Chung-Sang-Bo-Ha-Hwan (CSBHH) is an herbal prescription widely used to treat various chronic respiratory diseases. To investigate the system-level treatment mechanisms of CSBHH in respiratory tract diseases, we identified 56 active ingredients of CSBHH and evaluated the degree of overlap between their targets and respiratory tract disease-associated proteins. We then investigated the respiratory tract disease-related signaling pathways associated with CSBHH targets. Enrichment analysis showed that the CSBHH targets were significantly associated with various signaling pathways related to inflammation, alveolar structure, and tissue fibrosis. Experimental validation was conducted using phorbol-12-myristate-13-acetate (PMA)-stimulated NCI-H292 cells by analyzing the mRNA expression levels of biomarkers  derived from network pharmacological analysis, in addition to the mucin genes MUC5AC and MUC2, to investigate the phlegm-expelling effect of CSBHH. The mRNA expression levels of these genes were consistent with network pharmacological predictions in a concentration-dependent manner. These results suggest that the therapeutic mechanisms of CSBHH in respiratory tract diseases could be attributed to the simultaneous action of multiple active ingredients in the herbal prescription. Respiratory tract diseases represent a broad spectrum of illnesses and afflictions that compromise the body’s primary gas exchange system. These diseases impact the upper or lower respiratory tract and encompass conditions ranging from common ailments, such as influenza and rhinitis, to severe diseases, such as pneumonia and chronic obstructive pulmonary disease (COPD). The global burden of respiratory tract diseases is substantial and escalating, contributing to an array of socioeconomic and public health challenges due to emergent threats, such as novel viruses such as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of the coronavirus disease COVID-19) pandemic, and escalating environmental pollutants, such as fine particulate matter [9 pandemiHerbal medicines are widely used in Asian countries and are well received by patients. Chung-Sang-Bo-Ha-Hwan (CSBHH) or Chung-Sang-Bo-Ha-Tang (CSBHT) has been used for centuries to treat chronic lung diseases, such as asthma, in Korea ,3. Both Network pharmacology, which is derived from systems biology, is a powerful tool for identifying rational drug targets and repurposing drugs . This apIn the realm of traditional medicine, CSBHH has been employed for centuries to treat various respiratory diseases. Despite its historical use and observed benefits, the therapeutic mechanisms of CSBHH remain largely unclear. This study pioneered the application of a multiscale network-level approach with experimental validation to unravel the therapeutic effects and mechanisms of action of CSBHH in respiratory tract diseases. We collected information on the detectable components of CSBHH and their experimentally validated targets. By analyzing the collected dataset, we identified vital targets directly related to respiratory tract diseases that were significantly associated with disease-related signaling pathways. Furthermore, we investigated the effects of CSBHH on the targets related to respiratory diseases using phorbol-12-myristate-13-acetate (PMA)-stimulated human respiratory epithelial cells and comprehensively validated the protective effects of CSBHH in vitro. By doing so, we aimed to bridge the gap between traditional knowledge and modern scientific understanding, thereby providing a comprehensive strategy for elucidating the effects of CSBHH on various respiratory tract diseases and their system-level mechanisms.We identified the active ingredients of 18 herbs constituting CSBHH, which have been recorded and detected experimentally . We idenWe identified experimentally validated protein targets of CSBHH-active ingredients from the therapeutic target database (TTD), STITCH, DrugBank, and a dataset assembled by Huang et al. . We assep < 10−10).To investigate the association between CSBHH and respiratory tract diseases, we evaluated the degree of overlap between the CSBHH targets and respiratory tract disease-related proteins. The result showed that approximately one-third of proteins (34/107) were associated with CSBHH targets. We conducted a hypergeometric test to check whether the observed number of overlapping targets was higher than the random expectations. The values of random expectation were obtained by randomly selecting targets equal to the number of targets from the assembled CPI dataset and repeatedly calculating the number of overlapping targets between respiratory tract disease-related proteins and selected targets. We found that the observed value between CSBHH targets and respiratory tract disease-related proteins was significantly higher (2.80-fold) than the random expectations , Poria Sclerotium–NF-κB signaling pathway , Rehmanniae Radix Preparata–apoptosis (84.62), and Platycodonis Radix–JAK-STAT signaling pathway (78.94) pairs. These results suggest that CSBHH herbs are beneficial against respiratory tract diseases by comprehensively regulating the related signaling pathways.We investigated whether and which respiratory tract disease-related signaling pathways were associated with CSBHH targets. The results of the enrichment analysis showed that CSBHH targets were significantly associated with various respiratory tract disease-related signaling pathways involved in inflammation, the alveolar structure, and tissue fibrosis A. To ideA biological molecular network targeted by CSBHH was constructed to uncover the relationships among herbs, active ingredients, protein targets, and respiratory tract diseases. To elucidate the potential key mechanisms, we constructed the herb–active ingredient–target subnetwork focusing on targets that have a degree of overlap with CSBHH-active ingredients of at least seven (top 10% of the target degrees of overlap) . The netWe investigated the cytotoxicity on NCI-H292 cells to determine the appropriate CSBHH concentration range for additional experiments. Since CSBHH was not toxic at concentrations below 800 μg/mL, 200, 400, and 800 μg/mL CSBHH were used .To investigate the inhibitory effect of CSBHH on PMA-induced mRNA expression, the cells were pre-treated with 200, 400, or 800 μg/mL CSBHH and then treated with 100 nM PMA for 3 or 24 h. After 3 h of PMA treatment, IL-1β, TNF-α, mucin (MUC)5AC, and MUC2 mRNA expression levels were measured, and 24 h after treatment, glutathione S-transferase pi (GSTP1), glutathione S-transferase mu 1 (GSTM1), and phosphatase and tensin homolog (PTEN) mRNA expression levels were measured.p < 0.001), respectively, compared to that in the untreated cells. However, they were reduced by 0.27-fold and 0.50-fold (p < 0.001), respectively, in the 800 μg/mL CSBHH-treated cells compared to those in the PMA-stimulated cells (IL-1β and TNF-α mRNA expression in the PMA-stimulated cells increased by 34.75-fold and 14.60-fold (ed cells .p < 0.001) compared to that in the untreated cells. However, it was reduced by 0.43-fold (p < 0.001) in the 800 μg/mL CSBHH-treated cells compared to that in the PMA-stimulated cells. Interestingly, the mRNA expression of GSTM1 and PTEN in the PMA-stimulated cells decreased by 0.29-fold (p < 0.05) and 0.92-fold (p < 0.05), respectively, compared to that in the untreated cells. Additionally, the GSTM1 mRNA expression increased by 1.48-fold in the cells treated with 800 μg/mL CSBHH compared to that in the PMA-stimulated cells, and PTEN mRNA expression decreased by 0.61-fold (p < 0.001) (The GSTP1 mRNA expression in the PMA-stimulated cells increased by 34.75-fold (< 0.001) .p < 0.001) and 4.36-fold (p < 0.05), respectively, in PMA-stimulated cells compared to that in untreated cells. However, they decreased by 0.47-fold (p < 0.001) and 0.44-fold, respectively, in the 800 μg/mL CSBHH-treated cells compared to those in the PMA-stimulated cells .Network pharmacological analysis revealed complex relationships among the compounds, targets, and biological effects of CSBHH on respiratory tract diseases. This comprehensive analysis provides insights into the potential interactions between CSBHH-active ingredients and their protein targets in respiratory tract diseases. By constructing a compound–target network for CSBHH using various databases, we initially identified 3414 CPIs involving 56 CSBHH-active ingredients and 1081 protein targets. Among the 107 respiratory tract disease-associated proteins, approximately one-third (34/107) were associated with CSBHH targets, indicating the statistical significance of CSBHH, its individual herbs, and key active ingredients in respiratory tract diseases. Our subsequent analysis revealed that individual herb targets showed significant overlap with respiratory tract disease-related targets, such as Platycodonis Radix possesses anti-inflammatory, anti-cancer, anti-diabetic, and anti-obesity properties [Platycodonis Radix component, attenuates airway inflammation through the NF-κΒ signaling pathway in an ovalbumin (OVA)-induced asthmatic mouse model. Platycodin D inhibits eosinophilic inflammation, mucus production, and the production of T helper 2 (Th2) cytokines, such as IL-4, IL-5, and IL-13, in the bronchial mucosa of an OVA-induced asthmatic mouse model [operties . Platycose model ,16,17.Dioscoreae Rhizoma has anti-diabetic, antioxidant, and anti-inflammatory effects [Dioscoreae Rhizoma component, reduces the expression of inflammatory factors, such as IL-1β, TNF-α, and IL-6, in the OVA-induced asthmatic mouse model and primary tracheal epithelial cells and downregulates the NF-κΒ signaling pathway by activating glucocorticoid receptors [ effects ,19,20. Ieceptors ,22.Poria Sclerotium, used as an herbal medicine, has various activities such as anti-inflammatory, anti-bacterial, anti-tumor, and immune-enhancing properties [Poria Sclerotium induced nitric oxide (NO) production and iNOS transcription through NF-κΒ/Rel activation in RAW264.7 cells. Moreover, regulating the type 1 T-helper (Th1)/type 2 T-helper (Th2) response in OVA-induced mice has been observed to aid in improving immunodeficiency and preventing allergic diseases, including allergic asthma [operties ,24,25. Pc asthma ,28.Glycyrrhizae Radix has anticancer, antiviral, and anti-inflammatory effects [Glycyrrhizae Radix, inhibits allergic airway inflammation through the NF-κB signaling pathway [ effects ,30,31. 1 pathway . Rutin d pathway ,34. It h pathway ,36,37.Scutellariae Radix has antioxidant, anti-inflammatory, and anti-obesity effects [Scutellariae Radix, downregulates the NF-κB signaling pathway in LPS-induced macrophages [ effects . Oroxylirophages . It has rophages ,41,42. Crophages ,44,45. Wrophages ,47,48.Coptidis rhizoma has anticancer, anti-inflammatory, and expectorant effects [Coptidis rhizoma, downregulates the MAPK signaling pathway in breast cancer cells, induces apoptosis, and inhibits cancer cell proliferation. It suppresses the growth and invasion of breast cancer cells by downregulating the EGFR/MEK/ERK pathway [ effects ,50. Berb pathway ,52,53. I pathway ,55,56.Moutan Radicis Cortex has anticancer, antioxidant, and anti-inflammatory effects [Moutan Radicis Cortex, has been reported to have anti-inflammatory and anticoagulant effects in an LPS-induced acute lung injury rat model and to reduce IL-1β levels in the BALF of cigarette smoke-induced mice [ effects ,58. Paeoced mice ,60.Through an over-representation analysis based on the disease-related signaling pathways, we discovered that inflammation and the alveolar structure were the main categories, since all herbs have a significant relationship with PI3K–Akt, MAPK, NF-κΒ signaling, and apoptosis pathways. Furthermore, by focusing on 46 targets that interacted with seven or more active ingredients, we found that the key targets were both respiratory tract disease-related targets (direct relationship) and involved in enriched signaling pathways (indirect relationship), such as Akt1, TGFB1, TNF, IL2, TP53, and PARP1.Akt1, a serine/threonine-specific protein kinase, is involved in processes such as glucose metabolism, apoptosis, cell proliferation, transcription, and cell migration. Furthermore, CSBHH interacted with PTEN, a major negative regulator of the PI3K-Akt pathway. This interaction could potentially modulate PI3K-Akt signaling, thereby influencing cell survival, growth, and proliferation. In the MAPK signaling pathway, CSBHH appears to specifically interact with MAPK14 (p38-α), MAPK1 (ERK2), and MAPK8 (JNK1). These interactions suggest a potential modulation of cellular processes, such as the stress response, inflammation, cell proliferation, differentiation, and apoptosis, which could contribute to the treatment of respiratory diseases. Additionally, CSBHH’s interaction with TNF and CASP3 could influence the inflammatory response and apoptosis, respectively. Within the NF-kappa B signaling pathway, CSBHH demonstrates interactions with key targets—specifically, RELA and NFKB1. The modulation of cellular processes, such as immunity, inflammation, and cell survival, by CSBHH underscores its therapeutic potential. In the apoptosis pathway, CSBHH has been found to interact with several key targets, including CASP3, TNF, MAPK14, RELA, Akt1, MAPK1, MAPK8, BAD, NFKB1, IL6, PTGS2, NFKBIA, and CASP9. These interactions indicate that CSBHH may have the ability to modulate the apoptosis process, which could potentially be beneficial in the treatment of respiratory diseases. Altogether, these findings underscore the potential of CSBHH in treating respiratory diseases through a multi-targeted approach, influencing various signaling pathways and cellular processes.The potential targets of CSBHH related to the PI3K–Akt, MAPK, NF-κΒ signaling, and apoptosis pathways were summarized using KEGG Mapper . In the IL-1β, TNF-α, GSTP1, MUC5AC, and MUC2 gene expression increased in PMA-stimulated NCI-H292 cells. Our results showed that the PMA-induced increased IL-1β, TNF-α, GSTP1, MUC5AC, and MUC2 mRNA expression tended to decrease with CSBHH treatment. However, GSTM1 expression was decreased and increased in the PMA- and CSBHH-treated groups, respectively.These findings have guided the in vitro experimental trials while highlighting the complex and multifaceted nature of the potential for treating respiratory tract diseases. Considering the results of the in silico network pharmacological analysis, we investigated several inflammation- and apoptosis-related genes for experimental validation. We investigated the inhibitory effects of CSBHH on PMA-induced mRNA expression and found that In this study, IL-1 and TNF-α levels were evaluated as indicators of inflammation. The IL-1 family includes key cytokines involved in fever, inflammation, and the innate immune response . IL-1β iGSTP1, GSTM1, and PTEN play key roles in alveolar apoptosis ,68,69. GPTEN antagonizes the PI3K–AKT–mTOR pathway and regulates numerous cellular processes including cell survival and proliferation. Therefore, PTEN regulation is an important factor in cancer development because the impairment of PTEN function triggers mechanisms such as cell survival and proliferation ,76. SimiMUC5AC and MUC2 as well as five genes derived through network pharmacological analysis. Clinically, CSBHH has been widely used to treat various chronic respiratory diseases, particularly those related to phlegm, with the effect of dispelling phlegm and relieving panting [MUC5AC and MUC2 genes [MUC5AC and MUC2 expression may effectively alleviate excessive mucus production in respiratory diseases. Mucus is composed of mucin proteins, and MUC5AC is the major mucin protein secreted from airway epithelial cells, such as NCI-H292 cells. Therefore, we included the mucin genes, MUC5AC and MUC2, among the 21 mucin genes discovered to date [In addition, we experimentally evaluated the mucin genes  panting . The hypC2 genes . Therefo to date ,87.Numerous studies have reported the efficacy of CSBHT in expelling phlegm; CSBHT differs from CSBHH only in its formulation. In an OVA-induced allergic asthma model, CSBHT increased mucin sequestration from the bronchi and improved eosinophil infiltration into the tracheal mucosa ,89. In aMedications used to treat respiratory diseases such as asthma and COPD include controllers and relievers. Controller medications are used for routine maintenance and treatment to control the symptoms and reduce airway inflammation. Reliever medications are used as needed to relieve breakthrough symptoms or the exacerbation of symptoms . CombinaIn herbal medicine prescriptions, the active ingredients contained in each medicinal material are extracted through a decocting method for various medicinal materials. These active ingredients interact with each other to create a synergistic effect that is suitable for treating complex diseases through various activities and has few side effects. These active ingredients interact with each other to create a synergistic effect, which is suitable for treating complex diseases through various activities and has few side effects ,99. KangIn light of our findings, we believe that CSBHH has significant potential as a novel treatment option for respiratory diseases. Our study sheds light on the therapeutic effects and mechanisms of action of CSBHH and contributes to a deeper understanding of its role in the treatment of respiratory diseases. Looking ahead, we envision a future in which traditional medicines such as CSBHH are integrated into mainstream healthcare, providing patients with more diverse and effective treatment options. We anticipate that our findings will spur further research into the therapeutic mechanisms of traditional medicines, potentially leading to the discovery of new treatment strategies for respiratory diseases. However, we acknowledge that our study was at the beginning. The full potential of CSBHH and other traditional medicines can only be realized through continued research and rigorous clinical trials. We hope that our work will serve as a stepping stone for future studies aimed at exploring the untapped potential of traditional medicines in the treatment of respiratory diseases.A compound–target network is a bipartite network where nodes are defined as compounds or targets, and the edges between the compounds and targets are defined as interactions between them. A network was constructed by identifying active ingredients and their target proteins. We considered only the active ingredients of CSBHH herbs commonly recorded in TCM-mesh and TM-MC ,101. TheThe experimental targets of active CSBHH ingredients were obtained from data assembled by STITCH , DrugBanp-values and combined scores for the target genes. The combined score was calculated using the logarithm of the product of the p-value and the z-score. Bonferroni correction was applied to rectify the family-wise errors generated in multiple tests.The CSBHH target-associated pathways and biological processes were identified by enrichment analysis using Enrichr . EnrichrThe respiratory tract disease-related proteins and signaling pathways were retrieved to understand the mechanisms of action of CSBHH. The details of disease-related proteins were obtained from a comparative toxicogenomics database (CTD) . We consThe disease-related signaling pathway and its type were identified in the Safety and Effectiveness Evaluation Guide of Health Functional Foods for enhancing the health of the respiratory tract published by the Ministry of Food and Drug Safety (Republic of Korea) as follows: the NF-kappa B signaling pathway (hsa04064), MAPK signaling pathway (hsa04010), PI3K–Akt signaling pathway (hsa04151), and Jak–STAT signaling pathway (hsa04630) for inflammation; the apoptosis pathway (hsa04210) for the alveolar structure; and the TGF-beta signaling pathway (hsa04350) for tissue fibrosis.2 at 37 °C.Human mucoepidermoid pulmonary carcinoma cells, NCI-H292 , were cultured in RPMI-1640  containing 10% fetal bovine serum  and 100 U/mL penicillin–streptomycin  in 5% COPMA  was dissolved in 5 mM dimethyl sulfoxide stock solution , and the cells were stimulated for 3 or 24 h at a final concentration of 100 nM. CSBHH was dissolved in DMSO to a concentration of 200 mg/mL and diluted accordingly. For the cell experiments, 24 h-old NCI-H292 cells were starved overnight in serum-free RPMI-1640 medium and treated with PMA and CSBHH.4 cells/well) were seeded in 96-well plates and allowed to adhere for 24 h. The cells were then treated with different concentrations of CSBHH for 24 h. Next, 100 μL of 10% EZ-Cytox solution  in serum-free RPMI-1640 was added to each well and incubated for 1 h. The OD450 nm was measured using a microplate reader .NCI-H292 cells (2 × 105 cells/well) were seeded in six-well plates. The cells were then treated with a specific CSBHH concentration for 1 h after overnight starvation in a serum-free RPMI-1640 medium. The cells were then exposed to 100 nm PMA for 3 or 24 h. An RNeasy Mini Kit  was used to isolate the total cellular RNA. The RevertAid First Strand cDNA Synthesis Kit  was used to reverse-transcribe the RNA. PCR was performed using AccuPower® 2X GreenStar™ qPCR Master Mix  on a Quant Studio 3 real-time PCR system  with forward and reverse primers  and mucin genes (MUC5AC and MUC2) were consistent with network pharmacological predictions, substantiating the effect of CSBHH on inflammation and apoptosis, as well as its phlegm-expelling effects. These results highlight a multi-target, multi-ingredient strategy underlying the therapeutic efficacy of CSBHH in respiratory tract diseases. Future studies should focus on elucidating the specific molecular interactions between the active components and their targets to further optimize the therapeutic potential of CSBHH.


Aims: To determine the bioactive components of Hedyotis Diffusae Herba (HDH) and the targets in treating lupus nephritis (LN), and so as to elucidate the protective mechanism of HDH against LN.Methods and results: An aggregate of 147 drug targets and 162 LN targets were obtained from online databases, with 23 overlapped targets being determined as potential therapeutic targets of HDH against LN. Through centrality analysis, TNF, VEGFA and JUN were screened as core targets. And the bindings of TNF with stigmasterol, TNF with quercetin, and VEGFA with quercetin were further validated by molecular docking. By conducting Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) enrichment analyses for drug targets, disease targets and the shared targets, TNF signaling pathway, Toll-like receptor signaling pathway, NF-kappa B signaling pathway and HIF-1 signaling pathway, etc., were found in all these three lists, indicating the potential mechanism of HDH in the treatment of LN.Conclusion: HDH may ameliorate the renal injury in LN by targeting multi-targets and multi-pathways, including TNF signaling pathway, NF-kappa B signaling pathway, HIF-1 signaling pathway and so on, which provided novel insights into further researches of the drug discovery in LN. Systemic lupus erythematosus (SLE) is a multi-system autoimmune disease along with abnormally activated immune system, attacking practically any organ system in the human body . In SLE,Traditional Chinese medicine is one of the oldest healing systems and has been widely used for thousands of years . Due to Network pharmacology is an emerging interdisciplinary science that integrates virtual computing, high-throughput data analyses, network database retrieval, bioinformatic network construction and network topology analyses , and washttps://omim.org/), the DisGeNET database (https://www.disgenet.org/) and the DigSee database (http://210.107.182.61/geneSearch/). As a biomedical literature based authoritative database storing information about human genes and genetic phenotypes, OMIM database could provide us with phenotypes related targets. In the OMIM database, the term “Lupus nephritis” was used as a bait to retrieve LN related records and genes, and 75 records containing 35 LN related genes were obtained finally. Distinct with the OMIM database, the DisGeNET database and the DigSee database possess more massive data of human disease-related genes and scoring systems to help screening reliable diseases-related genes. By inputting “Lupus nephritis” into query box and setting “Score_gda” > 0.05 in DisGeNET database or “Evidence Sentence Score” > 0.5 in DigSee database, 51 LN associated targets from the DisGeNET database and 115 targets obtained from the DigSee database were acquired. All genes were then transformed into unified names by UniProt (https://www.uniprot.org/). A total of 162 genes were identified as LN targets by removing duplicates.LN related targets were retrieved from the OMIM database  (https:/database  (https:/database  (http:// UniProt  (https:/https://tcmspw.com/tcmsp.php) is a platform that captures relationship between Chinese herbal medicines, targets and diseases. And most importantly, it provides physical and chemical characteristics of herbal ingredients. To obtain bioactive chemical components of HDH, “Hedyotis Diffusae Herba” was used as the search term, and the components meeting following criterions were selected for further analyses: 1) oral bioavailability ≥40%; 2) drug-likeness ≥0.18; 3) number of rotatable bond <10; 4) molecular weight: 180–500; and 5) has corresponding Pubchem Cid. Poriferasterol, stigmasterol and quercetin were identified as bioactive chemical components of HDH.TCMSP database  (https:/http://stitch.embl.de/), the BATMAN-TCM database (http://bionet.ncpsb.org/batman-tcm/) and the TCMSP database (https://tcmspw.com/tcmsp.php), with the results being limited to homo species. Targets from the BATMAN-TCM database under the criterion of score >15 were selected for subsequent analyses. And for targets predicted from the TCMSP database, those displayed as originating from the DrugBank database were selected. All the returned targets were converted into standard names by UniProt , and further visualized by a data visualization website (http://www.bioinformatics.com.cn/). While the protein-protein interaction (PPI) networks for LN targets, HDH targets and the overlapped targets were constructed by the STRING database , a platform storing known PPIs originating from other databases and predicted PPIs that were obtained through computer algorithms. Further construction of HDH-bioactive components-targets network, LN targets network, and bioactive components-the overlapped targets-enriched KEGG pathways network were conducted by Cytoscape (version 3.7.2).The Gene Ontology (GO) enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways enrichment analysis were conducted by online function annotation tool DAVID  , while 3D-structures of stigmasterol and quercetin were downloaded from Pubchem database (https://pubchem.ncbi.nlm.nih.gov/). Molecular pretreatment, molecular docking simulation and the visualization of docking results were performed by PyMOL software (version 2.4.0) and AutoDockTools  were identified. And an aggregate of 147 targets of these components were predicted by the STITCH database, the BATMAN-TCM database and the TCMSP database . KEGG enIn total, 162 targets were identified as LN therapeutic targets from the OMIM database, the DisGeNET database and the DigSee database. KEGG pathway enrichment analysis illustrated that cytokine-cytokine receptor interaction, TNF signaling pathway, complement and coagulation cascades, Toll-like receptor signaling pathway participated in the course of LN . AdditioAs displayed in By setting “confidence level” to “≥ 0.900,” a more credible PPI network of the overlapped targets was acquired . SubsequTraditional Chinese medicine regards individuals as systems with various states, and has accumulated a large number of traditional Chinese medicine prescriptions . CompounIn this study, by applying the systematic integrated network pharmacology method to traditional Chinese medicine research, our analyses identified TNF, VEGFA and JUN as the most pivotal therapeutic targets of HDH against LN , with thTNF is a cytokine mainly secreted by macrophages, activating the transcription of several proinflammatory genes and is involved in the regulation of a wide spectrum of biological processes such as cell proliferation, differentiation and apoptosis . Since JIn the current study, we predicted that stigmasterol and quercetin, two bioactive components of HDH, might target TNF and thus influence the function of TNF. Stigmasterol is a plant sterol exhibiting anti-inflammatory activity . KangsamQuercetin is a flavonol widely distributed in plants . The effOur analyses also identified VEGFA as a key target in the course of HDH treatment against LN. It was evidenced that the plasma VEGFA level was reduced when SLE patients were treated with Mycophenolate mofetil . And YuaBesides, this study also elucidated that TNF signaling pathway, Toll-like receptor signaling pathway, NOD-like receptor signaling pathway, NF-kappa B signaling pathway, HIF-1 signaling pathway, and PI3K-Akt signaling pathway might be involved in the pharmacological effect of HDH against LN , 5A, C. in vitro and in vivo. However, it should be emphasized that even though the study identified several key targets, the multi-targets effect and multi-pathways effect of traditional Chinese medicine cannot be ignored, and the therapeutic effects of HDH in LN cannot be simply attributed to these binding. Anyway, the research provides us new insights into the therapeutic mechanism of HDH in LN. And the network pharmacology methods used in this study also offered guidance and inspiration for further researches and the development of new drugs.In general, our study mainly found that the active components of HDH, stigmasterol and quercetin, may play a protective role in LN patients by acting on TNF and VEGFA. Yet the interaction predicted in this study still needs to be tested


Astragalus membranaceus (AM) in molecular level in the oral ulcer (OU) treatment with reference to network pharmacology. Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform database was used in screening the AM active components and AM action targets; GeneCards database was used to screen OU targets; the common target were screened by Venny online tool; Cytoscape software was applied to construct the target gene regulation map of AM active components; STRING database was used to construct the protein–protein interaction network and the key targets were screened as per degree value; gene ontology enrichment and KEGG pathway enrichment of interactive genes were calculated through David database. There were 17 active ingredients and 429 target spots in Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform database. There are 606 target genes for OU in GeneCards database. There are 67 common targets, including 10 key targets: IL10, IL6, TNF, IL1B, CXCL8, CCL2, TLR4, IL4, ICAM1, and IFNG. It involves 30 gene ontology terms and 20 KEGG signal channels. The molecular docking results showed that quercetin and kaempferol had a good binding activity with IL6, IL1B, TNF, and CCL2. Network pharmacological analysis shows that AM can regulate multiple signal pathways through multiple targets to treat OU.To analyze the mechanism of  Oral hygiene, anti-inflammatory drugs, local anesthetic agents, oral decontamination, and mouth wash are some of the current therapies used to relieve pain and lessen disease severity in patients. Despite this, most of them are still supportive, and there is no effective treatment at present. Growth factors, immune modulators, and radioprotection agents have all been discovered in clinical trials and preclinical investigations as OU medicines. However, these drugs have limited effects, require intravenous injection or frequent administration, and may accelerate neoplastic growth. Therefore, there is an urgent need to identify OU drugs that are more cost-effective and have fewer side effects.Oral ulcer (OU) is the most common ulcer disease in human beings. OU is characterized by recurrent oral mucosal ulcer in healthy individuals, which occurs in oral mucosa .Astragalus membranaceus , a commonly used TCM, has been proved to have the medicinal function of reducing fatigue and enhancing immunity. Therefore, it is often used to treat cancer, frequent colds, and shortness of breath. According to modern pharmacological investigations, AM and its ingredients are thought to have immunomodulatory, antioxidant, anti-inflammatory, anti-diabetic, and antiviral properties. AM is also commonly used in the treatment of oral inflammatory diseases like OU and periodontitis. Yet, the mechanism of AM in the OU treatment in molecular level is still unclear. Some online databases, such as Ontology-Based Artificial Intelligence Model and online pharmacological database, are gradually being used to predict the pharmacological activity of TCM, to select the appropriate prescription of TCM, and to predict the side effects of drugs.,13 They have the advantages of high accuracy, simplicity and effectiveness. In this sense, the introduction of artificial intelligence, including network pharmacology, into TCM research has also attracted great attention. Network pharmacology employs omics, high-throughput screening and network analysis techniques. It elucidates the interaction between multiple compounds, targets, and pathways. Therefore, this study aims to explain the mechanism of AM in OU treatment in molecular level from a multidimensional perspective through network pharmacology.Traditional Chinese medicine (TCM) and natural medicine offer the distinct benefits of several targets and few adverse effects, making them ideal for long-term complementary and alternative therapy.This study does not need ethical approval because there is no personal data involved.http://tcmspw.com). On the basis of The Chinese Pharmacopoeia (2015 edition), there are 500 kinds of TCM and 30,069 kinds of TCM ingredients in TCMSP. TCM is mainly oral preparations following digestion, distribution, metabolism, and excretion to target organs and tissues, which is also called the ADME process. TCMSP predicts active compounds based on ADME parameters along with drug-likeness (DL) and oral bioavailability (OB). OB%≥30 and DL ≥ 0.18 criteria were set for screening active compounds identified in TCMSP database in this study. Subsequently, 17 active components of AM were selected. AM was input into TCMSP for obtaining the active ingredients and corresponding targets. The corresponding target gene name was converted into “GeneSymbol” format in STRING database  to establish the protein interaction relationship.The active compounds of AM were extracted through the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform . GeneCards is a comprehensive database of human genes, providing integrated genetic, genomic, and biological data. “Oral ulcer” was entered in the GeneCards database to retrieve related diseases and obtain all relevant target genes.OU related genes are from GeneCards: human gene database (https://bioinfogp.cnb.csic.es/tools/venny/index.html) was applied. For protein–protein interaction (PPI) analysis, the common targets were found through Venn diagram and entered into the STRING (http://string-db.org/), and the PPI results were visualized by using the software Cytoscape3.7.2 (http://www.cytoscape.org/). For integrating, visualizing and analyzing biological networks, software Cytoscape was applied.To make Venn diagram of target genes of effective compounds of AM and OU, Venny 2.1.0 . GO categories comprise biological processes (BP), cellular components (CC), and molecular functions. GO and KEGG enrichment analysis results (P < .05) were imported into the bioinformatics online mapping platform (www.bioinformatics.com.cn) for visual display.GO and KEGG pathway enrichment analysis were done on DAVID6.8 (http://www.rcsb.org/) were used to find the 2D molecular structures of active compounds and important proteins. ChemBio3D was used to minimize the molecule’s energy and store it in mol2 format. Autodock 1.5.6 and AutoDock Vina were used for docking validation, and Pymol (version 2.2.0) and Discovery Studio Client software (version 19.1.0) were used to display and evaluation the molecular docking patterns.High-value ingredients and targets were chosen for molecular docking investigations using AutoDock Vina based on the findings of the “Compound-Target” network. TCMSP databases and the RCSB PDB  bind hub gene IL6, IL1B, TNF, and CCL2 to various degrees Fig. . Accordi Most studies have shown that the recurrence of OU is related to immune inflammation. There is increased cytokines release such as IL-6 and TNF-α in the peripheral blood cells of patients having recurrent ulcer. Among rats with recurrent oral cancer, there was significant increase in the expression of NF-κBp65 and TNF-α.,22 AM has the effects of immune regulation, antioxidation, and anti-inflammatory. AM helps to reduce blood viscosity and platelet adhesion, improve microcirculation, improve immunity, and promote ulcer healing, as it has strong antioxidant activity by scavenging free radicals and activating antioxidant enzymes. Yet, the definitive AM mechanism in OU treatment is not clear.As a mild self-limiting disease, OU has a great effect on personal daily life due to recurrent and chronic nature. Studies have shown that oral probiotic tablets, whether in live or heat inactivated form, can have a beneficial effect on oral immunity through IL-10 and transforming growth factor-beta mediated IgA secretion. IL-6 is a cytokine produced by lymphocytes, monocytes, macrophages and endothelial cells. IL-6 can not only stimulate T lymphocytes to produce interferon γ, it can also stimulate B lymphocytes to secrete immunoglobulin. IL-6 plays a crucial role in chronic inflammation and even 2019 coronavirus disease (Covid-19). Previous studies have shown that the level of IL-6 in saliva of patients with oral cancer is several times higher than that of normal people. The susceptibility risk of oral diseases can be estimated by the degree of genetic variation of IL-6, and new treatment strategies can be designed based on IL-6 inhibition.Various immune cells express tumor necrosis factor superfamily. At present, it comprises 19 members. It mediates a variety of BP by binding to receptors. Tumor necrosis factor superfamily members are involved in inflammatory response, antiviral response, apoptosis induction, secondary lymphoid organs development, immune cells differentiation and activation, osteoclast formation, and tumorigenesis and angiogenesis regulation.TNF-α polymorphism is associated with oral submucosal fibrosis, TNF-α polymorphic GA genotype (rs361525) significantly increases the risk of oral diseases. As a chemokine member of CC subfamily, CCL2 induces the movement and recruitment of monocytes and macrophages. Binding of CCL2 to its receptors (the most important of which is CCR2) triggers various signaling pathways, and eventually leads to various immune events, such as inflammation. Previous studies have shown that the level of CCL2 in gingival crevicular fluid in periodontal lesions is up-regulated compared with healthy sites. CCL2 has been considered as a potential candidate gene for oral cancer caused by chronic infection.Through network pharmacology, this study explored the possible mechanism of AM in OU treatment in molecular level. We found 67 common targets, which may be a target for the treatment of OUs. The PPI network prompts IL10, IL6, TNF, IL1B, CXCL8, CCL2, TLR4, IL4, ICAM1, IFNG, etc. All these cytokines play an important role in OUAs an important multifunctional anti-inflammatory cytokine. IL-10 inhibits the activation and function of various innate and adaptive leukocytes, so as to inhibit possible outbreak of inflammatory cytokines, and prevent host cell injury.GO and KEGG enrichment analysis showed that AM in OU treatment involved multiple BP. The enrichment of GO BP in prevention of OU by AM mainly involves inflammatory response, immune response, cellular response to lipopolysaccharide, and so on. This finding suggests that AM may inhibit OU by regulating BP and pathways related to the inflammatory response. The inflammatory response is a dynamic immune process implemented by the innate and adaptive immune systems to protect the organism from harmful factors. Besides, it involves multiple signal pathways, in which the core targets IL-6, IL1B, TNF, and CCL2 are the key molecules enriched in the TNF signaling pathway. Studies have shown that quercetin can not only inhibit nuclear factor kappa-B phosphorylation and translocation but also inhibit AP-1 and reporter gene transcription to fight inflammation. Quercetin treatment can also reduce TNF-α induced inflammation. Kaempferol, a common flavonoid, exists in a great number in tea, citrus fruits, grapes, beans, edible plants, and vegetables. It is used in treatment of a variety of diseases, like enteritis, mastitis, lung injuries, cardiovascular disease, peptic ulcer disease, cancer, leukemia, rheumatoid arthritis, liver injuries, and kidney injuries. Kaempferol has strong antioxidant, anti-inflammatory, and anti-apoptotic potential. It plays a role by inhibiting the production of reactive oxygen species, up regulating NF-E2-related factor 2 and antioxidant related genes, and inhibiting angiotensin II, nuclear factor kappa-B, and mitogen-activated protein kinase. In addition, kaempferol can reverse the increased TNF-α and IL-1β Level and down regulate its gene expression to prevent neuroinflammation. We hypothesized that these 2 active components may be key components of AM in OU treatment.The molecular docking results showed that the “Compound-Target” network’s top 2 active components quercetin and kaempferol had a good binding activity with IL6, IL1B, TNF, and CCL2. Among the active ingredients of AM, quercetin is the first and kaempferol is the second. A major bioflavonoid, quercetin commonly exists in fruits and vegetables. Quercetin has strong antioxidant and chelating abilities. In addition, it also interacts with antioxidant enzymes and has the characteristics of regulating antioxidant enzyme activity.TNF has the lowest and most stable binding energy with quercetin and kaempferol, the 2 main active ingredients in AM. So TNF is likely to be the key core target. The most critical targets showed good binding activity with the major compounds, and preliminary analysis indicated that the pharmacodynamic mechanism of AM had a sufficient material basis.In fact, AM and its secondary products have long been admired. AM granules can effectively improve the clinical symptoms of OU and shorten the healing time of ulcer. In addition, AM, as a formula in compound drugs, has been shown to have a positive effect in the treatment of OU. Huangqi Jianzhong Tang (HQJZ) in with AM as raw material can significantly shorten the ulcer area, average ulcer period, and pain index of OU. Astragalus mouthwash, made of AM, has been developed to relieve OU. Astragalus injection on the basis of local treatment can also significantly promote the healing of OU and delay the recurrence. In this paper, network pharmacology and molecular docking were used to predict the core targets of AM on OU, which is expected to promote the transformation of AM–OU products. But the demarcation point regarding the dosage of AM causing OU remains to be confirmed by more experiments. It is well recognized that the immune-enhancing and anti-inflammatory effects of AM contribute to its positive effect on OU. In addition, regarding the toxicity of AM, some scholars conducted animal experiments and gave AM injection 1 g /20 g (mouse weight) to mice without obvious abnormal toxicity, which was 75 times that of human clinical dose. Therefore, it is safe for AM to be used clinically as part of a pharmaceutical agent or prescription.But there are still limitations to this study, including the following: although network pharmacology has the advantages of low cost and high efficiency, it can only report plants that have already been discovered. Therefore, we cannot take into account all source plants of AM. We look forward to more basic experimental and clinical studies of drugs in the future to enrich the network pharmacology database. AM is often used as a drug. However, there are still a small number of scholars who studied that the AM properties are warmer, patients who often suffer from OU are generally manifested as syndrome of dampness-heat in the spleen and stomach. Excessive intake of AM in such patients may exacerbate OU.The active pharmacological constituents of AM and the mechanism of action of AM on OU were verified by network pharmacology and Molecular docking. This study enriched our knowledge on the therapeutic effects of AM active components on OU. AM could play the functions of anti-inflammatory and promote the healing of OU. TNF signaling pathway is the most abundant pathway in AM–OU targets, suggesting that it may play a key role in OU treatment.This research was funded by High-level Talents Project of Hainan Natural Science Foundation (821RC687), National Natural Science Foundation of China (82201080), Teaching Achievement Award Cultivation Project of Hainan Medical University (HYjcpx202217), Higher Education and Teaching Reform Research Project of Hainan Province (Hnjg2021-60), Course Construction Project of Hainan Medical University (HYZD202215), Health Science Research Project of Hainan Province (22A200041), Innovative Scientific Research Project for Postgraduates of Hainan Medical College (Qhys2022-278).Conceptualization: Zhu-Ling Guo.Data curation: Wan Zhong, Si-Yu Tao, Xiao-Fang Cheng, Qing Yuan, Hong-Yuan Tian, Song Yang, Diwas Sunchuri, Zhu-Ling Guo.Formal analysis: Si-Yu Tao, Qing Yuan, Chu-Xing Li, Song Yang, Diwas Sunchuri.Funding acquisition: Qing Yuan, Chu-Xing Li.Investigation: Wan Zhong, Xiang Guo, Xiao-Fang Cheng, Qing Yuan, Hong-Yuan Tian.Methodology: Wan Zhong, Qing Yuan, Hong-Yuan Tian.Resources: Si-Yu Tao, Xiang Guo, Chu-Xing Li, Song Yang.Software: Xiang Guo, Hong-Yuan Tian, Song Yang.Writing – original draft: Wan Zhong.Writing – review & editing: Si-Yu Tao.


Monkeypox outbreaks across the globe has aroused widespread concern. Ruyi Jinhuang Powder (RJP), a common formula in Chinese medicine, is used to treat pox-like illnesses. This study aimed to identify the molecular mechanisms and therapeutic targets of RJP for the treatment of monkeypox using network pharmacology and bioinformatics techniques. The bioactive substances and potential targets of each component of RJP were retrieved from the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP). The differentially expressed genes (DEGs) of the monkeypox virus (MPXV) were identified from the GSE24125 by GEO2R. Key signaling pathways, bioactive components, and potential targets were obtained by bioinformatics analysis, including gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG), disease ontology (DO), and protein-protein interactions (PPI) analyses. Finally, molecular docking was used to predict the interaction between active compounds and core targets. A total of 158 active ingredients and 17 drug-disease-shared targets of RJP were screened. Bioinformatics indicated that wogonin and quercetin might be potential drug candidates. Potential therapeutic targets were identified. Immune-related mechanisms that exerted antiviral effects included signaling pathways like TNF, age-rage, and c-type lectin receptor pathways. Our results illustrated the good therapeutic effect of RJP on monkeypox in terms of biological activity, potential targets, and molecular mechanism. This also offered a promising strategy to reveal the scientific basis and therapeutic mechanism of herbal formulas used to treat the disease. Although the rash passes through multiple stages before it eventually scabs over and falls off, there is a strong likelihood that it may leave scars. The death rate from monkeypox can reach 10% when sepsis, meningitis, and osteomyelitis are also involved. According to reports, over 9109 confirmed cases of monkeypox have been found in at least 40 non-African nations in Europe, Latin America, and Asia, as of July 9, 2022 (https://www.monkeypoxmeter.com/). There is no specific treatment for monkeypox. It has been estimated that smallpox vaccination with vaccinia virus (another orthopoxvirus) protects against monkeypox by approximately 85%. However, in 1980 routine vaccination against smallpox became obsolete due to its eradication, which recently contributed to the geographic spread of monkeypox cases. Therefore, it is imperative to seek out treatment for monkeypox aggressively.One of the 4 orthopoxviruses that are pathogenic to humans, along with the smallpox virus, is the monkeypox virus (MPXV). It is a double-stranded DNA virus belonging to the Orthopoxvirus genus of the family Poxviridae. The most important symptom of monkeypox is the rash. It may also result in clinical symptoms such as fever, chills, and muscle aches.–8 Kai Hu noted that RJP could shorten the wound healing time of perianal abscesses and relieve the pain. Skin soft tissue infections of the can be treated more quickly and with less infectivity-related indices when RJP is used in combination with antibiotics. It can reduce the symptoms of lesions in people with papulopustular rosacea and has a positive clinical impact on various phlebitis.–15 Therefore, RJP has accumulated a significant amount of clinical data for the treatment of several dermatophytosis diseases. Skin lesions are the most significant clinical sign of monkeypox and the one most likely to cause long-term effects. We concluded that RJP, following the theory of TCM discriminatory evidence and clinical research, is one of the preferred formulae for the treatment of monkeypox. However, its potential therapeutic mechanisms for treating monkeypox have not been fully understood.Since monkeypox is not a large-scale epidemic, there are no records of Chinese medicine treating monkeypox in the past and present. However, the Chinese have been treating pox diseases with traditional Chinese medicine (TCM) for thousands of years and have accumulated rich clinical experience. After careful screening, we proposed Ruyi Jinhuang Powder (RJP) as a potentially effective formula for treating monkeypox. RJP is derived from Chen Shigong “Surgical Authenticity” from the Ming Dynasty and is included in the 2015 edition of “Chinese Pharmacopoeia.” The ten different types of herbs that make up RJP are Baizhi (radix angelicae dahuricae), Dahuang (radix et rhizoma rhei), Houpu , Huangbai (cortex phellodendri), Chenpi (percarpium citri reticulatae), Jianghuang (rhizoma curcumae longae), Cangzhu (rhizoma atractylodis), Tiannanxing (rhizoma arisaematis), Tianhuafen (radix trichosanthis kirlowii), and Gancao (radix glycyrrhizae). Huangbai and Cangzhu are the Jun herbs in the entire formula, which are also ingredients in the traditional Chinese medicinal formula Ermiao San. They have the function of clearing heat and drying dampness. The Chen herbs, which include Chenpi, Tiannanxing, Dahuang, and Houpu, clear heat and dry dampness, resolve phlegm and move qi. Jianghuang breaks blood and moves qi. Tianhuafen and Baizhi clear heat, detoxify toxins, reduce swelling, and relieve pain as adjuvants. Gancao harmonizes all the herbs, clears heat, and detoxifies while astringes the sores. Combining all the herbs can detoxify and subdue swelling, clear heat and astringent sores. RJP is frequently used to treat dermatological diseases, including sores, ulcers, and psoriasis, and has the effect of clearing heat, detoxifying, and dispersing swelling and nodules. We selected RJP for the following reasons: First, in ancient Chinese medical texts, monkeypox, like smallpox, is classified as an epidemic disease. The goal of the therapy should be to clear the heat, detoxify the toxin, resolve the dampness, and move the qi. This is precisely aligned with the formula meaning of RJP. Second, clinical studies reported that RJP could lower the serum levels of cytokines such as c-reactive protein, interleukin-6, and soluble interleukin-2 receptor as well as significantly improve the clinical symptoms of mumps, senile skin ulcers, and acute herpes zoster. This systematic approach to drug research is consistent with TCM holistic viewpoint and the multi-component, multi-pathway, multi-target synergistic mechanism of action of TCM formulations.,18Network pharmacology refers to a systematic pharmacological research method that combines pharmacology, molecular biology, electronic technology, and bioinformatics and holds great promise in studying the network relationships between the active ingredients in herbal formulas and associated targets, metabolic pathways, and diseases.To determine the common target and potential mechanism of action of RJP in the treatment of MPXV and to develop new medications, this study combined network pharmacology, bioinformatics, and molecular docking technology to identify the bioactive components and targets of RJP. The technology roadmap for this study is presented in Figure https://old.tcmsp-e.com/tcmsp.php). Many medication ingredients cannot reach specific protein targets in cells due to the pharmacological properties of TCM. Therefore, selecting the appropriate screening index for network pharmacology was very important. The oral bioavailability (OB) value  is an essential pharmacokinetic parameter in the absorption, distribution, metabolism and excretion properties of drugs. The drug-like properties (DL) value  is a significant screening value for pre-judging a large number of compounds in a short time. Therefore, only compounds that passed the screening requirements of OB ≥ 30% and DL ≥ 0.18 were included in the study. After filtering the active ingredients, it was important to match the target of each ingredient. To obtain the official gene name of the protein, we logged into the UniProt database (http://www.uniprot.org/),,21 used the UniProtKB search function, and narrowed our search to “Homo sapiens.” Because this study does not involve the ethical issues of clinical and animal experiments, and it is a study about network pharmacology and bioinformatics, it does not need ethical approval.The components of RJP and their corresponding targets were obtained from the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform  database is an open-source database that stores microarrays of various diseases of organisms. We downloaded the human genome data of monkeypox GSE24125 from the GEO database. GSE24125 is the gene set of a person infected with the MPXV contributed by Rubins. According to the original data provided by the contributors, we selected the primary human macrophage data on the GPL10912 platform (SMD Print_1046 LC-48 Homo sapiens), including 16 MPXV groups as the disease group, and the remainder of the inactivated virus group and the simulated infection group as the control group. The GEO2R online analysis tool performed ID conversion, miss value processing, and force normalization to remove the batch correspondence. The GEOquery and limma packages provided by GEO2R were used to read the data and obtain the difference multiple. According to the regulation, we set the threshold of differentially expressed genes (DEGs) as | log2FoldChange | ≥ 0.5 and the P value <.01. The DEGs that satisfied the above conditions were used for further analysis. The ggplot2 package was used to draw the gradient volcano map to depict the distribution and expression of DEGs.The Gene Expression Omnibus (GEO) . Drug composition target maps were obtained by Cytoscape 3.9.0 software. org. Hs. e.g., db, enrichplot, ggplot2, pathview, DOSE, ggnewscale, stringr). Results with P values < .05 were normally regarded as significant. The 3 enrichment analysis methods displayed only the first ten results.After converting the collected common targets into related Entrez IDs, we carried out Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway, and Disease Ontology (DO) enrichment analyses. We used R 4.1.0 and related R packages , which has the most organisms and proteins and set the operating conditions as Homo sapiens, to determine the core therapeutic targets. The confidence level was set to 0.4, and the independent nodes were hidden. The relevant PPI data was then downloaded from the STRING database and loaded into Cytoscape 3.9.0, extensively used in the construction and visualization of topological networks, particularly in network pharmacology research. The MCODE plug-in in Cytoscape software was used to analyze the core network and identify its closely linked sub-modules. The key modules were selected for further analysis, and the Degree, BottleNeck, EcCentricity, Maximal Clique Centrality, and Stress in the Cytohubba plug-in were used for further screening. The intersection of the top ten targets of each component was then used to determine the core therapeutic targets.We submitted the common action targets derived from the intersection of RJP and MPXV to the STRING database  was used to obtain 3D structures of proteins . These structures were then imported into PYMOL software (https://pymol.org/2/) and modified by removing water molecules, co-crystal ligands, and ions. Additionally, AutoDock Tools 1.5.6 was used to add missing hydrogen bonds and kolman partial charges to incorporate the non-polar hydrogens into their carbon counterparts. They were stored as PDBQT protein receptor files. In the second step, the 2D structure of the compound was downloaded from the PubChem database (https://pubchem.ncbi.nlm.nih.gov/) and then loaded into Chem3D software to minimize and transfer the energy (MM2 force field) into the 3D structure. Subsequently, the resultant 3D structure was modified in the AutoDock tool 1.5.6 by adding hydrogen and protonation, and the non-polar hydrogen was rotationally merged with its corresponding carbon. The torsion was automatically set in the software. The structure was then saved as a PDBQT ligand file. In the third step, the PDBQT structure of the receptor and ligand were imported into the AutoDock tool 1.5.6 to construct the docking pairing pocket. When building the box, the center point was located on the macromolecule, and the number of points in the X, Y, and Z dimensions were set so that the box completely wrapped the protein. These parameters of the paired cassette were determined for each protein by the configuration file for the next interfacing with Vina, including box center , box size , massive docking (num mode) to output (set to 20), energy _ range (set to 5). Finally, Vina software was used for molecular docking to calculate the docking affinity. In this process, besides the configuration files, PDBQT files of proteins and ligands were also used because PDBQT files determined the information of docked ligands and proteins. After Vina operated, the results were exported as pdbqt * and log *.txt files. The binding affinity score of the target protein receptor to the small molecule ligand was established by scoring the free energy of docking.In the first step, macromolecular receptor files were prepared from the Protein Data Bank. Protein data bank .We used OB ≥ 30% and DL ≥ 0.18 as the screening threshold to obtain the screened drug components, which included 20 components of Baizhi (radix angelicae dahuricae), 4 components of Cangzhu (rhizoma atractylodis), 6 components of Tiannanxing (rhizoma arisaematis), 5 components of Chenpi (percarpium citri reticulatae), 7 components of Dahuang (radix et rhizoma rhei), 88 components of Gancao (radix glycyrrhizae), 2 components of Houpu , 24 components of Huangbai (cortex phellodendri), 3 components of Jianghuang (rhizoma curcumae longae), and 2 components of Tianhuafen (radix trichosanthis kirlowii). After combining them and removing the duplicates, 148 compounds were obtained, considered to be the active ingredients of RJP . The gradual volcano plot depicts the distribution of the top 10 DEGs , including 671 for BP, 23 for CC, and 60 for MF. The bubble graph in Figure As is common knowledge, GO analysis includes biological processes (BP), cellular components (CC), and molecular functions (MF). We obtained 754 entries . The first 3 statistically significant pathways were cellular senescence (hsa04218), hepatitis B (hsa05161), and leishmaniasis (hsa05140). The top 10 significant enrichment items in the pathway with the highest gene counts are displayed in the bubble plot in Figure The function and signal transduction pathway of the anti-target of RJP were studied using the KEGG pathway. In this study, we acquired 60 KEGG pathways . The top 10 DO results are exhibited in Figure Common targets and major diseases were identified using DO enrichment analysis, and 227 statistically significant related diseases were determined .Subsequently, 2 candidate bioactive compounds  were docked with 7 candidate target proteins, including STAT1 (PDB ID: 7nuf), FN1 (PDB ID: 6gue), IL1A (PDB IDs: 2kki), CXCL2 (PDB IDs: 1qnk), CDK2 (PDB ID: 1b38), PTGS2 (PDB ID: 5f19), and IRF1 (UniProt ID: AF-P10914-F1-model _ v2). The results are displayed in Figure Binding energies are often calculated to assess the degree of affinity of a component for a protein target. Generally, binding energies of less than −4.25, −5.0, or −7.0 kcal/mole represent fair, good, or strong binding activity between the ligand and receptor, respectively. The binding energy indicates the possibility that the receptor will bind to the ligand. Conversely, lower binding energy implies a higher ligand-receptor affinity and a more stable conformation. According to the results of binding energy docking in Table Activities aimed at reducing high mortality rates during the COVID-19 pandemic were emphasized. Smallpox is caused by a smallpox virus infection, which has high transmission and mortality rates. With the focus on vaccination, smallpox has been eliminated. As a result, modern humans have little immunity to smallpox. Monkeypox is an infectious disease that has gradually spread worldwide recently. It is closely related to the smallpox virus and can cause similar human clinical diseases. supports the potential of antiviral therapy for monkeypox. Recent studies have shown that Tecovirimat and Brincidofovir exhibit antiviral activity against double-stranded DNA viruses, including poxviruses. However, due to the mutability of viruses, specific medicine against viruses often develop drug resistance after prolonged use. Therefore, finding suitable medication to prevent and treat monkeypox in the future is crucial. In previous studies, we have found that antiviral treatment for MPXV may be better than vaccination.The finding that treatment with antiviral drugs beginning 24 hours after monkeypox infection significantly reduces the death rate and the number of monkeypox-related lesions;,35 Recently, network pharmacology research has provided an effective strategy for obscuring the molecular mechanism of multi-target drugs. We used target prediction, GO, KEGG, DO, PPI, and other analysis methods to explore the pathways, components, and targets of RJP in the treatment of monkeypox. 7 core targets were screened after merging the PPI network of related RJP and monkeypox targets. GO and KEGG enrichment analysis confirmed that RJP could regulate various immunological and antiviral-related signals to conduct antiviral therapy, including TNF, age range, c-type lectin receptor signaling pathways, Kaposi sarcoma-associated herpesvirus infection, etc. Modulating these signaling pathways was a promising area of research and it provided a new direction for treating monkeypox.Due to the multi-target effect and relatively safe characteristics, TCM has attracted growing interest in recent years. RJP is a regularly prescribed drug in TCM. It has a variety of drug components and is often used for treating herpes and fever in conventional treatments, with the advantages of multi-target synergistic therapy and low toxicity. In this study, the potential therapeutic targets of RJP for monkeypox were explored through network pharmacology and multiple databases, and the associated pharmacological mechanisms were predicted. As we previously mentioned, TCM has demonstrated great potential for viral suppression, which can be therapeutic in various viral infectious diseases, including HIV, COVID-19, and others. Additionally, some studies have revealed that it can inhibit herpes virus infection by regulating the cellular NF-KB and MARK pathways. Quercetin is a kind of polyphenolic flavonoid, and subsequent studies showed it has anti-viral activity against the influenza virus, hepatitis C virus, dengue virus, and other different viruses.–40 However, quercetin has historically had difficulty becoming a more practical clinical medicinal agent due to its poor solubility. According to some researchers, it can enhance the antiviral activity of cells after being specially prepared. Mechanism studies showed that quercetin was selected as an inhibitor against SARS-CoV-2 which was mediated by blocking the 3a channel, inhibiting protein kinase B and phosphorylating protein kinase.,43 Other studies showed that the compound also has the function of regulating immune responses by reducing pro-inflammatory cytokines and enhancing anti-inflammatory cytokines. These have been proven to affect COVID-19 treatment positively.–46 Wu et al found quercetin inhibited influenza infection by interacting with the HA2 subunit. According to previous reports, RJP can treat diabetic repair ulcers through the Wnt/β-catenin pathway and can play an anti-inflammatory role by down-regulating IL1A, which is consistent with the key nodes derived from our PPI protein interaction. Moreover, RJP wound healing is the reaction of cells to injury; fibronectin is one of the key proteins in dynamic wound healing. MPXV replicates at the initial infection site, which causes an inflammatory response. More specifically, the virus presence significantly affects T-cell-mediated cytokine responses. The bioactive compounds found in RJP might suppress monkeypox by directly inhibiting the virus and lowering inflammation caused by monkeypox infection.A total of 148 active components and 17 hub targets were identified in this research. In previous studies about these active ingredients, wogonin has been revealed to reduce inflammation by inhibiting the production of inflammatory factors by immune cells such as lymphocytes, macrophages, and neutrophils.Our study identified 7 potential therapeutic targets for monkeypox, including CDK2, FN1, STAT1, PTGS2, IRF1, IL1A, CXCL2. However, there has been insufficient research on therapeutic targets and biomarkers for monkeypox. Therefore, there has been no relevant experiment to prove the role of these 7 genes in monkeypox. Encouragingly, the molecular docking results showed that most of the hub targets and the corresponding ligands had a strong binding affinity, except that the docking scores of quercetin and STAT1 was more than −5 kcal/mol . On one hand, the results of molecular docking improve the accuracy of this study on the treatment of monkeypox with RJP. On the other hand, relevant literature discussions provide insights into the mechanistic studies of monkeypox. Catalase and other substances help remove excess superoxide dismutase and catalase in the body and slow aging. A significant role of FN1 in anti-inflammatory mechanisms and related diseases has been extensively reported in the literature.–53 FN1 is highly expressed in nodular disease, a systemic inflammatory disease manifested by pulmonary involvement and erythema nodosum of the skin.FN1 is a member of the FN protein family. The FN-containing EDA significantly affects skin wound healing. However, CDK2 also has prominent contributions to immunological diseases and anti-inflammatory mechanisms. Evidence suggests that CDK2 is involved in the expression of inflammatory mediators in the psoriatic epidermis and regulates the psoriatic epidermal cell cycle, suggesting that modulating CDK2 is an effective strategy for treating psoriasis. This information undoubtedly provides us insight into studying the control of monkeypox.CDK2 is involved in cell cycle regulation and is generally considered important in cancer pathogenesis. It can reduce the structural damage caused by influenza virus infection through the JAK1/2-STAT1 signaling pathway. This pathway induces inflammatory response and regulates immunological response by mediating the signal transduction of the IL-2, interleukin-6, IL-10, interferon families, as well as other cytokines and growth factors.STAT1 have been demonstrated to be involved in regulating cellular antiviral defense mediated by linear ubiquitination.IRF1 is a transcription factor that can be produced by the IFN-γ activated JAK/STAT1 signaling pathway. IRF1 expression is significantly elevated in IFN-γ-activated macrophages, which regulate the expression of IL-12 and Nitric Oxide. Zafar et al found that the STAT1-IRF1 signaling process regulated the expression of pro-inflammatory genes in atherosclerosis, a pathogenic factor in the disease, and that targeted knockdown of IRF1 expression was beneficial in limiting plaque development. COX-2 is one of the important rate-limiting enzymes that catalyze the production of prostaglandins from arachidonic acid. The expression of COX-2 is rapidly up-regulated when cells are stimulated by various factors, including growth factors, cytokines, inflammatory mediators, etc. PGE2 is the main product of COX-2-catalyzed arachidonic acid production.PTGS2 gene encodes the COX-2 protein in organisms associated with inflammation.CXCL2 is an inflammatory chemokine, and its receptor CXCR2 is mainly expressed on the surface of neutrophils and natural killer cells when CXCL2 binds to CXCR2, its expression activity increases, which induces inflammatory cells such as neutrophils, monocytes, lymphocytes, and natural killer cells to migrate to the injury site and accumulate in the inflammatory site, aggravating the inflammatory response at the injury site. Evidence suggests that IL1A plays an important role in inflammatory diseases such as cardiovascular disease, colonic inflammation, neuroinflammation, and human skin inflammation. Lukens et al found a critical role for IL1A in tissue damage and wound inflammation in the severe inflammatory syndrome of neutrophilic skin disease.A potent mediator of inflammation and immunological response, IL1A belongs to the interleukin 1 family and is synthesized by various cell types, including macrophages.Some of these proteins regulate the damage caused by the virus from the perspective of inflammation and immunity to reduce viral replication, and some others directly played an antiviral role with a variety of targets and ways to reduce the damage by MPXV on the human body. However, there are some limitations to our study. First, limited by the source problem of monkeypox samples, the MPXV genes in this study were derived from secondary mining of the GEO database. Second, we could not experimentally verify the effectiveness of RJP against the MPXV due to the specificity of the disease and the safety concern surrounding virus prevention and control. This study can provide some significance for future research on monkeypox control and mechanism as scientists continue to investigate monkeypox in greater detail.In conclusion, this study found that RJP has great potential in treating monkeypox using a network pharmacological approach and bioinformatic analysis. It has the characteristics of multi-component, multi-target, multi-pathway, and complex connection. Enrichment analysis revealed that RJP has various synergistic therapeutic effects on monkeypox. The specific mechanism of RJP in the treatment of monkeypox may play a positive role through anti-inflammatory and antiviral activities. The limitation of this study is that there is no experimental verification of the interaction, compatibility changes, and different contents of the components in the compound on monkeypox. Afterward important pharmaceutical components should be selected for further mechanism analysis. Nevertheless, it can provide ideas for future research and experimental verification of the corresponding molecular mechanism. It also provided great clinical significance for the follow-up search to find more clinical drugs for treating monkeypox and other viral infectious diseases.Conceptualization: Xi Zhang.Data curation: Xinping Yu.Methodology: Chengcheng Fan.Project administration: Shuo Zhang.Resources: Huan Li.Software: Yingkai Shen.Supervision: Shuo Zhang.Validation: Zijin Sun.Visualization: Yueming Li.Writing – original draft: Xi Zhang.Writing – review & editing: Zhichao Yu.


We analyzed a PPI network of potential angelicin targets in the treatment of osteosarcoma and identified hub targets. We systematically performed GO and KEGG enrichment analyses of the potential targets of angelicin, and we predicted it function in osteosarcoma treatment and the underlying molecular mechanism. Through molecular docking, the interactions between hub targets and angelicin were simulated, and then, the hub targets of angelicin were identified. Based on these results, we validated the effects of angelicin on osteosarcoma cells by conducting in vitro experiments. The PPI network analysis of potential therapeutic targets identified four apoptosis-related hub targets, namely, BCL-2, Casp9, BAX and BIRC 2. GO and KEGG enrichment analyses demonstrated that angelicin regulates osteosarcoma cell apoptosis. Molecular docking results indicated that angelicin can freely bind to the hub targets listed above. In vitro experiments showed that angelicin promoted osteosarcoma cell apoptosis in a dose-dependent manner and inhibited osteosarcoma cell migration and proliferation in a time- and dose-dependent manner. The RT-PCR results showed that angelicin simultaneously promoted the mRNA expression of Bcl-2 and Casp9 and inhibited the mRNA expression of BAX and BIRC 2. Angelicin promotes osteosarcoma cell apoptosis and inhibits osteosarcoma cell proliferation and migration by activating a signaling network that is composed of hub targets that link multiple signaling pathways. Angelicin could become an alternative drug for the treatment of osteosarcoma.To explore the antitumor effects of angelicin on osteosarcoma and the underlying mechanism. We aimed to elucidate the mechanism by network pharmacology, molecular docking, and  Osteosarcoma (OS) is the most common bone malignancy in children and adolescents , 2 and iAngelica and Angelica dahurica [Angelicin is one of the main active ingredients of dahurica . Existindahurica –12. Howein vitro experiments to explore whether angelicin affects the biological behavior of OS and its specific mechanism.Recently, network pharmacology has offered a new strategy for exploring the relationships between drugs and diseases by integrating systems biology, multidirectional pharmaceutical biology, bioinformatics, and computer science , 14. CurThe flow chart of the study design is shown in https://pubchem.ncbi.nlm.nih.gov/) and SymMap (http://www.symmap.org) databases with the keyword “Angelicin”. PubChem: that includes a substantial amount of data related to small organic molecule bioactivity and is funded by the National Institutes of Health (NIH). The SymMap database is a traditional Chinese medicine (TCM) evidence association database that includes an immeasurable amount of information about herbal medicines, their ingredients, and their drug targets; the TCM symptoms, Western medicine symptoms, and diseases associated with these symptoms that these medicines are used to treat; and correlations among these data. In this way, the SymMap database correlates TCM and Western medicine at levels ranging from the phenotypic level to the molecular level.All the active ingredients of Angelicin were obtained from the Pubchem (https://www.disgenet.org/) and the GeneCards database (https://www.genecards.org/) using the keyword “Osteosarcoma” and using “Homo sapiens” as the filtering criterion. In addition, the targets obtained from the DisGeNET database with a score >0.8 were screened. The GeneCards database is a search platform that retrieves genes that are associated with human diseases from 150+ web sources. Genes were searched on the two platforms using the keyword “Osteosarcoma”. The retrieved data include information about Osteosarcoma, such as gene names and gene IDs. The targets that were obtained from these two databases were then integrated, and duplicates were removed, ultimately yielding genes related to “Homo sapiens OS”.A search was performed in the DisGeNET database (Homo sapiens OS-related targets were imported into an online Venn diagram mapping platform (https://bioinfogp.cnb.csic.es/tools/venny/). The region where the two circles of the Venn diagram intersect indicates the potential targets of angelicin in the treatment of OS. The targets that were related to the mechanism by which angelicin treats OS were imported into the STRING database. A PPI network diagram was generated with a confidence level of 0.7, and it was imported into the STRING app in Cytoscape software for further processing and analysis.The identified angelicin-related targets and To further analyze the PPI network and identify hub targets, we imported the PPI network into Cytoscape 3.8.0 software and analyzed the network graph with Network Analyzer. Information on the degree, betweenness centrality (BC), and closeness centrality (CC) of the corresponding targets was obtained. We used degree≥degree median, CC≤CC median, and BC≥BC median as the screening criteria to identify the hub targets.To further analyze the potential mechanism underlying the function of angelicin in OS treatment, we enriched the targets mentioned above with the Bioconductor Cluster Profiler package in R software. According to the GO enrichment analysis, the potential therapeutic targets of angelicin in the treatment of OS are described in terms of biological processes, cellular components, and molecular functions. Additionally, the potential signaling pathways by which angelicin functions in the treatment of OS were explored by KEGG enrichment analysis.To investigate the potential interaction between angelicin and its potential targets for the treatment of OS, we performed molecular docking between angelicin and the hub genes based on the principle of semiflexible molecular docking. The leading software for molecular docking is AutoDock, which includes AutoDock tools and AutoDock Vina. Angelicin and hub targets were processed with AutoDock tools, and the docking center of the hub targets was determined. The 3D structures of the target proteins were obtained from the PDB online platform, and the 3D structures of angelicin were obtained from the PubChem online platform.The human OS cell line MG63 (Procell CL-0157) was kindly provided by Procell Life Science and Technology Co., Ltd. MG63 Cells were grown in 90% modified Eagle’s medium (MEM) supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin, and 100 μmol/L streptomycin, and incubated at 37°C in a humidified atmosphere of 5% CO2.MG63 cells were treated with trypsin, grown in 96-well plates (3000 cells/well), and cultured with media supplemented with various concentrations of angelicin. The cells were incubated in a 37°C cell culture incubator for 24 h, 48 h or 72 h. At each time point, the plates were removed, the medium in the corresponding wells was gently aspirated, and the cells were gently washed with sterile PBS. CCK8 and serum-free medium were mixed at a ratio of 1:9, and 100 μl of the mixture was slowly added to each well. The cells were incubated in a cell culture chamber at 37°C for 1 h. The OD value of each well was measured at 450 nm by a multifunctional enzyme marker. Cell activity (%) = / × 100%.5 cells per well; when the cells had completely adhered to the well, the original medium was discarded, and the cells were washed 2 times with PBS. Angelicin was added at different concentrations and incubated for 24 h. The cells were stained with propidium iodide (PI) and annexin V-fluorescein isothiocyanate (FITC). ACEA NovoCyteTM  equipment was used to perform the flow cytometry analysis. FlowJo  software was used to analyze the results.According to the instructions, we analyzed the effect of angelicin on the apoptosis of MG63 cells by flow cytometry. MG63 cells were seeded in 6-well plates at a density of 5 × 105 cells per well. Then, the cell culture plates were gently shaken to uniformly distribute the cells to form a monolayer, and the cells were prepared for scoring when they reached 95% confluence. A 200 μL pipette tip was used to form a vertical scratch along a sterile straightedge. Then, the treated wells were gently washed 3 times with PBS to remove the nonadherent cells, and the plates were imaged and photographed (0 h). Media supplemented with different concentrations of angelicin (50 and 200 μmol/L) were added, and the control group was incubated in the same volume of MEM supplemented with DMSO; the cells were cultured in an incubator at a constant temperature of 37°C with 5% CO2 for 24 h or 48 h, and then, the cells were photographed. The scratch area S was obtained using the scratch area analysis module of ImageJ software, and the scratch healing rate was calculated according to the following formula: wound healing rate = (1-(S24h/S0h)) × 100%.MG63 OS cells in the logarithmic phase of growth were seeded in 6-well plates at a density of 3 × 10Total RNA was extracted from cells using an RNA extraction kit, first-strand complementary DNA (cDNA) was synthesized from the total RNA using an RNA reverse transcriptase kit, and quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) was performed using SYBR Green reagent  according to the instructions. The primer sequences are shown in in vitro experimental data was conducted using GraphPad Prism 9. The difference between two groups was assessed using one-way ANOVA. The statistical significance of the CCK8 results was assessed using two-way ANOVA. The data are presented as the means ± standard errors of the means (SEM), and a p value < 0.05 indicates statistical significance.Statistical analysis of the bioinformatics data and mapping was performed by R ×64 4.0.3, Cytoscape 3.8.0, and PyMOL software. Statistical analysis of the The original contributions presented in the study are included in the article Material, further inquiries can be directed to the corresponding authors.Using “angelicin” as the keyword, we screened its targets in the PubChem and SymMap databases. We identified 36 targets of angelicin by integrating and deweighting the screened targets, as shown in The keyword “osteosarcoma” was used to screen the DisGeNET and GeneCards databases. A total of 4389 relevant targets were identified after integration and removal of duplicates.Angelicin targets and OS therapeutic targets are shown in a Venn diagram . In totaThe obtained potential targets of angelicin in the treatment of OS were subjected to GO and KEGG enrichment analyses. The results of biological processes, cellular components, and molecular functions are shown in Molecular docking of the identified hub genes was performed using AutoDock software. The molecular docking results were visualized using PyMOL software, and the docking results of the four hub targets are shown in P < 0.0001). The differences in cell viability between the 50 μmol/L and 200 μmol/L groups were statistically significant (P < 0.0001)  . The viaP < 0.05). The apoptosis rate of OS MG63 cells gradually increased with increasing angelicin concentrations . The wound healing rate of OS cells gradually decreased with increasing angelicin concentration , and the results are shown in To measure the mRNA expression of pivotal targets in OS MG63 cells after treatment with different concentrations of angelicin, we performed RT-PCR experiments, and the results demonstrated that the expression of BAX and Casp9 gradually increased, while the expression of the antiapoptotic proteins Bcl-2 and BIRC2 gradually decreased with increasing angelicin concentration. The differences in mRNA expression among the different concentration groups were statistically significant (Angelica archangelica and Paeoniae Radix Alba [Osteosarcoma is the most common malignant tumor, accounting for approximately 55% of all bone tumors . OS is mdix Alba , 21. Exidix Alba , 10. InsAngelica affects the biological behaviors of osteosarcoma. When the NF-κB signaling pathway is inhibited, the biological behaviors of osteosarcoma, such as proliferation, migration, and apoptosis, are suppressed [in vitro results further confirmed the network pharmacological results. It was also demonstrated that angelicin inhibits osteosarcoma cell proliferation and migration in a dose-dependent manner and promotes osteosarcoma cell apoptosis in a dose-dependent manner.In this study, we intersected angelicin therapeutic targets and OS-related targets that were identified in several databases and found potential therapeutic targets through which angelicin functions in the treatment of OS. Furthermore, we found the molecular hub targets of angelicin. Then, we screened the hub targets, including Casp9, BIRC2, BAX, and BCL2, of which BCL2 and Casp9 are proapoptotic proteins, and BAX and BIRC2 are antiapoptotic proteins . All fouppressed , 28, 29.ppressed , 30, 31.in vitro experiments described above show that angelicin inhibits OS cell proliferation and migration and promotes OS cell apoptosis upregulating Casp9 and Bcl-2 gene expression and downregulating BAX and BIRC2 gene expression, thereby regulating apoptosis in multiple species, apoptosis, the P53 signaling pathway, the NF-κB signaling pathway, and the TNF signaling pathway to form a signaling pathway network.The network pharmacology, molecular docking, and in vitro experiments that angelicin inhibits osteosarcoma cell proliferation and migration and promotes osteosarcoma cell apoptosis. We are the first to observe these results. Network pharmacology is an efficient tool that is used for the investigation of the mechanisms of action of drugs. However, it is clear that the present study still has some limitations. This study did not examine the specific signaling pathways that are regulated by angelicin in osteosarcoma cells, but we have predicted which pathways are targeted by angelicin via network pharmacology; molecular biological validation is our next step. In addition, in vivo experiments were not conducted in this study, and the inhibitory effect of angelicin on osteosarcoma in vivo was not explored; we will pursue these experiments in future work. Third, the exploration of the loci of apoptotic proteins on which angelicin acts is another focus of our future work.Through pharmacological and molecular docking assays, we discovered the regulatory effect of angelicin on osteosarcoma cells. Based on these results, we verified with We found that via the regulation of multiple signaling pathways, angelicin inhibited osteosarcoma cell proliferation in a time- and dose-dependent manner, inhibited osteosarcoma cell migration in a dose-dependent manner, and promoted osteosarcoma cell apoptosis in a dose-dependent manner. Angelicin could be an alternative medicine for the treatment of osteosarcoma, and it still needs to be explored in depth.Supplementary Tables 1-2 and 4Supplementary Table 3