The traditional Chinese medicine prescription Suhexiang Pill (SHXP), a classic prescription for the treatment of plague, has been recommended in the 2019 Guideline for coronavirus disease 2019 (COVID-19) diagnosis and treatment of a severe type of COVID-19. However, the bioactive compounds and underlying mechanisms of SHXP for COVID-19 prevention and treatment have not yet been elucidated. This study investigates the mechanisms of SHXP in the treatment of COVID-19 based on network pharmacology and molecular docking.First, the bioactive ingredients and corresponding target genes of the SHXP were screened from the traditional Chinese medicine systems pharmacology database and analysis platform database. Then, we compiled COVID-19 disease targets from the GeneCards gene database and literature search. Subsequently, we constructed the core compound-target network, the protein-protein interaction network of the intersection of compound targets and disease targets, the drug-core compound-hub gene-pathway network, module analysis, and hub gene search by the Cytoscape software. The Metascape database and R language software were applied to analyze gene ontology biological processes and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment. Finally, AutoDock software was used for molecular docking of hub genes and core compounds.Toll-like receptor, interleukin 17, Chemokine, and hypoxia-inducible factor 1 signaling pathways. SHXP may play a vital role in the treatment of COVID-19 by suppressing the inflammatory storm, regulating immune function, and resisting viral invasion. Furthermore, the molecular docking results showed an excellent binding affinity between the core compounds and the hub genes.A total of 326 compounds, 2450 target genes of SHXP, and 251 genes related to COVID-19 were collected, among which there were 6 hub genes of SHXP associated with the treatment of COVID-19, namely interleukin 6, interleukin 10, vascular endothelial growth factor A, signal transducer and activator of transcription 3 (STAT3), tumor necrosis factor (TNF), and epidermal growth factor. Functional enrichment analysis suggested that the effect of SHXP against COVID-19 is mediated by synergistic regulation of several biological signaling pathways, including Janus kinase/ STAT3, phosphatidylinositol 3-kinase (PI3K)-protein kinase B (Akt), T cell receptor, TNF, Nuclear factor kappa-B, This study preliminarily predicted the potential therapeutic targets, signaling pathways, and molecular mechanisms of SHXP in the treatment of severe COVID-19, which include the moderate immune system, relieves the “cytokine storm,” and anti-viral entry into cells. The clinical features of COVID-19 are diverse and range from asymptomatic to severe and critical type. Severe and critical cases represented 14% and 5% of laboratory-confirmed COVID-19 patients, leading to a large proportion of deaths.–5 The focus and difficulty in the treatment of COVID-19 are the severe type and critical type, as they are the main factors affecting the mortality rate. The current research data showed that various TCM products and their combinations are effective treatment options for COVID-19. In the clinically severely ill patients, the intervention of TCM or the combination of Chinese and Western medicine contributes to the relief of the disease and the reduction of mortality.,8In the struggle against diseases for thousands of years, traditional Chinese medicine (TCM) has accumulated rich experience and formulated many effective prescriptions, which provide a solid foundation for the application of TCM theories and methods to the treatment of COVID-19.Preions of the Bureau of Taiping People's Welfare Pharmacy (Taiping Huimin Heji Ju Fang) of the Song Dynasty, and Shen Kuo gave a high evaluation on its treatment of epidemic diseases in the Dream Rivulet Diary (Mengxi Bitan). The National Health Commission of China Diagnosis and Treatment Protocol for COVID-19 infection recommends SHXP to treat critically ill patients with COVID-19. SHXP, which promotes qi to relieve pain and aromatic resuscitation, consist of fifteen common Chinese herbal medicines. In the formula, Liquidambar orientalis Mill. (Suhexiang) and Styrax tonkinensis (Pierre) Craib ex Hart. (Anxixiang) are good at clearing filth, dispelling turbidity, and inducing resuscitation. Moschus moschiferus Linnaeus (Shexiang) and Dryobalanops aromatica Gaertn. f. (Bingpian) are adept at inducing resuscitation and removing blockages of the whole body. The above four herbal medicines are the “Monarch” medicines in the prescription. Cyperus rotundus L. (Xiangfu), Eugenia caryophyllata Thunb. (Dingxiang), Aucklandia lappa Decne. (Muxiang), Aquilaria Sinensis (Lour.) Gilg. (Chenxiang), Santalum album L.(Tanxiang), Boswellia carterii Birdw. (Ruxiang) can regulate qi and blood, warm up the meridians, fall qi inversion. They are regarded as the “Minister” medicine in the prescription. Piper longum L. (Biba) is suitable for warming the spleen and stomach for dispelling cold; Bubalus bubalis Linnaeus (concentrate powder) (Shuiniujiao) has the effect of clearing the heat and detoxifying; Cinnamomum camphora (L.). Presl. (Zhusha) soothe the nerves; Atractylodes macrocephala Koidz. (Baizhu) can invigorate the spleen and stomach and dry up dampness; Terminalia chebula Retz. var. tomentella Kurt. (Hezi) inhibits qi and prevents the spicy fragrance from being too diffused and dissipating. They can be called as “Assistant” medicines. The combined use of various drugs can strengthen resuscitation and qi movement to relieve pain and prevent gas consumption damage and have a therapeutic effect on COVID-19.Suhexiang pill (SHXP) was first recorded in the prescription of –13 Molecular docking is an important research technology that can quickly and accurately predict the conformation of the binding of the receptor-drug complex and predict the binding pattern and affinity to support the screening of active components.–16 Therefore, we chose the network pharmacology and molecular docking approaches to explore the mechanisms of SHXP in the treatment of COVID-19. The corresponding workflow is shown in Figure Network pharmacology can reflect and clarify the interactive relationship between multiple compounds, multiple targets, and multiple pathways, which coincides with the overall concept of TCM. Meanwhile, it abstracts the relationship into a network model and illustrates the effect of drugs on human biological network from a systematic perspective.22.1http://tcmspw.com/tcmsp.php) and Bioinformatics Analysis Tool for Molecular mechANis -m of Traditional Chinese Medicine, . Oral bioavailability > 30% and drug-like(drug similarity)>0.18 were used as screening thresholds to filter compounds. Meanwhile, potential target proteins were collected for these active ingredients in the above 2 databases.,18 UniProt database (http://www.uniprot.org/) was used to convert protein names into gene names.We screened active ingredients of SHXP by searching the pharmacological database of the Traditional Chinese Medicine System and Analyzing Platforms  to construct a compound-predicted target network map. Cytoscape is an open-source bioinformatics analysis software for constructing molecular interaction networks composed of proteins, genes, drugs, and other interactions for visual browsing and analysis.,20The deduplicated compounds and their relevant targets were imported into the Cytoscape 3.7.2 software (2.3https://www.genecards.org) database provides gene-centric information that is automatically mined and integrated from myriad data sources. The Online Mendelian Inheritance in Man  database is a comprehensive, authoritative, and timely knowledgebase of human genes and genetic disorders compiled to support human genetics research, education, and the practice of clinical genetics.–23 We used these 2 databases to mine genes related to COVID-19 by searching for the keywords “novel coronavirus pneumonia” “COVID-19” and then setting up a data set for COVID-19 targets.The GeneCards , which provides information on predictive and experimental protein interactions. The prediction method of this database is derived from the experiments, databases, and text mining of neighborhood, gene fusion, co-occurrence and co-expression. In addition, the database defines protein–protein interactions (PPIs) with confidence ranges for data scores . In this study, PPIs with combined scores >0.7 were reserved for further research. Then, we used the plugin CytoHubba in Cytoscape software to analyze the data in the PPIs network of overlapping targets with 3 algorithms “Closeness, Betweenness, BottleNeck,” aiming to find hub genes accurately. The parameters were set as top = 15 and were ranked by the 3 algorithms. Betweenness describes the number of shortest paths between pairs of nodes passing through the node; closeness represents the inverse of the sum of distances from nodes to other nodes; bottleneck genes are considered as highly central proteins connecting multiple complexes that are more likely to be part of signal transduction pathways. The higher the 3 quantitative values of a node are, the greater its importance in the network.–29PPI data of potential therapeutic targets were obtained from the Search Tool for the Retrieval of Interacting Genes database , a biological information annotation database, is a web tool used for gene function annotation that combines more than 40 independent knowledge bases, including functional enrichment analysis, interactive analysis, gene annotation, and member search. It provides a comprehensive gene list annotation and analysis resources, which can help users apply the currently popular bioinformatics analysis methods to batch gene and protein analysis to realize the cognition of gene or protein function. Potential therapeutic targets were imported into the Matescape database as a gene list. The minimum gene overlap (Min Overlap) was set to 3, the P value was less than .01, and the minimum enrichment factor (Min Enrichment) was set to 1.5. GO enrichment analysis illustrates the role of target proteins of TCM compounds in gene function, including 3 modules: biological processes, molecular functions, and cellular components. KEGG pathway enrichment analysis provides the functional annotations of pathways of a given gene set and pathways enrichment analysis. Finally, GO and KEGG enrichment analyses were performed for the protein of the combined protein in the network construction.–32Metascape , and the threshold value was set with P ≤ .05 as a screening standard. The results were visualized and output in bar graphs and bubble graphs.Molecular Complex Detection (MCODE) is based on complex algorithms that cluster objects with similar properties. We clustered the data in the potential target PPIs network of the SHXP in the treatment of COVID-19.2.8http://tcmatcov.bbtcml.com) is a platform to predict the efficacy of the anti-coronavirus pneumonia effect of TCM. TCMATCOV is based on the interaction network imitating the disease network of COVID-19. TCMATCOV utilizes a quantitative evaluation algorithm to analyze disease network disturbance after multi-target drug attacks to predict potential drug effects. The formula composition of SHXP was input into TCMATCOV V1.0 with pinyin form, and the confidence score of protein interaction was set as 0.5 to analyze the robustness of the COVID-19 disease network. The negative control group selected Banxia Tianma Baizhu decoction, while the positive control group selected Qingfei Paidu decoction. This platform uses four kinds of topology measurement values to measure network stability, including average degree, average shortest path, degree centrality, and closeness centrality. The total disturbance score is used as a comprehensive indicator to assess the degree of drug interference with the COVID-19 disease network's robustness.TCM Anti COVID-19 , the US data center for the global PDB archive, serves thousands of data depositors in the Americas and Oceania and makes available 3D macromolecular structure data free of charge. The RCSB PDB database was used to retrieve and download the PDB ID of the potential target proteins, whereas the structure of active compounds was downloaded in the traditional Chinese medicine systems pharmacology database and analysis platform database as a ∗mol 2 format. The compound ligand and protein were converted to the “PDBQT” format by AutoDock software. Grid Box was set with the appropriate size and location. Finally, AutoDock Vina was performed for molecular docking. Generally, the binding energy ≤−5.0 kj/mol can be considered an excellent binding effect. Pymol software was used to visualize molecular docking results.–39The Research Collaboratory for Structural Bioinformatics Protein Data Bank , kaempferol, luteolin, isorhamnetin, hyndarin, piperine, stigmasterol, cheilanthifoline, sesamin, ellipticine, seeing Table 3.2COVID-19 disease targeted genes/proteins were mined by GeneCards and OMIM databases. In GeneCards database, we obtained 324 COVID-19 related genes with relevance scores higher than zero, and among which 20 targets that scored higher than 2 were considered as essential COVID-19 targets, namely angiotensin-converting enzyme II (ACE2), dipeptidyl-peptidase 4, interleukin 2 receptor, alpha, cathepsin L, interleukin 6 (IL6), interleukin 10 (IL10), angiotensin-converting enzyme (ACE), vascular endothelial growth factor A (VEGFA). In the OMIM database, we gained 2 related genes of COVID-19.3.3P < .05) were chosen according to the P value, as shown in  of the 3 modules to gain insights into the cellular component, molecular function, and biological processes that are affected in COVID-19. The results indicated that Module 1 was highly correlated with cell transduction, enzyme activity, immunologic process, and metabolic process. Module 2 was highly associated with cytokine production, apoptosis, and immune regulation. Module 3 was highly linked to cell movement, angiogenesis, chemokine production, and immune system regulation. Overall, the potential targets were highly connected with cytokines and the immune system. Subsequently, we performed KEGG enrichment analysis  of the 3 modules to obtain essential pathways of SHXP in treating COVID-19. Enrichment analysis of the KEGG pathway showed that Module 1 screened 100 pathways, mainly related to signal transduction pathways and inflammatory pathways. A total of 55 pathways were screened in Module 2, related to inflammation, immune pathways, and viral infection pathways. Module 3 has 27 pathways, mainly related to cytokine, immune, and cancer signaling pathways. Further analysis shows that the three motabledules contain 3 identical pathways: the JAK-STAT, PI3K-Akt, and C-type lectin receptor signaling pathways, suggesting these 3 pathways may be key pathways SHXP against COVID-19. GO enrichment analysis results were visualized using the R package, and the top twenty pathways of each module were selected for ribbon visualization  have been reported as potential targets for anti-COVID-19, these 2 proteins are also selected for molecular docking with 2 core compounds that have the highest degree. The docking details are shown in Table 3.8To systematically and holistically explain the interaction mechanisms among the different drugs, compounds, and main targets in SHXP, a “drug-core compounds-hub targets-pathway” network was established based on Cytoscape software. As shown in Figure 4 The expression levels of various pro-inflammatory factors in plasma were significantly higher in severe patients, and studies have suggested that “cytokine storm” is the primary pathogenic mechanism in severe patients with COVID-19. Cytokine storm, also known as an inflammatory storm, is an overactive immune response to external stimulus. In such an overactive immune response, cytokines and leukocytes in tissues and organs may increase uncontrollably due to a specific positive feedback regulatory mechanism.,42 However, the molecular mechanisms underlying the effects of SHXP in the treatment of COVID-19 are not clear. Hence, in the present study, a set of network pharmacology methods were used to predict, elucidate and confirm the potential mechanisms of SHXP against COVID-19 by integrating target prediction, network construction, mechanism analysis, and molecular docking verification.In contrast to patients with mild and common types, patients with severe and critical cases of COVID-19 often develop dyspnea and or hypoxemia 1 week after the onset of the disease, and severe cases can rapidly develop into acute respiratory distress syndrome (ARDS). Cytokines include pro-inflammatory cytokines such as interleukin -6 (IL-6), IL-2, tumor necrosis factor-α (TNF-α) and anti-inflammatory cytokines such as interleukin-10 and IL-1. The balance between the 2 is a critical factor in maintaining normal immune function that resists disease. Cytokines interact with each other in the body and are jointly involved in the inflammatory response, which has antagonistic and promoting effects and forms a complex immune regulatory network.TCM has multi-targets and integral regulating effects. Some TCM components have specific antibacterial and antiviral effects. Their antiviral research pays more attention to the overall effect, explaining from a specific index and considering the interaction between various indexes or factors. The overall regulation effect helps prevent cytokine storm from the source. Studies have shown that quercetin can reduce the expression of transforming growth factor, beta 1, actin alpha 2, smooth muscle, and TNF-α, inhibit rat alveolar cell apoptosis, and reduce rat lung tissue inflammation and fibrosis damage. At the same time, it can reduce lung inflammation by inhibiting the activation of the JAK2/STAT3 signaling pathway. Quercetin can also inhibit the activation of signal transducer and activator of transcription factor NF-кB, inhibit the production of TNF-α and nitrous oxide by macrophages, microglia, and mast cells, reducing the fatal shock response. Luteolin has been found to have anti-inflammatory, anti-viral, and other pharmacological effects, and to exhibit lung-protective effects, which may be related to its inhibition of the release of IL-6 and TNF-α. Kaempferol can prevent the phosphorylation of PI3K and Akt from reducing the inflammatory reaction after influenza virus infection, inhibit the release of inflammatory factors such as IL-6 and TNF-α and reduce the damage of oxidative stress to the lung.–50 Pathak and colleagues found that piperine has an immunomodulatory effect and can improve cell apoptosis and immune function suppression in mice.,52 Piperine can regulate oxidative stress and apoptosis and relieve the inhibition of cell proliferation, thereby regulating the morphological changes and cytokine release of T cells and B cells. Besides, piperine can play an anti-inflammatory role by inhibiting the expression of endothelial cell adhesion molecules that TNF-α induced. The mechanism involved may be due to the fact that it interferes with the early signal transduction of TNF-α, thereby affecting the activation of nuclear factor kappa-B. Sesamin can significantly reduce IL-6 and IL-1 mRNA expression in human primary synovial fibroblasts, indicating that sesamin can block TNF-α induced pro-inflammatory cytokine mRNA expression.Our study screened out 5 main active components of SHXP, named quercetin, kaempferol, luteolin, piperine, and sesamin. They are core compounds with anti-COVID-19 effects after screening through the compound-target network and molecular docking verification. Quercetin has anti-inflammatory, antibacterial, antiviral, gastric protection, and immune regulatory functions. IL-6 is a crucial inflammatory factor in the initial stage of inflammation. Studies have shown that quercetin can reduce inflammation by inhibiting the expression of IL-6mRNA in neutrophils during the process of LPS-induced production of inflammatory cytokine IL-6, thereby decreasing the synthesis and secretion of IL-6. IL10 is a protein-coding gene that can encode cytokine. This cytokine has pleiotropic effects in immunoregulation and inflammation, which has profound anti-inflammatory functions, limiting excessive tissue disruption caused by inflammation. Interleukin-10 can block NF-кB activity and regulate the JAK-STAT signaling pathway. NF-кB, a critical nuclear transcription factor, widely exists in eukaryotic cells, which regulates many molecules in various early immune response and inflammatory response stages. NF-кB plays an essential role in the occurrence and development of diseases involving inflammatory mediators, cytokines, and proteases. The TNF family refers to a group of cytokines that can cause cell death (apoptosis), including TNF-α and TNF-β. TNF-α is mainly produced by activated macrophages, which may be involved in systemic inflammation. TNF-β is secreted by lymphocytes and mediates many inflammatory, immunostimulatory, and antiviral responses. VEGFA is a member of the PDGF/VEGF growth factor family. VEGF levels increase in SARS-CoV-2 infection and promote inflammation by increasing angiotensin II (Ang II) levels. In turn, Ang II elevates VEGF, creating a vicious cycle in releasing inflammatory cytokines. When SARS-CoV-2 infects the human body, it causes the immune cells in the body to secrete a large number of inflammatory factors, forming a cytokine storm that attacks the immune organs in the human body, seriously affects the respiratory, circulatory, and other systems, and even causes the death of patients. If left uncontrolled, the cytokine storm can eventually lead to respiratory problems such as breathing difficulties and even death. Combined with clinical studies, the expression levels of inflammatory factors in some severe patients with COVID-19 were significantly higher than those in general type patients. The results showed that the intensity of expression of these inflammatory factors was related to the degree of infection. The useful components in SHXP may further improve the clinical symptoms of patients by acting on inflammatory factors.,60Through 3 algorithms of hub gene screening and molecular docking verification, we obtained 4 potential therapeutic targets of SHXP, which are IL6, IL10, VEGFA, and TNF. Lipopolysaccharide (LPS) can stimulate the synthesis and secretion of various pro-inflammatory factors, including IL-6, by binding to TLR and other LPS receptors on cell membranes. Because JAK-STAT may have been involved in the control process of the M2-type activation and anti-inflammatory differentiation of macrophages, the pathway may significantly affect the start of macrophages’ proliferation. M2-type macrophages can secrete anti-inflammatory factors such as IL4 and IL10, which act in the elimination of inflammation and wound healing in the body. IL10 can inhibit the expression of TNF-α induced by LPS and reduce the production of pro-inflammatory cytokines. The essential compounds in SHXP may inhibit the M1-type differentiation of macrophages by regulating the JAK-STAT signaling pathway, reduce the production of pro-inflammatory factors, meanwhile promote the M2-type differentiation of macrophages to increase the production of anti-inflammatory factors, thereby preventing COVID-19 patients from being hurt by the “inflammatory storm.” The JAK-STAT pathway can interact with TLR to regulate M1-type polarization and macrophages’ inflammatory response. The TLR is called the bridge, which connects innate immunity and acquired immunity, whose negative feedback mechanism exerts anti-infection immunity advantages. The PI3K-Akt signaling pathway regulates cell differentiation, proliferation, activation, and antiviral activity by expressing a series of protein factors. In recent years, many in vivo and in vitro studies have demonstrated that the PI3K-Akt signaling pathway generates a critical negative regulatory in the pulmonary inflammatory response. PI3K inhibitors can alleviate pulmonary edema in mice and significantly alleviate pulmonary inflammatory response, suggesting that the core compound in SHXP may alleviate pulmonary inflammation in patients with COVID-19 by controlling the PI3K-Akt signaling pathway. Besides, HIF-1 signaling pathway, TNF signaling pathway, IL-17 signaling pathway, T cell receptor signaling pathway and other signaling pathways are also enriched in hub genes, which may be related to the effect of SHXP on COVID-19. The HIF-1 signaling pathway is involved in hypoxia regulation in the human body and can activate the transcription of multiple genes under hypoxia conditions to increase oxygen delivery. Autopsy results showed that the COVID-19 patient had diffuse alveolar injury in both lungs with exudation of cellular mucus and detachment of alveolar epithelium, clinically presented with dyspnea, and eventually died of respiratory failure. Therefore, regulation of HIF-1 signaling pathway could reduce clinical hypoxia symptoms in patients with severe COVID-19. TNF signaling pathway was an essential pathway in the inflammatory response, in which related factor receptors can also induce apoptosis. Quercetin acted as an anti-inflammatory by regulating TNF signaling pathway.–67KEGG enrichment analysis showed that the key targets were mainly concentrated in TNF signaling pathway, PI3K-Akt signaling pathway, MAPK signaling pathway, HIF-1 signaling pathway, and NF-кB pathway. Four hub genes, IL6, IL10, STAT3, and EGF, are enriched in the JAK-STAT signaling pathway, closely related to macrophages’ inflammatory differentiation. Inflammatory factors can activate the corresponding JAK-STAT, play the function of signal transduction and transcriptional activation, and then affect the M1/M2 type differentiation and inflammatory direction of macrophages. Under the stimulation of pro-inflammatory factors, macrophages can release a series of cytokines that can further promote the macrophages’ recruitment and M1-type polarization, resulting in the self-amplification effect of the body's inflammatory response. Many studies have shown that the JAK-STAT signaling pathway may play a vital role in regulating macrophage inflammatory initiation. SARS-CoV-2 3CL hydrolase is a vital protein of the virus itself, which can hydrolyze polyproteins into mature proteins with replication and transcription functions. It is required to realize RNA replication of the virus and is considered a useful target against the SARS-CoV-2 virus. According to the network diagram of “drug-ingredient-target,” quercetin and luteolin ranked the highest in the degree were selected for molecular docking with ACE2 target and SARS-CoV-2 3CL hydrolase, the result showed that all the key compounds in the network had a strong affinity with ACE2 protein and SARS-CoV-2 3CL protein.ACE is a receptor in the airway, alveoli, and vascular endothelium. COVID-19 uses ACE to enter type II pneumocytes cells to induce ACE2 internalization and shedding, resulting in the occurrence and development of ARDS. The combination of SARS-CoV-2 and ACE2 was the leading cause of COVID-19.Although our study explored the molecular mechanisms of SHXP, there are still some limitations. First, the data of the study were obtained from existing databases, so the authenticity and integrity of the result depend on the data. In addition, further experiments are needed to confirm the prediction results of SHXP because our study relied on data analysis.5In this study, we systematically analyzed the therapeutic targets, signaling pathways, the potential mechanisms by which SHXP is useful in treating COVID-19 based on network pharmacology, and molecular docking approaches. The therapeutic efficacy of SHXP against COVID-19 is likely mediated via the regulation of 4 targets, namely, IL6, IL10, VEGFA, and TNF. Furthermore, the molecular docking simulation demonstrated an excellent affinity of these 4 hub genes, as well as ACE2 and 3CLpro with quercetin, kaempferol, luteolin, and piperine in SHXP. KEGG enrichment analysis illustrated that SHXP may simultaneously act on various pathways, including JAK-STAT signaling pathway, PI3K-Akt signaling pathway, TNF signaling pathway, NF-кB signaling pathway, TLR signaling pathway, IL-17 signaling pathway, HIF-1 signaling pathway, and T cell receptor signaling pathway. Our study showed that SHXP, a TCM formula, might be useful in treating COVID-19 by mainly regulating the immune response, inhibiting the inflammatory storm, and inhibiting virus entry into cells replicating themselves. These findings may offer a reference basis for the clinical application and further mechanism exploration by which SHXP exerts effects against COVID-19.Conceptualization: Jialin Li, Peizhi Ye.Data curation: Jialin Li, Yingying Tan, Chao Wu, Yingying Liu, Xiaotian Fan.Formal analysis: Jialin Li, Yingying Tan, Bei Zhang.Funding acquisition: Jialin Li, Jiarui Wu.Investigation: Jialin Li, Jianping Yi, Yi Xu.Methodology: Jialin Li, Shan Lu, Xinkui Liu.Project administration: Xinkui Liu.Resources: Jialin Li, Zhishan Wu.Software: Jialin Li, Zhihong Huang, Xinkui Liu, Liangliang Shen.Supervision: Peizhi Ye.Validation: Peizhi Ye, Lu Yao.Visualization: Zhihong Huang, Haojia Wang.Writing – original draft: Jialin Li.Writing – review & editing: Hua Luo, Antony Stalin, Yanfang Mou.


The rats were intragastrically administered with 50% CCl4 for 9 weeks to induce hepatic fibrosis, followed by various agents for 6 weeks. Transcriptomic analysis was carried out to predict the potential targets, and then multiple examinations were performed to verify the prediction. The results showed that TA significantly alleviated liver injury and fibrosis, as evidenced by the ameliorative pathological tissue, low transaminase activity, and decreased collagen accumulation. Besides, TA markedly reduced hepatocyte apoptosis by regulating the expression of caspase-3 and Bcl-2 families. The transcriptomic analysis revealed 2,173 differentially expressed genes (DEGs) between the TA and model groups, which could be enriched in the metabolic pathways and the PI3K/Akt and NF-κB signaling pathways. The metabolomics analysis showed that TA could regulate the glycerophospholipid metabolism pathway by regulating the synthesis of phosphatidylserines, phosphatidylethanolamines and phosphatidylcholines. Moreover, the integrative analysis of the transcriptomics and metabolomics data indicated that TA inhibited the glycerophospholipid metabolism pathway by inhibiting the expression of LPCAT4, PTDSS2, PLA2G2A and CEPT1. In addition, the relevant signaling pathways analysis confirmed that TA inhibited HSCs activation by blocking the PI3K/Akt/mTOR pathway and ameliorated inflammatory injury by inhibiting the NF-κB pathway. In conclusion, TA significantly alleviates liver fibrosis in vivo by inhibiting the glycerophospholipid metabolism pathway and the PI3K/Akt/mTOR and NF-κB signaling pathways.This study aimed to investigate the effects and underlying mechanisms of tormentic acid (TA) on carbon tetrachloride (CCl Liver fibrosis caused by various chronic stimuli, such as viruses, alcohol, and autoimmune diseases, is a reversible pathological process. Without appropriate intervention, it can progress to cirrhosis, portal hypertension, and hepatocellular carcinoma, leading to high morbidity and mortality . Thus, iMulti-omics analysis has been considered a useful tool to study the pathogenesis of various diseases and drug discovery because of its advantage in providing insight into the potential biomarkers and mechanism as a whole. Transcriptome analysis can detect the complete set of RNA transcripts, which is helpful to expose the molecular mechanisms of the anti-hepatic fibrosis drugs . MetabolPotentilla chinensis Ser. is a folk herb for treating immune disorders, which has been found to have a protective effect on liver injury  twice a week for 9 weeks to induce liver fibrosis, followed by TA (3.0 or 1.5 mg/kg) or colchicine (0.2 mg/kg) for 6 weeks. At the end of the treatment, all animals were fasted for 12 h and then anesthetized by intraperitoneal injection of 3% sodium pentobarbital (1.0 ml/kg). Blood and liver samples were collected immediately.Male Sprague–Dawley (SD) rats  were provided by the Experimental Animal Center of Guangxi Medical University . The animal experiment was approved by the Institutional Ethical Committee of Guangxi Medical University. The treatment was performed as shown in Liver samples were fixed in 4% paraformaldehyde, embedded in paraffin and sectioned into 4 μm slices. Then the slices were stained with the hematoxylin-eosin (H and E) staining to observe the hepatic pathological changes. Sirius red staining was used to assess collagen deposition, and the semi-qualification for collagen area was analyzed using ImagePro Plus 6 . Moreover, hepatocyte apoptosis was evaluated by the TUNEL staining .The activities of the serum enzymes, including alanine aminotransferase (ALT), aspartate aminotransferase (AST), albumin (ALB) and total bilirubin (TBIL), were estimated with an automatic biochemistry analyzer .Hyaluronic acid (HA), laminin (LN), procollagen type Ⅲ (HPCⅢ) and collagen type IV (Col- IV) contents were tested by the enzyme-linked immunosorbent assay (ELISA) kits . The hepatic hydroxyproline (Hyp) content was determined using a commercially available kit .The activities of SOD, GSH-Px, GSH-Rd and MDA in the hepatic tissue were detected using commercially available kits . The contents of interleukin-6 (IL-6), interleukin-10 (IL-10) and tumor necrosis factor-α (TNF-α) were determined using the ELISA kits  according to the assay procedure. The content of monocyte chemoattractant protein 1 (MCP-1) was detected using the ELISA kit (Sigma-Aldrich).p-values<0.05 and | log2 (fold change) |>1 using R package DESeq2.Total RNA was extracted from liver tissues with Trizol reagent (Thermo Fisher Scientific). RNA sequences analysis was performed by Shanghai Jiayin Biotechnology Co., Ltd . BrieflyMetabolomics was analyzed using Ultra-performance liquid chromatography equipped with quadrupole time-of-flight mass spectroscopy  as previously described . Approxi+, capillary: 250 kV, source temperature: 150°C, desolvation temperature: 350°C, acquisition times: 15 min, acquisition mode: positive, low mass: 50 Da, high mass: 1,000 Da, scan time: 0.5 s, collision energy: 6 V, ramp collision energy 10–20 V, cone voltage: 40 V. LockSpray properties: “Acquire LockSpray-Do not apply correction.” Leucine enkephalin was used for LockSpray. Nitrogen was used as both cone gas (50 l/h) and desolvation gas (800 l/h). Argon was set as collision gas. The liquid chromatography conditions were: Acquity UPLC®BEH C18 column . The gradient elution was performed using HPLC-grade water and acetonitrile containing 0.1% formic acid . The RNA was reverse-transcribed using an RT-PCR kit  to obtain cDNA. The mRNA expression was performed using the Applied 7,300 Fast Real-Time PCR detection system and SYBR Green I  according to the manufacturer’s instructions. The expression of Col-I, Col-Ⅲ, TGF-β, Bax, Bcl-2, PI3K, Akt, P70S6k, mTOR, and NF-κB p65 were analyzed. GAPDH was used as an internal control. The primer sequences used in this study are shown in The proteins of liver samples were extracted with the RIPA buffer , followed by centrifuging at 12,000 g for 15 min. Subsequently, the concentration of each protein sample was detected using the BCA Protein Assay Kit . Finally, the protein sample was separately mixed with loading buffer and denatured in boiling water for 5 min.α-SMA, Col-I and Col-Ⅲ . The membranes were washed three times with TBST buffer, followed by incubation in the dark with fluorescence-labeled rabbit/mouse anti-goat IgG  at 1:10,000 dilution under agitation for 1 h at room temperature. Finally, the membranes were scanned and quantified by Image Studio Lite software .The electrophoretic separation of the prepared proteins was performed on the sodium dodecyl sulfate-polyacrylamide gels (SDS-PAGE) and transfered to polyvinylidene fluoride (PVDF) membranes . The membranes were incubated with the primary antibodies overnight at 4°C: Akt, p-Akt, PI3K, p-PI3K, FAK, p-FAK, mTOR, p-mTOR, NF-κB p65, p-NF-κB p65 , IκBα, p-IκBα, IKKα/β, p-IKKα/β, Bcl-2, Bax, Caspase-3, and GAPDH , p-value < 0.05 was considered statistically significant.The data were expressed as the means ± standard deviation. The statistical analyses were performed using SPSS 13.0 software . The one-way ANOVA was used to determine statistical differences between the groups. A 4, as evidenced by less necrosis.Liver tissue pathological change was observed by H&E staining. As shown in 4 exposure caused a significant increase in ALT, AST, and TBIL, while a decrease in ALB . However, the contents of these collagen-related biomarkers in the colchicine and TA-treated groups were decreased compared to the model group (p < 0.05). Also, the Western blot and qPCR analysis showed that TA markedly inhibited the expression of Col-I and Col-III , and the content of IL-10 was lessened; however, treatment with TA could reverse these abnormal expressions induced by CCl4. Monocyte chemoattractant protein 1 (MCP-1) is a marker of hepatic inflammation , there were 3,607 differentially expressed genes (DEGs) between them . The Gene ontology (GO) analysis indicated that the DEGs were mainly involved in cell proliferation, immune system process, biological process and metabolites; meanwhile, the KEGG analysis showed that the DEGs were clustered in the metabolism-related pathways, ECM formation, PI3K/Akt signaling pathway, et al.  plots revealed a significant difference among the normal, model and TA groups , which w, et al. .For the TA and model groups (TA VS Model), the Volcano plot revealed 2,173 DEGs between them , which wThe transcriptomics analysis predicted that the effect of TA on fibrogenesis might be associated with the regulation of the metabolism process. It is well known that metabolic disturbance causes excessive lipid accumulation, easily inducing fatty liver and fibrosis. Thus, the metabolism-related pathways were further analyzed.As shown in 4 administration decreased 151 metabolites content and up-regulated 41 metabolites level, suggesting that CCl4 treatment led to significant metabolic disturbance. TA treatment might restore CCl4-induced metabolic change, as evidenced by the significant variance between the TA and model groups in the OPLS-DA and VIP diagram (p < 0.05 and Impact >0.1). Further analysis indicated that TA might regulate the glycerophospholipid metabolism pathway by moderating the key metabolites, including phosphatidylserines, phosphatidylethanolamines and phosphatidylcholines  is a weighted average of the original scores, providing a good summary and displaying the separation between groups. As shown in  diagram . Importa diagram . Among tcholines .p < 0.05 and impact>0.5)  . Among tact>0.5) . Also, tact>0.5) .4 significantly increased the phosphorylation of Akt, PI3K, FAK and mTOR and the mRNA level of Akt, PI3K, P70S6K and mTOR compared with the normal control group (p < 0.05). However, after treatment with TA, the expressions of these proteins and genes above were significantly decreased (p < 0.05), suggesting that TA could inhibit the PI3K/Akt/mTOR signaling pathway.Based on the prediction of the transcriptomics analysis, the PI3K/Akt/mTOR signaling pathway was detected. As shown in 4 exposure significantly enhanced the phosphorylation of p65, IKKα/β and IκBα; while TA treatment decreased the phosphorylation of these proteins. Similarly, CCl4 increased the mRNA level of IκBα and NF-κB p65, but TA treatment reduced their mRNA levels  nearly to the normal level. In addition, the TUNEL staining revealed that TA treatment markedly decreased the positive apoptotic cells by regulating the expression of Bcl-2 and caspase families. Importantly, the Sirius staining revealed that TA treatment markedly reduced collagen deposition; also, TA treatment could decrease the content of Hyp, HA, and LN, as well as the expression of HPC III and col-IV. The data above indicated that TA could significantly alleviate CCl4-induced liver injury and fibrogenesis.The pathological examination showed that administration with CClTranscriptomics analysis can reflect the global change of genes, which is a promising technique to elucidate complex pathogenic mechanisms. In this study, transcriptomic analysis was carried out to predict the potential targets of TA on liver fibrosis. The analysis indicated that there were 2,173 differentially expressed genes (DEGs) between the TA and model groups, and these DEGs could be functionally annotated in the liver fibrosis-related pathways, including the metabolic pathways , PI3K/Akt signaling pathway, ECM formation, and NF-κB signaling pathway.p < 0.05 and Impact >0.1). Further analysis indicated that the targets of TA to regulate the glycerophospholipid metabolism pathway included phosphatidylserines, phosphatidylethanolamines and phosphatidylcholines. It has been confirmed that phosphoethanolamines (PEs) and phosphocholines (PCs) are the key phospholipids of cell membranes , resulting in the destruction of cell membrane structure. Also, CCl4 treatment easily causes the disorder of PEs and PCs metabolism in rats with hepatic fibosis (4 model group were significantly increased, indicating an altered membrane phospholipids metabolism and the damage to the cell membrane; however, TA treatment significantly reversed these changes. Additionally, the integrative analysis of the transcriptome and metabolome revealed that TA could regulate the genes expression of LPCAT4, PTDSS2, PLA2G2A and CEPT1 and then inhibit the biosynthesis of phosphatidylserines, phosphatidylethanolamines and phosphatidylcholines. Our finding suggested that TA could restore the abnormal glycerophospholipid metabolism, which might be contributed to alleviating hepatocyte damage and fibrosis.Based on the prediction of transcriptomics, the effects of TA on metabolic profile and the relevant pathways were investigated. The OPLS-DA analysis showed that TA treatment resulted in 211 down-regulated and 131 up-regulated metabolites. These differential metabolites were mainly enriched in four metabolic pathways; among them, the glycerophospholipid metabolism pathway was the most relevant one (embranes , which a fibosis . In thisα-SMA and TGF-β, suggesting that TA could suppress HSCs activation and proliferation. Importantly, CCl4 exposure markedly activated the PI3K/Akt pathway in the fibrotic liver tissue, as evidenced by the significant increase in the phosphorylation of p-PI3K, p-Akt, p-mTOR and p-FAK. While treatment with TA significantly inhibited the phosphorylation levels of these proteins. Similarly, the results of RT-qPCR also showed that TA significantly inhibited the expression of the key genes of the PI3K/Akt/mTOR signaling pathway. These results suggested that TA inhibited HSCs activation by suppressing the PI3K/Akt/mTOR signaling pathway, which was vital for alleviating ECM accumulation and fibrogenesis.As the prediction of the transcriptomic analysis, the PI3K/Akt might also be involved in the regulation of TA on fibrogenesis. The PI3K/Akt/mTOR pathway is one of the critical signaling pathways, which can regulate cell proliferation and apoptosis . When stα/β, and p-IκBα. Similarly, the RT-qPCR results showed that TA significantly reduced the mRNA level of IκBα and NF-κB p65. These data indicated that TA ameliorated inflammatory injury by inhibiting the NF-κB signaling pathway.In addition to the PI3K/Akt/mTOR signaling pathway, the NF-κB pathway might also be involved in the underlying mechanism of TA against liver fibrosis. NF-κB is a critical transcription factor in the inflammatory response, participating in the inflammatory response during liver fibrosis . Usually4-induced liver fibrosis, which may be attributed to the inhibition of the glycerophospholipid metabolism pathway and the PI3K/Akt/mTOR and NF-κB signaling pathways (TA treatment significantly ameliorates CClpathways .


This study systematically explored the underlying mechanism of Rhizoma Coptidis against type 2 diabetes mellitus (T2DM) by using network pharmacology and molecular docking and experimental validation. We retrieved and screened active compounds of Rhizoma Coptidis and corresponding T2DM-related targets across multiple databases. PPI networks of the genes were constructed using STRING, and the core targets were screened via topological analysis. GO and KEGG enrichment analyses were performed by using DAVID. Finally, molecular docking and experimental studies were performed after bioinformatic analysis for verification. There were 14 active compounds and 19 core targets of Rhizoma Coptidis-T2DM, of which quercetin was identified as the main compound and IL6, VEGFA and TNF were the most significant core targets. GO and KEGG enrichment analyses showed that Rhizoma Coptidis ameliorated T2DM by regulating multiple biological processes and pathways. Docking studies indicated that IL6, VEGFA and TNF could stably bind with all active compounds of Rhizoma Coptidis. The results of our experiments revealed that Rhizoma Coptidis could inhibit the expression of IL6 and TNFα and enhance islet cell viability. This study suggests anti-inflammatory therapeutic effects of Rhizoma Coptidis on T2DM, thereby providing a scientific basis and new insight for further research on the antidiabetic effect of Rhizoma Coptidis. T2DM is often caused by the complex interactions between genetic and/or environmental associations3. T2DM is increasingly recognized as a serious public health problem, with an estimated 700 million patients worldwide by 2045, and constitutes a significant financial burden6. Currently, six classes of oral antidiabetic drugs, including metformin, glimepiride, repaglinide, pioglitazone, sitagliptin, and acarbose, are available7. Despite their benefits, these drugs are focused on a single compound and reported to have adverse effects. Hence, complementary and alternative medicine may be prospective options for T2DM intervention. As a supplementary alternative medicine, traditional Chinese medicine (TCM) has a rich history and has shown good results in the treatment of T2DM.Type 2 diabetes mellitus (T2DM) is a metabolic disease triggered by insulin resistance or impaired insulin secretion and characterized by hyperglycaemiaCoptis chinensis Franch8. According to TCM beliefs, Rhizoma Coptidis possesses the functions of clearing heat, drying dampness, wasting thirst, and quenching fire toxins9. Thus, Rhizoma Coptidis is widely used by TCM physicians to prevent and/or treat diabetes10. Many famous formulas containing Rhizoma Coptidis, such as Ge-Gen-Qin-Lian Decoction11, Dahuang Huanglian Xiexin Decoction12, and Huanglian Decoction13, have also been shown to exert therapeutic effects on T2DM. Recently, some clinical trials based on Rhizoma Coptidis intervention indicated a good hypoglycaemic effect for T2DM patients and an improvement of symptoms15. Many chemical compounds of Rhizoma Coptidis, such as berberine and polyphenols, have been verified to play a notable role in regulating glycometabolism by reducing insulin resistance in liver, muscle, and adipose tissues, increasing insulin levels, improving β cell dysfunction16, and regulating the intestinal flora17. Thus, this treatment has the potential to ameliorate diabetes symptoms and suppress T2DM-induced lesions18. However, due to the multiple components and multiple targeting characteristics of Chinese herbal medicine, the active compounds and underlying mechanisms have not been fully elucidated.Rhizoma Coptidis is the dried rhizome of the family Ranunculaceae: 19 and is characterized by its systematic nature, relevance and predictability20. Network pharmacology can construct a multimolecular, multitarget and multilink network model of “drug-compound-target” to understand the mechanism of Chinese herbal medicine21. The treatment benefits of Chinese herbal medicine are attributed to multiple components and targets that produce combined or synergistic effects22. Thus, the characteristics of herbs fit well with network pharmacology, making it a useful tool to quantitatively represent the mechanisms of herbs. Recently, TCM network pharmacology was proposed by Li et al.24, which integrates TCM theory with network pharmacology. The holistic view of TCM coincides with the overall systematic characteristics of network pharmacology25. TCM network pharmacology is a methodology that aims to reveal the biological associations among complex diseases, syndromes and herb treatments26. With rapid progress in bioinformatics, TCM network pharmacology has had wide applications, such as drug development, herbal ingredient target prediction, and compatibility regularity of drug pairs. Therefore, a network pharmacology approach was utilized to predict the antidiabetic mechanisms of Rhizoma Coptidis in this study.Network pharmacology is an interdisciplinary discipline newly developed for systematic research on drugs based on artificial intelligence and big dataPresently, we performed a systematic study to clarify the therapeutic effect of Rhizoma Coptidis for T2DM. The steps were as follows: the active compounds of Rhizoma Coptidis were identified by the TCMSP database, and the compound targets were predicted by the STP and SEA databases. The GeneCards and DisGeNET system was used to screen disease targets, and the overlapping targets between herbs and disease were also identified. In addition, the overlapping targets were entered into String for protein–protein interaction analysis, and core targets were screened according to topological structure. Furthermore, GO and KEGG enrichment analyses were performed using the DAVID tool to identify the functions and pathways. Finally, molecular docking technology and in vitro experiments were used to verify the results of the network pharmacology analysis. The workflow used in this study is illustrated in Fig. https://tcmspw.com/tcmsp.php). According to literature reports and pharmacokinetic parameters27, the compounds with oral bioavailability (OB) ≥ 30% had good absorption and slow metabolism after oral administration. The compounds with a drug-likeness (DL) ≥ 0.18 were chemically suitable for drug development. Hence, OB ≥ 30% and DL ≥ 0.18 were employed to identify the potential active compounds in Rhizoma Coptidis28.The chemical compounds of Rhizoma Coptidis were harvested using the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform  and Swiss target prediction (STP) (http://www.swisstargetprediction.ch/) in “Homo Sapiens” mode. The UniProt database (https//www.uniprot.org/) was used to convert target protein names into target gene names to standardize gene names29.Targets of the active compounds of Rhizoma Coptidis were selected through both the similarity ensemble approach (SEA) (https://www.genecards.org/) and DisGeNET (https://www.disgenet.org/search) with “type 2 diabetes mellitus” and “T2DM” as the key words30. The species was set to Homo sapiens. Then, these collected targets were merged, and duplicates were removed. The overlapping targets between compounds of Rhizoma Coptidis and T2DM targets were identified and visualized by VENNY 2.1 (https://bioinfogp.cnb.csic.es/tools/venny/).T2DM-related targets were searched in the GeneCards platform (http://www.cytoscape.org/) was used to establish the network diagram between the active compounds of Rhizoma Coptidis and T2DM-related targets31.Based on the above analyses, the active compound and targets of Rhizoma Coptidis were matched with the disease target of T2DM to obtain the compound target. Cytoscape 3.7.2 (https://string-db.org/) to obtain the interaction results among the targets with the gene type selected as Homo sapiens32, and the visual analysis results were obtained by Cytoscape 3.7.2.The overlapping targets were input into String 11.0  is an online tool for gene annotation, classification, and enrichment and pathway analysis. GO and KEGG pathway enrichment analyses were carried out using the DAVID tool. A p value < 0.05 and a p value < 0.01 were used as the thresholds for GO terms and KEGG pathways, respectively, and some top entries were chosen for visualization36. GO enrichment analysis mainly considers the biological process, cellular composition, and molecular function of the target, whereas KEGG enrichment analyses consider the potential biological pathways and functions associated with the target37.The DAVID database (https://pubchem.ncbi.nlm.nih.gov/) as the small-molecule ligand file. The protein receptor structure file was obtained from the RCSB PDB database (http://www.rcsb.org/) in PDB format; the original ligand structure of the protein was extracted by using PyMOL 2.3 software (https://pymol.org/2/). Autodock Vina was used for molecular docking and to calculate the results. The binding degree of the receptor-ligand can be judged by the level of binding energy in the molecular docking results. If the binding energy is less than − 5 kJ/mol, it indicates that the target has certain binding activity with the compound38. The lower the binding energy is, the better the docking effect. After docking, ligands with the lowest binding energy were selected to visualize the ligand–protein interaction.To validate the effectiveness of the screened compounds and targets, molecular docking was carried out between the active compounds and top 3 core targets. Metformin, a commonly used clinical drug, was used as a positive control. The name, molecular weight, and 2D structure of the compounds were determined in the PubChem database  was purchased from TCM Pharmacy of Affiliated Hospital of Shandong University of Traditional Chinese Medicine . Rhizoma Coptidis was decocted for 30 min twice with boiling deionized water . The solution was filtered and concentrated and stored at − 20°40. The experimental groups were divided into the control group (5.5 mmol/L glucose), high glucose-model group (25 mmol/L glucose), Rhizoma Coptidis low-dose group (25 mmol/L glucose and 50 µg/mL Rhizoma Coptidis), and Rhizoma Coptidis high-dose group (25 mmol/L glucose and 100 µg/mL Rhizoma Coptidis).Cell experiments were conducted in accordance with the guidelines and regulations of Shandong University of Traditional Chinese Medicine. Min6 cells were obtained from the Cell Bank of the Chinese Academy of Sciences. Min6 cells were cultured in DMEM  supplemented with 10% FBS  and 1% penicillin/streptomycin  and maintained at 37 °C in a humidified atmosphere with 5% CO3 cells/well. After incubation for 12 h, the cells were treated with different concentrations of drug-containing medium for 48 h41. Then, 10 µL CCK-8 was added to each well, and the plates were incubated for 1–4 h. Finally, the absorbance was measured at a wavelength of 450 nm.Cell viability was determined by CCK-8  assay. Min6 cells were seeded in a 96-well plate at a density of 4 × 1042. All reactions were carried out according to the manufacturer's protocol. The β-actin gene was used as an internal standard gene, and the data were quantitatively analysed by the 2−ΔΔCt method. The value in the control group was set to 1.0, and all data are shown as relative mRNA expression43. All primers were designed and synthesized by Sangon, Ltd. The primer sequences of each gene were as follows: IL6 FORWARD, 5′-TGAACTCCTTCTCCACAAGCG-3′; IL6 REVERSE, 5′-CCGTCGAGGATGTACCGAAT-3′; TNFα FORWARD, 5′-GAGAGATGGGGGAGATAAGGAGA-3′; TNFα REVERSE, 5′-TTAGCCCTGAGGTGTCTGGTT-3′; β-actin FORWARD, 5′-CTACCTCATGAAGATCCTGACC-3′; and β-actin REVERSE, 5′-CACAGCTTCTCTTTGATGTCAC-3’.Total mRNA was extracted using TRIzol reagent , and then gDNA was generated using a reverse transcription kit . Quantitative PCR was then performed using the SYBR Green PCR Kit 44. Cells under coverslips were observed using a fluorescence microscope.Min6 cells were seeded on coverslips. After formaldehyde  fixation, 0.1% Triton X-100  penetration and 10% goat serum  blocking were performed. Then, the Min6 cells were incubated with antibody  at 4 °C overnight. The next morning, after washing steps, the cells were incubated with goat anti-rabbit IgG/FITC antibody  at room temperature for 30 min and then with DAPI  for 5 min at room temperaturep < 0.05 was considered to be significantly different45.Statistical analysis was performed by using SPSS 24.0. The data were collected and are shown as the means ± standard deviations. Statistical analysis was performed by one-way ANOVA. 46 , 25 entries of cell composition (CC) and 30 entries of molecular function (MF). According to the p value, the top 5 terms in the biological process category were response to drug (GO:0042493), positive regulation of nitric oxide biosynthetic process (GO:0045429), positive regulation of vasoconstriction (GO:0045907), phospholipase C-activating G-protein coupled receptor signaling pathway (GO:0007200), and response to hypoxia (GO:0001666). These biological processes were strongly connected with the following molecular functions: enzyme binding (GO:0019899), drug binding (GO:0008144), steroid binding (GO:0005496), serine-type endopeptidase activity (GO:0004252), and protein homodimerization activity (GO:0042803). Moreover, these processes occurred mainly in the extracellular space (GO:0005615), plasma membrane (GO:0005886), caveola (GO:0005901), membrane raft (GO:0045121), and integral compounds of the plasma membrane (GO:0005887). The top 10 GO terms were visualized and are displayed in Fig. GO enrichment analysis was performed on 107 targets, and a total of 165 GO terms with p < 0.01), mainly involving Neuroactive ligand-receptor interaction(hsa04080), Calcium signalling pathway(hsa04020), HIF-1 signalling pathway(hsa04066), Pathways in cancer(hsa05200), TNF signalling pathway(hsa04668), and PI3K/Akt signalling pathway(hsa04151). We selected the top 15 results to draw a bubble diagram . These results indicate that the screened core targets indeed stably combine with Rhizoma Coptidis. The docking results of the top 3 targets are shown in Fig. Molecular docking was applied to analyse the binding of the top 3 targets  and all 14 active compounds of Rhizoma Coptidis. Their binding energy scores were all smaller than − 5 kJ/mol, and the results are listed in Table p < 0.05), indicating that high glucose inhibited Min6 cell viability. Compared with the model group, different concentrations (50 µg/mL and 100 µg/mL) of Rhizoma Coptidis enhanced cell viability  (p < 0.05). These results suggest that Rhizoma Coptidis enhances β cell viability during high-glucose injury.The effect of Rhizoma Coptidis on cell viability was detected by the CCK-8 method. As shown in Fig. The network pharmacology results revealed that IL6 and TNF are the core targets of Rhizoma Coptidis in the treatment of T2DM, and the TNF signalling pathway is its main intervention pathway. Additionally, the molecular docking results showed that all active compounds of Rhizoma Coptidis effectively bound to IL6 and TNF. Therefore, we selected IL6 and TNF for further verification.p < 0.05). Rhizoma Coptidis treatment  helped decrease the expression levels of IL6 and TNFα compared to those in the model group (p < 0.05) . However, this increase was inhibited by multiple concentrations (50 µg/mL and 100 µg/mL) of Rhizoma Coptidis (p < 0.05) -Canadine, Berlambine, Corchoroside A_qt, Magnograndiolide, Palmidin A, palmatine, quercetin, coptisine, Worenine, and Moupinamide. The above results demonstrated the effectiveness and diversity of chemical compounds in Rhizoma Coptidis to play a pharmacological role in T2DM. The degree value of quercetin was highest, so this compound was identified as the main compound in Rhizoma Coptidis. Quercetin exerted hypoglycaemic effects potentially by inhibiting pancreatic iron deposition and pancreatic β cell ferroptosis52. IL6 also increases the expression of insulin-degrading enzymes, which are important in glucose metabolism53. Vascular endothelial growth factor A (VEGFA) is a growth factor that has a vital role in angiogenesis and endothelial cell growth. Hyperglycaemia results in overexpression of VEGFA, which is a critical factor in diabetic complications such as diabetic retinopathy and diabetic nephropathy55. Similarly, as a crucial proinflammatory mediator, tumour necrosis factor (TNF) induces insulin resistance by impairing insulin signalling through serine phosphorylation56. In addition, TNF is mainly related to diabetic vascular dysfunction and diabetic nephropathy58. Collectively, the results of this study suggest that the core targets of Rhizoma Coptidis are of great significance in antidiabetic treatment.After prediction and screening, 107 overlapping targets were found for drugs and diseases, indicating the multiple targets of hypoglycaemia in Rhizoma Coptidis. Based on the topological analysis, we further identified 19 core targets from the 107 overlapping targets for subsequent study. Further analysis of the PPI results indicated that IL6, VEGFA and TNF were the top 3 overlapping targets, which act as the core targets of Rhizoma Coptidis in T2DM. IL6, VEGFA and TNF are primarily involved in inflammation, which suggests that Rhizoma Coptidis may have an anti-inflammatory effect. Interleukin-6 (IL6) is identified as a key mediator of inflammation, fibrosis and glucose metabolism; it is the basis of insulin resistance in patients with T2DMFurthermore, the primary core targets  were verified by molecular docking and experimental studies. The molecular docking results were successful according to intermolecular interactions. Experimental studies have also confirmed that Rhizoma Coptidis can notably lessen the expression of the inflammatory cytokines IL6 and TNFα to protect islet β cells against high-glucose injury. All these results sufficiently indicate that Rhizoma Coptidis possesses good anti-inflammatory activity. Accordingly, we propose that Rhizoma Coptidis had a therapeutic effect on T2DM by regulating inflammatory cytokines.59. Studies have shown that some compounds of Rhizoma Coptidis, such as berberine, have antioxidant activity, which improves insulin resistance and promotes insulin secretion by scavenging free radicals60, inhibiting the activation of polyol bypass and reducing the formation of terminal glycosylation products61. Apoptosis is the key factor in the pathophysiology of T2DM62. STZ-induced diabetes significantly increased the expression of apoptosis biomarkers63. Other studies have also confirmed the involvement of Jun, CASP3 and EGFR in islet β-cell apoptosis64. In addition, treatment of T2DM with Rhizoma Coptidis is also related to cellular composition and molecular function, including mitochondria, endoplasmic reticulum and cytokine activity65. Some studies have shown ultrastructural abnormalities in the cells of patients with T2DM, such as endoplasmic reticulum dilation and mitochondrial swelling66. Overall, our GO enrichment results suggest that Rhizoma Coptidis regulates various biological processes to ultimately alleviate T2DM.GO enrichment analysis showed that 107 targets were enriched in biological process, cellular composition, and molecular function. Oxidative stress is the most common pathological damage process in the body and is related to the pathogenesis of multiple systems and chronic diseases such as diabetes2+ oscillations. The rapid increase in intracellular Ca2+ triggers insulin release67. Ca2+ signalling and its dysregulation have been associated with the development of DM. Ca2+/cAMP signalling modulates the release of insulin from pancreatic β-cells and participates in the homeostasis of β-cells by stimulating cell proliferation and differentiation68. Additionally, Ca2+ plays a crucial role in cadmium-induced β-cell dysfunction and apoptosis69. The HIF-1 pathway is a well-known regulator of cellular glucose70. HIF may be activated during the early stage of diabetic nephropathy under hypoxia and stimulate the proliferation and aggregation of inflammatory factors in the damaged kidney72. T2DM is recognized as a chronic, low-grade inflammatory disease accompanied by elevated TNF expression. TNFα-mediated inflammation, obesity, and insulin resistance are associated with T2DM73. It was found that the PI3K/Akt signalling pathway was activated in high glucose‑stimulated HKC cells and was related to islet cell proliferation74. Insulin receptor activation through insulin binding stimulates the PI3K/AKT signalling pathway. Collectively, the findings of this study show the mode of action of the hypoglycaemic effects of Rhizoma Coptidis act through multiple targets and multiple pathways.In the KEGG enrichment analysis, some disease-related signalling pathways  were identified, which were closely linked to the occurrence and development of T2DM. Pulsatile insulin release from pancreatic cells is related to the frequency of CaIn conclusion, with the help of a network pharmacology strategy and molecular docking and experimental validation, this study investigated the mechanisms of action by which Rhizoma Coptidis could be used to treat T2DM. The findings suggest that the antidiabetic properties of Rhizoma Coptidis can be attributed to 14 compounds that correspond to 19 hypoglycaemic targets and are strongly related to 88 signalling pathways and many biological processes. Furthermore, molecular docking and experimentation verified the beneficial activity of Rhizoma Coptidis against 3 inflammatory factors. Overall, this study confirms that Rhizoma Coptidis might be used to treat T2DM with anti-inflammatory effects.Network pharmacology can be used to reveal the mechanism of Chinese herbal medicine at the molecular level, and from a system perspective, it provides effective evidence and a basis to support the ameliorative effect of Rhizoma Coptidis on T2DM. However, there are also some limitations. For example, the data on various drugs, genes, and proteins are not comprehensive, and appropriate computing software has not been developed. Network pharmacology is only a reasonable predictor of the mechanism of action of Rhizoma Coptidis against T2DM. Hence, further scientific verification and more rigorous research strategies should be carried out.


Pishuang, Pishi, arsenolite, As2O3, and CAS 1327-53-3), a naturally occurring and toxic mineral as a drug for more than 2000 years in China, has been found to have a valuable function in hepatocellular carcinoma (HCC) in recent years. However, its exact mechanism remains to be elucidated. Therefore, this study was intended to explore the potential anti-HCC mechanism of arsenic trioxide through network pharmacology.  Arsenic trioxide  network of arsenic trioxide and HCC common targets was established using STRING. GO and KEGG pathway enrichment analyses were performed by the Database for Annotation, Visualization, and Integrated Discovery (DAVID). Finally, an arsenic trioxide-target-pathway-HCC network was built by Cytoscape 3.2.1, and network topological analysis was carried out to screen the key candidate targets. . Conclusions. Our study showed that key targets of arsenic trioxide were mainly involved in multiple biological processes and pathways. It provided a theoretical basis for the screening of drug targets. A total of 346 corresponding targets of arsenic trioxide and 521 HCC-related targets were collected. After target mapping, a total of 52 common targets were obtained. GO analysis showed that the biological process was mainly involved in the negative regulation of cellular senescence, response to tumor necrosis factor, and cellular response to hypoxia. Molecular functions included NF-kappa B binding, enzyme binding, p53 binding, and transcription factor binding. Cellular components mainly were replication fork, ESC/E(Z) complex, RNA polymerase II transcription factor complex, and organelle membrane. KEGG pathways were mainly enriched in the PI3K-Akt signaling pathway, VEGF signaling pathway, p53 signaling pathway, HIF-1 signaling pathway, TNF signaling pathway, AMPK signaling pathway, NF-kappa B signaling pathway, FoxO signaling pathway, ErbB signaling pathway, and MAPK signaling pathway. In the arsenic trioxide-target-pathway-HCC network, targets such as AKT1, RAF1, RELA, TP53, and PTEN had a higher degree Hepatocellular carcinoma (HCC) is a common malignant tumor with poor prognosis, characterized by strong invasion and rapid growth . AccordiPishuang, in Chinese Pinyin, As2O3, and CAS 1327-53-3), a natural and toxic substance be applied as a drug for more than 2000 years in China, has been found to have a valuable function in acute promyelocytic leukemia in recent years 2O[As]3O[As]1O[As](O2)O3” in TargetNet [http://targetnet.scbdd.com/). Targets in which prediction probability was 0 should be removed.We collected targets of arsenic trioxide by the following 3 ways: (1) by importing “CAS 1327-53-3” in PubChem  . Only targets with “relevance score ≥20” could be included. The collected information included gene full name, gene symbol, and UniProt ID.HCC-related targets were collected by retrieving “hepatocellular carcinoma” and “liver cancer” in GeneCards  . The OmicShare tools (https://www.Omicshare.com/) were used to draw Venn diagrams.By mapping arsenic trioxide targets and HCC targets, we obtained arsenic trioxide and HCC common targets. Then, PPI network was constructed (combined score ≥0.9) through STRING  . GO mainly included molecular function (MF), biological process (BP), and cellular component (CC). KEGG was used to identify the main anti-HCC signaling pathways of arsenic trioxide. In this study, we selected 20 GO terms and 10 KEGG pathways to be visualized by OmicShare tools.Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were performed by the Database for Annotation, Visualization, and Integrated Discovery  . Network topological analysis was carried out to screen the key candidate targets. The topological parameters  of every node in the network were analysed, and all the results were ranked by “degree.”The arsenic trioxide-target-pathway-HCC network was built by Cytoscape 3.2.1   complex, RNA polymerase II transcription factor complex, and organelle membrane .P values were less than 0.05  [In this study, we obtained 346 targets of arsenic trioxide and 52 arsenic trioxide and HCC targets in common, which suggested that arsenic trioxide might be effective on HCC suppression by multitargets. As shown in the networks of arsenic trioxide-target-pathway-HCC and core pathway-target, nodes of AKT1, RAF1, TP53, and PTEN have a higher degree, indicating that they might be the key targets of arsenic trioxide for HCC therapy. Interestingly, AKT1 could regulate GSK-3igration . Furtherer cells . As the er cells –25. Accoer cells . It was lls [μM) . The tumlls [μM) . Goussetlls [μM) . In the lls [μM) , which ells [μM) . The higlls [μM) .α [α, an important inflammatory mediator in immune responses, was demonstrated to induce tumor cell lysis [α expression was upregulated during the apoptosis of tumor cells by arsenic trioxide [α protein synthesis can inhibit hypoxia-induced angiogenesis and metastasis [GO analysis showed that the targets were mainly enriched in the regulation of cellular senescence, response to tumor necrosis factor, and hypoxia. Senescence is a cellular state in which cells lose their ability to proliferate . A studyα . Most fall lysis , 41. Furtrioxide . Notablytrioxide . Regulattastasis . Mutant tastasis . AMPK antastasis . Accorditastasis –50 and hin vitro and in vivo experiments.In conclusion, we undertake a network pharmacology approach to explore the underlying anti-HCC molecular mechanisms of arsenic trioxide, which could provide a theoretical basis for the screening of drug targets. Our study showed that key targets of arsenic trioxide were mainly involved in multiple biological processes and pathways. In the near future, the series of promising results need to be verified by additional research, including


Venous thromboembolism (VTE) is a condition comprising deep venous thrombosis (DVT) and pulmonary embolism (PE). The prevalence of this disease is constantly increasing and it is also a chief reason for morbidity. Therefore, the primary prevention of VTE remains a highly important public health issue. At present, its diagnosis generally relies on subjective clinical examination and ultrasound imaging. D-dimer is also used as a biomarker, but it is considered to be poorly specific and only moderately sensitive. There are also no reliable methods that could accurately guide the type of treatment and potentially identify patients who may benefit from more aggressive therapies without the risk of bleeding. The application of metabolomics profiling in the area of vascular diseases may become a turning point in early diagnosis and patient management. Among the most described metabolites possibly related to VTE are carnitine species, glucose, phenylalanine, 3-hydroxybutarate, lactic acid, tryptophan and some monounsaturated and polyunsaturated fatty acids. The cell response to acute PE was suggested to involve the uncoupling between glycolysis and oxidative phosphorylation. Despite technological advancement in the identification of metabolites and their alteration in thrombosis, we still do not understand the mechanisms and pathways responsible for the occurrence of observed alterations. Venous thromboembolism (VTE) is a condition comprising deep venous thrombosis (DVT) and pulmonary embolism (PE) . In the Many risk factors related to the development of VTE have been established , however, most biological mechanisms associated with this disorder remain unsolved . In manyThe term ‘metabolomics’ refers to the comprehensive and systematic profiling of all low molecular weight compounds, which are intermediate or final products of metabolism, generated as a product of biochemical and physiological processes in the body and present in differential abundance in biofluids, cells and tissues ,26. Due The study of metabolites can reveal the physiologic state before disease onset as well as the complex interplay between genes, environmental risk factors, and VTE . This teThe field of metabolomics has been recently extensively developed and used for the identification of potential biomarkers for the early diagnosis of diseases. The key to the advancement of medicine is the improvement of our understanding of disease pathophysiology . The useThe interpretation of obtained data in isolation is difficult, and therefore there is a need for cross-omics studies with validation through different experimental approaches. Information obtained with the use of metabolomics should be analysed in combination with other omics studies in order to allow the understanding of disease-related mechanisms. However, such data is still not available in the current publications. These factors make the establishment of universal reference values, which could be used in diagnostic tests, challenging. The creation of cumulative and reliable databases based on data from large populations studies, such as the human metabolome database, could help to overcome these obstacles. Moreover, performing epidemiological studies could enable the collection of data concerning the distribution of metabolites in different patient populations ,33. In this review, we would like to summarize the result of metabolic studies performed in animal models of DVT and PE and also sparse data obtained from the analysis of human samples.The results of metabolome profiling in animals demonstrated that levels of several molecules involved in energy metabolism may be used as discriminatory factors between DVT and control animals . Maekawa2+ release from intracellular stores [Maekawa et al.  observedr stores . Numerour stores . TMAO har stores ,59. In ar stores . The impr stores ,62,63. Tr stores . Some str stores ,66. Morer stores . Accordir stores ,68. Seler stores ,70. The In turn, Cao et al.  analysed2 and reduced forms of nicotinamide adenine dinucleotide (NADH) or flavin adenine dinucleotide (FADH2). In subsequent steps, reduced coenzyme groups are used by mitochondria to synthesize ATP via the oxidative phosphorylation pathway [Metabolomic studies were also used in pulmonary embolism models. Bujak et al. , analysi pathway . The acc pathway , altered pathway ,73. More pathway  results  pathway ,75. Apar pathway . Finally pathway  results  pathway . Another pathway . The lev pathway . Apart f pathway . In addi pathway . On the  pathway  suggesteIn turn, Ma et al.  analysedp = 0.01, 0.004, 0.001, 0.04, 0.02, and 0.03, respectively). AC circulating in plasma plays a vital role in mitochondrial energy metabolism [There are just a few human studies of the metabolite profile of thrombosis. The application of untargeted metabolomics in the search for biomarkers of VTE in 40 male idiopathic adult VTE cases and 40 age-matched male controls resulted in the identification of 9400 metabolic features, 257 of which markedly differed between controls and patients with VTE in terms of serum concentrations . Furthertabolism ,84. The tabolism . Moreovetabolism . Some sttabolism ,86. Degutabolism  suggestetabolism . This eftabolism . Jiang etabolism ,90. It wtabolism . Other stabolism . Sanmarctabolism  confirmetabolism ,96. Howetabolism ,99,100. The study of patients with different severities of pulmonary embolism (PE) has revealed the presence of considerable differences in the tricarboxylic acid cycle, fatty acid, and purine metabolite pathway between low- and intermediate/high-risk PE patients . Apart fThe complexity of the thrombosis phenotype could be associated with complex interactions of genetic, environmental and acquired factors, which contribute to disease susceptibility. There is still a long road ahead before we will be able to understand the complicated pathomechanisms of diseases. The utility of high-throughput “omics” in biological systems is great; however, there are still many obstacles to overcome. If we do this, such tools will help to decipher the complexity of human pathology, which in consequence will facilitate the diagnosis and treatment of patients. Among the most described metabolites possibly related to VTE, there are carnitine species, glucose, phenylalanine, 3-hydroxybutarate, lactic acid, tryptophan and some monounsaturated and polyunsaturated fatty acids, which accounts for energetic metabolism. In addition, some specific metabolic alterations have been reported, including TMAO for arterial thrombosis and AC for venous thrombosis. In turn, cell response to acute PE was suggested to involve the uncoupling between glycolysis and oxidative phosphorylation. Despite technological advancement in the identification of metabolites and their alteration in thrombosis, we still do not understand the mechanisms and pathways responsible for the occurrence of observed alterations. Therefore, drawing conclusions concerning various metabolites involved in the development of thrombosis must be preceded by large studies performed with the use of high-throughput analytical techniques and the analysis of their possible role in this disease process.


BanXia-YiYiRen  has been clinically proven to be an effective Chinese medicine compatible with the treatment of insomnia. However, the underlying mechanism of BX-YYR against insomnia remains unclear. This study aimed to explore the pharmacological mechanisms of BX-YYR in treating insomnia based on network pharmacology and experimental validation. The drug-disease targets were obtained using publicly available databases. The analysis revealed 21 active compounds and 101 potential targets of BX-YYR from the pharmacological database of Chinese medicine system and analysis platform (TCMSP) and 1020 related targets of insomnia from the GeneCards and Online Mendelian Inheritance in Man (OMIM) databases. Furthermore, 38 common targets of BX-YYR against insomnia were identified, and these common targets were used to construct a protein–protein interaction (PPI) network. The visual PPI network was constructed by Cytoscape software. The top three genes from PPI according to degree value are FOS, AKT1, and CASP3. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment were applied to reveal the potential targets and signaling pathways involved in BX-YYR against insomnia, especially the serotonergic pathway. In addition, molecular docking revealed that baicalein, beta-sitosterol, and stigmasterol displayed strong binding to AKT1, FOS, PRKCA, and VEGFA. Experimental study found that BX-YYR against insomnia might play a role in improving sleep by modulating the serotonergic pathway. In summary, our findings revealed the underlying mechanism of BX-YYR against insomnia and provided an objective basis for further experimental study and clinical application. It is oThe combination of BX-YYR is derived from the formula for insomnia in the ‘Yellow Emperor’s Classic of Internal Medicine’ – Pinellia Rice Decoction, which has a wide range of clinical applications and the effect of improving insomnia symptoms. A study confirmed the good sedative-hypnotic effect of Panicum semifolium soup . InsomniAlthough the combination of BX-YYR and other drugs against insomnia has good efficacy in clinical practice, the specific mechanism of BX-YYR against insomnia at the cellular, molecular and genetic levels is not clear. In recent years, the novel network pharmacology of Chinese herbal medicine has brought together multidisciplinary thinking and theories from traditional pharmacology, bioinformatics and network biology . NetworkTherefore, the present study was designed to determine the potential mechanisms of major active compounds in BX-YYR in treating insomnia by utilizing network pharmacology and experimental validation. Our research may provide evidence to support the clinical use of BX-YYR against insomnia, help screen effective active compounds of BX-YYR, improve the formulation of BX-YYR, and provide a reliable reference for exploring the pharmacological mechanism of other Chinese medicines treating insomnia .Figure 2.2.1.2.1.1.http://lsp.nwu.edu.cn/tcmsp.php). We screened the Chinese herbal medicine BX-YYR compounds based on absorption, distribution, metabolism and excretion. In the TCMSP databases, a pharmacokinetic information retrieval filter was used to retrieve bioactive compounds for further analysis under the conditions of oral bioavailability (OB) ≥ 30% and drug-likelihood (DL) ≥ 0.18 , at 46.89%. In addition, MOL006967 , MOL000449 (stigmasterol), MOL001323 , and MOL006937  were ranked in the top five for oral utilization, with MOL000449 (stigmasterol) being the active ingredient shared by the drug pair of BX-YYR; thus, presumably, these ingredients play an important pharmacological role in the treatment of insomnia by BX-YYR. There were 101 active ingredient targets corresponding to BX-YYR, among which 38 common targets were related to insomnia. The PPI results show that the key targets for the treatment of insomnia by BX-YYR are FOS, AKT1, CASP3, TP53, VEGFA, etc. These key targets involve 897 BP, 84 MF and 39 CC, mainly including the regulation of G protein-coupled neurotransmitter receptor activity, serotonin binding, catecholamine binding, etc. In addition, they are associated with neuroactive ligand–receptor interactions, serotonin synapses, calcium signaling pathway, amphetamine addiction, dopaminergic synapses, apoptosis, cAMP signaling pathway and other KEGG signaling pathway modulation.The molecular docking results revealed that baicalein, beta-sitosterol, and stigmasterol have strong binding interactions with four target proteins  with molecular docking energies less than −5 kcal/mol. Among them, the molecular docking interaction of baicalein with AKT1 improves the stability of the baicalein compound in the AKT1 protein pocket and is predicted to be a potentially active small molecule. As one of the flavonoids in many traditional Chinese medicine extracts, baicalein has pharmacological effects such as sedation and anti-anxiety effects . In the Several other compounds, such as beta-sitosterol and stigmasterol, have slightly weaker binding ability to proteins due to their large molecular weight and hydrophobicity but also have multiple interactions and contribute more to stabilizing small molecules, which are also potentially active compounds and small molecules for the treatment of insomnia with BX-YYR in terms of macromolecular protein structural domain resolution. It provides a pharmacological mechanism for the treatment of insomnia by BX-YYR in terms of protein structure domain resolution, interactions between macromolecules, and binding in the enzyme-substrate catalytic process, which is valuable for screening the active ingredients of Chinese herbal medicine for the treatment of insomnia.Studies have shown that the FOS protein is involved in the normal differentiation, growth, learning, memory and other processes of cells and is more highly expressed in the brain, cortex, hippocampus, and striatum . AKT1 isIn mammals, serotonin is widely known as a neurotransmitter and hormone produced by intestinal chromophores in the gastrointestinal tract and circulating platelets . 5-HT inIn summary, this study usednetwork pharmacology and molecular docking methods to systematically explore and verify the potential mechanism of BXYYR in the treatment of insomnia from multiple perspectives for the first time. Finally, the animal experimental verification results show that this method can better explain the network characteristics and the synergistic action principle of Chinese medicine compounds, indicating that BX-YYR is a multicomponent, multi-target and multi-pathway drug in the treatment of insomnia. Next, on the basis of this research, this research group will use Chinese medicine chromatography and mass spectrometry technology analysis methods to verify the data mining results of active ingredients, laying a theoretical and experimental basis for future in-depth exploration of molecular biology, cell experiments and pharmacology.5.By using the method of network pharmacology, we have studied the potential targets of BX-YYR and its underlying mechanism of improving insomnia symptoms, which reflects its characteristics of multiple components, multiple targets, and multiple pathways. FOS, AKT1, CASP3, TP53 and VEGFA may be potential targets of BX-YYR in the treatment of insomnia. According to the enrichment analysis results of GO and KEGG signaling pathways, we found pathways closely related to the pathological process of insomnia, mainly including serotonergic pathways, neuroactive ligand–receptor interactions, calcium signaling pathways, dopaminergic synapses, and apoptotic cell death. Preliminary validation of molecular docking showed that the small molecule compounds  contained in BX-YYR generally have excellent binding affinity for the large molecular target proteins  encoded by the first 4 genes. Experimental validation revealed that the treatment of insomnia with BX-YYR was associated with the regulation of serotonergic signaling pathways, which resulted in improved sleep. Therefore, this study identified that BX-YYR could treat insomnia through directly or indirectly regulating the abovementioned potential targets and pathways. BX-YYR may provide a promising direction for future insomnia therapy, but also relevant experimental studies, which are very important to reveal the exact pharmacological mechanism ofBX-YYR on insomnia, should been carried out in the future study.