Objective: Yinchen Sini decoction (YCSND), a traditional Chinese medicine (TCM) formula, plays a crucial role in the treatment of liver disease. However, the bioactive constituents and pharmacological mechanisms of action remain unclear. The present study aimed to reveal the molecular mechanism of YCSND in the treatment of acute liver injury (ALI) using integrated network analysis and metabolomics.Methods: Ultra-high-performance liquid chromatography coupled with Q-Exactive focus mass spectrum (UHPLC-QE-MS) was utilized to identify metabolites in YCSND, and high-performance liquid chromatography (HPLC) was applied to evaluate the quality of four botanical drugs in YCSND. Cell damage and ALI models in mice were established using CCl4. 1H-NMR metabolomics approach, along with histopathological observation using hematoxylin and eosin (H&E), biochemical measurements, and reverse transcription quantitative real-time PCR (RT-qPCR), was applied to evaluate the effect of YCSND on CCl4- induced ALI. Network analysis was conducted to predict the potential targets of YCSND in ALI.Result: Our results showed that 89 metabolites in YCSND were identified using UHPLC-QE-MS. YCSND protected against ALI by reducing the levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), and malondialdehyde (MDA) contents and increasing those of superoxide dismutase (SOD), and glutathione (GSH) both in vivo and in vitro. The 1H-NMRmetabolic pattern revealed that YCSND reversed CCl4-induced metabolic abnormalities in the liver. Additionally, the Kyoto Encyclopedia of Genes and Genome (KEGG) pathway enrichment analysis identified five pathways related to liver injury, including the PI3K-AKT, MAPK, HIF-1, apoptosis, and TNF signaling pathways. Moreover, RT-qPCR showed YCSND regulated the inflammatory response  and apoptosis , and inhibited PI3K-AKT signaling pathway (Pi3k and Akt1). Combined network analysis and metabolomics showed a link between the key targets  and vital metabolites  of YCSND in ALI.Conclusion: Overall, the results contribute to the understanding of the therapeutic effects of YCSND on ALI, and indicate that the integrated network analysis and metabolomics could be a powerful strategy to reveal the pharmacological effects of TCM. CCl4 istoxicity . CCl4-ingenesis. . In the genesis. . Howevergenesis. , indicat4-induced ALI  and aspartate aminotransferase (AST) levels, Smad3 and Smad7 mRNA expression, and collagen I and III expressions in the liver. However, the effectiveness of only YCSND in ALI treatment, and its molecular mechanism are yet to be elucidated.Traditional Chinese medicine (TCM) formulas are commonly used to treat ALI, with the advantages of fewer side effects and multiple metabolites with several targets . Severaluced ALI . Yinchenthe body . Recent the body , glycyrrthe body , and chlthe body  have beethe body  and was the body . HoweverTraditional network analysis is typically used to predict potential targets and molecular mechanisms of TCM in disease treatment, using data mining and scientific computing techniques based on public databases . Network1H-NMR technology is a beneficial tool for analyzing metabolites and has several advantages, such as reliable results, nondestructive sampling, and minimal sample preparation . Moreover, network predictions and metabolomics were integrated to elucidate the molecular mechanisms of YCSND in liver injury from multiple perspectives. The present study serves as a theoretical basis for the therapeutic use of YCSND in liver diseases, offering novel insights into the study of TCM formulations. The graphic abstract of this study is shown in Artemisia capillaris Thunb. , Aconitum carmichaelii Debeaux , Zingiber officinale Roscoe , and honey-fried Glycyrrhiza glabra L.  were purchased from the drug department of Yunling grass bouquet  and were authenticated by Prof. Yuguang Chen . YCSND was prepared using 10 g each of Artemisia capillaris Thunb. , Aconitum carmichaelii Debeaux , Zingiber officinale Roscoe , and honey-fried Glycyrrhiza glabra L. . Quality control for the four botanical drugs for YCSND met the standards of the Chinese Pharmacopoeia , and hydroxyl radical (·OH) scavenging activities were determined using previously described methods  for 12 h, except those in the normal group. Finally, the prepared cells were used for subsequent analyses.HepG2 cells in the exponential growth phase were seeded into cell plates for 24 h, followed by treatment with YCSND  for another 12 h. Cells in the normal and model groups were treated with equal volumes of DMEM instead of YCSND. Subsequently, the cells were treated with CClCell viability was determined using the MTT assay method . Briefly4-treated (CCl4), low-dose YCSND (YCSNDL), medium-dose YCSND (YCSNDM), and high-dose YCSND groups (YCSNDH). Mice in the CON and CCl4 groups were treated with physiological saline, whereas those in the three YCSND groups were treated with 400, 800, and 1,600 mg/kg body weight of YCSND powders per day, respectively. According to the description of the body surface area normalization method between humans and animals  2 h after the last administration, except for mice in the CON group, which were injected with an equal volume of olive oil. After 24 h, all mice were euthanized by carbon dioxide inhalation  were purchased from Liaoning Changsheng Biotechnology. All animal experimentation procedures were approved by the Animal Care and Use Committee of Jinlin University . The mice were housed under standard conditions at 23°C ± 2°C in a 12-h light/dark cycle and fed standard mice chow and water. After adaptive feeding for 7 d, the mice were randomly assigned to five groups (six mice per group): control (CON), CCl animals , the doshalation , and livALT and AST levels in cell culture supernatants and serum were determined using standardized kits , according to the manufacturer’s instructions. Superoxide dismutase (SOD) and glutathione (GSH) activities and malondialdehyde (MDA) contents of HepG2 cells and serum were determined using standardized kits , according to the manufacturer’s instructions. Cell protein contents were measured using a bicinchoninic acid (BCA) protein assay kit .A small piece of the cut right lobe of the liver was fixed in a 10% formalin solution, dehydrated, made transparent, paraffin-embedded, and cut into 2-mm thick sections. Thereafter, the liver tissue was stained with hematoxylin and eosin (H&E) and viewed using a light microscope .Gapdh) was selected as the internal control for RT-qPCR and subsequent normalization. The relative expression levels of target genes were calculated using the comparative CT method (2−ΔΔCT). The primer sequences of the genes are listed in Total RNA extraction of the liver tissue, cDNA synthesis, and qPCR were performed according to previously described procedures  RT-qPCR 1H-NMR was performed according to previously described methods with slight modifications , and metabolomics pathway analysis was performed using MetaboAnalyst .Sample preparation for ications . Briefly methods . Orthogohttps://www.omim.org/), DisGeNET database (https://www.disgenet.org/), and GeneCard database (https://www.genecards.org/). The species “Homo sapiens” was selected, and the target gene information related to liver injury was collected and integrated. Venn diagram was used to obtain intersecting targets from the active ingredient targets of YCSND and the liver-injury targets. An intersecting target network between the metabolites of YCSND and targets was constructed using Cytoscape 3.8.2. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways enrichment analyses were performed using the Metascape database (https://metascape.org/). The top 20 predicted KEGG pathways, related metabolites, and targets of YCSND for the treatment of liver injury were screened to construct a metabolites-target-pathway (M-T-P) network using Cytoscape 3.8.2. Furthermore, we screened five typical KEGG pathways to construct a target-pathway (T-P) network using an online website (https://www.bioinformatics.com.cn).Network analysis was used to predict the potential molecular mechanism of YCSND in liver injury. The targets of the active metabolites in YCSND were determined following previously described method . The canin vivo affected by YCSND were predicted using the SWISS database (http://www.swisstargetprediction.ch/). Overlapping targets were obtained among those from the metabolites and network prediction. These targets played a key role in YCSND against ALI.Targets of the metabolites t-test for two groups or one-way ANOVA for multiple groups. Differences were considered significant when p < 0.05.Each experiment was performed independently at least three times, and data are expressed as mean ± standard deviation (SD). Statistical analysis was performed using GraphPad Prism (version 8.0) and SPSS 22.0. Statistical significance of differences was assessed with Student’s 2− and ·OH (YCSND contained polysaccharides (408.55 ± 15.04 mg Glu/g), flavonoids (92.70 ± 3.37 mg RE/g), and polyphenols (39.76 ± 0.83 mg GAE/g) . Additio and ·OH , which w and ·OH . Moreove and ·OH . Further and ·OH . Overallp > 0.05) cytotoxic effect on HepG2 cells  was reliable  can accelerate apoptosis by inhibiting Bcl2 expression and inducing the activation of caspase family proteins , pathways related to ALI, and targets related to pathways affected by YCSND , based on the SWISS database. Additionally, we identified 19 overlapping targets from the metabolites altered by YCSND and five pharmacological prediction pathways related to ALI . We alsoby YCSND .Over the years, several clinical trials have demonstrated the hepatoprotective potential of YCSND in hepatobiliary diseases ; howeverThe metabolites of TCM formulas are not simple combinations of plant-derived metabolites; instead, they are influenced by the source of botanical drugs and processing techniques . There w4-induced ALI can lead to abnormal accumulation or depletion of hepatic endogenous metabolites in the body, affecting a range of metabolic pathways, such as amino acid metabolism, energy metabolism, and lipid metabolism  activity . TCM haspoptosis . Moreovein vitro . The PI3in vitro . Phosphoin vitro . Glycyrrin vitro , scoparoin vitro , isorhamin vitro , quercetin vitro , and lutin vitro  have beeVegfa, Egfr, and Stat3. We suggested that network analysis should be based on the actual constituents of the TCM, rather than botanical drug information from existing databases. However, network analysis still relies heavily on databases, and this inherent bias caused by the databases is a common issue in network analysis. The completeness and timeliness of the Swiss database can particularly impact our study results. For example, delayed data updates and the abundance of potential targets for well-known active metabolites like quercetin with the less targets of new metabolites can lead to result in false positives or false negatives. To narrow down these biases, continuous improvement and timely information updates of databases should be the common goal of future network analysis research. Moreover, network analysis predictions may not comprehensively reflect the intricate interactions and mechanisms of drugs within the body. Therefore, experimental verification is necessary to ensure the authenticity of network predictions. Meanwhile, regarding the connection between network analysis and metabolism, the target genes related to changes in differential metabolites require further investigation. We validated the highly significant PI3K signaling pathway predicted by network analysis through RT-qPCR analysis. In future studies, other signaling pathways predicted by network analysis should be further considered to expand the molecular mechanisms of YCSND in ALI.This study had some limitations. Although the accuracy of the network prediction has improved, there was still heterogeneity in the experimental validation and prediction results in the present study. YCSND had only minimal effects on some of the predicted targets, such as 1H-NMR metabolomics revealed key targets  and related metabolites  regulated by YCSND in ALI treatment. Our results indicated that YCSND ameliorated ALI by regulating the PI3K-AKT signaling pathway, reducing inflammatory response, and inhibiting apoptosis (In the present study, integrated network analysis and poptosis . Overall


NCA tool of Cystoscape 3.7.2 was used to carried out topology analysis on PPI network, core components and key targets were obtained. Gene ontology and Kyoto encyclopedia of genes and genomes enrichment analysis were carried out for the intersection targets by David database. AutoDockTools 1.5.6 software (https://autodock.scripps.edu/) was used to conduct molecular docking verification of key components and key targets. Ninety-one ingredients of the modified Huanglian Wendan decoction and 23,075 diseases targets were obtained, 155 Intersection targets of the drug and disease were obtained by R language, Veen plot was drawn. Gene ontology enrichment analysis obtained 432 biological processes, 67 cell components, 106 molecular functions. Fifty-four Kyoto encyclopedia of genes and genomes enrichment pathways (P < .05) including tumor necrosis factor, hypoxia-induced factors-1, calcium, and drug metabolism-cytochrome P450 signaling pathway. Molecular docking showed quercetin, luteolin, kaempferol, and baicalein were stable in docking with core targets. Network pharmacology and molecular docking were used to preliminarily study the mechanism of action of modified Huanglian Wendan decoction in the treatment of PCOS, which laid foundation for future experimental research and clinical application.To investigate the mechanism of modified Huanglian Wendan decoction in the intervention of polycystic ovary syndrome (PCOS) by network pharmacology and molecular docking. The ingredients and targets of modified Huanglian Wendan decoction were retrieved from the traditional Chinese medicine Systems Pharmacology database. Related targets of PCOS were screened by Comparative Toxicogenomics Database database. Cytoscape 3.7.2 ( Its clinical manifestations are mainly irregular menstruation and infertility, the incidence of miscarriage, hirsutism, obesity and acne is as high as 5% to 10% in China, and 50% to 70% in infertile patients. At present, the pathogenesis of PCOS is complex and the exact etiology is unclear, involving many aspects such as genetic, environmental and psychological factors.Polycystic ovary syndrome (PCOS) is the most common gynecological endocrine disease in women of reproductive age, characterized by reproductive disorders, endocrine abnormalities and metabolic disorders. Tutor in the clinical flexible use of different diseases with the concept of treatment, ingenious modified, cuscutae semen, cyperi rhizoma, poria cocos, pericarpium citri reticulatae, coptidis rhizoma, angelicae sinensis radix, persicae semen, aurantii fructus immaturus, and pinellia ternata, follow the principle of compatibility, the treatment of PCOS has a good effect, but its specific mechanism of action needs further study.PCOS belongs to traditional Chinese medicine menstruation delay, amenorrhea or hypomenorrhea. Huanglian Wendan Decoction is from “Liuyin Tiaobian of Heat Stroke,” according to modern pharmacology that has significant lipid-lowering, hypoglycemic, anti-inflammatory and other pharmacological effects.Network pharmacology is an emerging bioinformatics approach in recent years to study target molecules. Through biological function and biological active compounds of traditional Chinese medicine (TCM), combined with its mechanism of action in treating diseases, to generate the complex interaction of network. The manuscript adopted the research methods of network pharmacology, and molecular docking, analysis of modified Huanglian Wendan Decoction potential targets for the treatment of PCOS and mechanism of action, for further research on modified Huanglian Wendan Decoction treatment of PCOS provides theoretical basis Fig. . (https://tcmsp-e.com/tcmsp.php) based on the parameters related to drug metabolism kinetics; oral bioavailability ≥ 30% and drug similarity ≥ 0.18 were the selecting conditions.The ingredients of cuscutae semen, cyperi rhizoma, poria cocos, citrus reticulata, coptidis rhizoma, angelicae sinensis radix, persicae semen, aurantii fructus immaturus, and arum ternatum thunb were gathered from TCMSP (http://ctdbase.org/) to obtain the known target information related to this disease. Uniprot (https://www.rcsb.org/) database was used to standardize the names of target genes.Enter the keyword “polycystic ovary syndrome” into the the comparative toxicogenomics database (CTD) databaseThe intersection of drugs component targets and diseases were obtained by R language and Venn diagram was drawn. the topological properties of network were analyzed. To better clarify the mechanism of the treatment of PCOS with modified Huanglian Wendan Decoction, the core network was selected based on topological parameters, degree and the betweenness centrality were key indexes to estimate the importance of nodes, as the degree and betweenness centrality increase, the nodes are more important in the network.The targets of intersection between modified Huanglian Wendan decoction and PCOS were taken to establish the network diagram using Cytoscape 3.7.2, (http://string.embl.de/), the species was set as “Homo Sapiens,” the lowest interaction threshold was set as “medium confidence,” and the target protein interaction network was obtained.The target at the intersection of TCM ingredients and diseases was imported into the STRING databasehttps://david.ncifcrf.gov/) was used to GO and KEGG pathway enrichment  analysis on the intersection target proteins, obtained the signal pathways of modified Huanglian Wendan decoction for the intervention of PCOS.DAVID ( (https://pubchem.ncbi.nlm.nih.gov/). Search the PDB database (https://www.rcsb.org/) for the protein and its crystal structure. After dehydration and hydrogenation, the protein was stored in PDB format with Autodoc 1.5.6 software (https://autodock.scripps.edu/). PyMol software was used to analyze the important proteins in the active ingredients and target network whose degree value was greater than the average.The Cytoscape3.7.2 CNA widget was used to screen the core targets and effective active compounds according to the degree value. The chemical structure of active components in MOL2 format of TCMSP was retrieved from the database, which was re-retrieved in Pubchem database and saved in SDF formathttp://links.lww.com/MD/I636 and Table S2, Supplemental Digital Content, http://links.lww.com/MD/I637). After screening again to remove duplication, 91 modified active ingredients of modified Huanglian Wendan decoction were identified. A total of 164 targets were predicted through UniProt gene annotation search using these 91 compounds, including 9 Cuscutae Semen, 10 Cyperi Rhizoma, 13 Poria Cocos, 4 pericarpium citri reticulatae, 9 Coptidis Rhizoma, 2 Angelicae Sinensis Radix, 16 Persicae Semen, 16 Aurantii Fructus Immaturus, and 10 Pinellia ternata.From the TCMSP databases, obtained 650 ingredients, through absorption, distribution, metabolism, and excretion  screening (Table http://links.lww.com/MD/I638).Through CTD databases, obtained 23,075 disease targets of PCOS .Data on PCOS-related targets retrieved from CTD databases were integrated, resulting in the retrieval of 23,075 targets. These targets were compared with the predicted targets, and 155 common targets were filtered as the key targets for testing the anti-PCOS activity of the ingredients , consisting of 9 herbal nodes, 91 ingredients nodes, and 155 protein nodes. Among the 91 compounds, the active ingredients closely related to PCOS were quercetin, luteolin, kaempferol, baicalein, β-sitosterol, isorhamnetin, stigmasterol, and nobiletin.Cytoscape 3.7.2 constructed network of TCM, ingredients and targets , 67 cellular component (CC)  106 and molecular function (MF)  of the 155 targets. The Top 15 enriched BP, MF and CC were selected and represented by a bar chart  , mainly include: neuroactive ligand receptor interaction, tumor necrosis factor (TNF) signaling pathway, hypoxia-induced factors (HIF)-1 signaling pathway, Calcium signaling pathway, drug metabolism- cytochrome P450.KEGG pathway enrichment analysis at Molecular docking is a technique that mimics the interaction between small ligand molecules and receptor protein macromolecules, and the binding energy between the 2 counterparts can be calculated to predict their affinity. A binding energy lower than 0 indicates that the 2 molecules combine spont- aneously and that smaller binding energies lead to more stable conformations.In order to further verify the prediction of the results, and elaborate the mechanism and scientific connotation of modified Huanglian Wendan decoction in the treatment of PCOS, the 4 components with the highest content of modified Huanglian Wendan decoction were selected for molecular docking verification, namely quercetin, kaempferol, luteolin, and baicalein Table . IL-6, V,14 Luteolin was found to regulate the ovarian and hormone levels of rats, up-regulate Nrf2 signaling pathway, and improve antioxidant response, which is of great significance for the prevention and treatment of PCOS. Studies have shown that kaempferol regulated MAPK signaling pathway to reduce inflammatory response and apoptosis, PI3K signaling pathway is mediated to promote meiosis of oocytes. Baicalein regulation of InsR/IRS-1 signaling pathway, inhibits gluconeogenesis of hepatocytes to improve glucose metabolism, has strong anti-IR effect, it restore the dynamic balance of reproductive hormones and normal pregnancy micro-environment through immune regulation, endocrine hormone action network, and improve the antioxidant capacity of mouse ovarian tissues by up-regulating TrxR expression.,19 Molecular docking, according to the results of key ingredients and targets combination conformation stability, speculated that these ingredients may be the key ingredients and targets of modified Huanglian Wendan decoction in the treatment of PCOS.Through the 3.4 and 3.5, obtained the active ingredients of modifed Huanglian Wendan decoction for treating PCOS were identified as quercetin, luteolin, kaempferol and baicalein. Modern pharmacological studies showed that quercetin could inhibit TLR/ NF-κB signaling pathway and MAPK signaling pathway, reduce the expression of testosterone (T), estradiol (E2), IL-6 and TNF-α, and increase the expression of FSH, thus alleviating the hormone, metabolism, and ovulation abnormalities caused by PCOS, it can also significantly reduce PCOS-IR and induce expression of GLUT4 and ERa genes in the uterus, therefore effectively treating PCOS and its complications: insulin resistance (IR) and infertility.,20 Studies have shown that VEGFA is expressed in ovarian GC and membrane cells, as well as in follicular fluid. Abnormal VEGFA expression leads to irregular angiogenesis, which increased ovarian interstitial vascularization and perifollicular angiogenesis, involved in the development and progression of PCOS. In addition, it affects the quality of follicular mature oocytes, during pregnancy and embryo growth and development, high VEGFA concentration in follicular fluid in patients with PCOS can lead to an increase in immature follicles and a decrease in fertilized eggs.–23 EGFR is the most abundant expression in human GC and follicular fluid, has been shown to be an ovulation induction factor. When EGFR and P-EGFR are down-regulated, the induction of oocyte maturation is significantly inhibited; EGFR can also delay the meiosis of oocytes by inducing ERK1/ERK2 phosphorylation, thereby regulating ovulation time.–26 MAPK8 is a member of the protein mitokine family, which is involved in cell proliferation, cell differentiation, cell apoptosis, and other mechanisms, meanwhile, acts as an integration point for multiple biochemical signals.,28 Experiments have confirmed that MAPK8 is closely related to obesity and IR, expression increase in IR in obese mice, higher expression of MAPK increase the start-up process of adipocyte differentiation, promote mature adipocyte differentiation; MAPK8 known as JNK1 gene, involved in initiating the signaling pathway of JNK1/SAPK1 of cell cycle, studies have shown that IR and hyperlipidemia in PCOS patients are related to the high expression of JNK1 mRNA caused by JNK1, speculated that it may be the factor affecting the pathogenesis of PCOS.,30 CCND1 regulates cyclinD1 protein and promotes cytoplasmic proliferation, Cyclin D1 and its inhibitors are associated with GC proliferation at specific follicular stages, CCND1 promoted E2 secretion of PCOS ovarian GC, increased the expression level of CYP19A1, inhibited the expression level of Caspase 3 in apoptotic GC, and reduced CASP3 cleavage.,32 CASP3 is a regulatory factor of apoptosis signaling pathway, the early ovarian stage of PCOS patients is characterized by increased number of follicles, and the apoptosis rate of sinus follicles is related to increased expression of CASP3 in GC. Hyperandrogenemia in the late stage is closely related to excessive GC apoptosis. GC is the only cell type that can express FSH receptor, and a large number of GC apoptosis occurs. In the process of T conversion to estrogen, androgen will gradually accumulate, and CASP3 will be overexpressed. Similarly, an excess of androgens can lead to an increase in CASP3 and GC, thus leading to a sustained increase in PCOS hyperandrogenemia.–37 FOS is nuclear phosphoprotein produced by c-FOS transcription, which plays an important role in regulating cell proliferation, differentiation and apoptosis. The silencing of FOS expression in GC resulted in a significant increase of CYP17 mRNA, suggesting that the FOS family regulates ovarian androgen production.,40 FOS can down-regulate the expression of CYP17 in cell membranes and particles, Inhibition of FOS expression in GC resulted in a significant increase in CYP17 expression and androstenedione production, even in this typical non-androstene-derived cell type, The abnormal down-regulation of FOS expression in androgen-producing tissues is the potential mechanism of excessive androgen production in PCOS. FOS is the proximal promoter of FSH subunit (FSHB), regulates the expression of FSHB gene. FOS with high expression can induce FSHB (31), FOS expression is low in PCOS patients, while FSH low level is general in PCOS patients, FOS is also involved in p38MAPK/ERK1/2 signal transduction and can be induced by GnRH in gonadotropins, which proves that GnRH regulation of FSHB gene expression may be induced by FOS,,43 in addition, some studies have confirmed that the expression of FOS is positively correlated with the expression of inflammatory adipocytokine  genes, and the mRNA level of the hormone producing factor CYP19A1 is also closely correlated with FOS mRNA , therefore, it is speculated that the high expression of FOS in PCOS may have an anti-inflammatory effect and have a certain influence on steroid production in adipose tissue, inhibiting the expression of FOS may accelerate the overall development of PCOS, FOS expression was inhibited to accelerate the development of PCOS.–46PPI results showed that IL-6, VEGFA, EGFR, MAPK8, CCND1, CASP3, and FOS showed the highest correlation among the core targets. Molecular docking proved that quercetin, luteolin, kaempferol, and baicalin had stable binding to these core targets, suggesting that network pharmacology has a high reliability for mechanism analysis of modified Huanglian Wendan decoction in the treatment of PCOS. IL-6 is a group of cytokines secreted by granulosa cells (GC), Studies have shown that IL-6 plays different roles in follicular generation, ovulation and luteal function, and is involved in the local inflammatory immune response of ovary, which is an influential factor of non-ovulation in PCOS patients. The follicle undergoes many structural changes during growth, including changes in the vasculature cell proliferation and differentiation and formation of fluid-filled cavities, and these changes together create a hypoxic environment within the follicle so that the oocyte itself develops in a potentially hypoxic environment Survival of hypoxic tissues is controlled by HIF, which are activated in hypoxia. Understanding of the HIF signaling pathway is growing in all areas of biology, and its role in ovarian development is steadily being clarified, one of the genes upregulated by HIF is vascular endothelial growth factor, which is a major inducer of angiogenesis and is essential for ovulation and body formation. Ovulation is also intrinsically linked to HIF activity, increasing HIF expression through a surge in luteinizing hormone (LH) through ovulation. The early activation and growth initiation of dormant follicles induced by hypoxia provides evidence that these changes are associated with reduced stromal cell proliferation, suggesting that hypoxia-induced damage to the serst-cell pool may be the mechanism of accelerated follicular activation. The reversal of hypoxia can cause the decrease of SIRT1 activity in mitochondria and during hypoxia.–50 The ovarian GC of PCOS patients show abnormal proliferation, which can be reversed by reducing cell proliferation and inhibiting apoptosis with inhibitors. Nitric oxide (NO) plays a role in the physiology of the reproductive system, follicular development and selection, is associated with angiogenic events, play a regulatory role in steroid production in ovarian cells by stimulating cyclooxygenase-2 activity, NO induces increased production of prostaglandin E2, which appears to be a common ovulation mechanism, NO level is negatively correlated with IR in PCOS patients, and the effects of NO and IR are mutually reinforcing, many of the effects of insulin depend on NO, which is an effector in insulin information transduction pathway.,53 ERK1 and ERK2 genes are closely related to IR in PCOS. The ERK2-PI3K/AKT and MAPK/ERK pathways in classic insulin-sensitive tissues target ovarian tissue, and disruption of the signaling pathway can lead to decreased insulin sensitivity and compensative hyperinsulinemia in PCOS rats.,55 GABA and positive-feedback GABA receptor regulatory steroids, such as isoprogesterone, stimulate food intake and weight gain. In women with PCOS, high serum isoprogeolone concentrations are associated with uncontrolled eating and disturbance of sensitivity to isoprogeolone. Receptor-gated ion channels play an important role in ovarian physiology and pathology. For example, inhibition of volt-gated Ca2+ channels can reduce extracellular Ca2+ concentration, prevent Ca2+ from transmembrane flow, and destroy LH stimulated steroid production in isolated ovarian cells, adenylate cyclase- activating polypeptide stimulates melatonin synthesis and melatonin can increase the expression of SIRT1 to inhibit mitochondrial autophagy and protect GC from mitochondrial damage, it can also regulate dehydroepiandrosterone-induced PCOS autophagy through PI3K-Akt pathway, thereby improving ovarian dysfunction and restoring female fertility.–60 Studies have shown that GC in patients with PCOS are in an abnormal proliferation state, TNF-α activates JNK and other signal transduction pathways to promote the proliferation of follicular membrane cells, resulting in ovarian white membrane thickening and follicular membrane cell hyperplasia in PCOS patients, and can promote the occurrence of PCOS-IR.–63 The binding of acetylcholine (ACH) to the miAChRs receptor in GnRHergic neurons activates the phospholipase C protein, which hydrolyzes phosphatidylinositol and stimulates the formation of inositol triphosphate (IP3). The binding of IP3 to its receptor in the membrane of the rough endoplasmic reticulum moves Ca2+ into the cytoplasm, the phosphorylated IP3 receptor decreases its response capacity when IP3 increases, induced by ACH, which results in a reduction in the release of calcium into the cytoplasm by the endoplasmic reticulum and a lower secretion of GnRH. ACH and choline acetyltransferase are present in adult rats, Primates and women mature ovarian follicle in granular cells, can serve as the role of FSH partial mediator role in the growth of follicle, stimulation of GC proliferation and growth of ovarian ACH levels increase, sinus increase our secondary follicle and corpus luteum number, before the ACH to stimulate the manner and ruptured follicle and corpus luteum formation.,66 In conclusion, Modified Huanglian Wendan decoction exert antioxidant stress response, regulate gated ion pathway, inhibit abnormal cell proliferation and other ways to achieve IR, regulate steroid hormone disorder, reduce ovarian cell apoptosis, and finally play the role of anti-PCOS.The functional enrichment results of GO biological process showed that the biological process of modified Huanglian Wendan decoction in treating PCOS mainly focused on oxidative stress, cell proliferation, Regulation of gated ion channels, and other aspects and other aspects. Hypoxia affect human health through regulating system and cell physiology of oxygen can produce reactive oxygen species, cause oxidative stress, and by changing the mammalian ovary oocytes and the physiological function of impact women’s reproductive health, hypoxic conditions induced by a variety of apoptosis signaling pathways, such as autophagy and apoptosis of ovarian follicle mammal necrosis, hypoxia inducing autophagy is through 5’ Amp-activated protein kinase- mammalian target of rapamyciner stress/unfolding protein response and protein kinase C-delta-C-JUNn terminal kinase 1 signaling pathway are performed in a variety of somatic cells,,68 TNF signaling pathway is an inflammatory signaling pathway, which is involved in the pathogenesis and development of PCOS. The inflammatory factor IL-6 enriched in this pathway is involved in the local inflammatory immune response of ovary, TNF-α can inhibit FSH synthesis, and both IL-6 and TNF-α can prevent a large number of follicles from growing, Regulation of TNF signaling pathway can play an anti-PCOS role.–73 HIF-1α is a multidirectional factor that controls intracellular communication, sterogenic activity and development rate of oocytes, and affects blastocyst rate, inhibition of HIF-1α signaling pathway can not only regulate ovarian cell function, but also reduce inflammatory response and prevent oxidative stress, activation of HIF-1α signaling pathway can stimulate the expression of VEGFA gene, increase endometrial vascular hyperplasia and repair, and provide conditions for implantation of fertilized eggs.,75 Calcium signaling pathways involved in oxidative stress reaction, oxidative stress can promote the development of inflammation, induce IR and androgen secretion, studies have shown that calcium disorders can cause ovarian follicles block, cause reproductive and menstrual disorders, resulting in PCOS, when calcium signaling pathways activated, intracellular calcium ion concentration increases, the activation of calmodulin, prompting designed the gonadotropins release, gonadotropins indirectly regulates oocyte maturation through paracrine mechanism, resulting in ovulation, Therefore, for PCOS patients, regulating calcium ion signaling pathway can resist oxidative stress, and then play a role in resisting insulin resistance and inhibiting androgen secretio.–80 P450 aromatase is a key enzyme that catalyzes the conversion of androstenedione and T to estrone and E2, increased androgen level in serum of PCOS patients suggests that there may be defective aromatase activity, Sustained high serum levels of LH in PCOS women stimulated the production of large amounts of and rostenedione, in ovarian follicular membrane cells and stromal cells and stimulated the expression of P450 aromatase mRNA, In the study of the effect of insulin sensitizer on PCOS, it was found that metformin can directly act on the ovary, reduce the activity of P450 aromatase, reduce the synthesis of estrogen, and remove the negative feedback of estrogen on FSH in follicular fluid, increase the expression of FSH receptor and promote follicular development.–83The KEGG enrichment pathway indicated that the main pathways of midified Huanglian Wendan decoction in treating PCOS were neuroactive ligand receptor interaction, TNF signaling pathway, HIF-1 signaling pathway, Calcium signaling pathway, drug metabolism -cytochrome P450. Neuroactive ligand receptor interaction plays an important role in the reproductive process, studies have confirmed that endometrial gene expression in PCOS patients is related to neuroactive ligand receptor interaction, in addition, neuroactive ligand receptor interaction pathway is related to abnormal ovarian follicular secretion. In this pathway, IL12B and F2R genes participate in the pathophysiological mechanism of PCOS by regulating cell death and cells. Moreover, studies have confirmed that the neuroactive ligand receptor interaction pathway can regulate endocrine and emotion, and emotion regulation has obvious therapeutic effect on PCOS patients.In summary, this study adopted network pharmacology and molecular docking methods to identify potential therapeutic targets of modified Huanglian Wendan decoction for PCOS. The relationship between modified Huanglian Wendan decoction and PCOS disease was preliminarily clarified, and the relationship between active molecules and target proteins was visualized, revealing that modified Huanglian Wendan decoction in treating PCOS is a multi-active ingredient that plays an anti-inflammatory and antioxidant stress role through multi-target and multi-pathway However, the prediction is only based on the existing database at present, which can only provide reference for the study of its mechanism of action. The subsequent research group plans to design animal experiments to conduct in-depth discussion on the mechanism of action of modified Huanglian Wendan decoction in the treatment of PCOS, so as to clarify its pharmacological mechanism more systematically and scientifically and provide more reference for its clinical use.In summary, this manuscript uses network pharmacology to analyze the effective targets and pathways of adding and modified Huanglian Wendan decoction, and preliminarily explores the target action pathway and molecular mechanism of adding and modified Huanglian Wendan decoction in the treatment of PCOS. This study indicated that the mechanism of modified Huanglian Wendan decoction in treating PCOS was related to its anti-inflammatory, antioxidant stress, regulation of in vivo hormone production and resistance to insulin. The main active components are quercetin, luteolin, kaempferol, and baicalein, and the key targets are IL-6, VEGFA, EGFR, MAPK8, CCND1, CASP3 and FOS. Neuroactive Ligand receptor interaction, TNF Pathway, HIF-1 pathway, Calcium signaling pathway, Drug metabolism- Cytochrome P450 and other signal pathways are correlated, which again confirms that TCM is effective in treating diseases through multiple targets and multiple pathways. Due to many subjective and objective factors, experimental verification needs to be carried out in the next step, which will open up a new way for the treatment of PCOS.This is work was supported by the fourth batch of National Outstanding Talents of Traditional Chinese Medicine  Research and Training Program of State Administration of Traditional Chinese Medicine , Shandong Provincial Traditional Chinese Medicine Science and Technology Plan Project (2020M042).Data curation: Zhaojng Wu, Tiantian Yang.Formal analysis: Zhaojng Wu.Funding acquisition: Hongbo Ma.Validation: Zhaojng Wu.Visualization: Zhaojng Wu.Writing – original draft: Zhaojng Wu.Writing – review & editing: Zhaojng Wu.


Eucommia ulmoides (EU) and its diverse extracts have demonstrated antioxidative, anti-inflammatory, and cytoprotective properties against hepatic ischemia–reperfusion injury (HIRI). However, the primary constituents of EU and their putative mechanisms remain elusive. This study aims to explore the potential mechanisms of EU in the prevention and treatment of HIRI by employing network pharmacology and molecular docking methodologies. The main components and corresponding protein targets of EU were searched in the literature and TCMSP, and the compound target network was constructed by Cytoscape 3.9.1. Liver ischemia–reperfusion injury targets were searched in OMIM and GeneCards databases. The intersection points of compound targets and disease targets were obtained, and the overlapping targets were imported into the STRING database to construct the PPI network. We further analyzed the targets for GO and KEGG enrichment. Finally, molecular docking studies were performed on the core targets and active compounds. The component-target network unveiled a total of 26 efficacious bioactive compounds corresponding to 207 target proteins. Notably, the top-ranking compounds based on degree centrality were quercetin, β-sitosterol, and gallic acid. Within the PPI network, the highest degree centrality encompassed RELA, AKT1, TP53. GO and KEGG enrichment analysis elucidated that EU in HIRI primarily engaged in positive regulation of gene expression, positive transcriptional regulation via RNA polymerase II promoter, negative modulation of apoptotic processes, positive regulation of transcription from DNA templates, and drug responsiveness, among other biological processes. Key pathways included cancer pathways, RAGE signaling pathway, lipid metabolism, atherosclerosis, TNF signaling pathway, PI3K-Akt signaling pathway, and apoptotic pathways. Molecular docking analysis revealed robust affinities between quercetin, β-sitosterol, gallic acid, and RELA, AKT1, TP53, respectively. This study reveals EU exhibits substantial potential in mitigating and treating HIRI through multifaceted targeting and involvement in intricate signaling pathways. It is a common clinical issue in diseases such as liver transplantation, liver surgery, shock, and trauma3. End-stage liver disease is a significant contributor to increased mortality rates associated with liver diseases worldwide, and liver transplantation remains the optimal treatment method for various end-stage liver diseases4. Despite the continuous development and replacement of precise minimally invasive hepatobiliary surgery and surgical techniques, HIRI remains a major cause of postoperative liver dysfunction and even multi-organ failure6. The occurrence of HIRI during surgery significantly increases the risk of acute and chronic rejection reactions in liver transplantation, substantially affecting the success rate of transplantation, exacerbating the shortage of available livers, and being closely related to early graft dysfunction8. Therefore, preventing and mitigating HIRI during surgery has been a persistent concern for hepatobiliary surgeons, and pharmacological preconditioning and targeted gene interventions have emerged as key research topics in both domestic and international HIRI prevention and treatment studies. Research has demonstrated that energy metabolism regulators, antioxidants, adenosine receptor agonists, calcium channel blockers, and other agents exhibit protective effects against HIRI10. Although there are literature reports indicating that pre-operative and post-operative pharmacological interventions can alleviate HIRI, such approaches have proven effective primarily in patients with limited liver tissue resection and prolonged ischemic time11. Moreover, many of the aforementioned drugs have substantial toxic side effects, hindering their development and clinical application. To date, there has been limited breakthrough progress in the prevention and treatment of HIRI. Therefore, the prevention and treatment of HIRI necessitate the exploration of safer and more effective therapeutic strategies.Hepatic ischemia–reperfusion injury (HIRI) refers to the pathological phenomenon where the liver undergoes additional damage upon the restoration of blood flow after a period of ischemia, during which liver cells experience a transient ischemic environmentEucommia ulmoides (EU), Salvia miltiorrhiza (SM), and resveratrol have clear preventive and therapeutic effects against HIRI. EU, a representative kidney-tonifying herb in China, is described in the classic TCM text "Ben Cao Gang Mu" as a liver-regulating herb that nourishes the liver, moistens dryness, tonifies liver deficiency, and possesses medicinal properties that were previously undiscovered. EU has a purple color and a sweet and slightly spicy taste, with a neutral and warm energy. It can enter the liver to tonify the kidneys. EU contains numerous active components, including phenylethanoid glycosides, lignans, terpenoids, polysaccharides, and flavonoids, which have pharmacological effects such as anti-tumor, anti-inflammatory, disinfectant, hepatoprotective, choleretic, and antioxidant properties. Additionally, EU can effectively treat conditions such as lumbago and knee pain, weak fetal retention, and scrotal itching caused by liver and kidney deficiency13. In recent years, there have been literature reports indicating that EU and its chemical constituents have protective effects against liver fibrosis, fatty liver, HIRI, and liver damage caused by carbon tetrachloride18. However, in our early studies on rats, we discovered that EU and some of its monomer components  could alleviate HIRI when administered as pretreatment to the rats. The mechanism behind this effect involves reducing the generation of reactive oxygen species (ROS) during HIRI and inhibiting the sterile inflammatory response mediated by HMGB1. It is evident that the role of EU in HIRI is worth acknowledging20.Traditional Chinese medicine (TCM) has been widely used in China for thousands of years to treat common and complex diseases, and its therapeutic efficacy has been well recognized throughout the long history of medicinal practices in China. TCM possesses advantages such as multiple targets, a wide safety range, and minimal toxicity and side effects. Research has found that TCM exhibits protective effects against HIRI, making the development of TCM monomers and herbal formulations with protective effects against HIRI a promising research direction in the near future. Interestingly, previous studies have shown that preoperative treatment with extracts of 21. Network pharmacology and molecular docking are powerful computational methods that can reveal the interactions between drugs and biological systems24. Network pharmacology helps uncover the complex interactions between multiple components of a drug and the corresponding target pathways in disease25. On the other hand, molecular docking can predict the binding affinity and structural interactions between active compounds in EU and protein targets related to HIRI26. The integration of network pharmacology and molecular docking can provide a comprehensive understanding of the potential mechanisms of EU in HIRI27. By identifying key targets and pathways, this approach can help elucidate the molecular basis of EU’s therapeutic effects and guide the development of new treatment strategies for HIRI.Due to the complex composition of TCM, with multiple targets and a wide range of therapeutic signaling pathways, in-depth research on traditional Chinese medicine poses challenges that cannot be addressed by traditional research methods alone, as they cannot accurately elucidate the molecular mechanisms of drug actionsIn this study, our aim is to explore the potential mechanisms of EU in HIRI using network pharmacology and molecular docking techniques. We will identify the active compounds in EU, predict their targets, and construct a drug-target interaction network. Additionally, we will use molecular docking simulations to validate the predicted targets and assess the binding affinity and stability of compound-target interactions. Understanding the molecular mechanisms of EU in HIRI can help in the development of new therapies and target mechanisms for the prevention and treatment of this severe disease. It provides a more favorable theoretical basis for the clinical application of EU in intervening HIRI. Furthermore, this research can provide scientific evidence for traditional medicine and promote the integration of traditional Chinese medicine with modern medicine in the field of liver diseases.https://old.tcmsp-e.com/tcmsp.php)28 and relevant literature review. Filtering criteria including oral bioavailability (OB) ≥ 30% and drug-likeness (DL) ≥ 0.18 were applied to obtain active compounds. The related targets of these active compounds were retrieved from the "Related Targets" section. For compounds without target information, the Canonical SMILES of the active substances were obtained from the PubChem database (https://pubchem.ncbi.nlm.nih.gov/)29 using their CAS numbers. The obtained Canonical SMILES were then input into the Swiss Target Prediction database (http://www.swisstargetprediction.ch/) to retrieve target information. The UniProt database (https://www.uniprot.org/)30 was further utilized to obtain gene symbols and IDs for the target proteins.The active components of EU were collected using the Traditional Chinese Medicine Systems Pharmacology Database 32 and GeneCards database (https://www.genecards.org/)33. A search using the keyword “Hepatic ischemia–reperfusion injury” was performed to obtain HIRI-related targets from each database. The target results from both databases were merged and duplicates were removed. The remaining genes were collected and used to investigate the information of HIRI disease target genes. Venn analysis of the potential targets of EU and HIRI was performed using an online website (www.bioinformatics.com.cn) to visualize the intersection.Information on the disease targets related to hepatic ischemia–reperfusion injury (HIRI) was collected from the Online Mendelian Inheritance in Man (OMIM) database (https://cn.string-db.org/)34 for PPI analysis. The analysis was performed using the “Homo sapiens” organism and a minimum required interaction score of 0.900. The resulting information on protein–protein interactions was used to construct a protein–protein interaction network. The exported node–node data was visualized and analyzed using Cytoscape 3.9.1. The network analysis plugin was employed to analyze the network's degree centrality (DC), betweenness centrality (BC), and closeness centrality (CC) to investigate the network's topological properties.The overlapping targets of EU and HIRI were imported into the STRING database . GO analysis was used to identify biological processes (BP), cellular components (CC), and molecular functions (MF). KEGG pathway analysis helped identify important signaling pathways involved in biological processes. The GO and KEGG data were uploaded to the bioinformatics platform (http://www.bioinformatics.com.cn/) for visualization and analysis.To study the biological functions of potential targets in the anti-HIRI effects of EU, the Gene Ontology (GO) analysis and KEGG pathway data were collected using the Database for Annotation36. It is a theoretical simulation method that combines modeling and affinity to investigate molecular interactions and predict binding modes. AutoDock 4 (1.5.6) was used to perform molecular docking of the active components of EU with the key targets, predicting their binding modes and binding affinities. The following process was conducted:The active components downloaded from the Protein Data Bank (PDB) were saved in mol2 format and imported into Chembio3D software for energy minimization. AutodockTools-1.5.6 software was used to add hydrogens, calculate charges, and assign charges. The ligand flexibility was set in the Ligand option and saved as a “pdbqt” format file.http://www.rcsb.org/) website. The proteins were imported into PyMOL (2.3.0) software to extract the original ligands and remove water molecules. The proteins were then imported into AutoDockTools (v1.5.6) software to add hydrogens, calculate charges, and assign charges. The atom types were set as Macromolecule and saved as a "pdbqt" format file.The key target proteins were downloaded from the PDB (In AutoDockTools (v1.5.6) software, the grid box was centered on the original ligand of the protein or around the reported key amino acid residues if no suitable original ligand was available.The docking box size was set to 80 × 80 × 80 (grid spacing of 0.375 Å), and other parameters were kept at their default values.The results were analyzed and processed using PyMOL and Ligplot software.Molecular docking is a commonly used method in drug discovery to study the recognition and interaction between receptors and ligandsEucommia ulmoides” or “Eucommia ulmoides bark” in the TCMSP database resulted in 147 Eucommia ulmoides-related active components. After filtering based on drug-likeness (DL) ≥ 0.18 and oral bioavailability (OB) ≥ 30%, 26 effective active components were selected. By analyzing the “Related Targets” information of these active components, a total of 479 target proteins were obtained. After removing duplicates, 207 target proteins were found to be associated with EU. Given the diverse active components and multiple target interactions of EU, we constructed a network of EU components and targets, as shown in Fig. A search for the keywords “Based on the Venn diagram results, there were 112 overlapping genes between the potential targets of EU and the target genes associated with HIRI. These common genes were imported into the STRING database to construct the protein–protein interaction (PPI) network. After removing disconnected nodes, the protein interaction network was obtained, as shown in Fig. p-value < 0.05, and the results were sorted in descending order based on the count of hit genes. The top 10 enriched terms are shown in Fig. The overlapping 112 target genes of EU and HIRI were subjected to Gene Ontology (GO) enrichment analysis using the DAVID database. A total of 712 biological processes, 65 cellular components, and 113 molecular functions were obtained under the GO terms. The enrichment analysis was performed with a significance threshold of Based on the previous PPI analysis and compound-target network analysis, we selected the top three active compounds: quercetin (MOL000098), paeonol (MOL000422), and β-sitosterol MOL000358), along with three key target proteins RELA (PDB ID: 1NFI), AKT1 (PDB ID: 3O96), and TP53 (PDB ID: 6MXY). The molecular docking was performed to predict the binding affinity between the active compounds and the target proteins. The docking scores are shown in Table 58, alongThe molecular docking results revealed the interactions between quercetin and RELA, AKT1, and TP53 Fig. A–C. In FPaeonol was found to interact with RELA, AKT1, and TP53 Fig. D–F. In FSimilarly, β-sitosterol showed interactions with RELA, AKT1, and TP53 Fig. G–I. In FThe lower the binding energy, the higher the affinity and stability of the ligand-receptor complex. The docking results indicated that all of these compounds had binding energies below − 5 kcal/mol, suggesting strong affinities with the target proteins. Among them, the docking between paeonol and RELA showed the lowest binding energy , while the docking between paeonol and AKT1 exhibited the lowest binding energy . The docking between paeonol and TP53 had the lowest binding energy . These three compounds  demonstrated good binding with the three core targets . Based on these findings, we predict that these compounds may play a key role in the treatment of HIRI.38. In severe cases, it can lead to liver failure and even mortality. Unfortunately, currently, there are no approved pharmaceutical treatments or interventions available for the prevention and management of HIRI. Previous investigations have indicated that calcium channel blockers, adenosine receptor agonists, energy metabolism regulators, and antioxidants demonstrate protective effects against HIRI10. However, the considerable adverse effects associated with these medications have limited their research to animal models, lacking clinical evidence, and impeding their application in clinical practice. Research on pharmacological pre-conditioning for the prevention of HIRI has yet to achieve significant breakthroughs. Therefore, it is of paramount importance to explore effective and innovative approaches for the prevention and management of HIRI. Traditional Chinese medicine (TCM) has garnered considerable attention in the scholarly community in recent years due to its wide safety range, multitarget effects, and minimal adverse effects39.Hepatic ischemia–reperfusion injury (HIRI) is a common complication observed during liver surgeries (such as liver transplantation and hepatectomy) and in the treatment of ischemic liver diseases (such as hepatic artery occlusion and liver infarction). HIRI is also a significant factor that impacts liver function and patient survival following liver transplantation. It gives rise to severe pathological and physiological alterations in the liver, including impaired liver function, hepatocyte apoptosis, inflammatory response, and cellular necrosis40. We noticed the anti-inflammatory pharmacological effects of EU in our effort to solve the problem of HIRI41, and we thought about the pathogenesis of sterile inflammation in HIRI and the anti-inflammatory effects of EU. Interestingly, we found that pre-treating rats with EU and some of its monomer components  could significantly reduce HIRI, and the mechanism involved reducing the generation of ROS during HIRI and inhibiting the sterile inflammatory response mediated by HMGB1 during HIRI42, thereby exerting a protective effect against HIRI. It can be seen that the role of EU in HIRI is worth affirming. However, the composition of EU is complex21, and there are many potential targets and mechanisms of action, many of which are still unknown. Traditional research methods cannot quickly and accurately elucidate the effective active ingredients and specific molecular mechanisms of drug action, so in-depth research faces some challenges. Therefore, it is very important to use computer algorithms such as network analysis and molecular docking to comprehensively strengthen the research on the prevention and treatment of HIRI with EU and its mechanisms. Exploring the protective effect and mechanism of EU on HIRI is an important supplement to drug pretreatment for the prevention and treatment of HIRI, and also lays a theoretical foundation for improving the prognosis of liver resection patients and promoting the development of liver transplantation.HIRI is an inevitable consequence of major liver surgery, and the sterile inflammatory response that endangers organ vitality in the early stage of reperfusion is a very important phase of HIRI43. Previous studies have also demonstrated that quercetin alleviates HIRI by inhibiting ERK/NF-κB pathway-induced apoptosis and autophagy44. Furthermore, in a rat model of HIRI, quercetin liposomes were found to be a valuable tool for addressing the current limitations in HIRI treatment45. However, it should be noted that the dosage of quercetin must be carefully considered, as different dosages can lead to varied outcomes46. Regarding kaempferol, a previous study on HIRI models suggested that its protective effects are mediated through the activation of the Nrf2/HO-1 signaling pathway, leading to the inhibition of oxidative stress and inflammation47. Additionally, kaempferol has demonstrated beneficial effects in liver, brain, lung, kidney, and myocardial ischemia–reperfusion injuries, primarily by suppressing oxidative stress and sterile inflammation53. Although no specific reports regarding the effects of β-sitosterol on HIRI were found, β-sitosterol has been shown to prevent myocardial ischemia–reperfusion injury (MIRI) both in vivo and in vitro, potentially through the regulation of the PPARγ/NF-κB signaling pathway54. Another study demonstrated that β-sitosterol pretreatment increased glutathione activity and superoxide dismutase levels while reducing malondialdehyde expression in the heart and kidneys, thus exerting protective effects against MIRI and renal ischemia–reperfusion injury (KIRI) by limiting inflammatory responses and oxidative stress55. Furthermore, β-sitosterol pretreatment reduced ischemic brain injury in rats after unilateral common carotid artery occlusion and exhibited therapeutic effects on damaged blood vessels57. These findings suggest the significant importance of these active components  in the prevention and treatment of HIRI using EU, warranting further exploration.Although several studies have demonstrated the anti-inflammatory effects of EU in hepatic ischemia–reperfusion injury (HIRI), the underlying molecular mechanisms of EU in preventing HIRI remain largely unclear. The aim of this study was to systematically investigate the potential mechanisms of EU in the prevention and treatment of HIRI. Initially, a total of 26 active components of EU, including quercetin, β-sitosterol, and kaempferol, were collected from the TCMSP database and relevant literature. In the component-target network, quercetin exhibited the highest degree, indicating its extensive connectivity. β-sitosterol ranked second in terms of, followed by kaempferol. Quercetin, the most abundant active component in EU, is considered an antioxidant and HO-1 inducer. Its strong HO-1 induction ability contributes to its prominent role in preventing HIRIWe collected HIRI disease targets from databases and found that EU shares 112 common targets with HIRI. These target genes are considered potential targets for EU in the prevention and treatment of HIRI. To further identify core target genes from these numerous targets, we constructed a protein–protein interaction (PPI) network. The results revealed that RELA, AKT1, TP53, MAPK1, TNF, MAPK14, FOS, CTNNB1, MYC, and IL6 may be potential core target genes, especially RELA, AKT1, and TP53.58. Recent research has also found that inhibiting NF-κB signaling can reduce inflammation, liver injury, cell apoptosis, and oxidative stress in HIRI mice58. Interestingly, in other studies investigating the intervention of HIRI with other herbal monomers, such as chlorogenic acid (CGA) and resveratrol, it was found that they can prevent HIRI by inhibiting TLR-4/NF-κB signaling pathway-mediated inflammation and mitochondria-mediated cell apoptosis60. TNF-α and IL-6, as typical inflammatory factors and key mediators of HIRI, were also found among our predicted potential targets, along with RELA, TNF, and IL-6. Therefore, it can be inferred that the prevention of HIRI by herbal monomers may primarily occur through the inhibition of RELA (NF-κB/p65) phosphorylation, thereby suppressing the expression of inflammatory factors, oxidative stress reactions, and cell apoptosis.RELA, also known as NF-κB/p65, is one of the five components of the NF-κB transcription factor family. Studies have shown that HIRI significantly induces phosphorylation of NF-κB/p65 and increases the expression of pro-inflammatory cytokines, including IL-6 and TNF-α. However, pretreatment with kaempferol can dose-dependently reverse these effects, thereby alleviating HIRI65.Akt, a serine/threonine kinase, is an oncogene that has attracted significant attention in the medical field due to its important role in regulating cell growth, metabolism, transcription, proliferation, protein synthesis, and survival. Among the three Akt isoforms  predominantly expressed in mammals, Akt1 exhibits widespread expression in tissues. Studies by Gao et al. have shown that limb ischemic preconditioning (LIPOC), acidic microenvironment, microRNA-218-5p, and microRNA-494 can alleviate HIRI by modulating the PI3K/Akt signaling pathway, which involves reducing cell apoptosis, inflammatory responses, and oxidative stress66. It has also been reported that NO can inhibit TP53 gene expression, thereby reducing the secretion of pro-inflammatory and chemotactic factors such as TNF-α and IL-1 to alleviate HIRI67. It is evident that inflammation plays a central role in the pathogenesis of HIRI.TP53 is a crucial tumor suppressor gene. Interestingly, previous studies have suggested that TP53 is upregulated in HIRI, but treatment with hydrogen-rich saline can significantly reduce TP53 expression at the mRNA and protein levelsInterestingly, EU exhibits strong anti-inflammatory effects, and previous studies have found that pretreatment with EU can improve HIRI in rats. Therefore, research focusing on inflammation may be a promising strategy for the prevention and treatment of HIRI using EU.68. The PI3K/AKT pathway is a complex and crucial signaling pathway that dynamically regulates various processes such as inflammation, metabolism, cell apoptosis, cell survival, and cell cycle71. Various preoperative interventions act on the PI3K/AKT signaling pathway to reduce cell apoptosis, sterile inflammation, and oxidative stress, attenuating liver injury caused by transient liver ischemia and preventing the occurrence of HIRI65.To explore the underlying mechanism of EU in preventing and treating HIRI, we conducted GO enrichment analysis and KEGG pathway analysis. GO enrichment analysis revealed significant enrichment in processes such as positive regulation of gene expression, positive regulation of transcription from RNA polymerase II promoter, negative regulation of apoptotic process, positive regulation of transcription, DNA-templated, and response to drug. In the top 30 enriched pathways identified by KEGG analysis, pathways involved included cancer pathway, AGE-RAGE signaling pathway in diabetic complications, lipid and atherosclerosis, TNF signaling pathway, PI3K-Akt pathway, and apoptosis. Inflammation, cell apoptosis, and oxidative stress play important roles in HIRI72. In addition, our previous research has found that chlorogenic acid can alleviate liver ischemia–reperfusion injury by suppressing HMGB1/TLR-4/NF-κB-mediated inflammation, oxidative stress, and cell apoptosis60. Subsequently, more studies have demonstrated the association between the mechanisms of preoperative intervention in preventing HIRI and the HMGB1/TLR-4/NF-κB-mediated inflammatory response75. Therefore, targeting the signaling pathway mediated by this pathway has great potential for the prevention and treatment of HIRI using EU.As mentioned earlier, RELA (NF-κB/p65) is a core regulatory factor of inflammatory mediators in HIRI, and mTOR activity in liver cells is dependent on the NF-κB pro-inflammatory signaling pathway mechanism to reduce the susceptibility of the liver to injuryIn this study, we employed a network pharmacology approach to identify the main active ingredients and target proteins of EU in preventing and treating HIRI. We then utilized protein–protein interaction (PPI) network analysis to identify key target proteins involved in EU's effects on HIRI. Furthermore, we conducted biological enrichment analysis to explore the potential molecular mechanisms underlying EU's effects on HIRI. Our findings revealed that EU exhibits a multi-component, multi-target, and multi-pathway characteristic in its effects on HIRI, with a focus on targets and pathways associated with inflammation, oxidative stress, and cell apoptosis. We further validated our predictions through molecular docking, which showed binding energies of less than 5.0 kcal/mol, indicating that these three compounds can effectively bind to their target proteins. Among them, β-sitosterol exhibited the lowest binding energy with Akt1, suggesting a more stable interaction between β-sitosterol and Akt1 protein.76. This research on these compounds is mostly limited to cellular and animal models. In particular, there is currently no reported research on the intervention of HIRI using β-sitosterol. Therefore, further investigation in this direction is crucial in the search for preventive drugs for HIRI, and we believe that the significance of research in this area is substantial.However, it is important to acknowledge some limitations of this study. Firstly, since our study relied on network databases, there may be some active ingredients and target proteins that were not included due to incomplete or unverified information in the databases. Secondly, further experimental validation is lacking for this assertion. There are research studies that have reported quercetin and naringenin were identified as the most important active ingredients of EU in preventing and treating IRIIn this study, we employed network pharmacology methodologies to investigate the underlying mechanisms behind the protective effects of EU against HIRI. Through the screening of active compounds and target proteins, construction of protein–protein interaction networks, and biological enrichment analysis, we discovered that EU exhibits a complex profile with respect to its multi-component, multi-target, and multi-pathway characteristics in combating HIRI, primarily focusing on targets and pathways associated with inflammation, oxidative stress, and cellular apoptosis. Molecular docking results further corroborated our predictions, indicating that quercetin, β-sitosterol, and paeonol can effectively bind to their respective target proteins. Through this study, we have the opportunity to direct our future experiments, screen various drugs, and delve deep into the specific molecular mechanisms. We hold the belief that, one day in the near future, EU or one of its potent active compounds can be utilized in clinical settings to alleviate HIRI, thus eliminating its hindrance to the selection of liver transplantation for individuals with end-stage liver disease.Supplementary Information.