The results of this work may provide a theoretical basis for further research on the molecular mechanism of QYSXC in the treatment of ONFH.Osteonecrosis of the femoral head (ONFH) is a chronic and irreversible disease that has a risk of eventually developing into a joint collapse and resulting in joint dysfunction. Quyushengxin capsule (QYSXC) is an effective and safe traditional Chinese medicine used in the treatment of ONFH. In this present study, an integrated approach was used to investigate the mechanism of QYSXC in the treatment of ONFH, which contained systems pharmacology, molecular docking, and chip experiment. In the systems pharmacology, target fishing, protein-protein interaction (PPI), Kyoto encyclopedia of genes and genomes (KEGG) pathway enrichment analysis, and herbs-compounds-targets-pathways (H-C-T-P) network construction were performed to study the mechanism of QYSXC in the treatment of ONFH. The results showed that 15 key compounds, 8 key targets, and 8 key signaling pathways were found for QYSXC in the treatment with ONFH. Then, molecular docking was performed to further explore the interaction between some key compounds and key targets. After that, the chip experiment was performed to verify some target factors, including ICAM-1, IL-6, IL-1 Osteonecrosis of the femoral head (ONFH) is a chronic and irreversible disease that has a risk of eventually developing into a joint collapse and resulting in joint dysfunction  and is aChemical drugs, such as mobike, fenbid, and other anti-inflammatory drugs, clofibrate, lovastatin, and other lipid scavenger drugs, were used in the early clinical treatment of ONFH . AlthougONFH belongs to the category of “bone arthralgia” and “bone erosion” in Chinese medicine. Quyushengxin capsule (QYSXC) is a TCM based on the theory of etiology and pathogenesis of TCM and formed in long-term clinical practice. It is composed of Astragali radix (Huangqi), Cinnamomi ramulus (Guizhi), Sparganii rhizoma (Sanleng), Curcumae rhizoma (Ezhu), Bolbostemmatis rhizoma (Beimu), and Strychni semen (Maqianzi). Clinical studies have shown that QYSXC was able to improve the overall treatment efficiency and improve the clinical indicators with ONFH patients . In basiIn the present study, an integrated approach was adopted to investigate the mechanism of QYSXC in the treatment of ONFH, which contained systems pharmacology, molecular docking, and chip experiment. Firstly, target fishing, protein-protein interaction (PPI), Kyoto encyclopedia of genes and genomes KEGG pathway enrichment analysis, and herbs-compounds-targets-pathways (H-C-T-P) network construction were performed to thoroughly analyze the mechanism of QYSXC in the treatment of ONFH during the systems pharmacology; then, molecular docking was carried out to further explore the interaction between some key compounds and key targets; finally, the chip experiment was performed to verify some target factors. In short, the results showed that QYSXC was able to treat ONFH through multiple components, multiple targets, and multiple pathways. This work may provide a theoretical basis for further research on the molecular mechanism of QYSXC in the treatment of ONFH.https://tcmsp-e.com/) [https://www.ncbi.nlm.nih.gov/), and the Encyclopedia of Traditional Chinese Medicine (ETCM), http://www.tcmip.cn/ETCM/index.php/Home/) [The chemical components contained in the herbs were collected by Traditional Chinese Medicine Systems Pharmacology (TCMSP) database and analysis platform (-e.com/) , Nationap/Home/)  traditioThe computer ADME virtual screening was performed to obtain the potential active ingredients with good ADME properties. The pre-OB and pre-Caco2 prediction models were selected to calculate the OB and Caco-2 values –17. The https://sea.bkslab.org/) [https://www.uniprot.org/) and corrected to the official shorthand.An integration of various approaches was used to predict the most likely protein targets of the potential active compounds, which was proposed by Yu et al. . Firstlyab.org/) ; finallyhttp://db.idrblab.net/ttd/) [https://ctdbase.org/) [https://www.pharmgkb.org/) [All obtained targets were mapped to the diseases in therapeutic target database (TTD), et/ttd/) , comparase.org/) , and phakb.org/)  databasehttps://string-db.org/) [The target genes obtained by the above screening were imported in STRING 11.0 (db.org/)  to constThe KEGG pathway enrichment analysis was performed by using clusterProfiler package of program R, and the results were visualized by program R.The H-C-T-P network was constructed by Cytoscape 3.8.0 software , and thehttps://pubchem.ncbi.nlm.nih.gov). The structures of the targets were obtained from the PDB database (http://www.pdb.org). The docking simulation was conducted with AutoDock 4.2.6 software [Molecular docking was carried out to investigate the interactions between some key components and targets. The structures of the key components were obtained from the PubChem database (software . During Sprague-Dawley (SD) rats weighing 180–220 g  were purchased from the Experimental Animal Facilities of Xi'an Jiaotong University. The certificate number was SCXK2012-003. The rats were fed with a standard pellet diet and tap water at random, and they were housed and maintained in 12 h light/darkness with standard humidity and temperature in the laboratory. The principles of laboratory animal care guidelines, approved by the Animal Ethics Committee at the Shaanxi academy of traditional Chinese medicine, have been strictly observed. All efforts were made to minimize animal suffering and reduce the animals used for experiments.The drug of QYSXC was produced by Shaanxi academy of traditional Chinese medicine (lot number: 20181213). Xianlinggubao Capsule (XLGBC) was purchased from Sinopharm Group Tongjitang (Guizhou) Pharmaceutical Co., Ltd. (lot number: 180503). LPS was purchased from Sigma-Aldrich . Methylprednisolone was purchased from Pfizer .μg/kg) at an interval of 24 hours; 24 hours later, the rats received three doses of intramuscular injection of methylprednisolone (40 mg/kg) at an interval of 24 hours. After 8 weeks, QYSXC and XLGBC groups were administered by intragastrically (i.g.) daily for 4 weeks according to the above specified dose. The control group and model group were given by an equal volume of deionized water in parallel.The interior powder (0.3 g/capsule) after being removed from the shells of QYSXC was blended with deionized water as a working mixture for use. The doses selected were subject to the conversion of clinical adult dosages. According to the instructions, the clinical adult dose was four capsules for once, three times a day. After acclimatization for a period of three days, they were randomized into 6 groups: blank control group, model group, QYSXC high dose group (0.48 g/kg), QYSXC medium dose group (0.24 g/kg), QYSXC low dose group (0.12 g/kg), and XLGBC positive control group (0.4 g/kg). Model group received two doses of intraperitoneal injection of lipopolysaccharide , with the agent being Wayen Biotechnologies , Inc. The Bio-Plex software calculated cytokine concentrations on the basis of fluorescence values derived from a recombinant cytokine standard included in the 96-well plate, and the software then generated a separate standard concentration curve for each of the 8 cytokines sampled. Each standard curve was created by means of eight concentration points. A curve fitted by a five-parameter logistic equation was generated by a nonlinear least-squares minimization algorithm, and the high and low limits of detection were determined. None of the cytokine levels were above or below the detection range. The following cytokines GM-CSF, IL-1P < 0.05 was considered statistically significant, and P < 0.01 was considered to have an extremely significant difference.The heatmap was drawn by Me V software. All data were expressed as the mean ± standard deviation (SD). Graphpad Prism 5.01 software was used to ascertain statistically significant differences. The levels of significance were determined using a one-way analysis of variance (ANOVA) followed by Dunnett's multiple comparison test. A total of 551 compounds were obtained by means of TCMSP, NCBI, and ETCM databases, including 87 ingredients in astragali radix, 248 ingredients in cinnamomi ramulus, 30 ingredients in sparganii rhizoma, 81 ingredients in curcumae rhizoma, 43 ingredients in bolbostemmatis rhizoma, 62 ingredients in strychni semen. These compounds are mainly organic acid ester compounds, saponin compounds, flavonoid compounds, and phenolic compounds.β and TNF-α in synovia fluid and can inhibit the occurrence of joint inflammation [In this paper, the pre-OB and pre-Caco2 prediction models were used to screen the potential active compounds from 551 constituents obtained above . There were a total of 283 compounds had good OB and Caco-2 values, including 28 components in astragali radix, 165 components in cinnamomi ramulus, 18 components in sparganii rhizoma, 43 components in curcumae rhizoma, 12 components in bolbostemmatis rhizoma, and 17 components in strychni semen. After deduplication, 223 compounds can be used as the potential active ingredients for QYSXC . Then, all these targets were mapped to the diseases in TTD, CTD, and PharmGKB databases to further screen active ingredients and targets. The results showed that 137 constituents and 69 targets (after deduplication) were obtained in treatment with ONFH for QYSXC.The PPI was obtained by STRING 11.0 platform, and the results were transfer to Cytoscape 3.8.0 software for redrawing. In the network, there were 2 of all nodes that have no connection with other nodes and have been removed. The results showed that these 67 targets can be regarded as the main targets for QYSXC, which had good connectivity in the PPI network. As shown in P < 0.05. The obviously unrelated pathways were removed, such as “Salmonella infection,” “Influenza A,” “Leishmaniasis,” and “Pathways in cancer.” Besides, these pathways in the top 30 according to the P value were visualized in P value is 8.06E − 08. The numbers of enriched targets in HIF-1 signaling pathway and PI3K-Akt signaling pathway are both 8. EGFR, MAPK1, IL6, RELA, IFNG, NOS3, NOS2, and INSR were enriched, respectively, in HIF-1 signaling pathway, and EGFR, MAPK1, IL6, RELA, NOS3, IKBKB, INSR, and IL2 were enriched in PI3K-Akt signaling pathway.The 67 targets were significantly enriched in a total of 136 pathways with The herbs-compounds-targets-pathways (H-C-T-P) network was constructed for QYSXC in treatment with ONFH by Cytoscape 3.8.0 . The netβ and other proinflammatory ones in rat OA chondrocytes [The results of network analysis showed that quercetin had the best network topological properties, kaempferol next, and the third was linoleic acid. Quercetin and kaempferol were the main components of astragali radix. Quercetin has been used as an oral supplement for osteoarthritis (OA) because of its good anti-inflammatory and antioxidative effects, and it can alleviate symptoms and delay the progress of knee osteoarthritis . In addidrocytes . Wong etdrocytes . Linoleidrocytes .In the potential key targets, PTGS2 and PTGS1 exhibited the particularly high DC, BC, and CC values. We found that all 15 key compounds were related to PTGS2 and PTGS1. Messenger RNA microarray analysis was carried out to analyze the differential expressed genes (DEGs) in OA synovial tissues and normal counterparts, and the results showed that PTGS1 was overexpressed in OA synovial cells and tissues compared with normal synovial cells . There aα. The occurrence of ONFH was closely related to autoimmune response. Clinical studies have found that the levels of many inflammatory related factors were increased in chondrocytes or serum of ONFH patients, such as IL-1β, TNF-α, PTGS2, and IL6 [According to the results of KEGG pathway enrichment analysis and network analysis, the pathways of neurodegeneration-multiple diseases and TNF signaling pathway may be the main pathways for QYSXC in treating ONFH. There are 10 targets were enriched in the pathways of neurodegeneration-multiple diseases, including PTGS2, NOS2, MAPK14, RELA, MAPK1, IL6, GRIN1, GRIN2A, NDUFS8, and NDUFV1. Studies have shown that neurodegeneration is related to some bone diseases, such as degenerative osteoarthritis and osteoporosis , 42. In  and IL6 , 45. In  and IL6 .To further explore the interaction between the key compounds and key targets, molecular docking was performed in this present study. The molecular docking was carried out between 4 key compounds  with the highest degree centrality for QYSXC and 3 core targets, namely, PTGS2 (PDB ID: 5IKR), PTGS1 (PDB ID: 6Y3C), and NOS3 (PDB ID: 1M9R), respectively. The empirical threshold  mentioned was taken as the evaluation standard for evaluating the binding ability of key components and targets. If the docking binding energy is lower than the threshold, it showed that the binding ability between the target and the compound was stronger. The results showed that the binding abilities of most key ingredients and 3 key targets were higher than the empirical threshold, except PTGS1 with quercetin and PTGS1 with eugenol see . MeanwhiAll the results of systems pharmacology and molecular docking showed that the QYSXC can treat ONFH through multiple components, multiple targets, and multiple pathways. Subsequently, some targets were selected for further experimental verification for TNF signaling pathway, which was reported by literatures.α, IL-1β, IL-2, IL-4, IL-6, IL-10, ICAM-1, and TNF-α were selected for further statistical analysis.The heatmap of 10 protein indexes in 50 samples was drawn by Me V software see . In thisα, IL-1β, IL-2, IL-4, IL-6, IL-10, ICAM-1, and TNF-α. As shown in α (0.12 g/kg QYSX group, P < 0.01 in the high and medium dose groups, and P < 0.05 in the low dose group, respectively. The results showed that QYSXC could inhibit the serum levels of IL-1α, IL-1β, IL-2, IL-4, IL-6, IL-10, ICAM-1, and TNF-α, and QYSX capsule may prevent and cure ONFH by inhibiting the expression of inflammatory factors. ONFH was a main cause of hip diseases in young adults and is frequently observed in patients treated with high dose of glucocorticoids. There have been various hypotheses about the pathogenesis of ONFH [ of ONFH . Studies of ONFH . A large of ONFH . This woIn the present study, an integrated approach was used to investigate the mechanism of QYSXC in the treatment of ONFH, which contained systems pharmacology, molecular docking, and chip experiment. The results showed that 15 potential key compounds, 8 potential key targets, and 8 potential key signaling pathways were determined for QYSXC in treatment with ONFH. And then, the molecular docking and chip experiment have been carried out to verify some key targets. In short, QYSXC can effectively treat ONFH by means of multiple components, multiple targets, and multiple pathways. The results of this work may provide a theoretical basis for further research on the molecular mechanism of QYSXC in the treatment of ONFH.


Drugs for the treatment of tumors could result in cardiotoxicity and cardiovascular diseases. We aimed to explore the anticancer properties of Huang yam as well as its cardioprotective properties using network pharmacology and molecular docking technology. The cardiovascular targets of the major chemical components of Huang yam were obtained from the following databases: TCMSP, ETCM, and BATMAN-TCM. The active ingredients of Huang yam were obtained from SwissADME. The cardiovascular targets of antitumor drugs were obtained using GeneCards, OMIM, DrugBank, DisGeNET, and SwissTargetPrediction databases. The drug-disease intersection genes were used to construct a drug-compound-target network using Cytoscape 3.7.1. A protein-protein interaction network was constructed using Cytoscape's BisoGenet, and the core targets of Huang yam were screened to determine their antitumor properties and identify the cardiovascular targets based on topological parameters. Potential targets were imported into the Metascape platform for GO and KEGG analysis. The results were saved and visualized using R software. The components with higher median values in the network were molecularly docked with the core targets. The network contained 10 compounds, including daucosterol, delusive, dioxin, panthogenin-B, and 124 targets, such as TP53, RPS27A, and UBC. The GO function enrichment analysis showed that there were 478 items in total. KEGG enrichment analysis showed a total of 140 main pathways associated with abnormal transcription of cancer, PI3K-Akt signaling pathway, cell cycle, cancer pathway, ubiquitination-mediated proteolysis, and other pathways. Molecular docking results showed that daucosterol, delusive, dioxin, and panthogenin-B had the highest affinity for TP53, RPS27A, and UBC. The treatment of diseases using traditional Chinese medicine encompasses multiple active ingredients, targets, and pathways. Huang yam has the potential to treat cardiotoxicity caused by antitumor drugs. Drugs for the treatment of tumors could result in cardiotoxicity and cardiovascular diseases, among other side effects . The rapAt present, cardiovascular disease is one of the main factors that affect tumor survival . Interesβ-sitosterol, and daucosterol. Huang yam is the main ingredient of Di'ao Xinxuekang Capsules, which is used to prevent and treat coronary heart disease, angina pectoris, and blood stasis caused by palpitations, dizziness, and other syndromes, and is available in the form of capsules. Studies have shown that it can improve myocardial ischemia, reduce myocardial infarction area, improve heart function, and enhance vasoconstriction. Additionally, it can lower blood lipid levels and reduce atherosclerosis, platelet aggregation, and thrombosis. Steroidal saponins, the principal component of Huang yam, have superior antitumor effects and are effective in leukemia and most solid tumors [Huang yam is a traditional Chinese medicine and a major ingredient of Di'aoxinxuekang, which is a Chinese patented medicine approved by the European Union . Pharmacd tumors . A meta-d tumors . In termAt present, there has been little research on the use of the main components of Huang yam in the treatment of tumors or antitumor drug-induced cardiotoxicity. Therefore, our current study used network pharmacology, molecular docking, and other methods to analyze the potential targets of Huang yam and explore its mechanism of action in tumors and antitumor drug-induced cardiotoxicity.Huang yam contains multiple components. Each component has many targets, and there may be synergistic or antagonistic effects among the targets. Therefore, it is difficult to identify the mechanism of action of Huang yam in the treatment of cardiotoxicity caused by antitumor drugs. As network pharmacology is based on the information obtained from various databases, it can provide information on the components of this traditional Chinese medicine (TCM) and its overall mechanism of action in treating diseases. The experimental framework of this research is shown in The keyword “Huang yam” was used to comprehensively search multiple databases to obtain the chemical components of Huang yam. Databases searched included Integrated Pharmacology-based Research Platform of TCM , TCM Syshttp://www.swissadme.ch/) to search for candidate compounds and predict the related parameters of drug-like properties. In the predicted results, if the gastrointestinal absorption of the compound was “high,” it indicated that the compound had good oral bioavailability; it was assumed that two of the five drug properties  and two or more compounds are “Yes.” A compound that satisfied both conditions was considered to be an active compound. The SDF format file of the selected active ingredients was imported onto the SwissTargetPrediction platform [http://www.swisstargetprediction.ch/), after which the species name was set to “Homo sapiens.” All predicted results were saved and the duplicate values were deleted.ChemDraw19.0  was usedplatform   and tyrosinase inhibitors were retrieved using PubChem . The Swik1 of connectivity was calculated, and all nodes with connectivity greater than 2× k1, namely, “Hit hubs,” were selected. The attributes of each node in the Hit hubs network were determined to obtain the degree centrality, closeness centrality, betweenness centrality, network centrality, and local average connectivity. All nodes whose attributes were greater than the five medians, k2, l2, m2, n2, and o2, were selected as the core targets.A PPI network was constructed using Cytoscape's BisoGenet , 22 plughttp://metascape.org) [The Metascape platform (ape.org)  can be uChemDraw19.0  was usedth potential candidate. The relevant target prediction technology was used to predict the target of all 11 identified active ingredients. Duplicate targets were excluded, after which 124 predicted targets were obtained.Thirty-eight compounds of Huang yam were obtained through a literature search using the following databases: TCMIP, China HowNet, and PubMed. Using ChemDraw19.0, the structural formulae of 38 compounds of Huang yam were drawn and converted into SDF files, after retrieving them from the SwissADME website. The targets were obtained using the SwissTargetPrediction platform. The Chinese Pharmacopoeia was searched, after which the active ingredient pseudoprotodioscin was identified as an 11Dioscorea panthaica Prain et Burk was drawn and analyzed.Using Cytoscape 3.7.1, the relationship network between active components and targets of Eleven candidate active compounds were obtained from the SwissTargetPrediction platform described in The total number of nodes is 139. The node size in After searching the GeneCards, OMIM, DrugBank, and DisGeNET databases, 2059 targets related to cardiotoxicity caused by antitumor drugs were obtained.It was discovered that the potential targets of Huang yam can interact directly or indirectly with 4557 targets, and the mutual relationships among these targets reached 107,763. PPI network mapping of the targets related to cardiotoxicity caused by antitumor drugs showed that there were 4590 related targets and 114605 interrelationships among these targets. The intersection network of the two is shown in To obtain richer node connection information in the PPI network, the efficiency of node information transmission was optimized to fully clarify the targets that play an important role in the network. The network topology characteristic attribute values of the abovementioned intersection PPI network graph were calculated. After two screenings, 124 key targets were obtained. The specific screening strategy is shown in P value, the biological processes of the three best scores in the PPI network and the module were retained to describe their functions, as shown in As the roles of proteins in the PPI network are mutual, they are usually classified as undirected graphs. The internal network of the module is a potential subnet of the PPI network. The density of the subnet connection was high, whereas that of the regional part of the connection was low. Therefore, the module is considered to have biological significance. This connection has two meanings: first, a complex comprising multiple proteins that play a biological role; secondly, a functional module, such as proteins located in the same pathway, which interact more closely. Therefore, to accurately analyze the mechanism of action of Huang yam in treating cardiotoxicity caused by antitumor drugs, it is necessary to further identify the internal modules of the core PPI network. The interactive relationship was analyzed using a complex molecular detection algorithm, and the module was obtained, as shown in The Metascape platform was used to perform gene enrichment analysis on the above 124 key nodes, including biological process, cellular component, and molecular function in GO and KEGG pathways. The results were saved, after which the R language was used to draw bubbles. P value after correction before selection, 20 bars were displayed. The left side of the figure shows the names of the top GOs. In biological processes, items such as SRP-dependent cotranslational proteins targeting the cell membrane, ribonucleoprotein complex biogenesis, RNA splicing, and ribonucleoprotein complex assembly were at the forefront. In molecular functions, items such as structural constituents of ribosomes, mRNA binding, protein domain-specific binding, and cadherin binding were at the top. In cellular components, items such as cytosolic ribosomes, cytosolic small ribosomal subunits, polysomal ribosomes, and catalytic step 2 spliceosome were in the dominant position.The GO analysis results of the candidate targets showed that 478 GO terms were enriched, and these were 316 biological processes, 78 cell components, and 84 molecular functions. According to the In KEGG enrichment analysis of candidate targets, 140 pathways were significantly enriched, which are represented using bubble mapping, as shown in The top 4 pharmacodynamic molecules with degree value and core genes in the PPI network for molecular docking were selected using AutoDock Vina software. The docking results show  that theTCM is an ancient system of medicine that originated in China and has a history spanning thousands of years. Several studies have shown that the combination of TCM and modern medicine can attenuate the toxicity of tumors. Syndrome differentiation and treatment is one of the core benefits of TCM. Huang yam has the functions of regulating qi , detoxifying, and relieving pain and swelling. A metastudy showed that Di'aoxinxuekang capsules, the main ingredient of which is Huang yam, are significantly better than isosorbide dinitrate for angina pectoris and can significantly improve symptoms in patients as observed in the ECG , 27. A rFrom the compound-target network of Huang yam, the top 4 compounds with degree values, namely, daucosterol, deltoside, dioscin, and panthogenin-B, were obtained, with degree values of 60, 58, 31, and 26, respectively. The degree values of daucosterol and deltoside far exceeded those of other ingredients; therefore, these were the potential core compounds with the highest degree of biological activity against tumors and cardiovascular diseases.β-catenin signaling pathway [Daucosterol can inhibit the migration and invasion of liver cancer cells through the Wnt/ pathway . Additio pathway . Further pathway . There aβ/mTOR pathways [Dioscin is a steroidal saponin. Pharmacological experiments have shown that dioscin has antitumor, anti-inflammatory, immune-regulatory, lipid-lowering, antiviral, and antiallergic properties. Furthermore, it has beneficial effects on the heart, liver, kidney, brain, and gastrointestinal system and has broad application prospects . Additiopathways .Dioscin can protect the liver and nephrotoxicity caused by the anticancer drug, doxorubicin . It inhiThe GO biological process of core genes is mainly concentrated in the process of protein targeting membrane, RNA shearing, and the assembly and generation of ribonucleoprotein complexes. Huang yam may induce RNA cleavage, interfere with the assembly and generation of ribonucleoprotein complexes, activate Nrf2 and Sirt2 signaling pathways, and reduce HO-1 levels. The expression of NQO1, Gst, GCLM, Keap1, and FOXO3a can interfere with the cardiotoxicity caused by antitumor drugs. Huang yam may intervene in the synthesis of corresponding proteins by influencing the production of ribosomes, thereby intervening in cardiotoxicity caused by antitumor drugs. There are many transcription factors in the gene targets of cardiotoxicity caused by antitumor components in Huang yam. It also involves the binding process of enzymes, transcription factors, and receptors, and achieves therapeutic effects by interfering with the synthesis of these proteins.KEGG enrichment analysis showed that the main regulatory pathways by which Huang yam interfered with anticancer drug-induced cardiotoxicity were abnormal transcription, PI3K-Akt signaling pathway, cell cycle, cancer pathway, and ubiquitination-mediated proteolysis. This indicates that Huang yam not only interferes with the cardiotoxicity caused by antitumor drugs but also plays a positive role in the treatment of cancer through the above pathways and can be used as an adjuvant therapy for cancer chemotherapy.The activation of the abnormal transcription signal pathway in cancer is related to the abnormal expression of genes related to the cell cycle and cell proliferation, leading to abnormal cell division and the inability of normal cell apoptosis to promote cancer cell proliferation. Cancer cells can use the misregulation mechanism of mRNA to produce proteins with abnormal domains , therebyAs one of the classic signaling pathways, the PI3K-Akt signaling pathway plays a key role in the occurrence and development of many diseases. It mainly affects the survival, apoptosis, metastasis, and infiltration of tumor cells. The specificity of Akt activation is one of the key factors that results in tumor proliferation . The PI3Changes in the cell cycle are closely related to heart diseases. Cardiomyocyte damage in many heart diseases is irreversible. Eghbali et al.  found thThe ubiquitination-mediated proteolysis pathway degrades intracellular proteins to regulate cell activities and plays a vital role in cell proliferation and growth. Xiao et al.  believe In this study, the method of network pharmacology and molecular docking was used to initially explore the biological mechanism of Huang yam in the treatment of cardiotoxicity caused by anticancer drugs. It was observed that Huang yam achieves therapeutic effects through a complex network of multiple components, pathways, and targets. This study showed that the main active ingredients of Huang yam are daucosterol, deltoside, dioscin, and Panthogenin-B, which mainly target TP53, RPS27A, and UBC. The main regulatory pathways were abnormal transcription, PI3K-Akt signaling pathway, cell cycle, cancer pathway, and ubiquitination-mediated proteolysis.This study showed that Huang yam may be effective in treating heart diseases caused by antitumor therapy by inhibiting ubiquitination-mediated proteolysis, PI3K-Akt signaling pathway, and cancer pathway. According to the research and prediction of network pharmacology, we found that Huang yam is not only efficacious in reducing cardiotoxicity caused by anticancer drugs but also has a positive effect on the treatment of tumors. Therefore, it may be a potential drug for tumor treatment.in vitro and in vivo experiments are required to corroborate our findings.The results of this study have been generated using a theoretical approach and the real-life situation may be slightly different. Thus, further studies using


Contributing to this gap is the fact that living organisms are complex systems whose emerging phenotypes are the results of multiple complex interactions taking place on various pathways. This demands powerful yet user-friendly pathway analysis tools to translate the now abundant high-throughput data into a better understanding of the underlying biological phenomena. Here we introduce Consensus Pathway Analysis (CPA), a web-based platform that allows researchers to (i) perform pathway analysis using eight established methods , (ii) perform meta-analysis of multiple datasets, (iii) combine methods and datasets to accurately identify the impacted pathways underlying the studied condition and (iv) interactively explore impacted pathways, and browse relationships between pathways and genes. The platform supports three types of input: (i) a list of differentially expressed genes, (ii) genes and fold changes and (iii) an expression matrix. It also allows users to import data from NCBI GEO. The CPA platform currently supports the analysis of multiple organisms using KEGG and Gene Ontology, and it is freely available at   Advanced high-throughput and sequencing technologies have transformed biological research by allowing scientists to monitor changes in living organisms and biological systems. Regardless of the assay technology used, a comparative analysis experiment often yields a set of differentially expressed (DE) genes or gene products. Though important, these lists of DE genes fail to reveal the mechanisms underlying the studied condition. To translate the differential expression to biological knowledge, researchers have been developing various knowledge bases that map genes and their products to functional modules and biological processes. These include KEGG , ReactomMore than 70 pathway methods have been developed thus far ,6. TheseIn spite of the availability of powerful pathway methods, understanding the phenomena that determine the measured changes is as challenging as ever, if not more so. First, the sheer number of methods makes it challenging for life scientists to choose the correct method for their data and purpose. In a recent publication , we haveRecognizing these challenges, many web-based tools have been developed to assist researchers in their analysis. Tools such as EnrichNet , GENAVi Here, we introduce Consensus Pathway Analysis (CPA), a comprehensive web-based resource that allows users to compare and contrast analysis results across different methods and experiments. Specifically, CPA allows researchers to: i) perform pathway analysis using eight popular methods, GSEA , GSA 1414, FGSEAThe CPA website is a cloud-computing service for pathway analysis. It provides functions to manage users’ data, supports multiple analysis sessions and visualizes results. All computations are performed on the CPA server hosted by the University of Nevada, Reno (UNR). Inputs, parameter settings, and analysis results are saved onto the user account and can easily be loaded and updated. Users can also switch between analysis sessions, as well as browse and export results at any time.Figure https://bioconductor.org/packages/3.12/data/annotation/).The CPA platform supports three different types of input including (i) a list of differentially expressed (DE) genes, (ii) genes and their fold changes and (iii) an expression matrix. The first two input types can be directly entered on the website or uploaded from users’ local machine as a .txt or .tsv file, in which each row represents a gene. For expression matrix input, a dataset can be represented by two .csv files (command-separated)—one for expression matrix and one for sample grouping. The sample grouping file has two columns in which the first column includes samples and the second column are their corresponding groups (e.g. control or disease). The sample grouping file is optional. If not provided, users need to manually select control and disease samples in the GUI . The plaCPA provides an easy-to-use file manager for users to upload and manage expression data . Users can upload expression data files from their local machine or import them from NCBI GEO. Data importation from GEO is based on the Bioconductor R package GEOquery . A datasFigure Currently, CPA supports the analysis of more than 1000 organisms that have KEGG pathways  and GO tP-value) and the number analyses in which the P-values are significant. By default, the significance threshold is set to 5% (after adjustment using FDR), and a node appears only if the pathway is significant in at least one analysis. A slice is colored if the pathway has a significant P-value in the corresponding analysis. When users hover the mouse over a node, a small window will appear and show the P-values of the pathway in all analyses. In Figure P-values of the Alzheimer’s disease pathway. All nine P-values of this pathway are significant  can be export as raster images (.png).While exploring the analysis results, users can export any graph as raster (.png) or vector (.svg) images. They can also export results obtained from differential analyses, genes information, and https://www.meteor.com/)—a full-stack JavaScript platform for modern web development – as the core web platform to create the web server and to communicate with user clients.Figure https://reactjs.org/). The website is designed to be user-friendly with three main pages: pathway analysis, pathway visualization, and data management. In the pathway analysis page, users can upload and choose datasets, select methods, and observe gene-level statistics. Gene-level plots are generated using the Plotly JavaScript graphing library (https://plotly.com/javascript/). In the visualization page, we implement the interactive network visualization using CytoscapeJS, which is a graph theory library (https://js.cytoscape.org/). Gene heatmaps are plotted using D3js (https://d3js.org/). In the data management page, we build the file manager using OpusCapita React File Manager (https://www.npmjs.com/package/@opuscapita/react-filemanager). Data exchange between user clients and back-end servers is accomplished using the Distributed Data Protocol (Meteor client) and a Nginx web proxy server (https://www.nginx.com).For the front end, we build the graphic user interface using React, which is a JavaScript library (https://nodejs.org). NodeJS is a JavaScript runtime environment built on Chrome’s V8 JavaScript engine that allows JavaScript code to run outside the browser environments. Input files for analysis are stored on the server’s storage for fast access. Other data including user information, analysis sessions, analysis configuration, and results are saved in a MongoDB database (https://www.mongodb.com). Once the requests for performing pathway analysis are received from clients and saved by the web server, they are passed onto R servers created by Rserve (https://www.rforge.net/Rserve/) to perform pathway analyses. Multiple Rserve instances can be created to perform multiple analyses concurrently. All software and packages in the back end run in containerized environments using Docker (https://www.docker.com/).For the back end, we build the webserver using Meteor and NodeJS   . The sigThe five pathways related to neurodegenerative diseases, Pathways of neurodegeneration - multiple diseases, Alzheimer’s disease, Huntington’s disease, Parkinson’s disease, and Prion disease, are consistently identified as significant in all of the nine analyses. The Amyotrophic lateral sclerosis pathway is significant in eight out of nine.P-values of the 14 pathways. The first column shows the pathway name while the next nine columns show the P-values obtained from the nine analyses. As the web interface also allows us to combine the P-values obtained for a pathway across multiple datasets, we use the addCLT method  belong to all of the five pathways and (ii) are differentially expressed in all three datasets  analyze gene/protein expression data using eight popular methods , (ii) perform meta-analysis of multiple datasets, (iii) combine methods and datasets to find consensus results and (iv) interactively explore significantly impacted pathways across multiple analyses, and browse relationships between pathways and genes. Our main objective is to help life scientists who are trying to understand the underlying biological mechanisms when comparing two phenotypes. The platform is user-friendly with rich features to explore and visualize pathway analysis results. More importantly, it allows users to see the differences, as well as the consensus results across many methods and experiments. At the same time, we also aim at helping bioinformaticians who are developing new pathway analysis methods.http://cpa.tinnguyen-lab.com. This web application is free and open to all users. The platform also includes a tutorial page with step-by-step instruction and example analyses.The Consensus Pathway Analysis (CPA) platform is available at gkab421_Supplemental_FileClick here for additional data file.


Preeclampsia (PE), one of hypertension-related disorders of pregnancy, is a common cause of maternal death worldwide. This study aimed to identify a circRNA-miRNA-mRNA-associated ceRNA network and related pathways in PE.via related databases. A circRNA-miRNA-mRNA regulatory network was constructed.We downloaded 3 microarray datasets from the Gene Expression Omnibus database, obtained 163 differentially expressed circRNAs (dif-circRNAs) (61 upregulated and 102 downregulated), 39 differentially expressed microRNAs (dif-miRNAs) (22 upregulated and 17 downregulated), and 271 differentially expressed mRNAs (dif-mRNAs) (168 upregulated and 103 downregulated) from placenta tissues of PE. Functional enrichment analysis and protein-protein interaction (PPI) network with module analysis of dif-mRNAs were performed. The regulatory relationship between dif-miRNAs and dif-mRNAs/circRNAs was predicted via the upregulation of GNB5 by binding to miR-1248 in PE. Meanwhile, hsa_circ_0052661 might upregulate IL2RB by binding miR-4303 to play a role in PE in the same way.A total of 53 pairs were obtained, including 13 circRNAs (10 upregulated and 3 downregulated), 9 miRNAs (3 upregulated and 6 downregulated) and 31 mRNAs (22 upregulated and 9 downregulated). GNB5 and IL2RB were obtained. KEGG enrichment analysis showed that both of them were closely related with the PI3K-Akt signalling pathway. Therefore, ceRNAs might affect the PI3K-Akt signalling pathway There is still a lack of predictive and diagnostic factors for preeclampsia, which is a common cause of maternal death worldwide.This study identified a novel circRNA-associated ceRNA network and related pathways in preeclampsia.GNB5 and IL2RB might be key genes in their related circRNA-associated ceRNA network, and probably take an important role in preeclampsia via PI3K-Akt signalling pathway, which made them to be potential markers of preeclampsia.GNB5 and IL2RB might be key genes involved in the PI3K-Akt signalling pathway in PE, and hsa_circ_0087208, hsa_circ_0035443, hsa_circ_0067557 and hsa_circ_0052661 might regulate these key genes in PE by binding miR-1248 or miR-4303. Preeclampsia (PE) has an increasing incidence worldwide and remains a high cause of morbidity and mortality in pregnant women and foetuses in developing countries . PE may via which the miRNAs and circRNAs take part in gene regulation. CircRNAs may serve as miRNA sponges to liberate mRNAs regulated by the miRNAs  GNB5 is closely involved in the PI3K-Akt signalling and other pathways in placental ischaemia, and it also took part in placental insufficiency and IUGR, which is consistent with the previous research results . More reThe protein encoded by the IL2RB gene represents the beta subunit (IL2Rβ) and is a type I membrane protein that is primarily expressed in the haematopoietic system. It also can be specifically expressed in placenta induced by some variants of an alternate promoter in an upstream long terminal repeat (LTR).  In placenta tissue, IL2RB is expressed primarily in trophoblast cells from the LTR promoter and that its expression is regulated by DNA methylation, DNA methylation levels were inversely correlated with expression level . IL2RB hvia the PI3K/AKT signalling pathway [via the PI3K/Akt/mTOR pathway [via the PI3K/AKT signalling pathway. Decreased expression of RBP4 in the placenta may contribute to PE development by decreasing the invasive ability of trophoblasts [via activation of the PI3K/Akt/eNOS pathway [Both GNB5 and IL2RB were highly associated with the PI3K-Akt signalling pathway. The PI3K/AKT signalling pathway regulates a wide range of cellular functions, including cell growth, proliferation, migration and invasion . Phospha pathway . As an i pathway . Li et ahoblasts . Annexin pathway . Further pathway .in silico study that builds on the material presented in the GEO database. All three analyzed data sets were obtained by microarray technology, which is not the most sensitive tool to detect the low abundant RNAs. So the analysis is limited by the methodology. And not all 3 datasets used the same array platform, which might also influenced the obtained conclusions. Further experimental studies are needed to validate and demonstrate the definite functions of these key factors in PE. Even more advanced methods could be used to profile RNA expression, such as next-generation sequencing (NGS) technology, and Droplet Digital PCR (ddPCR) for the quantification of low abundant microRNAs. A strength of our study is that it provides a new research direction on a ceRNA network in PE. Based on this novel genetic interaction, we can further investigate the related molecular mechanism in PE pathophysiology and potential direction for new biomarker discovery.We found that GNB5 and IL2RB were the key genes in the circRNA-miRNA-mRNA networks of PE. Hsa_circ_0087208, hsa_circ_0035443 and hsa_circ_0067557 with miR-1248 and hsa_circ_0052661 with miR-4303 were predicted as ceRNAs affecting GNB5 and IL2RB genes in the PI3K-Akt signalling pathway. This is a retrospective and Click here for additional data file.


Genome-wide association studies have identified a high number of genetic loci associated with hypertension suggesting the presence of an underlying polygenic architecture. In this study, we aimed to dissect the polygenic component of primary hypertension searching also for pathway-specific components.The polygenic risk score (PRS) models, based on the UK biobank genetic signals for hypertension status, were obtained on a target Italian case/control cohort including 561 cases and 731 hyper-normal controls from HYPERGENES, and were then applied to an independent validation cohort composed by multi-countries European-based samples including 1,284 cases and 960 hyper-normal controls.P = 9.01E-07) and 3.22, 95% CI =  (P = 6.47E-08) in the target and validation cohorts, respectively. The relatively high case/control ORs across PRS quantiles corroborates the presence of strong polygenic components which could be driven by an enrichment of risk alleles within the cases but also by potential enrichment of protective alleles in the old normotensive controls. Moreover, novel pathway-specific PRS revealed an enrichment of the polygenic signal attributable to specific biological pathways. Among those the most significantly associated with hypertension status was the calcium signaling pathway together with other mainly related such as the phosphatidylinositol/inositol phosphate pathways.The resulting genome-wide PRS was capable of stratifying the individuals for hypertension risk by comparing between individuals in the last PRS decile and the median decile: we observed an odds ratio (OR) of 3.62, CI =  (The development of pathway-specific PRS could prioritize biological mechanisms, according to their contribution to the genetic susceptibility, whose regulations might be a potential pharmacological preventive target. Essential hypertension is the major cardiovascular risk factor and its prevalence is constantly increasing worldwide in middle or old age . Currentper se are generally not informative for assessing disease risk. Advances in understanding the genetics of blood pressure (BP) confirmed that individual genetic loci have modest effects on BP (<1.0 mmHg) and no clinical meaning in the population  derived pathway-specific PRS for hypertension in a Caucasian case/control target cohort. The derived models were then applied to an independent validation cohort in association with hypertension status. Furthermore, the analysis of hypertension pathway-specific PRS in the target and in the validation HYPERGENES cohorts revealed some pathways characterized by a prominent genetic association.Data originate from the multicenter international study “HYPERGENES–European Network for Genetic-Epidemiological Studies: building a method to dissect complex genetic traits, using essential hypertension as a disease model,” funded by the European Union 7th Framework Programme (grant HEALTH-2007-201550) . For theSamples were genotyped using Illumina 1M-Duo array. As the data were already used for a GWAS on hypertension we refer the readers to the previous work for details on genotyping data generation . In the P-value of associations lower than a given threshold and retain only variants that are not in linkage with the indexed variants (variants with an r2 of correlations > 0.1 were considered in linkage). The process is repeated iteratively through all index SNPs, starting from the smallest P-value and allowing each SNP to appear only in one clump. A PRS so defined contains the most significantly phenotype-associated SNPs (according to the reference GWAS) for each linkage-disequilibrium (LD) filtered clump across the entire genome. The PRS is then computed as the weighted sum of the risk alleles, where the weights represent the strength of association with the phenotype as represented by the beta coefficients of the reference genome-wide summary statistics .PRS was computed using a clumping-thresholding approach by means of PRSice software . The pipP-values thresholds, from genome wide to full model, namely .Polygenic risk score for hypertension were built according to the variant effect sizes retrieved from the GWAS on hypertension performed on UK Biobank dataset including 144,793 cases and 313,761 controls . The sumThe PRS models are based on logistic regression analysis of hypertension case/control status considering PRS as explanatory variable and including sex, BMI and the first 4 principal components as covariates to adjust for potential residual population stratification. In order to assess the relative importance of PRS the partial Nagelkerke's pseudo-R2 is computed as the difference between the R2 of the full model including covariates and PRS and the only covariates model. The best fitting model was selected according to the higher partial R2. The flowchart for the study methodology is available as In order to test for the prediction performance of the models the receiving operating curves (ROC) were derived and the corresponding area under the curves (AUC) were computed. The DeLong's test was applied to compare the AUC of the full models and of the covariate-models and thus to assess the additional specific contribution in the prediction performance attributable to the PRS.http://www.prsice.info/prset_detail/). Both exonic and intronic variants were mapped to genes and considered in the pathway-specific PRS (intergenic variants were excluded). The genes associated with essential hypertension were analyzed for functional significance in the various metabolic pathways using the KEGG (Kyoto Encyclopedia of Genes and Genomes-http://www.genome.jp/kegg/) (https://www.gsea-msigdb.org/gsea/msigdb/).Beside genome-wide PRS also pathway-specific PRS were generated by associating variants to genes according to chromosome coordinates as implemented in PRSet , http://p/kegg/) . A totalP-value filtering) after clumping were generated for each pathway so to capture the overall genetic signal present in the pathway, an approach proved to be effective to obtain to study pathways-specific associations  k variants from the background set .Since pathway-specific PRS are based on a small number of variants, full models  used to assess to which extent the genetic signal is enriched in a given pathway with respect to the underlying genome-wide association signal.In the present work 10 k permutations were applied to derive the empirical The z-score for each pathway was derived as the ratio between the PRS effect size and standard error of the corresponding logistic regression while the Pearson correlation coefficient of the z-scores was used to assess the correlation of the pathway-specific PRS in the target and in the validation cohort.P-value thresholds . The distribution of the PRS in the two cohorts is shown in A logistic regression analysis including PRS and adjusted for sex, BMI and 4 Principal Components was implemented. A strong association between the PRS and hypertension status in the target Italian cohort was detected considering all the analyzed resholds . The besresholds  and reacIn order to test to which extent PRS can differentiate individuals for the hypertension risk we stratified the individuals in deciles according to the PRS and we compared the OR across the different groups. Both in the target and in the validation cohort we observed that the higher the decile the higher the OR .P = 2.47E-09 and P = 8.97E-12 in the target and in the validation cohort, respectively). Specifically, the AUC of the covariates-only model  reached and AUC of 0.68 and 0.66 in the target and validation cohort, respectively. The inclusion of PRS in the model led to an AUC of 0.75 and 0.73 in the target and in the validation cohort, respectively.The comparison of the ROC in full model and in the covariate-only model in both cohorts showed that PRS improves the discrimination between cases and controls . The AUCP-value. Pathway-specific PRS were also computed in the multi-countries validation cohort by considering the same variants.We further examined the pathway-specific PRS for enrichment of the polygenic signal by analyzing the polygenic signal that can be attributed to KEGG pathways by considering variants located in gene-region. A total of 175,488 variants across 11,950 genes were included in pathway analysis. Pathway-specific PRS in the target cohort detected a total of 21 significant (FDR adjusted) associations out of the 186 analyzed KEGG pathways derived-PRS see . The ideP-value = 1.67E-07). Of the 21 significant pathways identified in the target cohort, 7 were replicated . We also found evidence of enrichment for pathways involved in the phosphatidylinositol signaling system and the inositol phosphate metabolism, in the mammalian target of rapamycin (mTOR) and the vascular endothelial growth factor (VEGF) signaling pathways, and in the focal adhesion and the Huntington disease pathways (n = 4), four (n = 14), three (n = 18) or between two pathways (n = 89). The most common genes among the pathways were related to the phosphatidylinositol-4,5-bisphosphate 3-kinase, the phosphoinositide-3-kinase regulatory subunit, the inositol-trisphosphate 3-kinase gene families as well as the phospholipase C isoforms and the Akt serine/threonine kinases.A moderate correlation has been observed in the Z-scores of pathways-specific PRS in the target cohort  and in tated see . Among tpathways . Gene-enpathways , some ofIn the present study a strong association between PRS based on UK Biobank data and the hypertension status was detected in two independent case/control cohorts. The best fitting model was obtained considering 2,167 clumped variants confirming that hypertension is a highly polygenic trait as already verified in other larger cohorts , 9.A recent work describing the association of PRS for BP with incident hypertension in >200,000 of the FinnGenn population based-cohort showed that individuals with the highest 2.5% PRS have a 2.3-fold risk of hypertension and 10.6 years earlier hypertension onset compared to individuals with an average PRS . AnotherThe PRS analysis for hypertension reported in the present work regarded a target and validation case/control samples from HYPERGENES, collected over many years in different European regions. Compared to the above previous results in this framework, the peculiarity of the current samples was the inclusion of older hyper-controls in this cohort previously analyzed for a classical GWAS but not yet for a potential polygenic component . DespiteBeside the primary purpose in the definition of disease status, PRS may be also informative to gain insight into the potential disease-related biological pathways by addressing their genetic contribution in PRS .Our findings reporting the analysis of hypertension pathway-specific PRS in the target and in the validation HYPERGENES cohorts revealed some pathways characterized by a prominent genetic association. Strikingly, calcium signaling and phosphatidylinositol/inositol phosphate pathways were intuitive candidates that have been extensively implicated in hypertension, whereas others such as the mTOR, the VEGF, the focal adhesion signaling pathways, and particularly the Huntington disease pathways are unexpected. In hypertension, many of the mechanisms regulating intracellular calcium homeostasis are perturbed. Calcium intracellular concentration and signaling control the key functions of vascular smooth muscle cells (VSMCs), leading to activation of the phospholipase C (PLC)-mediated phosphoinositide hydrolysis, inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG) (PLC-IP3-DAG) pathway , 30, butThe phosphatidylinositol 4,5-bisphosphate 3-kinases (PI3Ks) are common to the inositol phosphate metabolism and to the phosphatidylinositol signaling system. These enzymes mediate intracellular transduction pathways by catalyzing the phosphorylation of phosphatidylinositol 4,5-bisphosphate (PIP2) upon several stimuli . FurtherIn this study, the KEGG pathway enrichment analysis showed that also VEGF pathway was associated with hypertension, generally considered to be the main pathway responsible for angiogenesis . The VEGFocal adhesions (FAs) are integrin-containing, multi-protein structures that form mechanical links between intracellular actin bundles and the extracellular matrix (ECM) in many cell types , and alsThe mTOR, a conserved serine/threonine protein , 43 is aIn this study the most apparently hypertension-unrelated KEGG pathway is the Huntington's disease (HD), as it is a monogenic neurodegenerative disease but also influenced by genetic and environmental modifiers . InteresDifferent limitations characterizes the present study aimed at dissecting the polygenic component of hypertension. The analysis has been performed only on European individuals from the UK Biobank and HYPERGENES projects . HoweverIn conclusion, the PRS reported here, which sum up the effect of numerous genetic variants, are capable of stratifying the individuals for hypertension status both in a target and a validation cohort. These findings provide insights into the clinically relevant performance of PRS for case–control discrimination, possibly before the clinical manifestation, as the genetic make-up of an individual is largely stable from birth; therefore, genetic information can potentially be used as an early risk predictor . MoreoveThe original contributions presented in the study are included in the article/CM, ES, LC, CL, and PM conceived and designed the project. MS, VG, CB, NG, LT, TK, FC, Z-YZ, JS, DC, CL, and PM recruited the subjects and performed the assessments of patients. CB performed the genotyping. CM and OB analyzed data. CM, ES, and LC wrote the manuscript. CL and PM revised critically the manuscript content. All the authors read and approved the final manuscript.http://www.appremed.org) received a non-binding grant from Omron Healthcare Co., Ltd., Kyoto, Japan.This work was supported by the HYPERGENES project , grant HEALTH-F4-2007-201550, funded by EU within the FP7. CM receives support by the BONFOR-program of the Medical Faculty, University of Bonn (Grant Number O-147.0002.1). APPREMED (URL: 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.


N-methyltransferase (NNMT), and quinolinate phosphoribosyltransferase (QPRT). Findings regarding target genes/proteins and pathways of medicinal herbs associated with alopecia treatment offer insights for further research to better understand the pathogenesis and therapeutic mechanism of medicinal herbs for alopecia treatment with traditional herbal medicine.Hair loss is one of the most common skin problems experienced by more than half of the world's population. In East Asia, medicinal herbs have been used widely in clinical practice to treat hair loss. Recent studies, including systematic literature reviews, indicate that medicinal herbs may demonstrate potential effects for hair loss treatment. In a previous study, we identified medical herbs used frequently for alopecia treatment. Herein, we explored the potential novel therapeutic mechanisms of 20 vital medicinal herbs for alopecia treatment that could distinguish them from known mechanisms of conventional drugs using network pharmacology analysis methods. We determined the herb-ingredient–target protein networks and ingredient-associated protein (gene)-associated pathway networks and calculated the weighted degree centrality to define the strength of the connections. Data showed that 20 vital medicinal herbs could exert therapeutic effects on alopecia mainly mediated via regulation of various target genes and proteins, including acetylcholinesterase (AChE), phospholipase A2 (PLA2) subtypes, ecto-5-nucleotidase (NTE5), folate receptor (FR), nicotinamide  This may lead to infrequent social interaction, lack of confidence, and may affect self-esteem2. Although many therapeutic interventions have been developed and adopted for alopecia treatment, their preventive and therapeutic effects are not satisfactory3. Minoxidil (MXD) and finasteride drugs approved by the US Food and Drug Administration (FDA) for alopecia treatment exhibit limited efficacy and are associated with recurrent cases after cessation4. Side effects, such as scalp dryness, skin irritation (MXD), and sexual and psychological disorder development (finasterid), are limitations of conventional therapy5. Owing to this reason, many patients undergo complementary and alternative medicine (CAM) interventions4.Approximately 50% of people experience hair loss throughout their life, which causes emotional and psychological problems and worsens the quality of life7. In a recent retrospective observational study, 142 of the 222 patients (64%) presented with > 90% hair recovery following subjection to traditional Chinese medicine (TCM) treatment6. In a recent systematic review conducted considering 30 TCM randomized clinical trials, adjunctive TCM therapy demonstrated an increased total effective rate, improved microelement level, and decreased symptom score of refractory alopecia compared to the Western medication-only group7. In terms of safety, the therapy presented with a tendency of reduced adverse events in the meta-analysis 7. Experimental studies conducted on alopecia have proposed several possible mechanisms of action of THM preparations, such as appropriate functioning of the hair growth cycle, inflammation, apoptosis, hormones, and angiogenesis5. Moreover, enhanced scalp blood circulation, nutritional support, and inhibition of 5α-reductase activity were reported as mechanisms mediating the activity of herbal preparations8. These studies indicate that THM may demonstrate potential therapeutic effects in alopecia treatment6.In East Asia, traditional herbal medicine (THM) is widely used for alopecia treatment in clinical practice5. Network pharmacology is a novel approach to investigate the system-level mechanisms of medicine9, and the method incorporates “the potential mechanism of multiple compounds” and “the pathways associated with the target of the compounds”10. The core concept of network pharmacology is appropriate for multi-component and multi-targeted agents and is suitable for comprehensively exploring the complex mechanisms of THM11. The network pharmacology-based approach exhibits advantages in the discovery of active compounds and the potential mechanism of THM13. Therefore, network pharmacology is widely used to explore the therapeutic mechanism of THM, including the herb-compound-target network, target interpretation, and related biological functions and diseases15. In the present era, network pharmacologic approaches are utilized to provide insights into a molecular basis for the formulation of experience-based TCM theory and treatment strategy13.However, system-level therapeutic mechanisms for combinations of medicinal herbs for alopecia treatment are not well established. Medicinal herbs may be more effective in alopecia associated with multiple pathogeneses because multiple components of the herbs affect multiple targets16. However, the therapeutic mechanism of hair loss by THM is not well established. In addition, we anticipate that the mechanism of hair loss treatment of THM might also have different mechanisms from that of conventional drugs. The present study aimed to determine the novel therapeutic mechanisms of action of THM preparations that could differentiate them from the conventional drugs for hair loss treatment.In a previous study conducted on the exploration of the modular characteristics of medicinal herbs for alopecia treatment in TCM using network analysis, we identified medicinal herbs frequently used for alopecia treatmentIn this study, we explored the biological pathways of medicinal herbs for alopecia treatment using network pharmacologic analysis. The workflow of this study is presented in Fig. 16, we investigated 312 herbal prescriptions for alopecia treatment, from which we derived and presented a network of 258 medicinal herbs via modularity analysis. Based on the analysis, we divided the 258 medicinal herbs into three modules . Those included in Module 1 were closely associated with the “Liver” and “Kidney” as per the TCM theory. Moreover, Module 2 herbs seemed to affect the body surface and were frequently used for external preparations, whereas Module 3 herbs were related to the “Stomach” (digestive function) as per the TCM theory. In the TCM theory, each medicinal herb in an herbal prescription can be categorized into the king, minister, assistant, or ambassador  group, which act as major (king), complementary (minister), neutralizing (assistant), and delivery/retaining (ambassador) components, respectively17. Most Module 1 medicinal herbs were categorized into the “king” or “minister” group in herbal prescription for alopecia treatment. Therefore, we selected Module 1 herbs for further network pharmacologic analyses. Previously, research based on a network analysis (association rule mining) of the top 20 medicinal herbs in each module in the order of frequency was conducted. Based on the results obtained, we selected the top 20 medicinal herbs of Module 1 for further investigation as presented in Table In a previous network analysis study19. Since, the TCMID includes data on 43,413 compounds derived from 8159 medicinal herbs, compound information was obtained from such a database18. We selected potential compounds based on oral bioavailability (OB ≥ 30) and drug-likeness index (DL ≥ 0.18) for integrative absorption, distribution, metabolism, and elimination (ADME) screening using default values obtained from the TCMID18. OB is an important pharmacokinetic index that is used to indicate the extent and speed of absorption into the blood circulation of orally administered drugs19, whereas DL aids estimation of the structural similarity between the related drug and ingredients of medicinal herbs. It also helps predict specific compounds that can be developed as drugs. Both OB and DL are utilized to perform screening of suitable compounds for drug development11. Since, BATMAN-TCM could provide target prediction information based on the existing drug-target interaction information, we used the target gene prediction score of compounds in 20 vital medicinal herbs based on the available data19. The drug prediction algorithm of BATMAN-TCM helps determine drug-drug similarity based on chemical structure and functional group based on similarity.To construct a tripartite network of compound (ingredient)-associated target gene-associated target pathway, we used data derived from the TCM Integrated Database (TCMID) and Bioinformatics Analysis Tool for Molecular Mechanism of TCM (BATMAN-TCM)20. KEGG helps provide target interpretation by highlighting high-level functions and molecular information for utilizing biological systems11. During this stage, PubChem ID was utilized as a unique gene ID that could be used to link extracted compounds whose information was derived from various databases21.Protein–protein similarity was predicted by considering protein sequences and gene ontology (GO) analysis. The final prediction scores were calculated based on the similarity ranking. We defined a compound (ingredient) with a target gene prediction score of > 55 as a positive interaction. We only extracted data and utilized compound–target gene interactions that met the positive interaction criteria. Next, to analyze gene–pathway associations, pathway data derived from the Kyoto Encyclopedia of Genes and Genomes (KEGG) database was used, which highlighted 29,039 interactions of 325 human pathways related to 7373 human proteins24. Classical degree centrality represents the simplest centrality measure and helps determine the number of direct connections of a specific node in the network. The advantage of degree centrality is the interpretability and comprehensibility of the results25. In this study, we evaluated the weighted degree centrality of individual associated pathways in a tripartite network consisting of compounds, target genes, and associated pathways. We presented only statistically significant weighted degree centralities compared to those obtained using the null model.One of the purposes of network analysis is to investigate the important nodes in a graph structure. A variety of centrality measures can be used to define the strength of the connections between the individual nodes in the graph structurei) is calculated as follows:1 is the total number of target genes, n2 is the total number of compounds.The weighted degree centrality of an associated pathway i  level to < 0.05, using the Benjamini–Hochberg procedure27. Table To better understand the analysis methods used, herb-compound-target gene network as a visualized network figure has been presented in this study. Data on the associated target genes and associated pathways of compounds in vital medicinal herbs, using the analysis described in “3The compounds included in 20 vital medicinal herbs are shown in Supplementary Material Based on the predefined OB and DL criteria, we explored the TCMID database and extracted information on 77 potential compounds isolated from 20 medicinal herbs. Based on the predefined prediction score, a further search using the BATMAN-TCM database resulted in the extraction of information on 25 associated potential target genes from 77 potential compounds. From the proteins targets, information on 54 associated potential target pathways was extracted from the KEGG pathway database, which presented with a statistically higher weighted degree of centrality than that obtained using the null model. The tripartite network comprising 77 compounds, 25 target proteins, and 54 target pathways of 20 vital medicinal herbs are presented in Table Twenty-five target proteins of potential compounds and fifty-four associated target pathways are presented in Table . The sequence in Table The 54 associated pathways of 20 vital medicinal herbs are presented in Table 3We have constructed and presented a herb-compound (ingredient)-associated gene network Fig. , and a lIn this study, we explored the possible mechanisms of action of 20 vital medicinal herbs for alopecia treatment using a network pharmacology analysis method for the first time. We extracted information on 77 potential compounds isolated from the 20 main medicinal herbs, and 25 potential target proteins/genes (from the 77 compounds) were found to be associated with 54 target pathways. AChE and PLA2G1B represented the largest nodes in the potential protein (gene) group. Metabolic, glycerophospholipid, and endocytosis pathways were the most frequently associated pathways for the mechanism of action of 20 vital medicinal herbs.The potential mechanism of action of 20 vital medicinal herbs for alopecia treatment may mainly be mediated via regulation of various target genes/proteins and target pathways, including glycerophospholipid metabolism in cholinergic synapses driven by acetylcholinesterase, lipid metabolism driven by various phospholipase PLA2 subtypes, the purine pathway driven by ecto-5-nucleotidase (NTE5) in the hair follicle cycle, macrophage-mediated inflammation via regulation of folate receptors (FRs), NNMT, and QPRT linking NAD + expression and activity, or DAB adaptor protein 2 (DAB2) inhibition of Wnt signaling.28, and the kidney possesses a high absorption capacity for lipid-binding proteins and lipid-regulating hormones29. Lipids are essential components of cellular membranes that act as skin barriers and demonstrate functions as bioactive lipid mediators30.In a previous study, we investigated the modular characteristics of medicinal herbs for alopecia treatment in TCM using network analysis and extracted information on 20 main medicinal herbs for alopecia treatment. These herbs are closely related to the “liver” and “kidney” function as per the TCM theory. The liver and kidneys play important roles in lipid metabolism. The liver is responsible for overall fatty acid synthesis and lipid circulationIn our study, we found that the main components of the 20 main medicinal herbs were closely associated with pivotal target proteins, including acetylcholinesterase (27 compounds), PLA2G1B (14 compounds), LPCAT1 (six compounds), NTE5 (five componds), fibroblast growth factor receptor 2 , FR-β and -γ (four compounds each), NNMT (four compounds), QPRT (four compounds), and DAB adaptor protein 2 (three compounds). These target proteins play a key role in mediating the therapeutic effects of medicinal herbs on alopecia by regulating the main pathways, including glycerophospholipid metabolism, choline metabolism, endocytosis, nicotinate/nicotinamide metabolism, ether lipid metabolism, RAS signaling pathway, glutamatergic synapse, purine metabolism, and pyrimidine metabolism involved in the hair follicle cycle.After comparing the data for the hair loss target protein extracted using the Genecard database with those of the target protein of herbal medicine for hair loss, only AChE and FGF-2 were found to overlap, whereas the other target proteins did not present with overlapping. These findings indicate that medicinal herbs used for alopecia treatment can act on target proteins, such as AChE and FGF-2, and demonstrate association with alopecia; however, they can exert a therapeutic effect on alopecia via the expression of distinct target genes, proteins, and pathways compared to those observed in conventional medicine. The findings reported in this study based on medicinal herbs that may be used for alopecia treatment can help provide insights into a new potential therapeutic target that may be distinct from the one reported in conventional medicine.31. Cholinergic neurons release ACh to induce chemical transmission via ACh nicotinic and muscarinic receptors32. ACh receptors play an important role in controlling the hair follicle cycle33.In our study, AChE was frequently associated with 27 ingredients in the 20 vital medicinal herbs. In cholinergic synapses, AChE is associated with glycerophospholipid metabolism and cholinergic synapses Table . AChE ca32. AChE has been reported to induce hair growth34. The glycerophospholipid metabolic pathway maintains cell membrane stability to confer protection to the cell against hypoxic stress-induced damage by upregulating mRNA, protein, and metabolite levels35. Therefore, the main medicinal herbs may affect alopecia by modulating AChE to regulate ACh signaling in hair follicle stem cells and to regulate the glycerophospholipid metabolic pathway. We suggest that AChE may be a key target active protein in medicinal herbs for hair loss treatment.Neuronal or non-neuronal ACh signaling or both lead to the depletion of stem cell populations in murine hair follicles in a complex neuroectodermal–mesodermal interaction system37 Particularly, FGF-2 is known to activate dermal papilla cell proliferation and to increase the size of hair follicles37. In our study, FGF-2 was identified as one of the major target proteins of main medicinal herbs for alopecia treatment. An experimental study showed that topical application of FGFs, including FGF-2, induced the anagen phase in telogenic C57BL/6 mice36. Adenosine stimulates the growth of the dermal papilla and prolongs the anagen phase by upregulating the cysteine levels modulated by FGF-2 and FGF-736. Therefore, the therapeutic effect of the main medicinal herbs on alopecia may involve the modulation of FGF-2 to promote dermal papilla cell proliferation and to prolong the anagen phase by increasing cysteine levels.Growth factors, including FGF, insulin-like growth factor (IGF)-1, vascular endothelial growth factor (VEGF), and epidermal growth factor (EGF), regulate hair morphogenesis and the hair cycle38. PLA2 family members were identified as important target proteins in our study  is a class of lipid biomolecules derived from the cleavage of phosphatidylcholine (PC) via the action of PLA2, the transfer of fatty acids to free cholesterol via LPCAT, or both41. High levels of NT5E (CD73) have also been found in hair follicle cells42. Dermal microcirculation is essential for supplying various growth factors and other bioactive molecules for hair maintenance43. Particularly, during the anagen phase, such a mechanism of dermal microcirculation is important for maintaining the high metabolic activity of hair follicle matrix cells.NT5E (CD73) is an enzyme expressed on free nerve endings in the epidermis and skin cells, which regulates purinergic signaling by desphosphorylating extracellular AMP to adenosine in nociceptive circuits43. In our study, NT5E (CD73) was one identified as of the major target proteins of the medicinal herbs for alopecia treatment. Therefore, 20 vital medicinal herbs for alopecia treatment may act by modulating NT5E (CD73) to promote dermal microcirculation through the regulation of purinergic signaling by desphosphorylating extracellular AMP to adenosine in hair follicles and dermal papilla cells.The mechanism of action of MXD, a topical application for alopecia, has not been completely elucidated. However, MXD is known to induce the release of adenosine in follicular keratinocytes and to promote the release of vascular endothelial growth factors in dermal papilla cells, thereby promoting dermal microcirculation44. A previous study has shown that FR-β is highly expressed on activated macrophages, cells which are found in various activated macrophage-mediated inflammatory diseases, including rheumatoid arthritis, psoriasis, Crohn's disease, and systemic lupus erythematosus44. Dermis-resident TREM2 + macrophages reportedly promote hair follicle stem cell quiescence and help maintain telogen via the regulation of Janus kinase (JAK)-signal transducer and activator of transcription 5 (STAT5) signaling45. Conversely, regulatory T cells are known to promote hair follicle stem cell activation and hair regrowth45. Therefore, the therapeutic effects of the vital medicinal herbs on alopecia may be involved in macrophage-mediated inflammation through the regulation of FRs, especially FR-β.In our study, FR-β and -γ were associated with four ingredients in 20 main medicinal herbs for alopecia treatment. The FR, which presents with three isoforms, namely FR-α, FR-β, and FR-γ in humans, is a cell surface glycosylphosphatidylinositol (GPI)-anchored glycoprotein with a high affinity for folic acidS-adenosyl methionine (SAM), which directly links one-carbon metabolism with the methylation balance and nicotinamide adenine dinucleotide (NAD +) levels of cells as therapeutic targets in addition to their metabolic function in detoxification47. NNMT activity and expression are modulated differently in various tissues. While NNMT in the adipose tissue is involved in obesity and insulin resistance, its expression in the liver demonstrates beneficial effects by regulating lipid parameters47. In the present study, the main medicinal herbs for alopecia treatment correlated closely with the liver and kidney as per the TCM theory. Although evidence to support the direct relationship between NNMT and alopecia is lacking, the vital medicinal herbs for alopecia treatment may exert a therapeutic effect by controlling lipid parameters in the liver.NNMT is a metabolic enzyme that catalyzes the methylation of nicotinamide to enable the formation of N-methylnicotinamide using the universal methyl donor 48. NAD + is synthesized via de novo synthesis, and the salvage pathway is an essential cofactor of the oxidation–reduction reaction48. NAD + activity is associated with various cellular functions, including calcium homeostasis, antioxidant activity, gene expression, and apoptosis49.QPRT is a key enzyme involved in the de novo synthesis of NAD + that presents with a restricted tissue distribution, including in the liver and kidney, which is involved in the effects of 20 main medicinal herbs for alopecia treatment in our study48. 20 vital medicinal herbs that affect liver function as per the TCM theory may act on alopecia by regulating NNMT and QPRT. Our findings suggest that NNMT and QPRT, which link NAD + expression and activity, may be deemed potential therapeutic targets for alopecia treatment.An experimental study has suggested that QPRT acts as a suppressor of spontaneous cell death by inhibiting the overproduction of active caspase 350. Additionally, DAB2 plays a vital role in regulating adipocyte cell size, number, and T cell function in the immune system51. Several studies have reported that DAB2 inhibits canonical Wnt signaling52 and plays a pivotal role in the initiation of hair follicle placode formation and development54. Androgen is known to downregulate the expression of dermal papilla cell-secreted factors associated with hair follicle stem cell differentiation through the inhibition of canonical Wnt signaling55. Therefore, the main medicinal herbs may exert an effect on alopecia via modulation of DAB2, leading to the inhibition of Wnt signaling.In this study, DAB2 was identified as a target protein associated with three compounds involved in 20 main medicinal herbs for alopecia treatment. DAB2 is a cargo-binding endocytic adaptor protein that controls cellular homeostasis and is implicated in several receptor-mediated signaling pathways, cell adhesive function, hematopoietic cell differentiation, and angiogenesis19 and TCM-ID18 databases were considered. However, these are databases that are widely considered in TCM network pharmacology research. Thus, we focused on the database specified in the TCM. Although there is a disadvantage of limited information in the database, a drawback due to which the search results may be omitted, it is already known that TCM-related databases present with high redundancy when information on medicinal herbs is explored. Moreover, search terms for medicinal herbs in the database must be narrowed for the TCM network pharmacologic research, as many medicinal herbs have different aliases in general databases56. Therefore, despite several shortcomings, a TCM-specific database was selected. In future studies, it is therefore recommended to use a more extensive database. Second, Presently, molecular docking methods are utilized to explore and visualize the interaction between the candidate target and the compound57. Even though we did not adopt molecular docking analysis in the present study, such an approach along with experimental study in further studies may deepen the understanding of the mechanism. In addition, a draft for method evaluation guidelines for network pharmacology is under development58. In future studies, expectedly, a more rigorous research methodology will be adopted using the developed guideline58. Finally, our results should be verified as these findings suggest a probable and possible mechanism via a network pharmacology analysis approach based on TCM medicinal herbs.This study presents with several limitations. First, only the BATMAN-TCMDespite such limitations, we confirm that this is a novel approach to use a network pharmacologic method to investigate the effect of medicinal herbs on the treatment of alopecia. Selection of medicinal herbs was performed based on the information presented in previous studies and network analyses. Here, exploration of a possible mechanism of medicinal herbs, which is different from a conventional alopecia drug mechanism, has been implemented. Our findings using a network pharmacological approach may help better understand the system-level mechanisms of action of multi-component and multi-target THM preparations for alopecia treatment. However, we anticipate that the mechanism of action of 20 vital medicinal herbs against alopecia may be associated with complex pathways and warrants further proof of concept of the target genes/proteins and target pathways suggested by our research. This is because network pharmacology is only a predictive tool.In this study, we analyzed a novel possible mechanism of action of 20 main medicinal herbs for alopecia treatment using a network pharmacological approach. We also determined the medicinal herb-ingredient-target protein network and constructed an ingredient-associated protein (gene)-associated pathway network. In conjunction, our data suggest that the effects of the vital medicinal herbs for alopecia treatment may be mediated mainly through the regulation of various target genes/proteins including AchE, PLA2 subtypes, NTE5, FR, NNMT, QPRT, and DAB2, and target pathways including glycerophospholipid metabolism, choline metabolism, endocytosis, nicotinate/nicotinamide metabolism, ether lipid metabolism, RAS signaling pathway, glutamatergic synapse, purine metabolism, and pyrimidine metabolism involved in the hair follicle cycle.These findings regarding target genes/proteins and target pathways of vital medicinal herbs associated with alopecia treatment will provide a novel foundation and will help support further studies to enhance our understanding of the therapeutic mechanism of medicinal herbs for alopecia treatment in TCM and to further elucidate the pathogenesis of alopecia.Supplementary Information 1.Supplementary Information 2.


Paeoniae Radix Alba (PRA), the root of the plant Paeonia lactiflora Pall., has been suggested to play an important role for the treatment of asthma. A biochemical understanding of the clinical effects of Paeoniae Radix Alba is needed. Here, we explore the phytochemicals and therapeutic mechanisms via a systematic and comprehensive network pharmacology analysis.  Through TCMSP, PubChem, GeneCards database, and SwissTargetPrediction online tools, potential targets of active ingredients from PRA for the treatment of asthma were obtained. Cytoscape 3.7.2 was used to determine the target of active ingredients of PRA. Target protein interaction (PPI) network was constructed through the STRING database. The Gene Ontology (GO) biological process and Kyoto Encyclopedia of Genes and Genes (KEGG) pathway enrichment analysis were analyzed through the biological information annotation database (DAVID).  Our results indicate that PRA contains 21 candidate active ingredients with the potential to treat asthma. The enrichment analysis of GO and KEGG pathways found that the treatment of asthma by PRA may be related to the process of TNF (tumor necrosis factor) release, which can regulate and inhibit multiple signaling pathways such as ceramide signaling.  Our work provides a phytochemical basis and therapeutic mechanisms of PRA for the treatment of asthma, which provides new insights on further research on PRA. Asthma siveness . This inPaeoniae Radix Alba , the root of the perennial herbaceous plant Paeonia lactiflora Pall. of the buttercup family, has the effects of nourishing blood and regulating menstruation, restraining yin and antiperspirant, softening liver and relieving pain, and suppressing liver-yang [Traditional Chinese medicine (TCM) has a long-lasting history of using herbal medicine in the treatment of various respiratory diseases . There iver-yang . Our prever-yang . Thus, wPaeoniae Radix Alba” as the key word. The ingredients that meet the criteria with oral biological degree (OB) > 20% and drug − like properties (DL) > 0.18 at the same time were selected as the active ingredients of PRA. Effective active ingredients of PRA contained in the database were queried and screened.TCMSP database was used to obtain the pharmaceutical ingredients with “http://www.Uniprot.org/) database, screened targets are converted into gene names, full protein names are corresponded to the gene abbreviations, and the component target-gene data table is made.Based on the TCMSP and DrugBank databases, target proteins corresponding to small molecules were sorted out, while components without target proteins and duplicate targets were deleted. Using the UniProt  and TTD data (https://db.idrblab.org/ttd/) database were searched for asthma-related targets. Duplicate targets were deleted, and information acquired were collected.The UniProt database (https://david.ncifcrf.gov/) and Metascape database (https://metascape.org/) were used to analyze the GO function and KEGG pathway enrichment analysis of the potential targets of PRA for the treatment of asthma. GOTERM_BP_DIRECT , GOTERM_CC_DIRECT (cell composition), and GOTERM_MF_DIRECT (molecular function) are saved in Gene_Ontology; KEGG_PATH-WAY was saved in Pathways.The DAVID database . Based on the DAVID tool, the GO function and KEGG pathway enrichment analysis of the target genes of PRA were performed, and the significance threshold was selected as the correction , DrugBank, and DisGeNET, and a total of 153 asthma-related targets were obtained. The intersection of these targeted genes with the previous targets of the active substance of PRA was analyzed. Finally, 13 genes are both target genes . KEGG enAsthma is a chronic disease characterized by inflammation hyperreactivity of the airways . It is aα gene is about 2.76 kb in length and consists 4 exons and 3 introns. It is closely linked to the MHC gene group and is located on the 6th and 17th chromosomes, respectively. The natural form of TNF-α exerts its biological effects is a homotrimer. TNF-α is mainly produced and secreted by activated alveolar macrophages with a variety of biologically active cytokines, which can stimulate neutrophils to cause degranulation and an increase in respiratory burst activity. TNF-α can promote the adhesion of neutrophils to endothelial cells [α is the key target of PRA for the treatment of asthma. Among them, TNF-related active ingredients are kaempferol and paeoniflorin. Kaempferol suppresses eosinophil infiltration and airway inflammation in airway epithelial cells and in mice with allergic asthma through inhibition of TNF-α [α, reduce the transcription of MMP genes, and reduce the expression of MMP. Due to the reduced expression of MMP, it can effectively protect the extracellular membrane (ECM) and basement membrane in the lung from dissolution and avoid asthma symptoms [α [The human TNF-al cells , and it al cells . The phaof TNF-α . Kaempfesymptoms . Paeonifptoms [α . Thus, pCHRM, COX, and PTGS2. Kaempferol has anti-inflammatory effects by inhibiting interleukin-4 (IL-4) kinase and downregulating the NF-κB pathway [Furthermore, there are many other genes which play roles in the coacting target genes, such as  pathway . By inhi pathway .2+ through the calcium ion pathway and inhibit the activation of CHRM2 and CHRM3 on the bronchial smooth muscle, thereby inhibiting the contraction of the bronchial smooth muscle and reducing cough [2+ produced by this reaction can directly bind to ion channels and can indirectly participate in the process of other signaling pathways related to the cough and asthma reaction [There are many highly sensitive cough receptors in the throat and trachea. When the mucosa of respiratory tract is stimulated by foreign bodies or secretions in the respiratory tract, it will produce a cough response through a series of neural reflexes, with the intention of expelling the secretions in the respiratory tract. A series of mechanical or chemical stimuli, such as the inhalation of harmful gases, the invasion of pathogenic microorganisms, the inflammatory factors produced by airway inflammation, and the contraction of airway smooth muscles, can all be used as inducements to cause the receptors to transmit signals to the cough center after the body is stimulated. Through network pharmacological analysis, it is found that PRA can act on key targets in cough response-related pathways. Beta-sitosterol and sitogluside, the active ingredients in PRA, can regulate the concentration of Cang cough . The stireaction .εRI” is expressed on the surface of a variety of immune cells . The massively secreted IgE can trigger allergic inflammation cascades by activating inflammatory cells, leading to airway smooth muscles contract, triggering asthma. Through network pharmacology analysis, it is found that β-sitosterol can bind to key targets on the pathways related to asthma response [PTGS2 gene is closely related to the inflammation of the asthmatic response, and the cyclooxygenase encoded by the PTGS2 gene has an airway remodeling effect [α signaling pathway.Asthma is mainly related to allergic inflammation and neurological reactions. When the mucous membrane of the respiratory tract tissue is stimulated by allergens, it will promote the production of large amounts of IgE antibodies, triggering type I allergies. IgE high-affinity receptor “Fcresponse . Chan etg effect . Kaempfeg effect . From thIn summary, this study used network pharmacology to analyze the main active ingredients of PRA. The main active ingredients and their possible molecular mechanisms for the treatment of asthma have been clarified. These results provide a theoretical basis of PRA for the treatment of asthma and provide a scientific basis for its better clinical application.