Our aim is to recommend the appropriate Chinese medicinals in clinical treatment of insomnia, which are  Based on network pharmacology, the active molecules and mechanism of these four Chinese medicinals treating insomnia were sought and analyzed. The components of the four Chinese medicinals with potential activity were collected and screened. Moreover, the recollected human disease-related targets were correlated through Cytoscape 3.8.2, and the network diagram of drug component disease targets was drawn. Based on the human protein-protein interaction database, the above network diagram was imported to establish the protein-protein interaction (PPI) and composite target pathway (C-T-P) networks. After selecting important information, the pathway analysis was carried out to show the biological process, core target, and core pathway of insomnia treatment.  In this study, 44 active components and 81 drug-disease common targets were obtained; 307 key targets were found in the PPI network; a core cluster composed of 14 nodes and 50 functional associations was found. SuānZăoRénTāng (Sour Jujube Decoction), which is commonly used in insomnia, they have similar effects on the immune system and HPA axis, while the focus of the four Chinese medicinals is mainly on endocrine regulation, and SuānZăoRénTāng (Sour Jujube Decoction) is mainly on anti-inflammatory effect. In summary, the four Chinese medicinals' effective components and main mechanism of in the treatment of insomnia may be related to their participation in the regulation of endocrine. Compared with the existing network pharmacological analysis results of  Insomnia, as a standard clinical symptom associated with neurological or psychological disorders, describes sleep latency or sleep disturbance, as well as the condition of subjective poor sleep quality . It is dSuānZăoRénTāng (Sour Jujube Decoction), GuīPíWán (Spleen-Restoring Pill), and ĀnShénDìngZhìWán  are recommended. Chinese medicinal is more effective in the treatment of insomnia, and there are fewer reports of negative effects.At present, there are three main ways to treat insomnia: cognitive behavior therapy, drug therapy, and traditional Chinese medicine (TCM) intervention . Among tsuānzăorén (Semen Ziziphi Spinosae), zhĭqiào (Fructus Aurantii), báizhú , fúlíng (Poria), báisháo (Radix Paeoniae Alba), chuānxiōng (Rhizoma Chuanxiong), and zhīmŭ (RhizomaAnemarrhenae). According to the domestic literature, the most frequently used drugs for insomnia are suānzăorén (Semen Ziziphi Spinosae), gāncăo (Radix et Rhizoma Glycyrrhizae), yèjiāoténg (Caulis Polygoni Multiflori), cháihú (Radix Bupleuri), fúlíng (Poria), dāngguī (Radix Angelicae Sinensis), chuānxiōng (Rhizoma Chuanxiong), and báisháo (Radix Paeoniae Alba) [SuānZăoRénTāng (Sour Jujube Decoction), which plays a role under the guidance of TCM theory. However, the main components and mechanism of the relevant combinations are still unknown, making it difficult to make a direct comparison with SuānZăoRénTāng (Sour Jujube Decoction). This results in a lack of evidence for clinical choice. Based on the theory of “sour medicinals boost yin,” on which SuānZăoRénTāng (Sour Jujube Decoction) is based, four commonly used Chinese medicinals, namely, suānzăorén (Semen Ziziphi Spinosae), chuānxiōng (Rhizoma Chuanxiong), fúlíng (Poria), and báisháo (Radix Paeoniae Alba), were selected in our study. The network pharmacological flow chart in This study is based on the in-depth discussion of the molecular mechanism of TCM formulas and medicinals for the effective treatment of insomnia. After analyzing 6765 TCM prescriptions for the treatment of insomnia , medicinae Alba) . It is oThe network pharmacology of Chinese medicinals provides a new method to study the protective effect of medicinals during the course of the disease and its possible mechanisms based on network biology and multidirectional pharmacology and makes it possible to explore the medical biological networks and further clarify the human complex network system. The construction of networks of drug and drug targets not only provides a prospective framework for clarifying the relationship between medicinal plants and diseases but also promotes drug research, development, and improvement to a certain extent .Therefore, this study is aimed at analyzing the biomedical targets and mechanism of the four Chinese medicinals for insomnia treatment by network pharmacology and to clarify the components and related properties of them to provide a reference for further pharmacological research and a new perspective for clinical treatment and drug research and development.http://lsp.nwu.edu.cn/tcmsp.php) and PharmMapper [http://www.lilabecust.cn/pharmmapper/), we searched for all chemical components of the four Chinese medicinals. The molecular structures were determined by literature mining and comparison, and the drug compound database was established after screening. Extracting the active target proteins of drugs from the UniProt [http://www.uniprot.org/) database, the target information database was established after processing such as duplicated data elimination.From the platforms of TCMSP  (http://rmMapper  (http:// UniProt  (https:/A), distribution (D), metabolism (M), and excretion (E) to reach the target cells, tissues, and organs to play their roles. Oral bioavailability (OB) and drug-likeness index (DL) are the key parameters of ADME. OB, reflecting drug utilization of oral administration, is the relative amount and rate of absorption of drugs into the blood circulation. As it is the key index to determine the drug characteristic of molecules with pharmacodynamic effects , OB ≥ 30% was taken as one of the screening conditions. DL, a data combing drug compounds and drug database, can be used not only to evaluate whether a compound is suitable for drug design but also to indicate the pharmacodynamic and pharmacokinetic characteristics of drug-like molecules. A compound with DL ≥ 0.18  is considered similar to the drugs in the DrugBank database. Therefore, OB ≥ 30% and DL ≥ 0.18 were taken as the filter conditions in this study. The target compounds of the four Chinese medicinals were predicted from TCMSP, PharmMapper, and UniProt databases. Then, preliminary screening was carried out, such as removing the compounds whose corresponding targets have not been determined, deleting the repeated targets and diseases, etc. Then, using GeneCards [https://www.genecards.org/) platform, the relevant targets of insomnia were predicted. After processing, association and analysis were made between these relevant targets and the target compounds of the four Chinese medicinals for the drug-disease targets, the basis of later pathway analysis, and topology network establishment.Clinically, these four Chinese medicinals are mostly administered orally. They need to go through absorption (eneCards  (https:/http://www.hprd.org/), the human protein-protein interaction database was obtained. And it was imported into Cytoscape [From HPRD  (http://ytoscape  3.8.2 soytoscape  stands fhttps://metascape.org/gp/indexhtml#/main/step1) containing multiple databases such as GO and KEGG can enrich biological pathways and infer protein complex functions. Confirming the species setting (Homo sapiens), the key targets of the four Chinese medicinals for insomnia were imported into the database, and GO enrichment analysis was performed, including biological process analysis (GO-BP), biological cell component (GO-CC), and MF. Next, key targets and core clusters were introduced for analysis of KEGG metabolic pathway. The signal paths of the top 10-log 10 (P) value were selected as the core path.Metascape  platformsuānzăorén (Semen Ziziphi Spinosae)—American tea acid, carotene, wild Jujuboside A, and phytosterol; 15 in fúlíng (Poria)—Poria neoacid B, Poria acid A, and ivy saponin; 7 in chuānxiōng (Rhizoma Chuanxiong)—Yang Chuanxiong quinone, Chuanxiong indole, and Chuanxiong nafurolactone; and 13 in báisháo (Radix Paeoniae Alba)—paeoniflorin, benzoyl paeoniflorin, and sitosterol. After processing, the predicted targets of the four Chinese medicinals were obtained: 213 in suānzăorén (Semen Ziziphi Spinosae), 31 in fúlíng (Poria), 43 in chuānxiōng (Rhizoma Chuanxiong), and 124 in báisháo (Radix Paeoniae Alba). After deleting the duplicated ones, the aggregate amount of predicted targets was 331.A total of 44 active ingredients were found in the four Chinese medicinals, including 9 in Insomnia-disease targets were retrieved through GeneCards platform, and 528 disease targets were obtained. A total of 81 drug-disease common targets were obtained after the 331 active component targets of the four Chinese medicinals were intersected with 528 disease targets. The drug-disease common target PPI network was derived by Cytoscape software, with 1121 nodes and 6231 action associations, as shown in The top 10 key nodes in the PPI network suggested that the mechanism of the four Chinese medicinals in the treatment of insomnia is related to genes such as ESR1, AKT1, SRC, AR, MAPK1, TP53, NR3C1, CREBBP, and EP300. A core cluster composed of 14 nodes and 50 functional associations was exported through the plug-in of MCODE, and the result suggests that these targets play a key role in PPI network connection, including targets like gene RET, PTK2, ERBB2, PTPN2, MET, SHC1, PDGFRB, STAT5B, INSR, PIK3R1, IRS1, CBL, GRB2, and PTPN6, as shown in According to the HPRD information, drug-disease targets and their neighbor targets were introduced to obtain 1121 nodes. The colors from dark orange to light orange were showing the degree value of nodes in descending order.Selecting nodes above the average degree value, we got 307 key targets. Among the 307 targets, diamonds are the top 10 in regard of the degree value, and the others are circular, of which colors appear from dark green to light green in descending order.The 14 circular nodes represent the core cluster, whose colors appear in green black to azure in descending order of degree value. And the 50 edges are shown in gray.The results are shown in Figures P) values from large to small, and the top 10 were selected as the core path. It was indicated that the key targets notably abounded in the AGE-RAGE signal pathway and cGMP-PKG signal pathway in neuroactive ligand-receptor interaction, cancer pathways, tuberculosis, and diabetes complications.As shown in Figures By introducing core clusters for pathway analysis, it was found that the four Chinese medicinals played a therapeutic role in the ErbB signal pathway, cancer pathway, EGFR tyrosine kinase inhibitor resistance, and insulin signal pathway to treat insomnia.Epidemiological evidence shows that insomnia is not only a trigger factor for many accidents but also a contributing factor to human diseases with high incidence rates such as diabetes, hypertension, and malignant tumors . FurtherChinese medicinals have been reported to have a good curative effect and less side effects in the treatment of insomnia . TherefoIn this study, 44 active components and 81 drug-disease common targets were obtained; 307 key targets were found in PPI network; a core cluster composed of 14 nodes and 50 functional associations was found. In addition, gene function annotation and signal pathway analysis were carried out on them.Referring to the node degree value, ESR1, AKT1, SRC, AR, MAPK1, TP53, etc. were chosen as the key targets. ESR1  gene encIn the KEGG pathway analysis, it was found that the key targets were significantly abounded in the neuroactive ligand-receptor interaction, and the core clusters had a mutual relationship with the ErbB signal pathway. Neuroactive ligand-receptor interaction  is a colsuānzăorén (Semen Ziziphi Spinosae), chuānxiōng (Rhizoma Chuanxiong), fúlíng (Poria), and báisháo (Radix Paeoniae Alba) is related to SuānZăoRénTāng (Sour Jujube Decoction). Yet, compared with SuānZăoRénTāng (Sour Jujube Decoction), the heat-clearing effect of this combination is weaker, and the yin-enriching and blood-nourishing effect is stronger. In other word, the combination of the four Chinese medicinals calms the heart and mind by regulating liver qi and nourishing liver blood. From this perspective, it can relate to the premenopausal syndrome featured by deficient blood and floating yang, which is in accordance with the dysfunction of HPA axis and autonomic nerve [From the perspective of TCM, the combination of the four Chinese medicinals of ic nerve .SuānZăoRénTāng (Sour Jujube Decoction). SuānZăoRénTāng (Sour Jujube Decoction) contains 139 active components, among which jujube kernel saponin is one of the core components, and 1386 targets with 27 significant correlated diseases [SuānZăoRénTāng (Sour Jujube Decoction) plays an effect on cell junction tissue paths like regulation of TRP path, adhesion junction, and vitamin B6 metabolism by inflammatory mediators. What is more, this formula had the regulation of ischemic heart disease, psychiatric diseases, neuropathic pain, and other diseases.Comparison was made between the network pharmacology of these four Chinese medicinals and of diseases . CorrelaSuānZăoRénTāng (Sour Jujube Decoction). For target and mediation center degrees above the mean value, estrogen receptor, calmodulin, androgen receptor, and heat shock protein 90 (HSP90) were associated with more than 70 compounds. It is suggested that the mechanism of SuānZăoRénTāng (Sour Jujube Decoction) lies in the multicomponent and multitarget interactions and has many potential therapeutic effects [Flavonoids such as quercetin, kaempferol, and 7-methoxy-2-methyl isoflavone account for the largest proportion and are predicted to be the core components of  effects . Studies effects . Due to  effects –39.SuānZăoRénTāng (Sour Jujube Decoction), the main inflammatory factors related to sleep are tumor necrosis factor (TNF), interleukin-1 (IL-1), interleukin-2 (IL-2), etc. These inflammatory factors can promote sleep to a certain extent, but their high expression level will affect the sleep quality [SuānZăoRénTāng (Sour Jujube Decoction) can produce an antidepressant effect. The mechanism for this may be to reduce the performance of hippocampal TNF-α and IL-1β to regulate the function of the immune system, restrain the apoptosis of hippocampal neurons, and lower brain injury [SuānZăoRénTāng (Sour Jujube Decoction) may affect the immune system by downregulating the expression of cytokine related pathways to achieve anti-insomnia and antidepression effects.In  quality , 41. The quality , while Sn injury . The secn injury –46. In cSuānZăoRénTāng (Sour Jujube Decoction) shows that they have similarities in downregulating the expression of cytokine related pathways, affecting the immune system and regulating the HPA axis. The combined use of the four Chinese medicinals in this study is more likely to play a role in antitumor effect and endocrine regulation, while SuānZăoRénTāng (Sour Jujube Decoction) may be more related to anti-inflammatory effects and central nervous system regulation. However, the network method has its own limitations, and there are differences in the analysis of its target and interaction relationship, so practical research is still needed to verify the information, such as key nodes.The correlation analysis between the conclusion of this study and suānzăorén (Semen Ziziphi Spinosae), fúlíng (Poria), and báisháo(Radix Paeoniae Alba) and chuānxiōng (Rhizoma Chuanxiong) may entail the regulation of the central nervous system and endocrine system and protection of brain function and immune system in patients with insomnia. Compared with SuānZăoRénTāng (Sour Jujube Decoction), these four Chinese medicinals may be more targeted for insomnia patients complicated by endocrine disorders such as climacteric syndrome and insomnia patients complicated by tumor.In conclusion, the related use  of


R 4.0.2, including associated packages. Subsequently, molecular docking analysis was performed with AutoDock Vina 1.1.2, with intro experiments involving SH-SY5Y cells used to further investigate the mechanism of SHCJ against AD. Finally, a total of 56 active compounds of SHCJ and 192 SHCJ-AD-related targets were identified. Quercetin was identified as the top potential candidate agent. HSP90AA1, AKT1, and MAPK1 represent potential therapeutic targets. The PI3K-Akt signaling pathway potentially represents a core one mediating the effects of SHCJ against AD. Additionally, molecular docking analysis indicated that quercetin could combine well with AKT1 and multiple apoptosis-related target genes. During cell experiments, a significant increase in cell viability along with a decrease in Aβ25-35-induced apoptosis was observed after treatment with SHCJ. Furthermore, SHCJ significantly increased the phosphorylation of PI3K and Akt while reversing Aβ25-35-induced apoptosis-related protein expression downregulation.The traditional Chinese medicine (TCM) formula, Sheng Huang Chong Ji (SHCJ) is largely applied for treating Alzheimer's disease (AD), but not much is known regarding its active compounds, molecular targets, and mechanism of action. The current study aimed to predict the potential molecular mechanism of SHCJ against AD based on network pharmacology combined with in vitro validation. Using public databases, SHCJ's active compounds, their potential targets, and AD-related genes were screened, while Cytoscape Version 3.7.2 was used to build protein–protein interaction (PPI) and compound-disease-target (C-D-T) networks. Analysis of enriched Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways and Gene Ontology (GO) terms was then carried out in  The elderly population around the world is being increasingly affected by Alzheimer's disease (AD), with memory loss and a progressive decline in the cognitive function being two main characteristics of this neurodegenerative disease. In 2020, an estimated 59 million people were affected by AD, according to the World Alzheimer´s Report, and with the ageing population increasing and the lack of effective interventions, there is no doubt that the number is projected to triple by 2050 . At presAnemarrhenae and Astragalus, have been demonstrated to have definite effects on neurodegenerative diseases [Traditional Chinese medicine (TCM) has been reported to be effective in treating cardiovascular diseases, neurological diseases, respiratory diseases, and tumours –5. In addiseases , 7, suggAstragalus 1:1, has achieved curative effects since its development. SHCJ is known to improve serum nitric oxide (NO) levels, reduce serum superoxide dismutase (SOD) levels, scavenge free radicals, and enhance endogenous antioxidant capacity in ageing patients [Shen Huang Chong Ji (SHCJ), combined with clinical experience, is made up of Ren Shen Gu Ben Wan, and its information is recorded in a TCM book under the name of Yi Fang Kao. SHCJ, which is composed of Ginseng Radix etRhizoma, Polygonati Rhizoma, Epimedii Folium, Rehmanniae Radix Praeparata, and Polygoni Multiflori Radix Praeparata at a ratio of 2 : 2:1 patients . In addipatients , which iNetwork pharmacology, based on principles of systems biology, basically analyses biological networks to identify specific signal nodes that would allow the design or identification of multitarget drugs. So far, this method has proved to be useful in clarifying the multitarget effect of TCM in several diseases , 11. Molhttps://www.megabionet.org/tcmid/), the Bioinformation Analysis Tool for Molecular Mechanism of Traditional Chinese Medicine , the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform , and a database which uses text mining to obtain information on traditional Chinese medicine and associated genes and diseases , were used to extract data regarding the chemical components of five herbs present in SHCJ. These databases are widely used to explore compounds in network pharmacology as they can provide comprehensive information on TCM herbs and their ingredients [Four publicly available databases, the Traditional Chinese Medicine Integrated Database  parameters using the TCMSP database. The bioactive components of SHCJ were then identified by specifying the parameters for oral bioavailability (OB) and drug-likeness (DL). As an important pharmacokinetic parameter of oral drugs, OB reflects the percentage of an orally administered drug that is absorbed and reaches the circulatory system in the human body. On the other hand, DL considers how similar a compound is with respect to conventional medicine and is used to evaluate the “drug-like” state of the prospective compound, which helps to optimize pharmacokinetics and drug characteristics , 19. In https://db.idrblab.net/ttd/), the Online Mendelian Inheritance in Man , PharmGKB (https://www.pharmgkb.org/), DrugBank (https://go.drugbank.com/), and Gene Card (https://www.genecards.org/) [https://www.uniprot.org/), which provides protein sequences of high quality and their functional annotations [Homo sapiens genes. A Venn diagram was then used to represent overlapping portions between drug and disease targets, which reflected the common targets involved in AD's treatment with SHCJ.AD-related genes were obtained using “Alzheimer Disease” as the keyword in public databases, five of which were the Therapeutic Target Database  –24. The otations , allowedhttps://string-db.org/), a PPI network was built, with only Homo sapiens selected as the species [https://cytoscape.org/), a widely used tool for constructing and visualizing PPI networks, topological characteristics were analysed with the “CytoNCA” plugin to obtain six topological parameters of nodes: local average connectivity (LAC), network centrality (NC), eigenvector centrality (EC), degree centrality (DC), betweenness centrality (BC), and closeness centrality (CC). The core target was subsequently identified based on the median of these parameters. Within the network, node significance was indicated by the values of the parameters, with higher ones indicating greater significance. Eventually, the CytoHubba plugin and the Maximal Clique Centrality (MCC) algorithm were used to screen hub genes independently to assess the results' reliability.Using the Search Tool for the Retrieval of Interacting Genes (STRING) database  and other associated packages, an analysis of enriched Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways and Gene Ontology (GO) terms was then performed, with only those results for which the p values and q values were <0.05 considered to be of statistical significance.To determine associated biological processes, Entrez gene IDs were first created from the approved symbols of identified SHCJ-AD target genes. Using The compound-pathway-target (C-P-T) network was built with Cytoscape Version 3.7.2 to identify links between active compounds, target genes, and enriched pathways, which were represented by nodes with different colors and shapes. Visual analysis of the network is achieved by combining the size of the node and edge reaction with the binding index. The C-T-P network eventually allowed the core compounds to be obtained.X, Y, and Z side lengths were adjusted to construct the box and fully wrap the whole protein. The size parameters  of the grid box are shown in For molecular docking, the AutoDock Vina 1.1.2 software was used. Before docking, ligand structures were energy-minimized employing an MMFF94 force field, and all receptor proteins were hydrotreated by PyMol. ADFRsuite1.0 was then used to convert small molecules and receptor proteins into PDBQT format for easy identification and docking by AutoDock Vina1.1.2 software. The docking grid was centered on the centroid of proteins, and the center coordinates of the proteins are shown in SHCJ granules were weighed in a 50 mL centrifuge tube and dissolved to 0.5 g/mL with ultrapure water. Then, 2 mL of solution was placed into 5 mL centrifuge tubes on an ultraclean table, and the centrifuge tube mouth was sealed with aluminium foil. The sealed centrifugal tube was freeze-dried for 48 h  to make a dry powder, which was sterilized for 30 min with ultraviolet light. The freeze-dried powder was stored at −80°C after preparation.2.Neuroblastoma cell lines SH-SY5Y were obtained from the cell library of the Chinese Academy of Sciences . Cells were cultured in Dulbecco's modified Eagle's medium  containing 1% of penicillin/streptomycin (Invitrogen) and 10% of foetal bovine serum  prior to incubation at 37°C and under 5% CO3 SH-SY5Y cells into each well of 96-well plates, incubation was performed for 24 h for cell attachment before treating them with different concentrations of SHCJ  for 24 and 48 h. A Cell Counting Kit-8  was then used to assess cell proliferation, with absorbance (OD) values determined at 450 nm using an enzyme labelling instrument .After seeding 6 × 106/mL and after seeding them into each well of six-well plates, cell treatment was performed for 24 h with 40 μM of Aβ25–35  and SHCJ at different concentrations . Cell apoptosis was then measured with an Annexin V-FITC/PI Apoptosis Detection Kit  by following the manufacturer's instructions. Finally, apoptosis was determined by flow cytometry , with data acquisition and analysis carried out using an MXP Cytometer and CXP Analysis 2.1.An SH-SY5Y cell suspension was first prepared at a density of 2 × 10μM Aβ25–35, 400 and 800 μg/mL of SHCJ, and 100 μM of quercetin. The concentration of total proteins was subsequently determined with a Pierce BCA Protein Assay Kit (Thermo Fisher Scientific) and a concentration of 1 μg/μL was used for protein expression analysis. After protein separation by SDS–PAGE and subsequent transfer onto polyvinylidene fluoride (PVDF) membrane, the latter was blocked before being incubated overnight at 4°C with primary antibodies against PI3K , p-PI3K , AKT , p-AKT , Bcl-2 , BAX , cleaved caspase-3 , and β-actin . This was followed by a 1 h incubation with the secondary antibody . After being washed, the membranes were eventually developed with Immobilon Western Chemiluminescent HRP  for visualization using the ImageJ software (NIH), with greyscale values being also calculated using the same software to determine protein expression.A cell suspension of similar density to that of flow cytometry analysis was seeded into six-well plates and left for 24 h for cell attachment. This was followed by a 24 h treatment with 40 P < 0.05.Results from at least three separate experiments were presented as the mean ± SEM. SPSS 20.0 was used for data analysis, with the means of groups compared by one-way ANOVA to determine significance at SHCJ consists of Ginseng Radix et Rhizoma, Polygonati Rhizoma, Rehmanniae Radix Praeparata, Epimedii Folium, and Polygoni Multiflori Radix Praeparata. A total of 439 ingredients were recognized in SHCJ , kaempferol , beta-sitosterol , luteolin , and stigmasterol . The top 5 gene nodes were PTGS2, PTGS1, HSP90AA1, PRKACA, and ADRB2, with degree values of 39, 31, 26, 22, and 20, respectively. These genes may represent critical nodes within the network.A C-D-T network with 242 nodes and 947 edges was used for visualizing the link between 56 active compounds of SHCJ and 192 hub target genes . Node coAfter submitting overlapping core target genes to the STRING database, a PPI network was built. In total, 158 nodes and 644 edges were obtained  and impop value and the redder color represent greater enrichment of the GO terms. In this study, regarding AD treatment, the hub target genes of SHCJ were mostly enriched in the ubiquitin-like protein ligase binding (GO: 0044389), the response to oxidative stress (GO: 0006979), RNA polymerase II-specific (GO: 0001228), the response to lipopolysaccharide (GO: 0032496), membrane regions (GO: 0098589), membrane microdomains (GO: 0098857), the response to molecules of bacterial origin (GO: 0002237), endopeptidase activity (GO: 0004175), and membrane rafts (GO: 0045121).The 192 hub target genes were annotated based on three functions: molecular function (MF), cellular component (CC), and biological process (BP). Of the enriched GO terms identified, 2826 were statistically significant and included 2486 from BP, 120 from CC, and 220 from MF. The top 30 ones are presented in a bubble diagram . The lowP ≤ 0.05), with the top 30 significantly enriched ones having high gene counts shown as the core pathways in n = 43), Kaposi sarcoma-associated herpesvirus infection (n = 34), chemical carcinogenesis-receptor activation (n = 34), PI3K-Akt signaling pathway (n = 34), and hepatitis B (n = 33). These results indicated that SHCJ could treat AD via different mechanisms, with the PI3K-Akt signaling pathway for which most genes were enriched, representing a core pathway in the treatment of SHCJ. Notably, the PI3K/AKT signaling pathway map further demonstrated the regulatory mechanism of SHCJ-AD-related genes represented by red nodes, suggesting that AKT activation could regulate the expression of downstream apoptosis-related genes, such as Casp9, Bcl2, and BAX , Aβ25-35-treated cells displayed improved cell proliferation. Accordingly, 400 and 800 μg/mL SHCJ were selected as the doses for use in subsequent experiments.After treatment with different concentrations of SHCJ, the viability of Aiability . Howeverβ25-35 treatment resulted in a significantly higher percentage of apoptotic cells, with the subsequent inhibitory effects of SHCJ on Aβ25-35-induced apoptosis being dependent on the SHCJ concentration. These results pointed towards SHCJ's neuroprotective effects on Aβ25-35-treated cells, with these effects being better than those of quercetin  to interfere with the innate apoptotic signals of the upstream apoptosome [Neuronal loss caused by intracellular neurofibrillary tangles and amyloid deposits is the main pathological feature of AD, whereas apoptosis has been demonstrated to be an important mechanism leading to neuronal loss . In AD, optosome . Endoplaoptosome .β-amyloid 25-35 (Aβ25-35)-induced neurotoxicity in PC12 cells [β-site APP cleaving enzyme 1 (BACE-1) and amyloid precursor protein (APP), both of which cause AD [In modern clinical treatment, SHCJ displays good therapeutic effects and various pharmaceutical active ingredients have been found to have neuroprotective effects. Ginsenoside has a good protective effect on cerebral ischaemia injury, and the neuroprotective mechanism is linked to SIRT1 signaling pathways and TLR4/MyD88 activation . Polysac12 cells . Icariincause AD . Therefoβ aggregation and reverses Aβ-induced neurotoxicity by forming hydrophobic interactions and hydrogen bonds with the β-sheet structure of Aβ [Using the different public databases, 56 active SHCJ compounds and 192 SHCJ-AD-related targets were identified. The results of this study not only showed that quercetin was the most important natural compound of SHCJ against AD but also that, within the C-D-T network, this compound was associated with most genes. Quercetin is a flavonol bioactive substance that displays various pharmacological activities and its ability to cross the blood-brain barrier (BBB) has been demonstrated, thereby highlighting its potential as a drug to prevent neurodegenerative diseases , 35. Quere of Aβ . In agedre of Aβ . All theThe PPI network was constructed to reveal that HSP90AA1, AKT1, and MAPK1 might represent core targets in the anti-AD process of SHCJ, given that these genes were linked to most target nodes. We mainly focused on the AKT1 gene, which codes for one type of serine/threonine-protein kinase called AKT kinase , as one of the critical nodes in the networks. AKT1 activation relies on the PI3K pathway, which mediates a variety of biological processes through serine/threonine phosphorylation of a series of downstream substrates. Studies have demonstrated that synaptic dysfunctions in AD were the result of oxidative modification of AKT1 by ROS species, and the possible mechanism may be caused by a subsequent reduction in the AKT1 target of mTOR signaling, leading to deficiencies in activity-dependent protein translation . AKT1 kiβ interactions with the PI3K/AKT pathway. For instance, it has been shown that Aβ could increase Tau phosphorylation and induce neurotoxicity by inhibiting the PI3K/AKT pathway in neuronal cells [The GO enrichment analysis annotated target genes from BP, CC, and MF. The results suggested that enrichment was mostly observed in membrane regions, membrane microdomains, and membrane rafts. Target genes were also enriched in response to lipopolysaccharide, in oxidative stress, and in molecules of bacterial origin. Of note, target genes were also enriched in neuron death and regulation of neuron death in the BP annotation. The analysis of enriched KEGG pathways yielded 172 signaling pathways for which the target genes were significantly enriched, thus reflecting SHCJ's ability to exert therapeutic effects against AD via different mechanisms. Combined with the results of the C-T-P network analysis, the PI3K-Akt signaling pathway, for which most genes were enriched, was identified as a key signaling pathway in AD treatment. These analyses and the above results mutually corroborate each other. As a vital cell signal transduction pathway, the PI3K/AKT signaling pathway has attracted great attention in neurodegenerative diseases due to its inhibition of apoptosis, promotion of cell proliferation, and maintenance of cell cycle regulation. It was reported that the response of the PI3K/Akt signaling pathway was significantly weakened in the hippocampus of AD patients . AD's paal cells . Meanwhial cells .β deposition is a major neuropathological feature of AD, so we established an AD model whereby 40 μM of Aβ25-35 was used for treating SH-SY5Y cells for 24 h, as previously reported [β25-35. SHCJ and quercetin were confirmed to upregulate the phosphorylation of AKT and PI3K while increasing the level of the antiapoptotic protein Bcl2. At the same time, being proapoptotic proteins, the levels of cleaved caspase-3 and BAX were reduced. It is noteworthy that quercetin, as a positive control drug for SHCJ in the treatment of AD [β25-35-induced SH-SY5Y cell apoptosis compared to SHCJ. These findings reflect the therapeutic advantages of traditional Chinese medicine, for which multiple components act on multiple targets.Validation of the results from network pharmacology was achieved by molecular docking, with the docking results showing that quercetin not only bound to the top 12 core target genes but also combined well with apoptosis-related genes, such as BAX, Bcl2, Caspase-8, Caspase-9, and Caspase-3. Hence, we designed in vitro cell experiments to verify the results obtained after using both approaches . Areported . The resnt of AD , has an In summary, the main active ingredients of SHCJ, their target genes, and the potential underlying mechanisms of their activities against AD were predicted using a network pharmacology approach. PPI and C-T-P networks were constructed. This network revealed that quercetin was the most important active compound, AKT1 potentially represented a key target gene, and the PI3K-AKT signaling pathway was potentially of great significance. In addition, in vitro experiments and molecular docking confirmed the accuracy of the predictions made by network pharmacology. Finally, within the context of a multidisciplinary intervention strategy, this study provides an innovative and reliable method to explore the active compounds of traditional Chinese medicine, its molecular targets, and potential mechanisms, especially in providing conclusive evidence regarding the therapeutic effects of SHCJ on AD.


Despite their importance for several biological functions, the existing signaling pathway databases do not include information on miRNA and lncRNA. Here, we redesigned a novel pathway database named NcPath by integrating and visualizing a total of 178 308 human experimentally validated miRNA–target interactions (MTIs), 32 282 experimentally verified lncRNA–target interactions (LTIs) and 4837 experimentally validated human ceRNA networks across 222 KEGG pathways (including 27 sub-categories). To expand the application potential of the redesigned NcPath database, we identified 556 798 reliable lncRNA–protein-coding genes (PCG) interaction pairs by integrating co-expression relations, ceRNA relations, co-TF-binding interactions, co-histone-modification interactions, cis-regulation relations and lncPro Tool predictions between lncRNAs and PCG. In addition, to determine the pathways in which miRNA/lncRNA targets are involved, we performed a KEGG enrichment analysis using a hypergeometric test. The NcPath database also provides information on MTIs/LTIs/ceRNA networks, PubMed IDs, gene annotations and the experimental verification method used. In summary, the NcPath database will serve as an important and continually updated platform that provides annotation and visualization of the pathways on which non-coding RNAs (miRNA and lncRNA) are involved, and provide support to multimodal non-coding RNAs enrichment analysis. The NcPath database is freely accessible at http://ncpath.pianlab.cn/. The code and manual to use NcPath can be found at https://github.com/Marscolono/NcPath/.NcPath database is freely available at Bioinformatics online. Understanding the mechanisms of gene regulation is a major challenge in molecular biology and bioinformatics. The rapid development of high-throughput sequencing technologies .miRNAs are small non-coding RNAs with around 18–26 nucleotides that are transcribed from DNA sequences into primary miRNAs, and subsequently processed into precursor and mature miRNAs in animal and plant species. Since the first miRNA gene Lin-4 was discovered in 1993  sets will be useful for analyzing non-coding RNA lists of interest submitted by different users.In order to facilitate the study of pathway-related miRNA, we reported the first version of the NcPath database named miR+Pathway , which fNcPath bridges the gap between biological pathways and human lncRNAs/miRNAs to enhance our understanding of lncRNAs/miRNAs function, particularly the investigation of the roles played by these types of RNAs in physiological and pathological processes. In summary, NcPath constitutes a more complete platform that provides two types of non-coding RNA sets in pathways for users, and performs visualization and enrichment analysis of non-coding RNA sets submitted by users.Homo sapiens, which were obtained from the latest release of org. Hs.eg.db (Release 3.11), miRBase (v.22.1)  between lncRNA and PCG, respectively. We then used the mean PCC of 27 tissues as the final co-expression score of lncRNA and PCG. The higher the absolute value of the average correlation coefficient, the stronger the interaction of lncRNA and PCG.Co-expression relation between LncRNAs and PCGs. We used the annotation file of the whole transcriptome downloaded from GENCODE V33  and their target genes were downloaded from seven distinct databases, specifically: StarBase  and TF (j) represent the TF set interacting with the mRNA i and the lncRNA j, respectively.TF  during evolution and are therefore considered to be functionally related. The higher the co-TF score and the co-HM score, the stronger the interaction of lncRNA and mRNA.lncRNA cis-regulation relation. Genes located 20KB upstream or downstream of an lncRNA were considered cis-regulated by the lncRNA. LncRNA and mRNA annotation files were downloaded from GENCODE  and operates on a Linux-based Aliyun Web server. Adopting the idea of separating front and back ends, the web server uses Golang Gin v1.7.7 and Python V3.6 mixed development. NcPath was built using microservice architecture, and gRPC was used for communication between microservices. The frontend is built using common HTML, CSS and Javascript libraries, including the Vuetify framework for styling, Apache Echarts for the visualization library and the Vue.js framework for the side. Vue.js is a popular front-end technology and the mainstream progressive framework for building user interfaces. We chose this software due to better performance advantages compared to other front-end mainstream frameworks.The current version of NcPath was organized using MySQL 5.7.17  set enrichment analysis for users that includes a total of two input modes . In the RNAs set . The botRNAs set . Users cThe ‘Search’ page is organized as an interactive table that allows users to quickly search for non-coding RNA sets and pathway-related information. Users can search for non-coding RNAs of interest using multiple features, including gene symbol, entrez ID or accession . The resAs the first use case, we studied the target genes and pathway networks of CDKN2B-AS1, a gene that has been previously described in humans and associated with various functions  in coronSecondly, based on the research of coronavirus disease-COVID-19, we are able to search for this pathway in the NcPath database to obtain the total interaction set of all non-coding RNAs involved in the target pathway . We alsoThe above operations are only to browse and search the information obtained by searching the NcPath, providing users with interactive information recorded in the database. But, how to obtain the CDKN2B-AS1 target gene enrichment pathway? We input CDKN2B-AS1 into the lncRNA enrichment frame for enrichment analysis, and the list of results is shown in P-value of 3.82 × 10−11 was the ‘Cell cycle’, and a previous study showed that silencing SHMT2 inhibits the progression of tongue squamous cell carcinoma through cell cycle regulation (P-value of 1.04 × 10−8, followed by ‘Adherens junction’, and ‘Cellular senenscence’. Importantly, numerous experiments demonstrated these pathways affect the occurrence and development of tongue squamous cell carcinoma (The regulation of gene expression in physiological and pathophysiological processes is a central question in biomedical and life science research. To explore the role played by differential miRNAs, lncRNAs and mRNA expression patterns simultaneously, it is necessary to implement enrichment analysis of these genes and non-coding RNAs. We used NcPath to perform functional analyses on the genes differentially expressed in tongue squamous cell carcinoma , with thWe present a significant update of our web server NcPath that allows for the integrative analysis of miRNA, lncRNA and target genes with pathway interaction networks. While the original version was focused on miRNA, we now offer support for lncRNA and a higher number of pathways. NcPath is the first database providing an enrichment analysis on miRNAs, lncRNAs and mRNAs together as input. The development of new technologies and the accumulation of experimental data resulted in the generation of an increasing number of miRNA-, lncRNA- and ceRNA-related information. We will also include additional experimental sets to extend our data sources and support more powerful enrichment analysis tools. In the future, NcPath will continue supplementing more non-coding RNA classes and new features to further enhance functional interpretations, following the developments in biology. In addition, we will focus on expanding the number of species and collections, and providing users with more efficient enrichment analysis methods in the future.btac812_Supplementary_DataClick here for additional data file.


Identification of new markers with high sensitivity and specificity will help in risk reduction in acute myocardial infarction (AMI) patients with chest pain and also prevent future adverse outcomes. Hence the aim of this study was to perform a detailed miR profiling data was extracted from GSE148153 and GSE24591 datasets using the GEO2R gene expression omnibus repository and analyzed using limma algorithm. Differentially expressed miRs were obtained by comparing MI patients with corresponding controls after multiple testing corrections. Data mining for identifying candidate miRs from published literature was also performed. Target prediction and gene enrichment was done using standard bioinformatics tools. Disease specific analysis was performed to identify target genes specific for AMI using open targets platform. Protein-protein interaction and pathway analysis was done using STRING database and Cytoscape platform.The analysis revealed significant miRs like let-7b-5p, let-7c-5p, miR-4505, and miR-342-3p in important functions/pathways including phosphatidylinositol-3-kinase/AKT and the mammalian target of rapamycin, advanced glycation end products and its receptor and renin–angiotensin–aldosterone system by directly targeting angiotensin II receptor type 1, forkhead box protein O1, etc. With this approach we were able to prioritize the miR candidates for a prospective clinical association study in AMI patients of south Indian origin. India has accounted the highest mortality rate of approximately 21% due to cardiovascular disease in 2010 and majority of the deaths was due to coronary artery disease (CAD). The most critical outcome of CAD is myocardial infarction (MI or acute MI) .via regulation of apoptotic, necrotic and autophagic cell death. Many miRs have been reported as ideal biomarkers (diagnosis and prognosis) and therapeutic targets in MI. Some of these include miR-1, miR-21, miR-30a, and miR-499  . The abe miR-499 . Howeverp-value cut-off of < 0.01.The miR profiling dataset for AMI was retrieved from GEO database using search terms “myocardial infarction’ AND “Homo sapiens” AND “microRNA” AND “serum” “non-coding RNA profiling by array” OR “non-coding RNA profiling by high-throughput sequencing” . The search revealed 241 possible datasets which were further filtered to exclude other diseases, sample type, methylation and candidate miR assays. Two datasets (GSE148153 and GSE24591) were found to be appropriate for the study –10. GSE1A literature search for the key terms “microRNA” AND “myocardial infarction” OR “acute coronary syndrome” was performed using Pubmed search engine. Full-text articles published till date  for miRs as biomarkers or prognostic markers in MI/AMI/ACS were collected.miRNet2.0  and miRWA disease-specific analysis was done using the Open targets platform . The tarThe pipeline of outlier miR analysis (POMA) , a netwop < 0.05). They were further filtered using multiple testing adjusted p-value less than 0.01, which resulted in 14 miRNAs . STRING database revealed that of the 25 miRs obtained from both the datasets, only 8 miRs  possessed target genes with significantly enriched functions. The graphical representation of the significantly enriched gene ontology terms for the predicted targets is shown in Some of the most significantly enriched terms included positive regulation of cardiac muscle tissue growth, lipoprotein metabolic process, regulation of brown cell differentiation, oxidation-reduction process, advanced glycation end products and its receptor (AGE-RAGE) signaling pathway, relaxin signaling pathway, extracellular membrane (ECM)-receptor interaction, signaling by receptor tyrosine kinases, interleukins (IL) −1, −4, and −13 signaling and activation of nuclear factor kappa B (NFKB) in B-cells. Further exploration using Cytoscape revealed several relevant pathways for the predicted targets including BRCA1 associated RING domain 1 (BARD1) signaling events, Notch signaling pathway, nuclear receptors in lipid metabolism and toxicity, role of calcineurin-dependent nuclear factor of activated T cells (NFAT) signaling in lymphocytes, receptor activator of nuclear factor kappa-B ligand (RANKL/RANK) signaling pathway, fibrosis, protease-activated receptor-1 (PAR-1) mediated thrombin signaling, adipogenesis, cardiac hypertrophic response, glypican-3 network, calcium regulation in cardiac cells, myometrial relaxation and contraction pathways, fibrin complement receptor-3 signaling pathway, brain-derived neurotrophic factor (BDNF) signaling pathway, angiotensin II receptor type 1 pathway, Phosphoinositide 3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) signaling pathway, RhoA signaling pathway phosphoinositides metabolism, and renin-angiotensin-aldosterone system (RAAS).The open targets platform revealed 1570 target genes specific for AMI, which on further filtering resulted in 12 genes, namely—HMG-CoA reductase (HMGCR), coagulation factor II (F2), prostaglandin G/H synthase 1 (PTGS1), peroxisome proliferator activated receptor gamma (PPARG), peroxisome proliferator-activated receptor alpha (PPARA), beta-3 adrenergic receptor (ADRB3), angiotensin II receptor type 1 (AGTR1), interleukin 1 receptor type 1 (IL1R1), cycling dependent kinase inhibitor 2B (CDKN2B), membrane metallo endopeptidase (MME), adenosine A2B receptor (ADORA2B), and cyclin-dependent kinase inhibitor 2A (CDKN2A) that were overlapping with the predicted targets for the miRs from both the datasets used in this investigation. Among these, 9 genes were targeted by let-7b-5p and 3 genes  were transcription factors uniquely targeted by let-7b-5p and miR-342-3p.in silico work flow along with findings obtained in this study.The literature survey provided 40 studies that have reported miRs as biomarkers for diagnosis and prognosis of AMI/ACS. About 22 miRs have been reported to possess biomarker properties for both diagnosis and prognosis –53, as sIn the present study 2 datasets (GSE148153 and GSE24591) , 10 weren = 18) compared to controls (n = 30) and exhibited a moderate predictability for AMI diagnosis with AUC value ranging between 0.85 and 0.89 (via SRY-box transcription factor 6 (SOX6)-mediated apoptosis and transcription factor EB (TFEB)-mediated autophagy. In addition, in vitro analysis revealed that the dysregulation of miR-324-3p impaired its cardioprotective ability by targeting SOX6 and TFEB (via this investigation.let-7b-5p was significantly up regulated (fold change ∼2.5) in AMI patients in GSE148153 dataset. On the contrary, the expression of let-7b-5p was reported as significantly down regulated in AMI patients (and 0.89 . Similarand 0.89  reportedand TFEB . The expp < 0.01) namely, miR-494-3p, miR-7641, miR-3195, let-7b, miR-4485, miR-21, and miR-6088. Except miR-21 and miR-6088, all the miRs were significant in the initial analysis . However, a reverse expression pattern was observed in this sub-analysis, whereby those miRs upregulated in the initial analysis  showed downregulation after removing female sample. Similarly, the downregulated miRs in the initial analysis  were upregulated after removing female sample. In the initial analysis, miR-21 and miR-6088 were not statistically significant. GSE148153 dataset analysis was performed on available data from four males and one female patients with MI. The data was re-analyzed after removing the female sample, to assess any discrepancies in the miR expressions. The sub-analysis revealed 7 differentially expressed miRs , NFKB1 and RANKL. FOXO1 is a transcriptional factor which plays vital roles in the regulation of cell metabolism in various tissues, particularly in the heart and endothelium. It plays a major role in glucose and lipid metabolic pathways, atherosclerosis and cell survival. A recent study  evaluateThe predicted target genes of the differentially expressed miRs among the datasets analyzed were found to be involved primarily in leptin and insulin signaling, PI3K/AKT/mTOR, AGE-RAGE, and RAAS signaling pathways. Leptin is essential for maintaining body weight and energy balance. A deficiency of leptin or defects in the components of this signaling pathway can lead to obesity, one of the major risk factors of AMI . Leptin via oxidative stress and pro-inflammatory response  using plasma samples as the input source.One major limitation of the study is that we could identify only 2 datasets that was relevant to our analysis criteria. Moreover, systematic literature search has not been incorporated in this investigation. Due to a lesser number of samples from available datasets, there is a greater chance of missing out vital miRs that play a contributory role in the pathogenesis of MI.in silico study has prioritized miR candidates, predicted their targets, and their functional enrichment to establish the molecular mechanisms and role in the pathogenesis and progression of AMI. The analysis revealed the regulation of significantly expressed miRs like let-7b-5p, let-7c-5p, miR-4505, and miR-342-3p in important functions/pathways including PI3K/AKT/mTOR, AGE-RAGE, and RAAS by directly targeting AGTR1, FOXO1, NFKB1, RANKL, HMGCR, and AT-1. Functional studies on these identified signaling pathways are required to understand and prove the role and mechanism of action of these miRs and target genes.In conclusion, this GSE148153 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE148153), GSE24591 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE245 91), GSE60993 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE60993), and GSE48060 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE48060).Publicly available datasets were analyzed in this study. This data can be found here: The gene expression omnibus (GEO) repository, accession numbers: Ethical review and approval was not required for the study on human participants in accordance with the local legislation and institutional requirements. Written informed consent for participation was not required for this study in accordance with the national legislation and the institutional requirements.PV and CRSU designed and conceived the study. PV contributed to the literature review, data collection, bioinformatics analysis, and wrote the manuscript draft. KB, MG, and SK reviewed the manuscript and provided inputs for data interpretation. CRSU contributed to the final approval of the manuscript and agreed to be accountable for all aspects of the work. All authors contributed to the article and approved the submitted version.


Overview: Idiopathic pulmonary fibrosis (IPF) is a disease caused by many factors, eventually resulting in lung function failure. Jinbei oral liquid (JBOL) is a traditional Chinese clinical medicine used to treat pulmonary diseases. However, the pharmacological effects and mechanism of the action of JBOL on IPF remain unclear. This study investigated the protective effects and mechanism of the action of JBOL on IPF using network pharmacology analysis, followed by in vivo and in vitro experimental validation.Methods: The components of JBOL and their targets were screened using the TCMSP database. IPF-associated genes were obtained using DisGeNET and Drugbank. The common targets of JBOL and IPF were identified with the STRING database, and a protein–protein interaction (PPI) network was constructed. GO and KEGG analyses were performed. Sprague–Dawley rats were injected with bleomycin (BLM) to establish an IPF model and treated orally with JBOL at doses of 5.4, 10.8, and 21.6 ml/kg. A dose of 54 mg/kg of pirfenidone was used as a control. All rats were treated for 28 successive days. Dynamic pulmonary compliance (Cdyn), minute ventilation volume (MVV), vital capacity (VC), and lung resistance (LR) were used to evaluate the efficacy of JBOL. TGF-β–treated A549 cells were exposed to JBOL, and epithelial-to-mesenchymal transition (EMT) changes were assessed. Western blots were performed.Results: Two hundred seventy-eight compounds and 374 targets were screened, and 103 targets related to IPF were identified. Core targets, including MAPK1 (ERK2), MAPK14 (p38), JUN, IL-6, AKT, and others, were identified by constructing a PPI network. Several pathways were involved, including the MAPK pathway. Experimentally, JBOL increased the levels of the pulmonary function indices  in a dose-dependent manner and reduced the RL level in the BLM-treated rats. JBOL increased the epithelial marker E-cadherin and suppressed the mesenchymal marker vimentin expression in the TGF-β–treated A549 cells. The suppression of ERK1/2, JNK, and p38 phosphorylation by JBOL was validated.Conclusion: JBOL had therapeutic effects against IPF by regulating pulmonary function and EMT through a systemic network mechanism, thus supporting the need for future clinical trials of JBOL. Idiopathic pulmonary fibrosis (IPF) is a severe lung disease with the pathological characteristics of greatly diminished lung function, excessive fibroblast proliferation, and extracellular matrix deposition . The 5-yAstragalus membranaceus (Fisch.) Bge, Codonopsis pilosula (Franch.) Nannf, Glehnia littoralis Fr. Schmidt ex Miq, Salvia miltiorrhiza Bge, Angelica sinensis (Oliv.) Diels, Ligusticum striatum DC, Fritillaria cirrhosa D. Don, Pinellia ternata (Thunb.) Breit, Glycyrrhiza uralensis Fisch, Lonicera japonica Thunb, Scutellaria baicalensis Georgi, and Forsythia suspensa (Thunb.) Vahl.  is a traditional Chinese herbal medicine  Vahl. . JBOL is.) Vahl. . Most of.) Vahl. . Our pre.) Vahl. . HoweverNetwork pharmacology is a valuable strategy to elucidate the mechanisms of action of traditional Chinese medicines (TCM), especially to specify their characteristics, including multiple components and numerous pathway targets . The appin vitro and in vivo experimental methods.In the present study, an integrated investigation of JBOL was employed using active ingredient screening, target prediction, and network construction. The pharmacological mechanisms of JBOL on IPF and its potential targets were verified using http://lsp.nwu.edu.cn/tcmsp.php) was used. Additional reports were identified using the PubMed database searching the plant names in references that were published in the last 10 years. The JBOL active compounds were screened based on the properties of absorption, distribution, metabolism, and excretion (ADME). Two ADME-related models were developed. 1) Oral bioavailability (OB) is a critical pharmacokinetic parameter representing the percentage of a dose of an orally administered drug that reaches the systemic circulation. 2) DL is an index used to estimate the “drugability”  of potential drugs and is calculated using the Tanimoto coefficient. An OB > 20% and DL > 0.10 were used as criteria in the TCMSP screening. We also screened the available literature using the Latin names of the 12 herbs as keywords in the PubMed database to identify additional chemical components.The Traditional Chinese Medicine System Pharmacology (TCMSP) Database, The drug–target network was mapped based on the source of the TCMSP database. For experimental validation of the compound targets, information was retrieved from the HIT database . Compounhttps://www.disgenet.org/) (https://www.drugbank.ca/) databases. Genes identified in DisGeNET were scored, and the IPF-related genes were screened based on scores greater than the median score (Target genes related to IPF were collected by searching the keyword “IPF” in the DisGeNET (et.org/)  and Druget.org/)  (https:/an score .http://www.bioinformatics.com.cn/) (Bioinformatics (com.cn/)  was usedhttps://string-db.org/) was the primary tool used to construct the PPI network. The interaction relationships between the functional proteins were included in this database . The GO analysis of the biological processes, molecular functions, cell component annotation, and the KEGG pathway enrichment analysis were performed based on a p-value < 0.05  was purchased from the Hanhui Pharmaceuticals Company . Pirfenidone was obtained from the Beijing Continent Pharmaceuticals Company . TGF-β1 was purchased from MedChemExpress . Antibodies, including E-cadherin, vimentin, phospho-P38, phospho-ERK1/2, phospho-JNK, and GAPDH, were obtained from Cell Signaling Technology (MA. USA). The reference compounds, including adenosine (110879-201703), guanosine (111977-201501), chlorogenic acid (110753-202018), loganin (111640-201808), ferulic acid (110773-201614), imperialine (110767-201710), peimine (110750-201311), peiminine (110751-201712), imperatorin (110826-201918), and 18β-glycyrrhetinic acid (110723-201715) were provided by the National Institutes for Food and Drug Control. Rutin (120025-202004), liquiritigenin (070002-202011), and formononetin (130035-202003) were purchased from Shandong WoDeSen Bioscience Technology, Ltd. . Isoliquiritigenin (B21525) was obtained from Yuanye Biotech Co., Ltd. . Tanshinone I (17122704) was obtained from Chengdu Pufei De Biotech Co., Ltd. . The purity of all reference compounds was greater than 98%.Jinbei oral liquid (JBOL) (batch number: 2102001) was provided by Shandong Hongjitang Pharmaceutical Co., Ltd. . The doses of each herb in 1,000 ml of JBOL were as follows: 66 g ® BEH C18 column  . The column temperature was 30°C. The mobile phase was an elution composed of 0.1% formic acid water (A) and acetonitrile (B). One-μl samples were injected into the system, and the mobile phase flow rate was 0.3 ml/min.An Agilent 1290 UHPLC system  was used to analyze JBOL, and separation was achieved with a Waters ACQUITY UPLCad libitum. The rats were divided into six groups: 1) treatment with deionized water (control group); 2) treatment with 3 mg/kg BLM administered intratracheally ; 3) treatment with 3 mg/kg BLM (i.t.) and intragastric administration  of JBOL (5.4 ml/kg); 4) treatment with 3 mg/kg BLM (i.t.) and JBOL ; 5) treatment with 3 mg/kg BLM (i.t.) and JBOL ; 6) treatment with 3 mg/kg BLM (i.t.) and pirfenidone  as a positive control. The doses of JBOL were chosen based on our previous reports  rats, weighing approximately 200 g, were obtained from the Vital River Laboratory Animal Technology Co., Ltd. . The animal experiments were approved by the Animal Ethics Committee at Shandong University of Traditional Chinese Medicine. All rats were acclimated in an animal holding room at 25°C and provided food and water  reports . The comThe rats were anesthetized and fixed in a supine position on a board. A trachea tube was inserted into the trachea and connected with the body plethysmograph. The change in gas pressure was measured to calculate the lung volume indirectly. The indices, including dynamic pulmonary compliance (Cdyn), minute ventilation volume (MVV), vital capacity (VC), and lung resistance (LR) data were determined.2 in a humidified chamber.Human type II alveolar epithelial A549 cells were purchased from the Type Culture Collection of the Chinese Academy of Sciences . Dulbecco’s modified Eagle’s medium  with 10% fetal calf serum (FCS), 100 μg/ml streptomycin, and 100 U/ml penicillin was used for cell culture. The A549 cells were maintained at 37°C with 5% COThe A549 cells were treated with culture medium (control) or 5 ng/ml TGF-β1 with or without 0.05, 0.5, or 1% JBOL. JBOL was diluted using a culture medium. After incubation for 48 h, the cells were observed using an inverted phase-contrast microscope . The cell morphology, including characteristics of mesenchymal–epithelial transition (EMT), was observed, and images were captured at ×200 magnification. For the protein expression assay, JBOL and 5 ng/ml TGF-β1 were added to the cells and incubated for 72 h. Then the cells were lysed for analysis.Lung tissue samples or A549 cells were treated for 40 min with RIPA lysis buffer containing phosphatase inhibitors, then centrifuged  at 4°C. The protein concentration of the supernatant was assessed using bicinchoninic acid. Individual protein samples (10–30 μg) were loaded onto SDS-polyacrylamide gels for separation and then transferred to PVDF membranes . The membranes were incubated with different primary antibody solutions and shaken overnight. After the membranes were washed with Tris-buffered saline with Tween-20 (TBST), HRP-labeled secondary antibodies were added, and the membranes were incubated for 2 h. The protein bands were visualized using ECL-Plus detection . The intensity of the protein bands was quantified using Image Pro-Plus 6.0 software.t-test was used to compare the differences between the two groups. A p-value < 0.05 was considered statistically significant.All data were expressed as means ± standard error of the mean (SEM). The one-way analysis of variance (ANOVA) was used for multiple comparisons. The Student’s Astragalus membranaceus, AM), Dangshen , Beishashen , Danshen , Danggui , Chuanxiong , Chuanbeimu , Banxia , Gancao , Jinyinhua , Lianqiao , and Huangqin . The TCMSP database was used to determine the compounds present in JBOL and predict their targets. Two hundred seventy-eight candidate compounds were identified in JBOL, including 15 in AM, 20 in CP, 10 in GL, 27 in SM, 5 in AS, 9 in LS, 40 in FC, 29 in PT, 48 in RU, 15 in LJ, 22 in FS, and 97 in SB  and DrugBank databases  network was constructed using a confidence score of 0.4. The PPI network nodes represented the selected targets, and the edges represented target interactions. As seen in p-value < 0.05, 1,956 enriched biological processes were identified, including response to lipopolysaccharide, response to steroid hormones, regulation of oxygen levels, response to growth factors, and others  during pulmonary fibrosis . We evalSince the hub nodes in the PPI network included MAPK proteins, and MAPK pathways are thought to act downstream of many of the aforementioned identified pathways , the phoIPF is a chronic, progressive pulmonary disease with an unclear etiology and the pathological characteristics of progressive EMT transition, excessive extracellular matrix protein deposition, and, ultimately, respiratory system failure and death . AlthougJBOL currently is prescribed based on traditional Chinese medicine theory and has been used as a clinical treatment for interstitial lung disease . RecentlIn this study, systematic network pharmacology was used to discover the pharmacological mechanisms of JBOL underlying the IPF treatment. The process included JBOL chemical component screening, compound–target prediction, network construction, and experimental validation. Two hundred seventy-eight identified compounds in JBOL targeted 374 proteins. Among these targets, 103 targets were strongly associated with IPF . The keyThere are numerous chemical components in JBOL. The active components are challenging to identify and were confirmed by the network pharmacology analysis. Therefore, we further identified major chemical components and determined their content in JBOL using UHPLC. By combining information from the TCMSP database and the UHPLC analysis, seven identified components, guanosine, formononetin, liquiritigenin, isoliquiritigenin, imperatorin, tanshinone I, and 18β-glycyrrhetinic acid, were confirmed as major active components. Although the content of most of the identified compounds was low, the efficacy of JBOL might be attributed to the combined and additive effects of these compounds.Chlorogenic acid is thought to prevent IPF but has poor oral bioavailability . Isoliquin vivo experiment to confirm that JBOL protected pulmonary function in BLM-induced rats. Previously, a histopathological assessment was performed to verify the protective effect of JBOL in the BLM-induced lung impairment , MAPK14 (p38), MAPK8 (JNK1), and JUN were key hub nodes in the PPI network. Simultaneously, they commonly act as downstream molecules of various signaling pathways to execute signaling functions, such as the PI3K/Akt and HIF-1 signaling pathways . ERK1/2,This study is the first to show that JBOL could improve pulmonary function and inhibit EMT in the TGF-β1–induced A549 cells. The pharmacological mechanisms underlying the efficacy of JBOL might be related to systematic network regulation, including multiple components targeting several pathways centered on inhibiting phosphorylation of MAPK ERK1/2, p38, and JNK. Thus, these findings provide a theoretical and experimental basis for promoting the clinical application of JBOL.


Osteoporosis is the most common metabolic disease of skeleton with reduced bone density and weaker bone. Qianggu Capsule as a traditional chinese medicine has been widely used to treat osteoporosis. The potential pharmacological mechanism of its active ingredient Gusuibu is not well understood. The purpose of this work is to analyze the anti-osteoporosis function of Gusuibu based on network pharmacology, and further explore the potential mechanism of Qianggu Capsule. The active compounds and their corresponding targets of Gusuibu were obtained from TCMSP, TCMID, and BATMAN-TCM databases. Potential therapeutic targets for osteoporosis were obtained through DisGeNET, TTD, GeneCards, MalaCards, CTD, and OMIM databases. The overlapping targets of Gusuibu and osteoporosis were obtained. GO and KEGG pathway enrichment analysis were performed. The “Gusuibu-active compounds-target genes-osteoporosis” network and protein-protein interaction (PPI) network were constructed, and the top hub genes were screened by using the plug-in CytoHubba. Molecular docking was used to verify the binding activity of hub genes and key compounds. We identified 21 active compounds and 140 potential therapeutic targets that may be related to Gusuibu and 10 hub genes . Molecular docking analysis demonstrated that four key active small molecules in Gusuibu  have excellent binding affinity to the target proteins encoded by the top 10 hub genes. Our new findings indicated that one key active compound kaempferol activated the expression of osteoblast specific transcription factor OSX through JNK kinase pathway. Osteoporosis is the most common metabolic disease of skeleton with reduced bone density and weaker bone structure. Patients are more likely to have fractures with the pain and other complications, and the quality of life is dramatically affected . PostmenIt is well known that TCM has a long history of practice in China and other Asian countries to treat a wide range of diseases, including osteoporosis . Among tIn recent years, many researchers have started to use bioinformatics and network pharmacology to analyze the multi-component, multi-target and multi-pathway characteristics of TCM in order to elucidate mechanisms of its action, thus providing directions for further research . GusuibuIn this study we used a network pharmacology approach to analyze the key genes, active compounds and pathways in the anti-osteoporosis of Gusuibu, and further explored possible underlying molecular mechanisms. We identified one key active compound kaempferol in Gusuibu as an upregulator of osteoblast specific transcription factor OSX.https://tcmspw.com/tcmsp.php) database (http://bionet.ncpsb.org/batman-tcm/) (http://www.megabionet.org/tcmid/) (https://www.uniprot.org/) for “Homo sapiens”   and TCM /tcmid/) , the cor/tcmid/) . The datsapiens” .https://www.genecards.org/) (Relevance score ≥10)   (Score ≥ 0.01)   (Inference score ≥ 15)  (The targets related to osteoporosis were obtained through retrieving GeneCards (ore ≥10) , MalaCards.org/) , DisGeNe ≥ 0.01) , Therapene 2020) , Comparare ≥ 15) , and Onler 2020)  using thhttp://www.bioinformatics.com.cn/) to obtain the common targets. These were the candidate targets of Gusuibu to treat osteoporosis. Cytoscape software (version 3.7.2) was used to construct the network diagram of “Gusuibu-active compounds-target genes-osteoporosis”  was used to perform a PPI network analysis on the common targets (Homo sapiens” with a confidence level greater than 0.4. Cytoscape software (version 3.7.2) was used to construct the PPI network, and the plug-in 12 CytoHubba algorithms  were used to find the top 10 hub genes  was used to perform Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis to explore biological processes and signaling pathways in Gusuibu for osteoporosis . An adjuhttp://sankeymatic.com/) reveals the correspondence between herbs, components, and targets by constructing the interrelationship between the active compounds of Gusuibu and the top 10 hub genes. AutoDock Vina was used to perform molecular docking between the top 10 hub genes and key active ingredients to predict their binding affinity (http://www.rcsb.org/) and the PubChem database (https://pubchem.ncbi.nlm.nih.gov/) (The Sankey diagram (affinity . The PDBih.gov/) . The smaih.gov/) .C2C12 mesenchymal stem cells (ATCC) were cultured in DMEM with 10% fetal bovine serum , 100U/mL penicillin G sodium and 100 μg/ml streptomycin sulfate in a humidified atmosphere at 37°C. Cells were plated in 6-well plates, cultured to 60–80% confluence and treated with kaempferol.−ΔΔCt method was used to compare the RNA expressions. RNA levels were normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) levels.Kaempferol  was used to stimulate C2C12 cells for 24 h before harvest. TRIzol  was used to extract total RNA from cultured cells as previously described . RNA purp < 0.05 being considered as statistically significant.All qPCR experiments were performed in triplicate. Data were expressed as the mean ± SD. Comparisons were made between groups by Student’s t test with Rhizoma Drynariae) were obtained through the TCMSP database (Number: 71), TCMID database (Number: 53), and BATMAN-TCM platform (Number: 7). Based on the respective standards of TCMSP database (OB ≥ 30% and DL ≥ 0.18) and BATMAN-TCM platform  along with the existence of target proteins, a total of 24 active compounds of Gusuibu were screened. The corresponding target proteins were deduplicated, and a total of 331 target proteins were obtained. The UniProt database was used to convert the target proteins predicted by Gusuibu’s bioactive compounds into gene names. A total of 131 compounds in Gusuibu  were obtained, including 2,398 biological process (BP) items, 75 cellular component (CC) terms and 128 molecular function (MF) items. As shown in The Cluster Profiler package in R was used to perform GO enrichment analysis on 140 potential targets of Gusuibu in the treatment of osteoporosis. 2601 GO items  were obtained through the above R package. As shown in A total of 166 KEGG enriched pathways  and four key active compounds  using AutoDock Vina. As shown in To explore the possible role of one key active compound kaempferol in osteoblast genes, we examined the effect of kaempferol on osteoblast differentiation related gene expressions. C2C12 cells were cultured and treated as indicated with kaempferol  for 24 h. Total RNA was isolated and measured by real time RT-PCR. As shown in We asked which pathway was involved in kaempferol induced OSX gene expression. To explore molecular mechanisms of kaempferol effect on OSX expression, we used a loss-of-function approach to examine possible pathways involved. The selective inhibitors were used: SB203580 as a specific inhibitor for p38 MAPK pathway, and SP600125 is a specific inhibitor for JNK kinase pathway. C2C12 cells were treated with kaempferol. Different inhibitors were added in the culture medium. As shown in Rhizoma Drynariae (Gusuibu)  . StudiesGusuibu) . The rawBased on the principle of ADME (setting OB ≥ 30 and DL ≥ 0.18) , we searin vitro and in vivo -catechin, (R)-naringenin, Aureusidin, Digallate, Eriodyctiol (flavanone), Eriodictyol, DavalliosideA_qt, Dihydrocaffeic acid, Kurarinone, Chitranone, Stigmasterol, Xanthogalenol]. Among them, Luteolin targeted nine hub genes, Naringenin targeted five hub genes, Kaempferol targeted five hub genes, Beta-sitosterol targeted three hub genes, and the others targeted one hub gene each. Luteolin is a flavonoid found in many herbal extracts with antioxidant, anti-inflammatory and anti-tumor activities, and has been shown to reduce glucocorticoid-induced osteoporosis by modulating the ERK/Lrp-5/GSK-3β signaling pathway  in vivo . We summ in vivo  in TableWe further analyzed the potential therapeutic genes of Gusuibu against osteoporosis by GO and KEGG pathway enrichment analysis. We used osteoporosis as a search term in the KEGG database and obtained ten directly related pathways, including osteoclast differentiation (hsa04380), AGE-RAGE signaling pathway in diabetic complications (hsa04933), Endocrine and other factor-regulated calcium reabsorption (hsa04961), Mineral absorption (hsa04978), Wnt pathway (hsa04310), MAPK pathway (hsa04010), Apoptosis (hsa04210), Chemokine pathway (hsa04062), T cell receptor pathway (hsa04660), and B cell receptor pathway (hsa04662). Five of the top 10 hub genes  are enriched in osteoclast differentiation. Eight of the top 10 hub genes  are enriched in AGE-RAGE pathway in diabetic complications. Eight of the top 10 hub genes  are enriched in MAPK pathway. Six of the top 10 hub genes  are enriched in Apoptosis. Three of the top 10 hub genes  are enriched in Chemokine pathway. Five of the top 10 hub genes  are enriched in T cell receptor pathway. Four of the top 10 hub genes  are enriched in B cell receptor pathway. The three pathways are not enriched including Endocrine and other factor-regulated calcium reabsorption, Mineral absorption, and Wnt pathway.To search for potentially relevant pathways, we obtained 50 enriched pathways as osteoporosis-related pathways and used Cytoscape software to construct the “pathway-gene” network. Studies have shown that there are many pathways associated with osteoporosis, which are mainly related to bone metabolism, inflammatory response, immune response, endocrine metabolism and apoptosis . Based oAs an osteoblast-specific transcription factor OSX is required for bone formation and osteoblast differentiation . OSX wasIn summary, through network pharmacology, we explored the potential mechanisms by which Gusuibu exerts its anti-osteoporosis effects with a multi-component, multi-target, and multi-pathway profile. Potential therapeutic targets for Gusuibu to exert anti-osteoporosis effects include AKT1, IL6, JUN, TNF, MAPK3, VEGFA, EGFR, MAPK1, CASP3, PTGS2. Most of the key active compounds in Gusuibu are flavonoids, mainly including Luteolin, Naringenin, Kaempferol, and Beta-sitosterol. Interestingly, kaempferol was identified as an upregulator of OSX.


Lagynocystis pyramidalis and Jaekelocarpus oklahomensis using virtual models based on high-resolution X-ray tomography scans of three-dimensionally preserved fossil specimens. Multivariate analyses of the size, spacing and arrangement of the internal bars in these fossils indicate they are substantially more similar to gill bars in modern enteropneust hemichordates and cephalochordates than to other internal bar-like structures in fossil blastozoan echinoderms. The close similarity between the internal bars of the stylophorans L. pyramidalis and J. oklahomensis and the gill bars of extant chordates and hemichordates is strong evidence for their homology. Differences between these internal bars and bar-like elements of the respiratory systems in blastozoans suggest these structures might have arisen through parallel evolution across deuterostomes, perhaps underpinned by a common developmental genetic mechanism.Deuterostomes comprise three phyla with radically different body plans. Phylogenetic bracketing of the living deuterostome clades suggests the latest common ancestor of echinoderms, hemichordates and chordates was a bilaterally symmetrical worm with pharyngeal openings, with these characters lost in echinoderms. Early fossil echinoderms with pharyngeal openings have been described, but their interpretation is highly controversial. Here, we critically evaluate the evidence for pharyngeal structures (gill bars) in the extinct stylophoran echinoderms  Pharyngeal openings, which are outlets of the pharynx, are the only morphological character widely accepted as a deuterostome synapomorphy , inc, inc17])ill bars ,19, compill bars . These iill bars ,25,26 wiill bars –29, whicill bars .j) [figure 1e) [Ontogenetic studies of modern deuterostomes reveal that pharyngeal bars first appear as simple pores in early developmental stages and are subsequently added posteriorly as growth continues . The phafigure 1j) . In blasj) ,34, withgure 1e) . In rhomgure 1e) . The numgure 1e) . UnfortuL. pyramidalis and J. oklahomensis. For comparison, we also examine gill bars in extant enteropneust hemichordates and a cephalochordate, as well as morphologically similar bar-like elements of the respiratory systems in three fossil blastozoan echinoderms. We use linear discriminant analysis (LDA), principal component analysis (PCA) and pairwise analysis of variance (ANOVA) to quantify the similarity between these structures. The results provide new insights into stylophoran palaeobiology, with important implications for the appearance, function and evolution of internal bar-like structures in extinct echinoderms.To evaluate the evidence for gill bars in stylophorans, we use X-ray tomography to measure and describe the morphology of the internal bars in the stylophorans . 2 (a)L. pyramidalis  were obtained on loan from the Natural History Museum, London (NHMUK). In addition, the Pennsylvanian stylophoran J. oklahomensis (UWBM 74305) was incorporated into our analysis using existing data available on DigiMorph (http://digimorph.org/specimens/Jaeckelocarpus_oklahomensis/). Complete adult specimens of the extant enteropneust hemichordates Balanoglossus sp.  and Schizocardium sp.  and the cephalochordate Branchiostoma floridae  were collected and preserved in 70% ethanol. These samples were stained with phosphotungstic acid for 17 days.Three specimens of the Middle Ordovician stylophoran Cryptoschisma sp. (MGM-3383D) and Hyperoblastus reimanni (CMC IP 37404) and the Late Devonian rhombiferan Strobilocystites polleyi (CMC IP 36209), were selected for inclusion in our study. Specimens were obtained on loan from the Museo Geominero (MGM) and the Cincinnati Museum Center (CMC).Additionally, specimens of three Devonian blastozoan echinoderms, the fissiculate and spiraculate blastoids The preservational characteristics of the fossil samples are discussed in the electronic supplementary material, information. (b)L. pyramidalis, Balanoglossus sp., Schizocardium sp., B. floridae and S. polleyi were imaged with X-ray micro-tomography using the Nikon Metrology HMX ST 225 micro-CT scanner at the Natural History Museum, London. Jaekelocarpus oklahomensis was scanned at the University of Texas High-Resolution X-Ray Computed Tomography Facility in 2001 Tomographic datasets were used to digitally reconstruct the anatomy of 10 specimens using the SPIERS software suite . This in (d)post hoc testing (Holm–Bonferroni and Tukey 95% confidence tests) using the stats  package. 3 (a)L. pyramidalis  and two bilaterally symmetrical complexes in J. oklahomensis . The pharynx walls of the hemichordates and cephalochordate are lined with pairs of parallel bars differentiated into primary and secondary bars that bifurcate ventrally . The blastoids contain five pairs of hydrospires divided into a variable number of hollow folds, which connect to the exterior via slits or pores adjacent to the ambulacra . The thecal cavity of the rhombiferan is connected to the exterior through rhomb-pore openings distributed within three complexes . The major morphological characteristics of these structures are summarized in The thecal cavities of the stylophorans house a series of repeating elongate bars, which are divided into three elliptical fields in  (b)a). These trends are consistent with the PCA results (b). In LDA and PCA plots, depth and length are the main drivers of the disparity between groups . Squared cosines of PCA indicate that the importance of depth, width and spacing are high in the first dimension, while the second dimension is largely dominated by length . Boxplot and LDA partition plots further support these results for each grouping .The LDA plot shows that some taxa can be differentiated based on the dimensions of their internal bars or bar-like structures: the blastoids, cephalochordate and rhombiferan occupy distinct regions of the morphospace clearly separated from each other and other taxa, whereas the enteropneusts and stylophorans occupy a large region of the morphospace with a strong overlap a. These  results b. In LDAL. pyramidalis) show no statistically significant differences in any of the measurements analysed .ANOVA indicates that there are no statistically significant differences in the width or length of the bars among the enteropneusts and stylophorans . Conversely, the length of the folds in the blastoids and pores in the rhombiferan are significantly different from the length of the internal bars in all other groups . The depth of bars in all the groupings shows statistically significant differences . The spacing between bars in the stylophorans is not significantly different from the spacing of gill bars in the cephalochordates and enteropneusts . Specimens of the same species . There are also many fewer internal bars in the stylophorans  than in the cephalochordate (approx. 250) and enteropneusts . These differences may be the product of heterochrony because in the stylophorans the number of bars in adult animals remains much lower and the secondary bars do not appear to develop at any ontogenetic stage. In early developmental stages of enteropneusts the gills begin as a single pair of pores, with subsequent pairs added posteriorly during growth [L. pyramidalis and J. oklahomensis versus those of hemichordates and cephalochordates is the composition. Stylophoran internal bars are thought to have originally composed of extracellular calcite [L. pyramidalis or J. oklahomensis. Pharyngeal bars in cephalochordates and enteropneusts are made from extracellular collagen [L. pyramidalis and J. oklahomensis, subsequently if the internal bars had different composition (e.g. collagen) they would have not been preserved. Invertebrate collagen is not known to precede the development of mineralized tissue [There are some notable differences between the internal bars of the stylophorans and the gill bars of extant deuterostomes. For instance, the secondary gill bars of cephalochordates and enteropneusts are not present in g growth . In cephg growth . These pg growth . Stylophg growth . The mos calcite . In othe calcite , but thecollagen ; howeverd tissue , but in d tissue .a,b). In addition, the blastoids show almost no overlap in the morphospace (b). This is most likely a result of the complex hydrospire morphology that is well documented as highly variable across taxa [figure 1d,e and electronic supplementary material, figure S2d,e), which differs from the arrangement of the internal bars in the stylophorans, enteropneusts and cephalochordate . There is little information available on the ontogeny of the internal bars in stylophorans, making direct comparisons with other echinoderms unfeasible; however, the structures preserved in adults greatly differ from blastozoans in their relative positioning, the nature of any associated external openings and overall morphology.Multivariate analyses indicate that the gill bars and gill bar-like structures in the cephalochordate, enteropneusts and stylophorans show statistically significant differences from the bar-like elements of the respiratory systems in the blastoids and rhombiferan a,b. In aphospace , particuphospace b. This ioss taxa ,29. The J. oklahomensis were lined with cilia, similar to gills in many modern animals, and they are close to large gaps in the theca that were interpreted as atrial openings [L. pyramidalis are apparently not closely associated with any thecal openings , suggesting a different function to filter feeding. The placement of the internal bars within the theca adjacent to the main appendage, which is assumed to have housed extensions of a water vascular system in life [The function of the internal bars in the stylophorans is unclear. The gill bars of extant enteropneusts and cephalochordates are used in filter feeding ,8,31,45.openings ,20, cons in life ,23,25, p in life ,19, coul in life ,22).L. pyramidalis and Carboniferous J. oklahomensis are inferred to be derived within Stylophora. Earlier stylophorans, such as the Cambrian Ceratocystis, lack evidence of internal bars [L. pyramidalis and J. oklahomensis specimens are recurrently found with internal bars preserved. This may be because the internal bars were composed of collagen in all other stylophorans (similar to the gill bars of extant deuterostomes), and hence would have had a much lower preservation potential than the calcitic bars of L. pyramidalis and J. oklahomensis. Collagenous gill bars are more decay resistant than other internal structures in extant hemichordates [The morphological characters shared by the Ordovician nal bars , and simnal bars ,52, wherhordates ,54 and hhordates –57, but hordates ,16. The These results bring us a step closer to resolving deep evolutionary traits within the deuterostome tree. Pharyngeal openings were present in the common ancestor of deuterostomes and secondarily lost in all echinoderms sometime after the Carboniferous. This has important implications for the sequence of acquisition of the fundamental echinoderm characters, with pharyngeal openings lost after the acquisition of a functional echinoderm-type water vascular system.Click here for additional data file.