Objective: Aloe-emodin (AE) is an anthraquinone compound extracted from the rhizome of the natural plant rhubarb. Initially, it was shown that AE exerts an anti-inflammatory effect. Further studies revealed its antitumor activity against various types of cancer. However, the mechanisms underlying these properties remain unclear. Based on network pharmacology and molecular docking, this study investigated the molecular mechanism of AE in the treatment of hepatocellular carcinoma (HCC), and evaluated its therapeutic effect through in vitro experiments.Methods: CTD, Pharmmapper, SuperPred and TargetNet were the databases to obtain potential drug-related targets. DisGenet, GeneCards, OMIM and TTD were used to identify potential disease-related targets. Intersection genes for drugs and diseases were obtained through the Venn diagram. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses of intersecting genes were conducted by the website of Bioinformatics. Intersection genes were introduced into STRING to construct a protein-protein interaction network, while the Cytoscape3.9.1 software was used to visualize and analyze the core targets. AutoDock4.2.6 was utilized to achieve molecular docking between drug and core targets. In vitro experiments investigated the therapeutic effects and related mechanisms of AE.Results: 63 overlapped genes were obtained and GO analysis generated 3,646 entries by these 63 intersecting genes. KEGG analysis mainly involved apoptosis, proteoglycans in cancer, TNF signaling pathway, TP53 signaling pathway, PI3K-AKT signaling pathway, etc. AKT1, EGFR, ESR1, TP53, and SRC have been identified as core targets because the binding energies of them between aloe-emodin were less than -5 kcal/Mol.The mRNA and protein expression, prognosis, mutation status, and immune infiltration related to core targets were further revealed. The involvement of AKT1 and EGFR, as well as the key target of the PI3K-AKT signaling pathway, indicated the importance of this signaling pathway in the treatment of HCC using AE. The results of the Cell Counting Kit-8 assay and flow analysis demonstrated the therapeutic effect of AE. The downregulation of EGFR, PI3KR1, AKT1, and BCL2 in mRNA expression and PI3KR1, AKT,p-AKT in protein expression confirmed our hypothesis.Conclusion: Based on network pharmacology and molecular docking, our study initially showed that AE exerted a therapeutic effect on HCC by modulating multiple signaling pathways. Various analyses confirmed the antiproliferative activity and pro-apoptotic effect of AE on HCC through the PI3K-AKT signaling pathway. This study revealed the therapeutic mechanism of AE in the treatment of HCC through a novel approach, providing a theoretical basis for the clinical application of AE. Sorafenib, a molecularly targeted agent with good efficacy against liver cancer, is another treatment option. This drug can inhibit the proliferation of cancer cells, and is indicated for the treatment of patients with advanced liver cancer. Nevertheless, an increasing number of patients develop tolerance to chemotherapeutic drugs, and only a minority of patients with HCC benefit from continued chemotherapy  is an anthraquinone compound extracted from the roots, stems, and leaves of aloe vera, rhubarb, cassia, and other plants. It exerts a broad range of antibacterial, antiviral, and anti-inflammatory effects. Previous study has demonstrated that AE could restrain the lipopolysaccharide-induced production of proinflammatory cytokines in RAW264.7 macrophages via downregulation of nuclear factor-κB (NF-κB), mitogen-activated protein kinase (MAPK), and phosphatidylinositol 3 kinase (PI3K) . AE alsoproteins . Studiesproteins . Furtherproteins , gastricproteins , colon cproteins , liver cproteins , and othNetwork pharmacology is an emerging method which not only combines network biology and poly-pharmacology to validate drug-actionable targets through computational software, but also explores potential mechanisms of drug therapeutic actions . NetworkThe workflow of this analysis is shown in Liver cancer-related genes were recognized using four genetic databases, namely, DisGenet , GeneCarGO is a biological system used for studying the effects of individual genes on an organism at different biological levels. KEGG provides evidence indicating the potential involvement of individual genes in biological signaling pathways . GO and The drug-target-pathway network was established by introducing intersection genes and KEGG signaling pathway items into Cytoscape 3.9.1. Cytoscape is a platform that integrates complex network structures with data and presents them in graphical form . The celHomo sapiens” and a medium confidence score of 0.4 to ensure more protein-protein information would be included. The PPI network was updated after removing disconnected nodes. The tab-separated values (tsv) format file was downloaded and imported into Cytoscape 3.9.1 for subsequent visualization. The strength of interactions between proteins in Cytoscape would be calculated in order to screen for core targets.PPI is a method utilized to study the mechanism through which proteins function harmoniously in cells . Using tUsing the “CytoNCA” plug-in in Cytoscape 3.9.1, we determined the core targets according to their degree values. “CytoNCA” was a plug-in for calculating the strength of interactions between proteins, some proteins with higher interaction strengths with other proteins could be screened out as core targets . SeveralWe sought to further understand the relationship between candidate proteins and AE, as well as their mechanism of action. Therefore, molecular docking was performed to determine the strength of the interaction between receptors and ligands. The SDF  of AE was downloaded from PubChem, and the Protein Data Bank (PDB) database was used to obtain the SDF format file of the original ligand. Moreover, the pdb format files of receptor proteins were obtained from the PDB database . The SDFversus normal hepatocytes using the Gene Expression Profiling Interactive Analysis (GEPIA) database  was calculated using TIMER. And genetic alterations and genetic mutation sites of core targets were detected using cBioPortal database.When cancer occurs, it is especially prominent to know about gene mutations and changes in the immune system . The TumSCSP-526,Shanghai, China) were maintained in DMEM supplemented with 10% fetal bovine serum (Gibco), 1% penicillin and streptomycin (100 U/mL), 1% glutamax, and 1% sodium pyruvate 100 mM solution at 37°C in a humidified incubator with 5% CO2.AE (100 mg) was purchased from TCI Chemicals , dissolved in dimethyl sulfoxide (60 mg: 1 mL), and stored at −20°C. The final concentrations of AE used in different experiments were reached with Dulbecco’s modified Eagle’s medium (DMEM) . HepG2 cells  and cultured. AE  was used to treat HepG2 cells cells for 0, 12, 24, 48, 72 h. Subsequently, CCK8 reagent (10 μL) was added to each well containing DMEM (90 μL). CCK8 and DMEM were also added to one well not containing cells (blank controls). Thereafter, a microplate reader was used to measure the optical density (OD) at 415 nm wavelength. Percentage viability was calculated using the following formula: % Viability = (OD of treated cells − OD of blank control)/(OD of negative control − OD of blank control) ×100.Cell Counting Kit-8  assay was used to analyze cell viability according to the instructions provided by the manufacturer. Cells were seeded into 96-well microplates (3 × 105cells/well) and cultured overnight. Following adhesion, the cells were exposed to different concentrations  of AE for 24 h. Subsequently, the cells were collected and incubated with Annexin V-FITC/PI at room temperature in the dark for 15 min according to the user manual. The proportion of apoptotic cells was determined using BDAccuri™ C6Plus flow cytometry .The Annexin V-fluorescein isothiocyanate/propidium iodide (Annexin V-FITC/PI) Apoptosis detection Kit  was used to verify the effect of AE on HCC cell apoptosis. Briefly, HepG2 cells were seeded into six-well plates (2 × 105cells/well in 1 mL of DMEM medium) and cultured. Previous studies have shown that AE exerted its greatest inhibitory effect on HCC at the concentration of 100 μM was used to conduct the DNA amplification.We also evaluated the effect of AE on the transcription of core genes in the PI3K-AKT signaling pathway. Cells were seeded into six-well plates  at 150 ul per well for 20 min. The lysed cells were subsequently transferred to 1.5 mL centrifuge tubes, followed centrifugation  at 4°C for 10 min. Then the centrifuged supernatant was extracted and mixed with 20% loading buffer for Agarose Gel Electrophoresis. Protein samples were then separated on the 10% PAGE gel and transferred to a nitrocellulose membrane. The membrane was incubated with a blocking solution of 5% skim milk powder and Tris-buffered saline with Tween-20 (TBST) for 2 hours at room temperature to block non-specific antibody binding, and then washed three times with TBST for 10 min. Then the membrane was incubated overnight at 4°C using primary antibodies with dilutions of 1:5000. PI3KR1 antibody (60225-1-Ig), AKT1 antibody (30203-2-Ig),p-AKT1 antibody (66444-1-Ig) and GAPDH antibody (60004-1-Ig) were purchased from Proteintech Group . The primary antibody was washed 3 times with TBST at the end of the incubation, followed by incubation with the secondary antibody conjugated to horseradish peroxidase  with dilution of 1:10000 for 1 h at room temperature. Then the protein bands was added with ECL luminescence reagent  for visualization by ChemiDoc XRS + System . The Western blot results were quantified using Fiji (2.14.0) software , respectively. The mean ± standard deviation was used to plot the data. p-values < 0.05 indicate statistically significant differences. The analysis was performed using the GraphPad Prism software version 9.0   .A total of 127 genes were identified from Pharmmapper according to a norm fit score ≥0.4. In addition, 109 and 52 genes were selected from SuperPred and TargetNet, respectively, based on a probability ≥0.5. Moreover, 47 genes were selected from the Comparative Toxicogenomics Database. Following the removal of duplicates, a total of 306 drug-related genes were obtained.1143 disease-related genes were identified from Disgenet, Genecards, OMIM, and Therapeutic Target Database. As shown in the Venn diagram , 63 genep-value, and the genes contained in each type are shown , cellular component (n = 194), and molecular function (n = 336). The top 10 entries of each type were filtered based on the re shown . Red nod. In addition, we also performed a correlation analysis of the mRNA expression of core targets with the progression of HCC. We found that the expression of ESR1 was markedly changed with the development of HCC .The chemical structure of AE is presented in p < 0.05) (Annexin V-FITC and PI were designed to detect apoptosis in the early and late stages respectively. Cells in the lower right corner of the cross door were early apoptotic cells stained by FITC. Those in the upper right corner of the cross door were late apoptotic cells stained by PI . The pro < 0.05) .EGFR, PI3K, AKT1, and B-cell lymphoma 2 (BCL2) (p < 0.05).The sequences of primers used in this study are shown in 2 (BCL2) . Abnormap < 0.05)  . TogetheIn vitro experiments were also utilized to confirm the therapeutic effect and mechanism of aloe-emodin.HCC is the most common type of liver cancer and associated with an extremely high mortality rate . The manin vitro experiments directly demonstrated that AE could its inhibitory effects on HCC by downregulation of PI3KR1, AKT1, p-AKT. The TNF signaling pathway includes two types of receptors  that play different roles by binding to TNF. TNFR1 binds to TNF to form a complex that can directly recruit CASP8 and initiate a protease cascade reaction. In addition, the receptor-interacting protein (RIP) in this complex induces the activation of NF-κB, which plays an important role in cell survival, proliferation, inflammation, and immune regulation  were selected by protein-protein network. Proteins do not function as single substances, but as whole groups in biological processes . Proteinin vivo . Patientin vitro experiments. Through the decreased expression of PI3KR1, AKT1, and BCL2 mRNA levels compared with control group, we demonstrated that the inhibition of hepatocellular carcinoma by aloe-emodin could be mediated through the PI3K-AKT signaling pathway. The downregulation of PI3KR1, AKT and phospho-AKT in protein levels further reinforced our findings. And through network pharmacology, we also verified the involvement of AE in other signaling pathways for the treatment of HCC. Meanwhile, our article has some limitations. Our utilization of the database is incomplete to the extent which leads to reduced credibility of the selected genes. The signaling downstream of the PI3K-AKT signaling pathway need to be further explored. Still and all, our research affirms the therapeutic role of aloe-emodin on hepatocellular carcinoma through the regulation of PI3K-AKT signaling pathway.Our study confirmed the inhibitory effects of AE on HCC and the aberrant activation of PI3K-AKT was also affirmed in our This study elucidated the regulatory mechanism of AE in the treatment of HCC through network pharmacology and cell experiments. The results demonstrated that multiple signaling pathways are involved in this process. AE could exert its therapeutic effect by binding to EGFR, SRC, AKT1, TP53, and ESR1. Our findings also verified the importance of the PI3K-AKT signaling pathway through which AE might exert its therapeutic effect on HCC. However, the regulatory effects of AE on signaling targets downstream of the PI3K-AKT pathway remain unknown, and more scientific practice needs to be engaged to improve the understanding of the therapeutic effects of AE. In conclusion, our study provides a more solid theoretical basis for the clinical application and exploration of the mechanism of AE in the treatment of HCC.


Objective: To explore the functional mechanisms of Suanzaoren decoction (SZRD) for treating insomnia using network pharmacology and molecular docking.Methods: The active ingredients and corresponding targets of SZRD were obtained from the Traditional Chinese Medicine Systems Pharmacology database, and then, the names of the target proteins were standardized using the UniProt database. The insomnia-related targets were obtained from the GeneCards, DisGeNET, and DrugBank databases. Next, a Venn diagram comprising the drug and disease targets was created, and the intersecting targets were used to draw the active ingredient-target network diagram using Cytoscape software. Next, the STRING database was used to build a protein-protein interaction network, followed by cluster analysis using the MCODE plug-in. The Database for Annotation, Visualization, Integrated Discovery , and the Metascape database were used for Gene Ontology (GO) enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses. AutoDock Vina and Pymol software were used for molecular docking.Results: SZRD contained 138 active ingredients, corresponding to 239 targets. We also identified 2,062 insomnia-related targets, among which, 95 drug and disease targets intersected. The GO analysis identified 490, 62, and 114 genes related to biological processes, cellular components, and molecular functions, respectively. Lipid and atherosclerosis, chemical carcinogen-receptor activation, and neuroactive ligand-receptor interaction were the most common pathways in the KEGG analysis. Molecular docking demonstrated that the primary active components of SZRD for insomnia had good binding capabilities with the core proteins in PPI network.Conclusion: Insomnia treatment with SZRD involves multiple targets and signaling pathways, which may improve insomnia by reducing inflammation, regulating neurotransmitters. Insomnia is a common sleep disorder primarily manifesting as difficulty falling asleep, decreased sleep quality, and shortened sleep duration . The annMany research groups have investigated the mechanisms involved in the prevention and treatment of insomnia, however, there are several causes, and the mechanisms are complex. Insomnia-related mechanistic studies have explored the hypothalamic-pituitary-adrenalin axis dysfunction, vagal tone changes, melatonin system function decline, the influence of inflammatory response factors, central neurotransmitter disorders, and functional or structural abnormalities of the limbic cortical system circuits . In addiTraditional Chinese medicines (TCM), such as Suanzaoren decoction (SZRD), are safe and effective treatments for insomnia. SZRD is prepared with Suanzaoren, Chuanxiong, Gancao, Fuling, and Zhimu. Ligustrin I, the active component of Chuanxiong, penetrates the blood-brain barrier , regulatTCM prescriptions comprise several components that exert their effects via several pathways, requiring multi-target coordination. Therefore, a comprehensive understanding of the pharmacodynamic material basis and mechanisms is essential. The multi-layer and multi-angle research strategy of network pharmacology coincides with the systematic and holistic view of TCM. Therefore, in this study, we used bioinformatics to predict SZRD-specific therapeutic targets and signaling pathways and explore potential insomnia-related mechanisms.https://old.tcmsp-e.com/tcmsp.php)  were identified using the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform  , filteremsp.php) . The acthttps://www.genecards.org) (https://www.disgenet.org) (https://www.drugbank.com) (The keyword “Insomnia” was searched in the GeneCards database (rds.org) , DisGeNEnet.org) , and Druank.com)  to identWe integrated the active ingredient (drug)-related and disease-related targets to identify overlapping targets. The active ingredient-target network was generated using Cytoscape 3.9.1 software; the nodes represent the active ingredients and targets, and the edges represent the interrelationships between them. The Network Analyzer is a built-in tool to calculate the degree value, which was used to screen the major active ingredients.https://cn.string-db.org) , betweenness centrality (BC), and neighborhood centrality (NC). Then, they were analyzed using the molecular complex detection (MCODE) plug-in.The intersection targets were uploaded to the STRING database (-db.org)  to consthttps://david.ncifcrf.gov)   . GO analape.org) , and 20 https://pubchem.ncbi.nlm.nih.gov) (https://www.rcsb.org) (http://www.pymol.org/pymol), and pre-processing, such as hydrogenation and identification of active pockets, was performed using AutoDock Tools (https://ccsb.scripps.edu/autodock/). Molecular docking was performed using AutoDock Vina software   and thencsb.org) . Water msoftware , and theIn total, 138 active ingredients were identified in the TCMSP platform: 9 for Suanzaoren, 7 for Chuanxiong, 92 for Gancao, 15 for Fuling, and 15 for Zhimu . AdditioThe active ingredients and the 95 overlapping targets were used to construct the active ingredient-target network , which cThe active ingredients were screened based on their degree values, and eight compounds had a degree value greater than two times the average: kaempferol, quercetin, 7-Methoxy-2-methyl isoflavone, naringenin stigmasterol, medicarpin, formononetin, and shinpterocarpin . The degThe 95 overlapping targets were uploaded to the STRING database to construct the PPI network and obtain the corresponding network data , which wMCODE is one of the most widely used algorithms for mining protein complexes by using the intrinsic relationships of proteins in the network to find protein clusters. Further analysis of the PPI network with MCODE yielded seven protein clusters, then a GO enrichment analysis of the seven protein clusters was performed .p-value -coclaurine) and the five core proteins from the PPI network . The binding energy of the nine active ingredients to the five core proteins was <5 kcal-mol–1 .Insomnia is a common sleep disorder that is believed to be mainly caused by HPA axis dysfunction, central neurotransmitters disorders, inflammatory response factors, and changes in vagal tone. This study explored the potential of SZRD in treating insomnia using network pharmacology and molecular docking methods.The KEGG enrichment results revealed various disease pathways unrelated to our study, probably due to shared molecular targets contributing to different diseases. Therefore, we chose to analyze the signaling pathways closely related to insomnia in the enrichment results. Our findings suggest that SZRD may exert its therapeutic effect on insomnia by modulating the neuroactive ligand-receptor interaction, serotonergic synapse, and PI3K/AKT pathway. The neuroactive ligand-receptor interaction pathway, which involves receptors and ligands on the plasma membrane associated with intra- and extracellular signaling pathways, is physiologically relevant to neural function. . The neuThe orexin belongs to class A peptide subclass in the neuroactive ligand-receptor interaction signaling pathway. The orexin increases neuronal excitability by raising intracellular calcium levels through the classical phospholipase C cascade (PLC-IP2/DAG) and activates ERK 1/2 expression through Gq/PLC/PKC signaling. Orexins act through the activation of 2 G protein-coupled receptors (GPCRs), orexin receptor 1 (OX1R), and orexin receptor 2 (OX2R) . OX2R isSerotonergic synapse includes 5-hydroxytryptamine, a range of 5-hydroxytryptamine receptors, and post-receptor signaling pathways. 5-Hydroxytryptamine (5-HT) is an indole derivative catalyzed by tryptophan hydroxylase to produce 5-hydroxytryptophan, and then catalyzed by 5-hydroxytryptophan decarboxylase to generate 5-hydroxytryptophan, one of the most important neurotransmitters for sleep regulation with the highest levels in the cerebral cortex and synapses. Binding of 5-HT to its receptor activates genes associated with downstream signaling pathways, which in turn regulate sleep . Among tThe PI3K/Akt signaling pathway regulates the normal activity of neuronal cells, and elevated levels of phosphatidylinositol 3-kinase (PI3K) and protein kinase B (Akt) modulate the activity of genes involved in the transduction of inflammatory cytokines driving insomnia pathogenesis . The PI3The PPI network identified IL-6, AKT1, TNF, IL-1β, and VEGFA as the key proteins. IL-6, IL-1β, and TNF-α are common inflammatory factors, and significantly higher IL-6 and TNF-α levels are reported in patients with insomnia than in healthy individuals, whereas decreasing their levels had a calming effect . The NodIn summary, our results showed that the active ingredients in SZRD bind to specific proteins, which regulate certain signaling pathways and affect downstream processes, such as the inflammatory response and neurotransmitter regulation, thereby relieving insomnia. In addition, we confirmed the multi-target, multi-pathway, and synergistic involvement of SZRD for insomnia treatment. However, TCM ingredients are complex, and the network pharmacology is limited to theoretical discussions. Therefore, further clinical and laboratory experiments are needed to validate the results. Nevertheless, our results lay a strong foundation for subsequent in-depth studies.


Panax ginseng C.A. Mey. Products have significant therapeutic effects on HF. Therefore, it is of great significance to explore the mechanism of action of Processed Panax ginseng C.A. Mey. Products in the treatment of HF.Heart failure (HF) is the most common cardiovascular disease in clinics. Processed The saponin-like constituents of 3 different ginseng preparations were characterized by UPLC/QE-MS and the identified saponin constituents were subjected to network pharmacological analysis. Protein–protein interaction analyses of the targets of different ginseng preparations for the treatment of heart failure (HF) were performed using the STRING database, Gene Ontology enrichment analyses and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses were performed using the DAVID database, and the results of the network pharmacological analyses were validated using the Autodock software. Finally, the relative quantitative content of 5 major ginsenosides in 3 processed ginseng products was evaluated.A total of 40 saponin compounds were identified based on mass spectrometry data. Network pharmacology and molecular docking analyses were used to predict the major targets of these sapions compounds and the key pathways mediating their anti-HF effects. After conducting a thorough screening, the study identified 5 primary ingredients of ginseng products ginsenoside Rh4, ginsenoside Rk3, ginsenoside Rk1, ginsenoside Rg5, and ginsenoside CK that can potentially target 22 essential proteins: EGFR, AKT1, ERBB2, STAT3, TNF, ESR1, MTOR, HRAS, MMP9, and PIK3CA, etc. Additionally, the Kyoto Encyclopedia of Genes and Genomes pathway analysis revealed that ginseng products can be beneficial in treating HF by interacting with pathways such as the PI3K-Akt signaling pathway, the TNF signaling pathway, the mTOR signaling pathway, and others.The present study revealed that the treatment of HF with different processed ginseng products may be related to the regulation of the PI3K-Akt signaling pathway, TNF signaling pathway, apoptosis pathway, mTOR signaling pathway, etc, and that the key active ingredients may be concentrated in black ginseng, which provides a theoretical basis and direction for the further study of the mechanism of action of ginseng. This provides a theoretical basis and research direction for further in-depth study of its mechanism of action. In recent decades, Western medicine has made great strides in improving the prognosis and reducing the morbidity and mortality of patients with HF. However, the “single-gene-single-target” treatment mode of Western drugs also brings certain side effects, which limits its clinical application in some aspects. Chinese medicine has accumulated rich clinical experience in the prevention and treatment of HF, especially in controlling symptoms, improving cardiac function, and enhancing quality of life.Heart failure (HF) is a complex clinical syndrome caused by the progression of various cardiac diseases to the severe stage. It has become the leading cause of death worldwide, often associated with dyspnea, fluid retention, reduced exercise tolerance, etc, and HF accounts for 32% of all deaths worldwide, with a low cure rate. Patients with underlying heart disease are more likely to develop HF.Panax ginseng C.A. Mey.) has been a valuable medicinal material used in traditional oriental medicine for thousands of years and is now widely used as a healthy food in East Asia and worldwide. Furthermore, not only raw sun-dried ginseng but also several processed ginseng products have been used in the food industry and herbal markets. According to the different processing methods, there are 3 processed ginseng products, raw sun-dried ginseng (DG), red ginseng (RG), and black ginseng (BG). Ginsenosides are considered to be the main active substances of ginseng and ginseng products. Previous studies have demonstrated that ginseng has significant positive inotropic effects, which can significantly enhance myocardial contractility, improve ventricular remodeling, bring the hemodynamics of patients with heart failure (HF) to a stable state, and contribute to the improvement of cardiac function and structure. Wan found that black ginseng was stronger than RG and ginseng against HF. Cao showed that based on the TGF-β/Smad signaling pathway, black ginseng decoction had a stronger protective effect than white ginseng decoction on cardiac hypertrophy rats. Liu found that Rg5 significantly reduced cardiac dilatation and venous congestion and increased cardiac output and heart rate. It has also been shown that Rg5 improves metabolic disorders and cardiac dilatation in zebrafish with HF. Dai found that TSBG has an excellent therapeutic effect on DOX-induced HF in rats, probably by regulating the Akt/mTOR autophagy signaling pathway, resulting in the improvement of taurine and hypotaurine metabolism, arachidonic acid metabolism and sphingolipid metabolism, which may provide a reference for elucidating the potential mechanism of action of TSBG against HF. However, the key active components, targets of action and biological mechanisms of different processed products of ginseng to comprehensively regulate and improve the whole of HF from the overall network level are not clear.Ginseng  formulas with “multi-component, multi-pathway and multi-target.” This study intends to explore the bioactive components and mechanism of action of ginseng-processed products for HF at a systemic level by integrating network pharmacology and molecular docking techniques, to provide a theoretical basis and research direction for subsequent clinical and basic studies.Network pharmacology is a research methodology that integrates molecular docking, construction of drug-disease target networks, and network characterization for the holistic regulation of multiple targets, which is instructive for the development of new drugs as well as elucidating the molecular mechanisms behind drugs.DG, RG, and BG are 5 years old, Changbai county origin of ginseng processed products. In 2020, all proceed ginseng was purchased from Jilin Cidayan Traditional Chinese Medicine Distribution Co., Ltd.HPLC-grade acetonitrile and methanol were purchased from Fisher Scientific (USA). HPLC-grade formic acid was purchased from TEDIA (USA). Methanol, and alcohol  were purchased from Beijing Chemical Plant (China). The ginsenoside standards used in Shanghai Yuanye Biotechnology Co., Ltd . Rg1 (22427-39-0), Re (52286-59-6), Rb1 (41753-43-9), Ro (34367-04-9), Rc (11021-14-0), Rd (52705-93-8), Rg3 (14197-60-5), CK (39262-14-1), Rk1 (494753-69-4). The purities of these compounds were more than 98%.The DG, RG, and BG samples were crushed and sieved to obtain fine powder for later use. Fine powder (100 mg) from DG, RG, and BG samples was suspended in 25 mL of 70% (v/v) methanol, and ultrasonically extracted for 60 minutes at 55°C. After centrifugation , the solution was filtered by using a syringe filter (0.22 µm) and injected into the UPLC system.For metabolite profiling of the processed ginseng products, we used a previously constructed method based on UPLC-QE-OrbitrapMS.Chromatographic separation was performed on an Ultimate 3000 system  coupled with Waters BEH RP-18 column . The column temperature was maintained at 30 °C, and the mobile phases A and B were water with 0.1% formic acid and acetonitrile, respectively. The gradient elution program was as follows: 0 to 5 minutes (75%A), 5 to 8 minutes (75–64%A), 8 to 15 minutes (64–52%A), 15 to 20 minutes (52–30%A), 20 to 25 minutes (30–10%A), 25 to 28 minutes (10%A), 28 to 28.1 minutes (10–75%A), 28.1 to 34 minutes (75%A); the flow rate was set at 0.2 mL/min, and the sample injection volume was 1 µL for each run.Mass spectrometric detection was carried out on a Q-Exactive Orbitrap/MS  equipped with an ESI ion source operated in the positive or negative ion mode. The parameters of the ion source were set as follows: sheath gas flow of 40 Arb, aux gas flow of 12 Arb, and sweep gas flow of 1 Arb, e S-Lens RF level was 55%. Capillary voltage was set to +3.5 kV or −3.5 kV with a capillary temperature of 333 °C and an aux gas heater temperature of 317 °C. The high-accuracy MS data were collected from m/z 100 to 1500 Da in positive or negative ion mode.http://www.swisstargetprediction.ch) to predict identified different processed products of ginseng ginsenosides compounds of potential targets.Using the Swiss Target Prediction platform (https://omim.org/), DISGENET database (https://www.disgenet.org/) and Drug bank database (https://www.drugbank.com/), “Heart failure” was used as the search keyword, and SOCRE > 0.7 was used as the screening condition in the DISGENET database to retrieve the targets related to HF disease. After the integration of the disease database, the repeated targets were eliminated for screening.Using OMIM database (https://cn.string-db.org/), and relevant parameters were set. The species was selected as “Homo sapiens,” and the confidence score represented the PPI degree, and the confidence was set as 0.4. The generated files were imported into Cytoscape software (3.9.1) to screen the top 50 hub genes under MCC, and the PPI network diagram was visualized. At the same time, the network topology analysis is carried out to calculate the “degree,” “Betweenness centrality,” and “Closeness Centrality” values, and then to obtain the core target.The target sites of ginsenosides identified were mapped to the target sites of HF, and the Venn diagram was drawn to visualize the data. The intersection of component targets and disease targets was calculated to establish a candidate target library. The candidate targets were imported into the String database (https://metascape.org/gp/index.html). GO enrichment analysis selected biological process (BP), molecular function (MF) and cellular component (CC). Second, choose P < .01, the result of the database access data import microscopic letter to the platform (http://www.bioinformatics.com.cn/), which will access visual analysis, generate KEGG bubble chart and GO histogram analysis.Gene Ontology enrichment (GO) and Kyoto Encyclopedia of Genes and Genomes pathway analysis (KEGG) were performed on the targets using the Mescape database , and its ligands and water molecules were removed with Pymol. The 3D structure information of protein and compound structure was uploaded, and then molecular docking was carried out to obtain the docking results of the target protein and compound molecules.To further evaluate the interaction between target proteins and compound molecules, molecular docking between target proteins and compound molecules was performed using AutoDock Vina molecular docking software. The core protein structure of the PPI network was downloaded from the PDB database (The core components of ginsenoside in the treatment of HF were obtained through network pharmacology combined with molecular docking. The relative contents of the core components in the different processed products of 3 kinds of ginseng were compared by boxplot.70% (v/v) methanol was used to extract ginsenosides and other metabolites from the 3 samples including DG, RG, and BG. The extracts were then subjected to UPLC-QE-Orbitrap MS in positive and negative ion mode. Figure –19 Ginseng produces trace amounts of rare saponins during processing, which are the products of deglycosylation, dehydration and oxidation at the C-20 position of the original ginseng diol (triol) type saponin.After method analysis, a total of 40 ginsenosides were identified in ginseng processed products, of which 9 ginsenosides were qualitatively identified by standard products, and the remaining 31 ginsenosides were obtained by literature review, as shown in Table The targets were collected from SwissTarget Prediction, OMIM, Drug Bank and DISGENET database, and 3051 targets were finally obtained after deleting the duplicate targets. Finally, 401 related targets of ginsenosides and 3051 disease-related targets were obtained. The Venny diagram Fig.  was consCytoscape software was used to screen the top 50 hub genes from candidate target sites according to the method in Section 4.3.3, and the PPI network diagram was visualized , as shown in Figure To investigate the biological functions of the target genes, 3 GO functional analyses were performed on 50 hub genes, including BP, CC, and MF, and the results with significant enrichment were visualized (P < .01) with significant enrichment and high disease relevance were screened out from 50 hub genes as shown in Figure CC mainly involves receptor complex, phosphatidylinositol 3-kinase complex, class I, membrane microdomain, etc. MF mainly involves protein tyrosine kinase activity, protein serine/threonine/tyrosine kinase activity, transmembrane receptor protein tyrosine kinase activity, etc. KEGG pathway enrichment analysis can explain which pathway ginsenoside acts on in the treatment of HF. 15 The pathways , and the core components were found to have a strong binding force with mTOR , HF has brought a huge economic burden to the whole of society. Heart failure is the terminal point of most cardiovascular diseases and the leading cause of death in cardiovascular patients. Its high incidence and mortality have become a major public health problem threatening human health. Further mechanistic investigation revealed that Rk3 prevented H/R-induced injury and apoptosis in H9C2 cardiomyocytes via AKT/Nrf-2/HO-1 and MAPK pathways. These observations indicate that Rk3 has the potential to exert cardioprotective effects against H/R injury, which may be of great importance for clinical efficacy in acute myocardial infarction (AMI) treatment. Yang showed that ginsenoside CK intervention could significantly reduce the levels of myocardial necrosis and infarct fiber scar, and reduce the inflammatory factors TNF-α and interleukin in the serum of AMI mice. The secretion of IL-6 increases the secretion of the anti-inflammatory factor IL-10, and this mechanism reduces the inflammatory response by increasing the proportion of regulatory T cells, thereby reducing the injury caused by cardiac remodeling after AMI. The enrichment results of GO and KEGG suggested that the mechanisms of different ginseng derived products in the treatment of HF were mainly related to pathways in cancer, PI3K-Akt pathway, Ras pathway, apoptosis, and mTOR pathway and other pathways, which shows that different derived products of ginseng have the characteristics of multi-target and multi-pathway in the treatment of HF. The results of molecular docking showed that the active components had good binding activity with potential targets, and the results of network pharmacology analysis were reliable to a certain extent.Forty ginsenosides were identified by UPLC-QE-Orbitrap-MS/MS analysis, including ginsenosides Rb1, Rc, Rd, Re, and Rh1.Based on database screening, 124 candidate targets for the treatment of HF were obtained from different derived ginsenosides of ginseng, including 22 core targets such as EGFR, AKT1, ERBB2, STAT3, TNF, ESR1, MTOR, HRAS, MMP9, and PIK3CA. Composition-target-disease-pathway network analysis showed that compound K, ginsenoside Rh4, ginsenoside Rk3, ginsenoside Rk1, ginsenoside Rg5, ginsenoside F4, and other components were associated with more targets. It may be an important component of various derived products of ginseng in the treatment of HF. Rg5 inhibited Drp1 activation and promoted HK-II mitochondrial binding through Akt activation. Rg5 prevented the opening of the mitochondrial permeability transition pore and increased ATP production, thereby enhancing cardiomyocyte resistance to hypoxia/reoxygenation injury. Jin also used the HPLC method to determine the contents of 13 kinds of monomer ginsenosides in white ginseng, RG, and black ginseng. The results showed that the contents of ginsenosides Rg1, Re, Rg2, Rb1, Rc, Rb2, and Rd decreased significantly, but the contents of ginsenosides (20R)-Rg2, Rg3, (20R)-Rg3, Rh1, Rk1, and Rg5 increased significantly during the steaming process of RG and black ginseng.In conclusion, compared with raw sun-dried ginseng and RG, black ginseng contains more rare ginsenosides, such as ginsenosides Rg3, (20R)-Rg3, Rs3, (20R)-Rs3, Rk1, Rg5, Rh2, CK, etc, while the content of common ginsenosides is very low, such as ginsenosides Rg1, Re, Rb1, Rc, Rb2, Rb3, Rd, etc. However, the content of common ginsenosides is very low, such as ginsenosides Rg1, Re, Rb1, Rc, Rb2, Rb3, Rd, etc. From the research results reported in the literature, it was found that the ginsenosides in black ginseng were mainly rare ginsenosides . However, the contents of common ginsenosides  were relatively low. These results indicated that common ginsenosides were converted into rare ginsenosides during processing. mTOR is an evolutionarily conserved protein that is widely involved in cell growth and development, regulation of protein synthesis, ribosome biogenesis, T cell fate selection, sensing and integration of upstream signaling pathways of various extracellular stimuli such as growth factors, nutrients, amino acids, starvation, and hypoxia stimuli. mTOR interacts with specific adaptor proteins to form 2 distinct macromolecular functional complexes called mTORC1 (mTOR complex 1) and mTORC2 (mTOR complex 2). mTOR is sensitive to rapamycin and negatively regulates autophagy; mTORC2 has no direct effect on autophagy. Partial and selective inhibition of mTORC1 provides cardiac protection in a variety of cardiac pathological conditions, suggesting that pharmacological inhibition of mTORC1 may represent a potential therapeutic intervention target for the treatment of cardiovascular disease. Studies have shown that atorvastatin can improve left ventricular function and remodeling in spontaneously hypertensive rat models, which may be achieved by regulating cardiomyocyte autophagy through the Akt/mTOR signaling pathway. Rapamycin can reduce myocardial hypertrophy, activate cardiomyocyte autophagy and reduce myocardial infarction size by inhibiting mTORC1.Rapamycin plays an anti-cardiac hypertrophy role by activating Akt, promoting protein ubiquitination, and inhibiting cardiomyocyte apoptosis and cardiac fibrosis.,29Through network pharmacology and molecular docking, ginsenosides were found to have the strongest binding force with mTOR in the treatment of HF. mTOR (mechanistic target of rapamycin) is a ~290 kDa, highly conserved atypical filament/threonine kinase and a member of the PIKK family.This study firstly elucidated the mechanism of multi-component, multi-target and multi-pathway action of various ginseng derivatives in the treatment of HF. It was found that the representative saponins contained in black ginseng have a good prospect in the treatment of HF and cardioprotection based on the mTOR pathway and autophagy, but further pharmacological experiments are still needed to verify this. At the same time, another limitation of this study is that it did not include polysaccharides, proteins, and other macromolecular compounds, which also need further investigation.Conceptualization: Tingting Dai.Data curation: Tingting Dai.Formal analysis: Tingting Dai.Funding acquisition: Jiyu Gong.Investigation: Tingting Dai.Methodology: Tingting Dai.Project administration: Jiyu Gong.Supervision: Shuying Liu.Validation: Shuying Liu.Writing – original draft: Tingting Dai.Writing – review & editing: Tingting Dai.


To objectively evaluate the efficacy of the Zilongjin tablets in non-small cell lung cancer (NSCLC) and to explore its potential mechanism of action against NSCLC and COVID-19 based on network pharmacology.The database was searched for randomized controlled trials (RCTs) of the Zilongjin tablets for NSCLC published up to 22 August 2022. The quality of included trials was assessed using Cochrane standard guidelines, and a meta-analysis was performed using Rev Man 5.3. Gene targets for intersections of NSCLC and COVID-19 (the NC) and drugs were obtained from the TCMSP database, HERB database, GeneCards database, and the NCBI database for network pharmacology research.Meta-analysis included 14 articles with 2,430 patients. The meta-analysis showed that the Zilongjin tablets combined with conventional chemotherapy were significantly more effective than chemotherapy alone in the treatment of NSCLC. A total of 29 drug-disease intersecting targets were identified in the network pharmacology. The “ingredient-target-pathway” diagram component-target-pathway network contained 119 nodes and 429 edges, with the majority of targets associated with inflammatory responses.The efficacy and quality of life of the Zilongjin tablets combined conventional chemotherapy for NSCLC were significantly better than chemotherapy alone, alleviating various adverse effects. At the same time, the Zilongjin tablets may modulate the inflammatory response to alleviate NSCLC and COVID-19. Lung cancer has a high incidence and mortality rate, with approximately 1.8 million new cases and 1.6 million deaths per year, and the 5-year survival rate of lung cancer ranges from 4 to 17%. Moreover, it is the leading cause of cancer-related death among men; and the second leading cause of cancer-related death among women worldwide . Non-smaAt the time of the COVID-19 pandemic, a large number of lung cancer patients face difficulty in admission to a hospital for treatment, and their risk of COVID-19 infection is 2.31 times higher than that of the general population, due to immunosuppression and respiratory infections –9. When A large number of clinical studies have shown that traditional Chinese medicine combined with chemotherapy can improve not only the anti-tumor effect but also enhance the immune function of the body . The Zilhttp://www.who.int/ictrp/en/), and US National Institutes of Health Ongoing Trials Register . Meanwhile, eligible trials were retrieved from the initial date until August 22, 2022. The following keywords and phrases, and their abbreviations or derivatives, were used separately or in combination: “the Zilongjin tablets” and “non-small cell lung cancer” or “NSCLC” and “RCT”. The search terms were adapted to different databases with a reliable search strategy developed by the Cochrane Collaboration , Wan-fang Database, China BioMedical Literature (CBM), MEDLINE (PubMed), EMBASE, Google Scholar, World Health Organization International Clinical Trials Registry ; disease control rate (DCR); Quality of life; and Adverse reactions.Two reviewers independently screened the titles and abstracts of searching results against pre-specified inclusion criteria to identify potential relevance. Disagreements were resolved by consensus. The third reviewer judged all articles included. Two reviewers systematically extracted data regarding study design, participant characteristics, interventions, and outcomes independently. Similarly, discrepancies were resolved by discussion between the two reviewers and consultation with the third reviewer.We included randomized controlled trials (RCTs) irrespective of blinding or language. We excluded quasi-randomized trials.Two reviewers independently extracted data, including details of the study population, intervention, and outcome, using a predesigned data extraction form. The following data were extracted: gross trial characteristics ; baseline of the patient and disease data ; interventions ways; and outcomes (outcome measures and the rate of adverse events). We resolved differences in data extraction by consensus or a third party. One reviewer entered data into Cochrane software Review Manager 5.3 (RevMan5.3) , and cheThe risk of bias evaluation tool proposed by the Cochrane Collaboration was utilized to address the following seven domains: random sequence generation, allocation concealment, blinding of participants and personnel, blinding of outcome assessment, incomplete outcome data, selective outcome reporting, and “other bias” . The ris2 statistics to assess the heterogeneity. A fixed-effect (FE) model was used if there was no significant heterogeneity in the data (I2 < 50%), and a random-effect (RE) model was used if significant heterogeneity was present (I2 > 50%)  .https://tcmspw.com/tcmsp.php), HERB database (http://herb.ac.cn/). Furthermore, all the drug components were collated and retrieved in the PubChem database (https://pubchem.ncbi.nlm.nih.gov/) to obtain the CID numbers, Smiles numbers, and canonical molecular formulas of the active ingredients by Python. The detected drug compounds were imported into the STITCH database (http://stitch.embl.de/cgi/input.pl) to obtain the gene targets interacting with the active ingredient of the Zilongjin tablets, selecting “Homo sapiens”, and screening the targets with a combined score >0.95. We also obtained the gene targets from the HERB database (P < 0.05). Then, we collated all gene targets and screened out duplicate items.The names of all the ingredients of Zilongjin Tablets were retrieved through the TCMSP database (https://www.genecards.org) and the NCBI database (https://www.ncbi.nlm.nih.gov) respectively. Additionally, we de-duplicated them with scores >10 in the GeneCards database. Bioinformatics & Evolutionary Genomics (http://bioinformatics.psb.ugent.be/webtools/Venn/) was used to intersect the genes of NSCLC and COVID-19, and the two disease intersections are referred to as the NC.The disease targets about the NSCLC and COVID-19 were retrieved from the GeneCards database (http://string-db.org) for the protein-protein interaction (PPI) network. We selected the species as “Homo sapiens”, and the protein interaction score as “high confidence > 0.9”, hiding the unlinked nodes, and kept the rest of the parameters unchanged to obtain the PPI network map. Furthermore, we obtained the interactions information file “string_interactions.tsv” from the String database, imported the data into Cytoscape 3.8.2 software to restring the topology of analysis results by “Network Analyzer” function, and made the PPI network map according to the degree value.Using the Bioinformatics & Evolutionary Genomics website, the gene targets of the Zilongjin tablets were intersected with the NC disease targets to obtain the targets of the Zilongjin tablets for both diseases. The 29 targets obtained by screening were imported into the STRING database (https://david.ncifcrf.gov/home.jsp) for Gene Ontology (GO) analysis, including biological process (BP), cellular component (CC), and molecular function (MF), and Kyoto Encyclopedia of Genes and Genomes (KEGG). The results were visualized and analyzed using a microbiology letter (http://www.bioinformatics.com.cn/login/)  , 18. MeaWe obtained the drug components corresponding to the intersecting targets from the HERB database, and afterward, we docked it with the specific active ingredients of the Zilongjin tablets. Cytoscape 3.8.2 software was used to construct the active ingredient-the NC target network of the Zilongjin tablets, and “merge” and “group attributes layout” were used to cluster the active ingredients. The node size was described by “degree.”The flowchart of the study search results is shown in The characteristics of the included 14 trials are summarized in As shown in 2 = 12.62, DF = 10 (P = 0.25); I2 = 21%]   . Meta-an2= 5.82, DF = 7 (P = 0.56); I2 = 0%] ; I2 = 2%). The fixed effect model was used for meta-analysis (P < 0.00001].Platelet counts were reported in 9 studies (650 cases), and there was no statistical heterogeneity . The fixed effect model was used for meta-analysis. The results of the meta-analysis showed that the hemoglobin of the experimental group was significantly higher than that of the control group, and the difference was statistically significant .Hemoglobin was reported in 11 studies (849 cases), and there was no statistical heterogeneity . The fixed effect model was used for meta-analysis. The results of the meta-analysis showed that the leukocyte of the experimental group was significantly higher than that of the control group, and the difference was statistically significant .Leukocytes were reported in 11 studies (725 cases), and there was no statistical heterogeneity . The fixed effect model was used for meta-analysis. The results of the meta-analysis showed that there was no significant difference in diarrhea between the experimental group and the control group .Diarrhea was reported in 6 studies (389 cases), and there was no statistical heterogeneity . The fixed effect model was used for meta-analysis. The results of the meta-analysis showed that nausea and vomiting of the experimental group were significantly lower than that of the control group, and the difference was statistically significant .Nausea and vomiting were reported in 8 studies (450 cases) . The fixed effect model was used for meta-analysis. The results of the meta-analysis showed that there was no significant difference in hair loss between the experimental group and the control group .Hair loss was reported in 2 studies (158 cases), and there was no statistical heterogeneity [ChiAR . ASR . HSL . SNL . SLR . SBH . CR . DI .A total of 149 active ingredients of AR, 276 of ASR, 104 of HSL, 94 of SNL, 253 of SLR, 113 of SBH, 279 of CR, and 21 of DI. Additionally, a total of 787 after de-weighting. Then, we obtained the stitch-PPI networks of the drugs .We obtained 397 COVID-19-related targets and 4,307 NSCLC-related targets in total, of which 267 were associated with COVID-19 and NSCLC .There are 29 targets where the gene targets of the Zilongjin tablets intersect with the NC .p-value: < 1.0e-16)  . In Cyto1.0e-16) . TNF , positive regulation of MAP kinase activity  and positive regulation of chemokine production , positive regulation of interleukin-8 production , et al. , extracellular region , and secretory granule , et al. related , enzyme binding , identical protein binding , heme binding  et al. , Inflammatory bowel disease , African trypanosomiasis , Antifolate resistance , et al. . A total of 285 entries in total for BP, mainly involving lipopolysaccharide-mediated signaling pathway  et al. . KEGG pa, et al. .The network for visual topology analysis of KEGG pathways contains 79 nodes with 322 edges , the greThe component-target-pathway network consists of 119 nodes and 429 edges. Among them, the 42 green nodes represent the active ingredients of the Zilongjin tablets, with high degree values such as lupeol, curcumin, and apigenin; the 25 pink nodes represent the targets of the Zilongjin tablets on the NC; the 52 purple nodes represent the pathways of the Zilongjin tablets on the NC . Lupeol Lung cancer continues to be the most lethal disease in the world . The 5-yPhlegm buildup and blood stagnation, which prevent the Qi of the lung from spreading and purifying, are the fundamental pathogenic factors for lung cancer. Cancer patients were experiencing immunity declining as the disease and its treatments and were often in touch with medical facilities during the COVID-19 outbreak because they needed therapy. Owing to hypo immunity and the lung damage caused by their lung cancer, patients are more susceptible to the SARS-CoV-2 of the COVID-19 virus, which can further exacerbate their condition. According to some disease reports, COVID-19 can cause severe infections and even death , 41. WitAccording to the theory of traditional Chinese medcine (TCM), “Yili's qi” is the main cause of COVID-19. Moreover, COVID-19's occurrence and development are affected by several pathogenic variables, including moisture, toxicity, cold, heat, and stasis, and the interpretation of the cause is intricate. Many researchers have dissected the interpretation of the cause of COVID-19 from the perspectives of the “warm and turbid,” “heat-toxin,” “cold-dampness,” “cold-damp, the volatile-dry combo,” and “plague-toxicity”, but the conclusion has not been unified. Nowadays, the majority of studies categorize COVID-19 into four phases: (1) Early-stage symptoms might include cold-dampness invading the lungs, heat-dampness invading the lungs, etc. (2) Middle-late-stage symptoms gradually deepen the degree, “appearing to become heat due to depression” (the pathogenic factor transforms into the heat-toxin due to long-term stagnation), “the stagnation of pathogenic dampness and heat” (the combination of dampness and heat damages the body), “phlegm obstructs the lung” , and “syndrome of lung collaterals injured by heat” . (3) In the recovery period, there is “intermingled deficiency and excess”. Although the healthy qi expelling pathogen, the qi is insufficient and the qi, blood, yin, and yang are deficient. Meanwhile, there is a possibility of recurrence of the disease due to the small amount of pathogen vested in the body. (4) In the critical stage, there are several symptoms, such as “syndrome of epidemic toxin blocking lung” .Yet, there is no variation in the viral strains' capacity to infect various groups when people are infected with the same type of COVID-19 virus (SARS-CoV-2). The varying immunological capacities of various people are an influencing factor in disease severity. Because of high-level immunity, the general population with COVID-19 often has milder symptoms. In contrast, patients with COVID-19 combined NSCLC generally have more severe symptoms due to low-level immunity, and the disease symptoms are mainly intermingled deficiency and excess. The following explanations apply: (1) NSCLC patients have lung damage, so lung qi is weaker to resist pathogenic factors . Additionally, the DUXIE (pathogenic factor) invading their bodies may even be guided by the patient's inherent pathogenic factors, aggravating the inherent condition. Then the pathogenic factors directly cause serious damage to the lungs, resulting in symptoms such as “phlegm obstructs the lung”, “syndrome of lung collaterals injured by heat”, and “syndrome of epidemic toxin blocking lung”. (2) NSCLC patients who have undergone chemotherapy have a deficient constitution, an imbalance of qi, blood, yin and yang, and dysfunction of the viscera. They are less able to fend off the virus than healthy individuals, which causes their disease state to change more quickly and unstablely than healthy individuals and increases their risk of the disease.The primary goal of treatment in the early, middle and late phases of the disease in healthy individuals with COVID-19 is to eradicate the DUXIE (pathogenic factor), avoiding the use of tonic formulations or medications that may induce DUXIE (pathogenic factor) stacking. Patients with COVID-19 and NSCLC, on the other hand, require a mix of corrective (the application of tonics) and detoxifying  treatment.The Zilongjin tablets, as a TCM treatment for lung cancer, have the ability to benefit Qi and nourish the blood, clear heat and detoxify the blood, and regulate Qi and resolve stasis. In this study, the mechanism of action of the Zilongjin tablet on COVID-19 combined with NSCLC was discussed through network pharmacology. The top three major drug components of the Zilongjin tablets are lupeol, curcumin, and apigenin, as shown in the “Component-Target-Pathway” diagram. Lupeol is a natural product found in the AR and other organisms with data available. It acts through numerous signaling pathways such as Inflammatory bowel disease and HIF-1 signaling pathway, and target IL6, STAT3. Curcumin is a natural dyestuff found in the ASR and CR, and it has a role as an anti-inflammatory agent, an antineoplastic agent, a hepatoprotective agent, and an anti-inflammatory agent. Additionally, antifolate resistance, the HIF-1 signaling pathway, and legionellosis are a few of the many signaling pathways through which it functions, and it targets NOS2, IL6, IL1B, NFKB1, TLR4, STAT3, BECN1, HIF1A, FASN, SELP, and other molecules. A natural substance called apigenin is present in SLR, SBH, and other organisms for which data are available, and functions as a metabolite modulator and antineoplastic agent, targeting NFKB1, STAT3, TGFB1, IL2, IFNG, CAT, CD274, and IFNG. These genes are involved in the inflammatory response, and inflammation-triggered immunoreaction plays a significant role in both NSCLC and COVID-19. Moreover, COVID-19-induced endothelial dysfunction will further exacerbate pathological transformation and cause more pronounced inflammation. A cohort study reported that the Neutrophil-to-lymphocyte ratio and platelet-to-lymphocyte ratio were discovered to be separate predictors of mortality in patients with NSCLC neo-coronary pneumonia . The Zil2 + cisplatin 25 mg/m2; (ii) mitomycin (MMC) 6 mg/m2 + vincristine (VDS) 3 mg/m2 + cisplatin (DDP). The other 10 studies designed only one chemotherapy regimen, such as irinotecan 175 mg/m2 + carboplatin (AUC = 5), docetaxel 75 mg/m2 + cisplatin 20 mg/m2, gemcitabine hydrochloride 1,000 mg/m2 + cisplatin 60 mg/m2, etc. The chemotherapy regimens were not consistent across studies and may have an impact on treatment outcomes, as well as on the results of this study.There are some limitations to this study. Fourteen RCTs were national studies and may be subject to some bias. Three included studies explicitly stated that they used a randomization table to achieve randomization, while other studies did not report randomization procedures, and only 1 article reported a double-blind approach, factors that may contribute to implementation bias, and measurement bias. Although objective response rate analyses reported that the Zilongjin tablets in combination with chemotherapeutic agents were significantly more effective than chemotherapy alone in the treatment of NSCLC. According to objective response rate analyses, due to the small sample sizes of the included studies, there was some clinical heterogeneity that affected the analysis's outcome. The dosage of the Zilongjin tablets was consistent across the RCTs , but the chemotherapy regimens used varied, with two chemotherapy regimens set up in Wang and Yan study  (i) noreIn this study, we comprehensively evaluated the efficacy of the Zilongjin tablets in the treatment of NSCLC. The efficacy and the degree of improvement in the quality of life of the Zilongjin tablets combined with chemotherapy in the treatment of NSCLC were significantly better than those of chemotherapy alone, and they could attenuate various adverse effects brought about by the disease or chemotherapy. In the network pharmacology, it reported that the efficacy of the Zilongjin tablets against NSCLC combined with COVID-19 might be achieved through the bidirectional regulation of inflammation, while further laboratory analysis was required for subsequent efficacy verification.All data used in this article are from public databases. The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author.WY and XX contributed to the conception and design of the study. WY, YZ, and YY organized the literature review database and performed the statistical analysis. JG, PH, and XX organized the pharmacological database and performed the statistical analysis. WY, YZ, and XX wrote the first draft of the manuscript. JG wrote sections of the manuscript. JG and XX contributed to the manuscript revision. All authors read and approved the submitted version.


Traditional Chinese medicine (TCM) is widely used in the clinical treatment of hepatolenticular degeneration (HLD) and liver fibrosis (LF). In the present study, the curative effect was assessed using meta-analysis. The possible mechanism of TCM against LF in HLD was investigated using network pharmacology and molecular dynamics simulation.For literature collection, we searched several databases, including PubMed, Embase, Cochrane Library, Web of Science, Chinese National Knowledge Infrastructure (CNKI), VIP Database for Chinese Technical Periodicals (VIP) and Wan Fang database until February 2023, and the Review Manager 5.3 was used to analyze the data. Network pharmacology and molecular dynamics simulation were used to explore the mechanism of TCM in treating LF in HLD.p = 0.002]. It not only has a better effect on liver protection  but also had an excellent therapeutic effect on LF through four indexes . Concurrently, the liver stiffness measurement decreased significantly . The results of network pharmacological experiments and molecular dynamics simulation indicate that the three high-frequency TCMs  primarily act on the core targets  via the core components , regulate the signal pathway , and play a role of anti-LF.The results of the meta-analysis revealed that the addition of Chinese herbal medicine (CHM) in treating HLD resulted in a higher total clinical effective rate than western medicine alone [RR 1.25, 95% CI , Meta-analysis indicates that TCM is beneficial in treating HLD patients and improving LF. The present study successfully predicts the effective components and potential targets and pathways involved in treating LF for the three high-frequency CHMs of DH-HL-JH. The findings of the present study are hoped to provide some evidence support for clinical treatment.https://www.crd.york.ac.uk/PROSPERO, identifier: CRD42022302374. Hepatolenticular degeneration (HLD), also known as Wilson's disease (WD), is a rare chronic autosomal recessive disease that presents a hereditary copper metabolism disorder . A mutatHowever, under the guidance of the theory of TCM, individualized treatment is common in clinics. Although both patients are WD, the choice and compatibility of Chinese herbal medicine (CHM) differ. Simultaneously, some research reports have methodological defects, one-sided theoretical premises and a lack of experimental design, all of which reduce the effectiveness and universality of the research findings. In this systematic review, using an evidence-based research strategy, after a meta-analysis of clinically effective prescriptions in the treatment of WD, the three CHMs with the highest frequency of effective prescriptions were selected to investigate the mechanism of the treatment of liver fibers in WD using network pharmacology and molecular dynamics simulation. This is useful to determine future basic research direction and screen suitable active ingredients for clinical trials.https://www.crd.york.ac.uk/PROSPERO).This systematic review with meta-analysis was registered with the number CRD42022302374 , VIP Database for Chinese Technical Periodicals (VIP) and Wan Fang database without language restrictions. The search strategy of PubMed was used, but it was modified to work with other English or Chinese databases. The PubMed search strategy was as follows:#1*”[Text Word] OR “Pseudosclerosis”[Text Word] OR “wilson disease*”[Text Word] OR “wilson s disease*”[Text Word] OR “wilsons disease*”[Text Word] OR “westphal strumpell syndrome*”[Text Word] OR “copper storage disease*”[Text Word] OR “progressive lenticular degeneration*”[Text Word] OR “neurohepatic degeneration*”[Text Word]“Hepatolenticular Degeneration”[MeSH Terms] OR “hepatolenticular degeneration#2“Liver Cirrhosis”[MeSH Terms] OR “hepatic cirrhosis”[Text Word] OR “cirrhosis hepatic”[Text Word] OR “cirrhosis liver”[Text Word] OR “fibrosis liver”[Text Word] OR “liver fibrosis”[Text Word]#3#1 AND #2#4“drugs, chinese herbal”[MeSH Terms] OR “Chinese Herbal Drugs”[Text Word] OR “Chinese Plant Extracts”[Text Word] OR “Herbal Medicine”[MeSH Terms] OR “medicine herbal”[Text Word] OR “Hawaiian Herbal Medicine”[Text Word] OR “la au lapa au”[Text Word] OR “Herbalism”[Text Word] OR “la au lapa au”[Text Word]#5*”[Title/Abstract] OR “placebo”[Title/Abstract] OR “trial”[Title/Abstract] OR “groups”[Title/Abstract] OR “crossover”[Title/Abstract] OR “cross-over”[Title/Abstract]) NOT ).Subjects: patients with clinically confirmed WD LF  , Chinese , ChinesIntervention: patients in the control group will receive routine Western medicine, while patients in the intervention group will receive TCM based on control Western medicine.Outcome index: the main observation indexes include clinical effective rate  and liver manifestations .Trial group was only treated with TCM in the study design.Non-randomized grouping; Randomized method obvious error.In vitro or animal studies.Case reports, reviews, meta-analyses or laboratory studies only.To avoid the analysis of duplicate data, the latest study was included in the current study when two (or more) studies had the same cohort of patients.https://tcmspw.com/index.php) was used to search for active ingredients and potential targets of “high-frequency TCM” (https://pubchem.ncbi.nlm.nih.gov/) (http://www.swisstargetprediction.ch/) (https://www.uniprot.org/), standardize the protein name, limit the species to “homo sapiens,” and remove duplicate targets (https://www.genecards.org/) (https://www.omim.org/) (https://www.omicshare.com) was used to obtain the intersection targets of “high-frequency TCM” and LF. Cytoscape3.8.2 Software created the “TCM-active ingredient-potential target” network (TCMSP database (ncy TCM” . The actncy TCM” . The “Caih.gov/)  and impoion.ch/) . The tarion.ch/) . Then, u targets . The disds.org/)  and OMIMim.org/) . The tarim.org/) . The omi network .https://www.string-db.org/) (https://panglaodb.se/index.html) (The intersection target was imported into the STRING platform (db.org/) , and thedb.org/) . Cytoscadb.org/)  and MCODdb.org/)  plug-insdb.org/) . Becauseex.html)  was usedp-values < 0.05 and Q-values < 0.05 statistically significant.The omicshare platform enriched the core genes by Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG). The primary use of GO functional analysis is to describe the functions of gene targets, including biological processes, cellular components and molecular functions. KEGG enrichment analysis can determine the signal pathway of intersection target enrichment. The Fisher test considered The amber18 software package was used to perform the molecular dynamics (MD) simulation of the protein-small molecule complex. The protein uses the ff14SB force field parameter, whereas the small molecular ligand uses the gaff general force field parameter and calculates its AM1-BCC atomic charge using the ANTECHAMBER module. After loading the protein-small molecule complex into the tleap module, hydrogen atoms and antagonistic ions are automatically added to neutralize the charge. The TIP3P dominant water model is selected, and periodic boundary conditions are established. MD simulation workflow includes four steps: energy minimization, heating, balance, and production dynamics simulation. First, the energy of water molecules is minimized in 10,000 steps  by constraining the heavy atoms of proteins . The constraints are then released in 10,000 steps to optimize the energy of the entire system. After energy minimization, the system is slowly heated to 300 K at 50 ps time, and once heated, the system is balanced at 50 ps using the NPT ensemble. Finally, a 100 ns MD simulation is performed under an NPT ensemble with time step 2 fs. Every 20 ps, the trajectory data are saved, and the correlation analysis is performed using the CPPTRAJ module. The MMPBSA.py module calculated the binding free energy of ligand and protein.A preliminary search in the electronic database identified 91 studies. In addition, two potentially eligible records were identified through manual searches, of which 66 remained after the removal of duplicates. After screening titles and abstracts, 49 articles about non-clinical trials, animal trials, and case reports not focusing on the efficacy of TCM or not related to WD LF were excluded. Following a full-text evaluation of the remaining 17 articles, seven were excluded based on the research purpose and screening criteria . Finally, ten trials were identified .The included studies were all published in Chinese between 2000 and 2022. With 674 participants, all ten studies –18, 33 wThe quality of the method is assessed using seven criteria in the Cochrane collaboration tool. All of the included studies indicated randomization. The method of randomization was reported in five studies –16, 33, p = 0.57, I2 = 0%, using fixed effect model, total effect test n = 197, RR 1.25, 95% CI [1.09 to 1.44], Z = 3.31, p = 0.002 < 0.05; ; I2 = 74%) ; I2 = 91%) ; I2 = 95%)  index. Meta-analysis revealed that  . Based o2 = 91%) . Based o2 = 95%) . Within n = 569, SMD = −1.15, 95% CI [−1.76 to −0.53]; Z = 3.66, p = 0.0003; heterogeneity: chi-square = 88.87, df = 8 (p < 0.00001); I2 = 91%) ; I2 = 90%) ; I2 = 88%) ; I2 = 85%)  (2 = 91%) , procoll2 = 90%) , and col2 = 88%)  than the2 = 85%) .Although only one study used liver ultrasound as a liver function index for observation and analysis, combining traditional Chinese and Western medicine significantly improves liver cirrhosis compared to Western medicine alone. The improvement rate of integrated traditional Chinese and Western medicine was 54.0% (27/50), while the simple Western medicine group improved at a rate of 44.0% (22/50),  .n = 195, SMD = −1.06, 95% CI [−1.77 to −0.36]; Z = 2.95, p = 0.003)   . We anal < 0.05) . After e < 0.05) , the het < 0.05) .TCM treats WD by focusing on the concept, syndrome differentiation, and treatment. Several clinical and experimental studies revealed that TCM has an excellent effect on WD treatment. TCM believes that the cause of WD is a lack of congenital endowment and that the accumulation of copper toxin in the liver due to spleen deficiency is the leading cause of LF in WD. Numerous clinical data demonstrated that TCM could reduce the accumulation of copper in the body, protect and promote brain and liver function recovery, improve patient quality of life and prolong the life cycle , 35. CliBased on our review, we recorded and sorted the CHMs used in clinical trials that can effectively improve WD liver fibers . The thrTCMSP database yielded 16 active ingredients of Rhei Radix Et Rhizoma, 14 Coptidis Rhizoma, and two species of Curcumae Longae Rhizoma. Simultaneously, a literature search reveals that curcumin can effectively inhibit the occurrence and development of LF. Therefore, it is supplemented. The PPI network included 131 nodes and 1,212 edges . Nine tap-value. The findings of the biological process analysis revealed that the intersection targets were primarily enriched in the regulation of DNA replication, cell death, reactive oxygen species metabolic process, growth, and positive regulation of phosphorylation, as shown in We intercepted the first 30 terms, from smallest to largest, based on the adjusted p-value. The analysis revealed that these targets were mostly enriched in the PI3K-Akt, EGFR, MAPK, and VEGF signaling pathways, as presented in We intercepted the top 30 KEGG pathways from smallest to largest based on the Eight core targets and six key ingredients were docked molecularly. Rhein, eupatin, quercetin, berlambine, stigmasterol, and curcumin all had high binding energies to the core targets, with quercetin and JUN having the highest binding energy of −9.9 kJ/mol . The molMD simulations reveal information about the stability of protein-ligand complexes. The present study performed 100 ns MD simulations of JUN-quercetin, AKT1-Berlambine, JUN-Berlambine and SCR-Rhein based on the docking results to evaluate the molecular motion, trajectory, structural characteristics, binding potential and conformational changes. The root mean square deviation (RMSD) curve represents the fluctuation of the protein conformation. The RMSD of the protein in the complex is on the left , and theIn the present study, meta-analysis was used to assess the efficacy of traditional CHM in treating WD. We identified that interventional therapy with TCM brought positive results with few adverse reactions in ten clinical trials. Evidence of a more effective combination of traditional Chinese and Western medicine is emerging, which may assist in recommending clinical treatment. However, more high-quality trials are required to confirm this hypothesis. Simultaneously, based on the effectiveness of TCM in the clinical treatment of WD, we investigated its efficacy in treating WD LF. Nine trials using four indicators of LF reveal that integrated traditional Chinese and Western medicine can more effectively improve WD LF. Because clinical manifestations of LF lack specificity , the obsBased on the standard of “OB ≥ 30% and DL ≥ 0.18,” we searched the TCMSP database and identified 34 “DH-HL-JH” active ingredients via a literature supplement. Then, using the OMIM and GeneCards databases, we identified 881 LF disease targets. A total of 140 potential “DH-HL-JH”-LF targets were identified. According to the degree value of the nodes of the “DH-HL-JH” anti-LF target network, the main ingredients of Rhei Radix Rt Rhizome, Coptidis Rhizome, and Curcumae Longae Rhizome are rhein and eupatin, quercetin and berlambine, and stigmasterol and curcumin.4) / ethanol injured rats, as well as the expression of α-SMA and TGF-β1. Rhein can prevent LF by inhibiting TGF-β1 and activating HSCs (The liver of WD patients can exhibit various injury patterns, with LF being one of the pathological processes. LF is part of the tissue repair response to chronic liver injury . Whateveing HSCs . Li et aing HSCs  demonstring HSCs . The deving HSCs .4-induced LF (Curcumin, a diarylheptane derivative found in Curcumae Longae Rhizoma, is a powerful fat-soluble antioxidant . Curcumiduced LF . The actUsing cytoHubba and MCODE plug-in in Cytoscape software, eight core targets are obtained using different algorithms. The target AKT1 is an AKT kinase subtype. Reyes-Gordillo et al. demonstrate that AKT1 participates in ethanol-and lipopolysaccharide-mediated LF and inhibits hepatocytes and HSC fibrogenesis and proliferation, which plays a unique role in the fibrosis process . SRC kinGO function analysis revealed that various biological functions, including DNA metabolic process, cell death, reactive oxygen species metabolic process and signal transduction, were linked to the role of “DH-HL-JH” in LF. The results of the KEGG pathway analysis indicated that multiple pathways, including PI3K-Akt, MAPK, VEGF signaling pathways and EGFR tyrosine kinase inhibitor resistance, were linked to the role of “DH-HL-JH” in LF. Sun et al.  demonstrFinally, docking was performed on six core ingredients and eight key target proteins. The binding affinities of the docking results ranged from −6.4 to −9.9 kcal/mol, indicating that all targets may have good docking ability with active ingredients. The docking results revealed that the four JUN-quercetin, AKT1-berlambine, JUN-berlambine, and SRC-rhein groups had a low binding affinity. The interaction of the four groups mentioned above was studied further by MD simulation. The visualization of the RMSD curve in the JUN-quercetin group revealed that, despite fluctuations, the small molecules normally returned to their initial docking position, indicating that they could bind stably to the receptor. The visualization of the RMSD curve in the AKT1-berlambine group revealed that the protein and small molecules formed a stable complex conformation. Small molecule binding had an insignificant effect on the protein conformation. The visualization of the RMSD curve of the JUN-berlambine group indicated that the binding position of small molecules was shifted to some extent compared with the initial docking position.Similarly, the visualization of the RMSD curve in the SRC-rhein group demonstrated that the protein and small molecules formed a stable complex conformation. Small molecule binding initially affected the protein conformation but was eventually stable after the induced fit. The conformational comparison results of the four groups before and after kinetics demonstrate that the small molecules bind stably to the receptor protein and have good binding activity, indicating that these active ingredients may contribute to the therapeutic effect of “DH-HL-JH” in WD-associated LF. However, the mechanism of high-frequency TCM against LF remains unknown.In the present study, CHM is generally safe and well-tolerated in WD patients. However, it is still impossible to confirm the safety of using CHM because only some studies mentioned intervention safety or investigated adverse reactions. It is suggested that more effort be put into documenting and reporting these interventions' adverse effects. Simultaneously, WD is a chronic disease that requires lifelong treatment. Long-term efficacy and safety are important evaluation indicators for determining the clinical effectiveness of drug therapy. However, the duration of treatment in the present study was 32 to 336 days. The long-term safety of TCM in treating WD is unknown due to the short course of treatment. Related studies indicated that  treatmenThe combination of high-frequency CHMs may be an effective treatment for WD LF and may have potential therapeutic value for patients with various TCM syndrome types.For patients with WD LF, there has been a gradual increase in the use of integrated traditional Chinese and Western medicine over the last few decades. The meta-analysis has shown TCM to be beneficial in treating WD LF patients and improving LF. Through network pharmacological experiments and MD simulation technology, it was identified that the three high-frequency CHMs of Rhei Radix Et Rhizoma-Coptidis Rhizoma-Curcumae Longae Rhizoma, could act on the core targets of LF, such as AKT1, SRC, and JUN through the core ingredients such as rhein, quercetin and berlambine, regulate the signal pathway and play the role of anti-LF.The original contributions presented in the study are included in the article/PW and XY proposed and designed this study. XY, TW, YS, and YT jointly completed the data acquisition. XY and TW analyzed the data, interpreted the results, and contributed to writing the manuscript. PW and YG critically revised the manuscript for important intellectual content. All authors read and approved the final manuscript.


RT-PCR and Western blotting analysis confirmed that saffron treatment can prevent the CCl4-induced upregulation of HIF-1α, VEGFA, AKT, and PI3K, suggesting that saffron may regulate AKT/HIF-1α/VEGF and alleviate liver fibrosis.Liver fibrosis refers to a complex inflammatory response caused by multiple factors, which is a known cause of liver cirrhosis and even liver cancer. As a valuable medicine food homology herb, saffron has been widely used in the world. Saffron is commonly used in liver-related diseases and has rich therapeutic and health benefits. The therapeutic effect is satisfactory, but its mechanism is still unclear. In order to clarify these problems, we planned to determine the pharmacological effects and mechanisms of saffron extract in preventing and treating liver fibrosis through network pharmacology analysis combined with in vivo validation experiments. Through UPLC-Q-Exactive-MS analysis, a total of fifty-six nutrients and active ingredients were identified, and nine of them were screened to predict their therapeutic targets for liver fibrosis. Then, network pharmacology analysis was applied to identify 321 targets for saffron extract to alleviate liver fibrosis. Functional and pathway enrichment analysis showed that the putative targets of saffron for the treatment of hepatic fibrosis are mainly involved in the calcium signaling pathway, the HIF-1 signaling pathway, endocrine resistance, the PI3K/Akt signaling pathway, lipid and atherosclerosis, and the cAMP signaling pathway. Based on the CCl It is ufibrosis , while P pathway .Crocus sativus L., and the yield is low. It takes about 20 flowers to obtain 1 g stigma, which is a valuable medicinal material. Saffron is rich in carotenoids, carbohydrates, proteins, anthocyanins, vitamins and minerals, as well as in crocin, picrocrocin, and safranal. Saffron is considered to be an herb with a wealth of health benefits and nutritional value in folk medicine [Saffron is a traditional dual-use natural medicine with multiple pharmacological functions planted in China, Iran, Spain, India, and other countries. Saffron can be used not only as a medicine but also as a dye, spice, and condiment, which has been used for thousands of years . Saffronmedicine ,12. Saffmedicine . In addi4-induced liver fibrosis.Currently, saffron is mainly used for treating cardiovascular diseases and liver diseases in clinics. At present, there are many preparations containing saffron , such asUPLC-MS/MS technology was used to qualitatively analyze the chemical components of saffron, and the total ion current (TIC) diagram was obtained in positive and negative ion modes . The datAccording to the standard compounds, existing literature data, and obtained MS data, a total of 56 chemical components were identified. Based on the ADME properties of the components and their content in saffron, we identified nine components that can be used as the representative components of subsequent network pharmacology for analysis .Nine active ingredients in saffron were identified through screening, and 435 potential targets of nine active ingredients in saffron were obtained based on database screening. We performed a Venn analysis on 6134 candidate targets related to liver fibrosis in the database and potential targets of active ingredients in saffron, of which 321 overlapped A. We hypA total of 321 potential targets of saffron were imported into the string database and Cytoscape 3.7.1 to obtain the PPIs network A. The PPKEGG and GO enrichment analyses were performed on 255 targets obtained by PPI analysis. The biological processes involved in target enrichment are mainly related to chemical–organic substance reactions. In terms of the enriched molecular functions, the target proteins were mainly related to protein binding, signal transduction activity, and macromolecular complex binding A. This sp < 0.01) and liver index (−17.8% and −27.6%) (p < 0.05 and p < 0.01)  ( < 0.01) .4 treatment leads to serious liver injury. Importantly, saffron treatment significantly reduced serum AST (−33.1% and −34.1%) (p < 0.01), ALT (−49.9% and −63.4%) (p < 0.001), and TBIL (−19.7% and −22.1%) (p < 0.05) levels in a dose-dependent manner, indicating that saffron significantly reduces CCl4-induced hepatotoxicity , and the brownish-yellow area became darker, mainly distributed in the portal vein area and the fiber area, and the degree of hepatic fibrosis deepened in the model group. Compared with the model group, the positive expression area of α-SMA and Col II in the liver tissue of the saffron group were significantly reduced (p < 0.01), and the brown-yellow area was reduced in area and lightened in color . The mRNA levels of the saffron-treated group were significantly lower than those of the model group.The mRNA and protein expression levels of the genes involved in the AKT/HIF-1/VEGF signaling pathway obtained from the network pharmacological screen were determined by RT-PCR and Western blotting. RT-PCR was used to detect the mRNA expression levels of AKT/HIF-1α/VEGF signaling pathway-related genes . The resCompared to the model group, the saffron group decreased the expression ratio of p-AKT/AKT. This is a downstream molecule of PI3K that activates the VEGF/HIFA signaling pathway. The result suggests that saffron affected the expression of p-AKT in fibrotic liver tissue. This result demonstrates that saffron has anti-inflammatory activity by inhibiting the PI3K/AKT-mediated VEGF/HIFA signaling pathway, thereby reducing the release of cytokines and thus attenuating the inflammatory response in the liver fibrosis process and inhibiting pathological angiogenesis .4-induced liver fibrosis model, saffron inhibited the inflammatory infiltration in the process of liver fibrosis by regulating and inhibiting the inflammatory AKT/PI3K signaling pathway, while regulating HIF-1α/VEGF signaling pathway can improve the insufficient blood supply of hepatocytes, vascular diseases, and cell hypoxia caused by the destruction of liver tissue structure, as shown in Liver fibrosis is a complex disease, and current treatment options for liver fibrosis remain limited. The research results prove that saffron has a therapeutic effect on liver fibrosis, and its mechanism of action is related to the regulation of the AKT/HIF-1α/VEGF signaling pathway. In the CClThe existence of long-term inflammatory reactions and continuous hepatocyte damage are the main factors that can induce liver fibrosis. Through network pharmacology, we screened that Akt/PI3K signaling pathway is the main inflammatory pathway of saffron to improve liver fibrosis. Existing studies have shown that saffron can treat liver diseases through its anti-inflammatory effect. Safranal, the main bioactive component of saffron, can significantly inhibit the proliferation of liver cancer cells and induce apoptosis, showing anti-inflammatory properties, and having significant inhibition effects on inflammatory markers such as NF-κB, COX2, iNOS, and TNF-α . Some stLiver fibrosis and cirrhosis belong to the category of blood stasis in TCM. The methods of treating liver fibrosis in TCM mainly focus on herbs of promoting blood circulation and removing blood stasis . HepaticALT, AST, and TBIL kits were purchased from Wuhan Servicebio Biotechnology Co., Ltd. . Antibody human α-SMA and Col II are from Abcam .Saffron was purchased from Sichuan NEAUTUS Traditional Chinese Medicine Co., Ltd. . We prepared saffron sample powder by grinding accurately weighed 80 mg of saffron powder, placing it in a 50 mL brown centrifuge tube, adding 20 mL of pure water, and extracting it by ultrasound in 25 °C water for 30 min. After centrifuging the extract at 14,000 r/min for 10 min, we took the supernatant and put the saffron sample away for storage at −4 °C for future use.Chromatographic conditions of saffron extract: mobile phase A (0.1% formic acid water)—mobile phase B (acetonitrile), elution gradient 0~5 min, 95~70% A; 5~25 min, 70~20% A; Flow rate 0.2 mL/min; Column temperature 30 °C, injection volume 3 μL.m/z 50–1200 Da; scanning time 0.2 s.Mass spectrometry conditions: capillary voltage 2.5 kV; source temperature 120 °C; desolvent gas temperature 450 °C; desolvent gas flow rate 600 L/h; air flow rate in conical hole 50 L/h; spray 6.5 bar; source offset ion scanning range https://tcmspw.com (accessed on 28 May 2022)). Next, we searched PubChem (https://pubchem.ncbi.nlm.nih.gov (accessed on 30 May 2022)), Drugbank (https://www.drugbank.ca/drugs (accessed on 2 June 2022)), and used a molecular similarity matching tool based on Swisstargetprediction (http://www.swisstargetprediction.ch (accessed on 2 June 2022)) and a molecular similarity matching tool based on TCMSP to identify potential target proteins of saffron extracts. Then, through the Genecards server (https://www.genecards.org (accessed on 2 June 2022)), the related targets of liver fibrosis were determined by the Online Mendelian genetic man (OMIM) database. Based on the previous steps, two groups of targets, drug-related genes, and disease targets were prepared. Using the Venn map software (http://bioinformatics.psb.ugent.be/webtools/Venn/ (accessed on 2 June 2022)), cross genes were screened by R software (version 3.5.1). According to the interaction data, we constructed the interaction network of the active components and hypothetical targets of saffron extract in the treatment of liver fibrosis and visualized the PPIs network with Cytoscape software (version 3.7.1). The network analyzer plug-in and cytonca plug-in Cytoscape were used to measure the topological importance of nodes and networks. Finally, using the Bioconductor software package (version 3.17), including the Panther classification system, bioinformatics annotation evaluation was performed on proteins with overlapping expression patterns using R software (version 3.5.1), GO annotation database website (http://geneontology.org (accessed on 10 June 2022)), and KEGG pathway enrichment analysis (https://www.kegg.jp (accessed on 12 June 2022)).The active chemical constituents of saffron extracts were obtained by LC/MS analysis combined with TCMSP (4 dissolved in olive oil (twice a week) for 6 weeks. Meanwhile, the saffron group was gavaged with saffron extract once a day, and the control and model groups were gavaged with equal volume of 0.9% saline for 6 weeks.Male KM mice (8 weeks old) were purchased from Institute of Laboratory Animals, Chongqing Institute of Traditional Chinese Medicine, Animal License No. SCXK  2017-0003. This study was reviewed and approved by the Animal Ethics Committee of Chengdu University of Traditional Chinese Medicine (2014DL-023). The animals were acclimatized to the environment for 1 week before the experiment. Mice were housed in an environment with controlled temperature (22 ± 2 °C) and humidity (60 ± 10%), with a light/dark cycle of 12 h, and fed with standard chow. Mice were randomly divided into four groups (N = 8): control, model, saffron high-dose group (100 mg/kg), and saffron low-dose group (50 mg/kg). A mouse model of hepatic fibrosis was prepared by intraperitoneal injection of CClAt the end of the experiment, the weight of the liver and spleen were recorded. Calculation of liver coefficient and spleen coefficient (organ coefficient (%) = organ (g)/body weight (g) × 100%) was conducted.g for 15 min and subjected to biochemical analysis. The liver function indices  were measured by ELISA kit.Blood samples were collected at the end of the study. The serum was centrifuged at 3000× Livers were fixed with 4% paraformaldehyde for 48 h, embedded in paraffin, cut into 5 µm thick sections and stained with hematoxylin eosin. The slices were washed twice in acidified water and then dehydrated with 100% ethanol. The slices were immersed in Masson A solution overnight, and then washed with tap water slightly; the excess water was slightly drained. The slices were then immersed in Masson D solution for 6 min and the excess water was drained as much as possible. The slices were then treated with Masson E solution for about 1 min, they were not washed, and the excess Masson E solution was drained slightly. The slices were then directly dyed in Masson F solution for 2–10 s, and then dehydrated with 100% ethanol. The positive staining area was analyzed and calculated by the Image-Pro Plus 6.0 system.Paraffin-embedded liver tissue sections of 5 µm thickness were subjected to immunohistochemistry, deproteinization, and 0.3% hydrogen peroxide treatment for 15 min to block endogenous peroxidase activity. Liver sections were further blocked with 2% bovine serum albumin and then reacted with rabbit α-SMA and Col II and incubated at 4 °C for 16 h. After washing, the sections were incubated with BSA for 60 min at room temperature. Samples were analyzed by light microscopy and α-integrated optical density (IOD) values expressed as α-SMA and Col II were detected using Image pro + 6.0 software.The protein samples were loaded into 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to the PVDF membrane. Membrane culture in QuickBlock™ buffer  was blocked at room temperature for 1 h and then incubated with primary antibody AKT1, VEGFA, HIF1A, PI3K, P-AKT, and P-PI3K from Affinity  at 4 °C overnight. The membrane was then incubated with HRP binding secondary antibodies at room temperature for 1 h. The bands were observed using the chemiluminescence kit.Total RNA was extracted from liver samples by Trizol* Reagent and converted into complementary DNA using qscriptcdna super mixing reagent. Using platinum according to the manufacturer’s instructions, *SYBR* Green q-PCR was used to detect the gene expression at the target gene level. The PCR primer sequences used in this study are shown in p < 0.05 in different groups was considered significant.All data were expressed as mean ± SEM, and one-way analysis of variance (ANOVA) was performed, followed by the Bonferroni posttest. In conclusion, our results provide new evidence that saffron ameliorates inflammatory infiltration in liver fiber processes, as well as vasculopathy and cellular hypoxia in a model of fibrosis through AKT/HIF-1α/VEGF signaling. Our study shows that saffron can be used as a candidate drug and health food for the prevention and treatment of liver fibrosis.