceded by a progressive increase in vascular tone. Wogonin can inhibit aortic contraction induced by norepinephrine and potassium chloride in a dose-dependent manner by inhibiting extracellular calcium influx and intracellular calcium release [Wogonin, a flavonoid isolated from  release . On the  release . Hyperpr release ,33. Prev release ,35. Part release . Wogonin release . Wogonin release . 2O2-induced migration of HUVECs as well as microvessel sprouting from rat aortic rings by targeting the PI3K/Akt pathway and reducing VEGF expression [In addition to cell proliferation, cell migration and the production and degradation of the extracellular matrix are also the pathological basis of vascular structure changes. Vascular smooth muscle cells can also regulate the migration of self as well as vascular endothelial cells and induce the occurrence of vascular inflammation by secreting intercellular adhesion factors including ICAM and VCAM ,40. Wounpression . In the pression . Wogoninpression . In the In the early stage of hypoxic stimulation, the change of intracellular oxygen content in PAECs causes PASMCs to undergo phenotypic transformation through a paracrine pathway and promotes angiogenesis to adapt to the hypoxic environment . Under pIn the present study, a combination of network pharmacology and experimental validation was applied to unveil the biochemistry basis and underlying mechanisms of wogonin in the treatment of PH. The results of network pharmacology shed light on the clinical and pharmaceutical significance of wogonin in treating PH. The effects of wogonin in inhibiting PASMCs proliferation, migration, and phenotypic switching are also consistent with the results of network pharmacology. Moreover, the anti-proliferative effect of wogonin may be mediated by the regulation of the HIF-1/NOX4 pathway . It is a


Eucommia ulmoides Oliver (EU) is a plant used in Chinese medicine as a medicinal herb to treat autoimmune and inflammatory conditions. We used network pharmacology to examine the active ingredients and estimate the main targets and pathways affected by EU when it is used to treat ankylosing spondylitis (AS).  The Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform was used to search for active ingredients in EU and their target proteins. The GeneCards Database was used to find AS-related targets. The targets from the EU and AS searches that coincided were selected by constructing a Venn diagram. Then, a STRING network platform and Cytoscape software were used to analyse the protein-protein interaction (PPI) network and key targets. The strong affinity between EU and its targets was confirmed using molecular docking techniques. The Gene Ontology and the Kyoto Encyclopaedia of Genes and Genomes (KEGG) pathway enrichment analysis of overlapping targets was performed using the database for annotation, visualization, and integrated discovery online tool. α, and other signalling pathways.  The number of active ingredients against AS in EU was discovered to be 28. Major targets against AS in the PPI network and core targets analyses were identified as IL-1B, PTGS2, IL-8, nMMP-9, CCL2, MYC, and IL-2. Furthermore, molecular docking studies showed the strong affinity between EU's bioactive molecules and their AS targets. Enrichment analysis revealed that active ingredients from EU were involved in a variety of biological processes, including the response to molecules derived from bacteria, extracellular stimuli, nutrient levels, and the regulation of reactive oxygen species, all of which are mediated by interleukin-17, TNF- The therapy for AS using EU involves a multitarget, multipathway, and multiselection mechanism that includes anti-inflammatory and analgesic effects. This study provides a theoretical basis for future research into targeted molecular therapies for AS. However, long-term medication has been linked to several adverse effects, including cardiovascular, gastrointestinal, and renal concerns [Ankylosing spondylitis (AS) affects the spine and sacroiliac joints and is a chronic inflammatory illness. Due to the inflammatory damage induced by AS, patients have persistent back pain and morning stiffness, which limits their spinal mobility . Furtherconcerns , 5. Anotconcerns .Eucommia ulmoides Oliver (EU), known as ‘du zhong' in Chinese and native to China, is the only plant in the Eucommia genus of the Eucommiaceae family. The chemical constituents of EU leaves include phenylpropanoid compounds such as sitosterol (SIT), chlorogenic acid, and ursolic acid; iridoids such as eucommiol, geniposide, geniposidic acid, and aucubin; and flavonoids such as quercetin and kaempferol [Traditional Chinese medicine (TCM) divides AS into three categories: lower back pain, spinal stiffness, and kyphosis. Many researchers have studied the function of TCM in the treatment of AS . Eucommiempferol . As a naempferol . It is mempferol .The extract of EU also lowers the activity of the PI3K/Akt pathway, which helps to reduce the production of inflammatory mediators . One of Regarding biological networks, network pharmacology is a uniquely promising and cost-effective strategy for discovering bioactive components, predicting drug targets, and analysing mechanisms of drug action . FurtherIn this research, we used network pharmacology to examine the active constituents of EU for the treatment of AS and suggest the main targets and pathways involved for the first time. The targets of EU active ingredients in the treatment of AS were analysed and their enrichment analysis was carried out. The targets were then preliminarily confirmed using molecular docking, which might provide a theoretical basis for later research into targeted molecular therapies for AS.https://tcmspw.com/tcmsp) [The TCM Systems Pharmacology Database and Analysis Platform (TCMSP) (m/tcmsp)  was usedm/tcmsp) . As prevm/tcmsp) , the comhttps://www.uniprot.org/) was used to connect the target proteins and gene information. After entering the active ingredients and target proteins into the Cytoscape 3.8.0 program, an ‘active ingredient-target component' network was created. To select important active ingredients, a significance analysis was performed.Drug target proteins were discovered when the active ingredients identified as described in https://omim.org/), PharmGKB (https://www.pharmgkb.org/), TTD (https://db.idrblab.net/ttd/), DrugBank (https://go.drugbank.com/), and GeneCards (https://www.genecards.org/) databases were used to find AS-related targets. To specify the AS condition, the terms ‘ankylosing spondylitis' or ‘AS' were used; the term ‘Homo sapiens' was used to search for human disease targets related to AS. Using a Venn diagram, the pharmacological targets that were identified in The OMIM (https://string-db.org/) [Homo sapiens (0.400). The PPI network was built using Cytoscape 3.8.0 software, and the key targets were examined and selected.The chosen targets were entered into the STRING network platform (db.org/)  to creatP < 0.05 were presented [Homo sapiens. We used the ‘Analysis' function to create a graphic depiction of the top 10 elements in biological process (BP), molecular function (MF), and cellular component (CC) based on their relevance.The key mechanisms of the action of the targets were investigated using GO enrichment. The GO enrichment of EU's targets in treating AS was investigated using the ClueGO plug-in in Cytoscape. Only relevant GO keywords with resented , and thehttps://david.ncifcrf.gov/). The official gene symbols were used as the identifiers. GeneList was chosen as the list type. Homo sapiens was the only species available. Then, we looked for KEGG pathway information that differed significantly. We used R software, R 3.6.0 for Windows to create a bubble plot of the signalling pathways. We used the DAVID database to download the most enriched pathway, and the enriched genes were indicated with red stars.The KEGG pathway was used to further explain the function of the targets in metabolism, signal transduction, and other activities by monitoring their distribution throughout the network. For pathway enrichment analysis, the targets were loaded into the database for annotation, visualization, and integrated discovery (DAVID) database (https://www.rcsb.org/) was used to get the crystal structures of the protein targets. The imported crystal structures were used to build the receptor protein grid in the molecular operating environment (MOE), which was related to protonation, water removal, repeated structure building, structure preparation, and energy reduction. The ligand binding site was chosen using the receptor grid design. Finally, to acquire the graphical results of molecular docking, the three-dimensional BioData Mining (2020) 13 : 12 Page 5 of 18 chemical structure was altered using ChemBioDraw software, imported into the MOE, and docked with the protein construct.To represent the relationship between small molecule ligands and protein receptors, molecular docking technology developed based on the lock and key concept. In addition, based on the conformation change and energy matching procedure, the optimal binding mode and location were found. The SYBYL-X 2.1.1 for Windows was used in this work to confirm the compound-protein target interaction. The Research Collaboratory for Structural Bioinformatics Protein Data Bank database . There were 1604 BP, 105 MF, and 25 CC protein targets in all. Responses to lipopolysaccharide (LPS), bacterial molecules, extracellular stimuli, nutrient levels, reactive oxygen species metabolic processes, multicellular organism, biotic stimuli, and female pregnancy are all examples of BP. DNA-binding transcription factor binding, nuclear receptor activity, ligand-activated transcription factor activity, RNA polymerase II-specificDNA-binding, transcription factor binding, transcription coactivator binding, cytokine receptor binding, and repressing transcription factor binding are all examples of MF. The exterior side of the plasma membrane, membrane raft, membrane microdomain, ficolin-1-rich granule lumen, and secretory granule lumen are all part of the CC. All the top ten protein targets are shown in The FunRich 3.1.3 program screened a total of 1734 GO items (P < 0.05). Response to molecules of bacterial origin, LPS, response to extracellular stimuli, response to nutrient levels, reactive oxygen species, multi-cellular organism processes, cellular response to biotic stimuli, and regulation of reactive oxygen species were among the 139 KEGG-enriched signalling pathways discovered by searching the DAVID database  were chosen, and the active ingredient quercetin was rated first in the median of the ‘active drug-target component' network. For molecular docking, SYBYL-X 2.1.1 was employed . Higher The rational method of network pharmacology is used to investigate the possible biological mechanisms of TCM in the treatment and prevention of many illnesses, particularly chronic and recurrent disorders . PreviouAverrhoa carambola leaf extract depresses the central nervous system in the thiopental-sodium model of Swiss albino mice [Lannea coromandelica is potentially antidiabetic [Avicennia alba has antidiabetic, anti-inflammatory, analgesic, and antidiarrheal activity [Previous studies have found that many TCM components play important roles in many human diseases. For example, ino mice . Also, tdiabetic . In addidiabetic . As repoactivity . Furtheractivity , 55. Bioactivity , 57. In activity . Kaempfeactivity , 60. Alsactivity . The actactivity . The catactivity . The comactivity .Notably, these EU active ingredients work together to produce anti-inflammatory, anti-apoptotic, and cartilage homeostatic effects, all of which might be considered viable treatment options for AS.The PPI network analysis revealed that IL-1B, PTGS2, IL-8, and nMMP-9 may be significant targets for EU's effectiveness in the treatment of AS. These targets had the most contact with others in EU's overall active ingredient-AS target network, and they were most likely to cause a cascade of antagonistic effects on AS. Furthermore, gene targets working on several pathways were discovered in the CTP network, indicating the relevance of certain targets in the broader AS bioinformatics network. This also supported EU's ‘multiple targets-multiple routes' regulatory mechanism.α. The mechanism of IL-8 action in AS, like that of IL-1, is unclear. The levels of IL-8 are higher in patients with AS, according to existing research [The cytokine IL-1B is a proinflammatory cytokine generated primarily by activated macrophages and belongs to the IL-1 family. Monocytes and macrophages and B and T lymphocytes are all activated by IL-1B, which causes fibroblast proliferation and synovial pannus development. In addition, IL-1B has a role in chronic inflammation. Furthermore, the intracellular actions of IL-1A and IL-1B may cause inflammation in AS . There aresearch . In osteresearch . In addiresearch . While tα, coupled with IL-17 and interleukin-23 (IL-23), plays a crucial role. The cytokine IL-17 has been demonstrated to improve T cell priming and induce the production of proinflammatory mediators  by a variety of cell types, including fibroblasts, endothelial cells, macrophages, and epithelial cells [α are complicated and not completely understood. When TNF-α binds to the TNFR1, it activates proinflammatory and programmed cell death pathways, which are linked to tissue damage, according to results from in vitro and in vivo studies. The TNFR2 has been demonstrated to modulate tissue healing and angiogenesis signals [α has been found in the sacroiliac joints of individuals suffering from AS, especially in the early active stages of the condition [α levels are linked to the disease activity.Our KEGG study demonstrated that lipids and atherosclerosis, the IL-17 signalling pathway, and the TNF signalling pathway are all linked to EU's therapeutic mechanism for the treatment of AS. As a chronic inflammatory disease, AS affects the spine and sacroiliac joints. It has the potential to harm the skeletal system's structural and functional integrity. Several other organs, including the eyes, skin, gastrointestinal tract, and cardiovascular system (CVS) may also be damaged . Among Cal cells . The cytal cells . The sig signals . The sigondition , 75, andMolecular docking experiments also revealed that a representative selection of AS targets, IL-1B, PTGS2, and IL-8 had a high affinity for the active ingredients of EU. In this research, network pharmacology was employed to build on EU's possible anti-AS actions, which were then visualised using molecular docking. However, the findings from the prediction need additional experimental verification due to the limits of network pharmacology and molecular docking technologies.According to this research, treatment of AS using EU might involve a complex multitarget, multipathway, and multiselection mechanism. The proteins IL-1B, PTGS2, IL-8, nMMP-9, CCL2, MYC, and IL2 were identified as major targets against AS in the PPI network and core target analyses. The strong affinity between EU's bioactive molecules and their AS targets was confirmed. As a medicinal herb, EU may be used as an analgesic, immunomodulator, antimicrobial, and anti-inflammatory. This study might provide a theoretical basis for research into targeted molecular therapies for AS.


To systematically evaluate the clinical efficacy of modified Xiebai Powder or modified Xiebai Powder combined with Western medicine in the treatment of pneumonia and explore its potential mechanism of action.  Meta-analysis was used to screen the eligible literature on randomized controlled trials (RCTs) about Xiebai Powder in the treatment of pneumonia, and Review Manager 5.3 software was used for statistical analysis of the data. Based on the results of the meta-analysis, the active ingredients in Xiebai Powder and their therapeutic targets, disease-related targets, and intersection targets were screened using methods of network pharmacology, and their biological processes and key signaling pathways were analyzed using bioinformatics tools. Molecular docking was carried out to verify and predict the mechanisms for Xiebai Powder combined with Western medicine in the treatment of pneumonia.  A total of 16 papers were screened out, with a total of 1,465 patients. The results of the meta-analysis showed that modified Xiebai Powder or modified Xiebai Powder combined with Western medicine were superior to conventional Western medicine in terms of clinical efficacy, shortening the disappearance time of symptoms  and reducing the level of C-reactive protein, and the incidence of adverse reactions was significantly reduced. A total of 40 active ingredients in Xiebai Powder and 285 therapeutic targets of Xiebai Powder combined with azithromycin after deduplication were screened out from the database. KEGG enrichment analysis showed that Xiebai Powder combined with azithromycin might play a role in the treatment of pneumonia through the IL-17 signaling pathway, tumor necrosis factor signaling pathway, C-type lectin receptor signaling pathway, Toll-like receptor signaling pathway, and HIF-1 signaling pathway.  Modified Xiebai Powder or modified Xiebai Powder combined with azithromycin has better effects in treating pneumonia, and modified Xiebai Powder combined with azithromycin may play a role in treating pneumonia through several pathways such as the IL-17 signaling pathway. Pneumonia is an acute inflammation of the lower respiratory tract and lung parenchyma, and the main clinical symptoms are fever, cough, and shortness of breath . It is uKey to Therapeutics of Children's Diseases, written by Qianyi in the Northern Song Dynasty. Key to Therapeutics of Children's Diseases an early monograph on syndrome differentiation and treatment of pediatric diseases in China. It is also the first existing pediatric book preserved in the form of the original book in the world. It is available in Japan and some other countries and has a wide range of influence [liang each, licorice is one qian, and the amount of japonica rice is a zuo [Xiebai Powder is a classic prescription that comes from nfluence . Xiebai nfluence . In Xiebis a zuo . Cortex is a zuo . Modifieis a zuo , but theFor RCTs published in both Chinese and English, adult patients in the study should meet the diagnostic criteria published in the Chinese Journal of Tuberculosis and Respiratory Diseases in 2016 , and chiDuplicate publications, unobtainable literature, literature with incomplete or erroneous information, reviews, medical records, experience summaries, animal experiments, nonrandomized controlled experiments, inconsistent experimental designs, and patients with other comorbidities (such as heart failure) were excluded.Clinical efficacy, body temperature recovery time, cough disappearance time, pulmonary rales disappearance time, and C-reactive protein level were used as primary outcomes.Patients in the control group were treated with Western medicine. Patients in the experimental group were treated with modified Xiebai Powder or modified Xiebai Powder combined with Western medicine.The Chinese retrieval platforms used include CNKI, VIP, and Wanfang. The English databases used include PubMed, Cochrane Library, and Science Direct. Relevant Chinese and English literature from the establishment of the databases to January 2022 were obtained. The keywords of English and Chinese retrieval were “Xiebai Powder (泻白散),” “pneumonia (肺炎),” etc.After obtaining the eligible literature, relevant information such as title, first author, publication time, gender of patients, sample size, name of the disease, classification of disease, treatment measures, and outcomes were extracted. RCTs were reviewed using the Cochrane Handbook for random sequence generation, allocation concealment, blinding , completeness of outcome data, selective reporting, and other biases . The assP ≤ 0.05 indicated a statistically significant difference. The Chi-square test was used to evaluate statistical heterogeneity between studies. P°≥0.1 and I2°≤°50% indicated no heterogeneity, and analysis was performed using a fixed-effects model; otherwise, a random-effects model was used, combined with subgroup analysis or sensitivity analysis to find the source of heterogeneity.Review Manager 5.3 software was used for statistical analysis. Enumeration data were analyzed with an odds ratio (OR), mean difference (MD) for measurement data, and 95% confidence interval (CI) for efficacy analysis and statistics. https://old.tcmsp-e.com/tcmsp.php), and the active ingredients of the drugs were screened according to the oral bioavailability (OB) ≥ 30% and the drug‐likeness (DL) ≥ 0.18.According to the published literature , 12, andThe active ingredients of Xiebai Powder were obtained from the TCMSP database, and the names of protein targets were converted to gene names using the UniProt database. Azithromycin was retrieved in the TTD database, and the canonical SMILES structure was imported into the Swiss Target Prediction database and the SEA database to obtain the therapeutic targets. The Gene Cards database was used to search the keyword “pneumonia” to obtain pneumonia-related targets, and the DisGeNET database and the TTD database were used to complement pneumonia-related targets.The intersection targets of active ingredients of Xiebai Powder combined with azithromycin and pneumonia were identified using the Venny2.1 online tool and imported into the STRING database for PPI analysis. Limiting the species to “Homo sapiens,” a PPI network was constructed. The PPI network data was imported into Cytoscape 3.9.1 software and topologically analyzed using the Analyze Network function. The medians of degree centrality, closeness centrality (CC), and betweenness centrality (BC) were taken as the chi values to screen key targets.The intersection targets screened in Key targets with top degree values were selected and input into the PDB database. Their 3D structures were downloaded and saved in the PDB format, and water molecules and other ligands were removed using PyMol software. The top eight active ingredients in Xiebai Powder, according to the degree values, and the SDF format files of the active ingredients were downloaded from the PubChem database and imported into OpenBabel to be converted into PDB format. Proteins were hydrogenated using AutoDock. The Grid Box in the Grid module was set, and the vina program was used to semiflexible dock targets (receptors) with chemical ingredients (ligands). The combinations with the lowest binding energy were selected and visualized using PyMol software.A total of 138 related papers were retrieved, and 16 papers on RCTs were included according to the inclusion and exclusion criteria, with a total of 1465 patients, including 744 cases in the experimental group and 721 cases in the control group. The specific process of literature screening is shown in All RCTs were randomly grouped, of which two , 14 were(1) Clinical Efficacy. All studies [P° = 0.89, I2° = °0%), and a fixed-effects model was used for analysis. The results showed that the clinical efficacy in the experimental group (modified Xiebai Powder or modified Xiebai Powder combined with Western medicine treatment) was better than that in the control group  , P°<0.00001;  studies –28 repor(2) Body Temperature Recovery Time. A total of five papers [P°<0.00001, I2° = °97%). Subgroup analysis was performed according to whether the disease was clearly classified. The heterogeneity testing result of the three studies with unknown disease classification was P°<0.00001 and I2° = °96%, and the heterogeneity testing result of the two studies with well-defined disease classification was P°<0.00001 and I2° = °99%, indicating that disease classification was not the source of heterogeneity in this outcome. It is found by sensitivity analysis that the heterogeneity came from three studies [P° = 0.75, I2° = °0%) and analyzed by a fixed-effects model. The results showed that the body temperature recovery time in the experimental group  was shorter than that in the control group  , P°<0.00001; P°<0.00001; MD° = °−0.47, 95% CI , P°<0.00001; MD° = °−0.79, 95% CI , P°<0.00001. By analyzing and comparing the results, it was found that modified Xiebai Powder combined with conventional Western medicine had a better therapeutic effect than conventional Western medicine alone.e papers , 24, 25  studies , 24, 25, studies , 17 were(3) Cough Disappearance Time. Five papers [P°<0.00001, I2° = °97%). Subgroup analysis was performed according to whether the disease was clearly classified. The heterogeneity testing result of the two studies with unknown disease classification was P°<0.00001 and I2° = °97%, and the heterogeneity testing result of the three studies with well-defined disease classification was P°<0.00001 and I2° = °36%, indicating that disease classification was not the source of heterogeneity in this outcome. Sensitivity analysis showed that the heterogeneity came from three studies [P° = 0.89, I2° = °0%) and analyzed by a fixed-effects model. The results showed that the elimination of cough in the experimental group was more effective than that in the control group , P°<0.00001; P°<0.00001; MD° = °−5.60, 95% CI , P°<0.00001; MD° = °−2.20, 95% CI , P°<0.00001. By analyzing and comparing the results, it was found that modified Xiebai Powder combined with conventional Western medicine had a better therapeutic effect than conventional Western medicine.e papers , 20, 25  studies , 17, 18,(4) Pulmonary Rales Disappearance Time. A total of five papers [P° = 0.001, I2° = °77%). Subgroup analysis was performed according to whether the disease was clearly classified. The heterogeneity testing result of the three studies with unknown disease classification was P°<0.00001 and I2° = °82%, and the heterogeneity testing result of the two studies with well-defined disease classification was P°<0.00001 and I2° = °72%, indicating that disease classification was not the source of heterogeneity in this outcome. Sensitivity analysis showed that the heterogeneity came from two studies [P° = 0.15, I2° = °47%) and analyzed by a fixed-effects model. The results showed that the elimination of pulmonary rales in the experimental group was more effective than that in the control group , P < 0.00001; P < 0.00001; MD = −1.70, 95% CI , P < 0.00001. By analyzing and comparing the results, it was found that modified Xiebai Powder combined with conventional Western medicine had a better therapeutic effect than conventional Western medicine.e papers , 24, 25  studies , 24, whi studies , 18, 25 (5) C-Reactive Protein (CRP). A total of three papers [P°<0.00001, I2° = °100%). Sensitivity analysis found that the heterogeneity came from 1 study [P° = 0.18, I2° = °43%) and analyzed by a fixed-effects model. The results showed that the reduction of CRP level in the experimental group was more effective than that in the control group , P < 0.00001; P < 0.00001).e papers , 19, 25  1 study , 19 wereP° = 0.84, I2° = °0%), and a fixed-effects model was used for analysis. The results showed that the incidence of adverse reactions in the experimental group  was lower than that in the control group  , P = 0.003; A total of three papers , 18, 24 Publication bias analysis was performed on clinical effective rates, and funnel plots were drawn to observe symmetry. The number of points on the left side of Sensitivity analysis was performed on the included literature, and descriptive analysis was performed on the results. The pooled effect size of clinical effective rates in the 16 studies was excluded. The results showed no qualitative change in the pooled effect size, and the results of this study were relatively stable.38H72N2O12) had 55 target genes. Xiebai Powder combined with azithromycin had 285 therapeutic targets after deduplication. A total of 1359 pneumonia-related targets were retrieved through the Gene Cards database, 1032 through the DisGeNET database, and 17 through the TTD database. After deduplication, 1926 pneumonia-related targets were collected , and the top 20, according to the P value ranking, were plotted . The docking results were visualized by PyMol software, partially shown in The active ingredients and key targets with high degree values were selected, and AutoDock software was used for molecular docking. The results are shown in During the analysis of this study, a total of 138 papers were read, and only 16 papers in Chinese were eligible for this systematic review and meta-analysis, with a total of 1,465 patients. Clinical efficacy, body temperature recovery time, cough disappearance time, pulmonary rales' disappearance time, and C-reactive protein level were selected as primary outcomes. Meta-analysis results showed that all the outcomes were statistically significant, indicating that modified Xiebai Powder or modified Xiebai Powder combined with conventional Western medicine treatment had better effects than conventional Western medicine treatment.Due to the limitation of the overall level and quantity of the literature included in this study, more high-quality literature should be included in the follow-up to further verify the analysis results. The outcomes in the included literature were mainly clinical efficiency and clinical manifestation, and less attention was paid to the changes in vital signs  , procalcβ-sitosterol, quercetin, kaempferol, naringenin, isorhamnetin, and other compounds. Among them, β-sitosterol is a phytosterol, and the others are flavonoids. β-Sitosterol can inhibit proinflammatory cytokines such as TNF-α and IL-6. A number of in vitro and in vivo experiments have shown that quercetin has anti-inflammatory activity and can also inhibit apoptosis and repair damaged lung tissue by inhibiting the growth and metastasis of lung cancer cells [The network pharmacological analysis showed that the main active ingredients in Xiebai Powder in the treatment of pneumonia involved er cells . Kaempfeer cells . It can α and IL-6. Beta-sitosterol is one of the effective components of TCM. Maxing Shigan Decoction comes from the treatise on Shang Han Lun and is composed of Ephedrae herba, armeniacae semen amarum, gypsum fibrosum, and Glycrrhizae Radix et Rhizoma. Among the four TCM, Ephedrae herba, armeniacae semen amarum, and Glycrrhizae Radix et Rhizoma all contain β-sitosterol [Results of network pharmacology and molecular docking show that beta-sitosterol can inhibit the proinflammatory cytokines TNF-tosterol –36. Maxitosterol .Jin Kui Yao Lue. It is composed of Phragmitis rhizome, Coicis Semen, coix seed, and Persicae Semen. These four TCMs all contain human β-sitosterol [Qianjin Weijing decoction is derived from tosterol –40. Qiantosterol . From thΒ, TP53, CASP3, PTGS2, JUN, STAT3, etc. Among them, both TNF and IL-6 are inflammatory factors. A study showed that the levels of TNF-α and IL-6 in patients with pneumonia were significantly increased, indicating that TNF-α and IL-6 genes were involved in the onset of pneumonia [Β has been found to be a targeted treatment for COVID-19 [Β. It is speculated that the expression of the abovementioned transcription factor proteins is closely related to inhibiting the occurrence of the inflammatory response [The key targets of Xiebai Powder combined with azithromycin in the treatment of pneumonia were mainly TNF, IL-6, ALB, AKT1, IL-1neumonia . ALB is neumonia . TP53 isneumonia . IL-1Β hCOVID-19 . STAT3 presponse .The KEGG analysis showed that Xiebai Powder might play a role in the treatment of pneumonia through the hepatitis B-induced signaling pathway, cancer signaling pathway, toxoplasmosis-induced signaling pathway, Toll-like receptor signaling pathway, tumor necrosis factor signaling pathway, and pertussis-induced signaling pathway. The KEGG analysis showed that azithromycin might play a role in the treatment of pneumonia through the IL-17 signaling pathway, NOD-like receptor signaling pathway, Toll-like receptor signaling pathway, TNF signaling pathway, and PI3K-Akt signaling pathway.α in the tumor necrosis factor signaling pathway can not only lead to abnormal pathways but also promote inflammatory factors such as IL-1 and IL-8, stimulate the body's inflammatory responses, or even cause inflammatory chain reactions [The KEGG enrichment pathways of Xiebai Powder combined with azithromycin mainly involved the IL-17 signaling pathway, tumor necrosis factor signaling pathway, c-type lectin receptor signaling pathway, Toll-like receptor signaling pathway, and HIF-1 signaling pathway. The IL-17 signaling pathway is associated with the occurrence and development of pneumonia-induced sepsis. In addition, the upregulation of upstream and downstream molecules in the IL-17 signaling pathway indicates the activation of the IL-17 signaling pathway, which can promote apoptosis in pneumonia-induced sepsis . Excessieactions . The Toleactions .The main KEGG pathways of azithromycin in pediatric pneumonia are the IL-17 signaling pathway, NOD-like receptor signaling pathway, Toll-like receptor signaling pathway, TNF signaling pathway, and PI3K-Akt signaling pathway. NOD-like receptors can mediate inflammatory responses, activate NF-B p65 and p38 MAPK signaling pathways, and lead to the production of inflammatory factors . A NOD-lIn this study, meta-analysis combined with network pharmacology was used to evaluate Xiebai Powder in the treatment of pneumonia systematically and to predict its potential mechanism of action. In terms of clinical efficacy, body temperature recovery time, cough disappearance time, pulmonary rales' disappearance time, and C-reactive protein reduction, the experimental group was superior to the control group, with a lower incidence of adverse reactions.Also, the mechanism for Xiebai Powder combined with azithromycin in the treatment of pneumonia was analyzed using network pharmacology. Due to the complexity of chemical ingredients and targets of TCM and their effects on the human body, further studies are needed to provide more evidence. Based on the potential pathways obtained through meta-analysis and network pharmacological studies, the next step will be to obtain key pathways based on cell level or animal experiments to study the mechanisms of Xibai Powder in the treatment of pediatric pneumonia.Overall, the analysis results are credible. More high-quality papers will be included in the follow-up to verify the analysis results further. Considering dynamic changes in pneumonia studies, in future research, other relevant outcomes such as changes in vital signs , procalcitonin (PCT), and T cell population will be added to improve the accuracy of clinical efficacy evaluation further and enhance the quality of evidence.