Network pharmacology and molecular docking to explore the potential mechanism of Chlorogenic acid  in combined septic acute liver injury

Network pharmacology and molecular docking to explore the potential mechanism of Chlorogenic acid in combined septic acute liver injury | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Network pharmacology and molecular docking to explore the potential mechanism of Chlorogenic acid in combined septic acute liver injury shangping fang, Hui Su, Jiameng Liu, Kecheng Zhai, Yangmengna Gao, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4780298/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 02 Jan, 2025 Read the published version in Naunyn-Schmiedeberg's Archives of Pharmacology → Version 1 posted 9 You are reading this latest preprint version Abstract Objective To investigate the biological activities and mechanisms of chlorogenic acid (CGA) in treating septic acute liver injury (SALI) using network pharmacology, molecular docking, and in vivo studies. Methods Potential targets related to both chlorogenic acid and septic acute liver injury were searched from public databases. Protein-protein interaction (PPI), Gene Ontology (GO), and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were conducted. Molecular docking was performed to predict the binding affinity between the active compounds and core targets. Finally, in vitro and in vivo experiments were carried out for further validation. Results A total of 60 common targets were identified between acute septic liver injury and chlorogenic acid, among which 10 shared core targets were screened using Cytoscape. Molecular docking results indicated that these core targets had good binding activity with chlorogenic acid. In the SALI mouse model, chlorogenic acid demonstrated significant protective effects on the liver and anti-inflammatory properties, acting through the TLR4/NF-κB pathway. Conclusion CGA not only improves pathological damage in acute septic liver injury but also exerts its effects potentially through multiple pathways including TLR4. chlorogenic acid septic acute liver injury network pharmacology molecular docking TLR4 Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Sepsis is a life-threatening condition characterized by a dysregulated host response to infection or injury, leading to systemic inflammation and multiple organ dysfunction, which can progress to septic shock and organ failure [ 1 , 2 ]. According to the Global Sepsis Alliance, sepsis affects 47 to 50 million people worldwide annually, with at least 11 million deaths, resulting in one death every 2.8 seconds, and mortality rates ranging from 15% to over 50% annually [ 3 , 4 ]. Sepsis acute liver injury (SALI) is a common complication that directly contributes to disease progression and mortality, posing a significant challenge in ICUs due to its poor prognosis and high mortality rates [ 5 , 6 ]. Currently, there is no definitive therapeutic drug available in clinical practice, highlighting the importance of exploring novel treatment options. Chlorogenic acid (CGA), extracted from traditional Chinese medicines such as honeysuckle and eucommia bark, possesses a wide range of biological activities. Modern pharmacological studies have shown that CGA exhibits antibacterial, antiviral, hepatoprotective, cholagogic, antitumor, hypotensive, hypolipidemic, free radical scavenging, and central nervous system stimulating effects, making it a promising therapeutic agent for sepsis [ 7 , 8 ]. The Chemical structural formula of CGA in Fig. 2A.Research indicates that CGA can mitigate LPS-induced septic liver injury [ 9 ], but the underlying molecular mechanisms remain unclear. Therefore, this study aims to investigate whether CGA can improve CLP-induced septic acute liver injury in mice and delve into its specific mechanisms of action, providing experimental and theoretical foundations for the clinical application of CGA. Additionally, network pharmacology and molecular docking techniques were employed to further elucidate the therapeutic mechanisms of CGA. Materials and methods CGA Target Gene Analysis of CGA The molecular formula of CGA was obtained from the PubChem database ( https://pubchem.ncbi.nlm.nih.gov/ ). This compound was then imported into the Swiss Target Prediction database ( http://www.swisstargetprediction.ch/ ), with screening conditions set to target genes of the species Homo sapiens with Prob > 0, to identify potential targets of CGA. Based on the UniProt database ( https://www.uniprot.org/ ), the target names of the genes were standardized along with their corresponding protein IDs. By searching the Similarity Ensemble Approach database ( https://sea.bkslab.org/ ), the target genes of CGA were identified, with duplicates removed and merged based on the criterion of MaxTC > 0.4. Ultimately, the potential targets of CGA were identified. Target Prediction for Septic Acute Liver Injury Database Search: Using the keyword "septic acute liver injury," searches were conducted in Gene Cards ( https://www.genecards.org/ ), OMIM ( https://omim.org/ ), TTD ( http://db.idrblab.net/ttd/ ), Drug Bank ( https://go.drugbank.com/ ) and Pharm GKB databases ( https://www.pharmgkb.org/ ). The target information from these five databases was combined, and duplicate targets were removed. Construction of Overlapping Targets and Protein–protein interaction network The overlapping targets between CGA and acute septic liver injury were identified using the jvenn online tool ( https://jvenn.toulouse.inrae.fr/app/example.html)t o create a Venn diagram, yielding the intersection targets representing CGA's potential targets in treating acute septic liver injury. These intersecting targets were imported into the String 12.0 database ( https://cn.string-db.org/ ), with the species set to "Homo sapiens" and a minimum required interaction score > 0.9, to construct a PPI (protein-protein interaction) network. The top 10 core potential targets were selected using the CytoNCA tool in Cytoscape 3.9.1. GO and KEGG pathway enrichment analysis The biological processes (BP), molecular functions (MF), and cellular components (CC) of the core potential targets were enriched through GO analysis, while KEGG pathway enrichment analysis was also conducted using the bioinformatics platform ( http://www.bioinformatics.com.cn ). This process aimed to identify the potential biological processes and pathways involved in CGA's treatment of septic acute liver injury. Molecular Docking Protein crystal structures of the 10 core targets identified in Section of Protein–protein interaction network (EGFR(2xkn), ESR1(1xpc), GSK3B(1q5k), PTGS2(3nt1), LCK(1qpc), TLR4(2z62), PPARA(2p54), HSP90AA1(2qfo), ACE(3nxq), MMP9(4xct)) were retrieved from the RCSB PDB database ( https://www.rcsb.org/ ). Using PyMol software, water molecules were removed, ligands were detached, and duplicate sequences were deleted, saving the files in PDB format. AutoDock Tools was then used to add hydrogen atoms, calculate charges, and assign AD4 atom types to the protein receptors. Finally, PyMol was employed for visualization. Animal experiment Animal Model Establishment The septic acute liver injury mouse model was established following the cecal ligation and puncture (CLP) method described in reference [ 10 ]. Mice were acclimatized for one week prior to the procedure, with food withheld for 12 hours before surgery but water allowed ad libitum. Mice were anesthetized with an intraperitoneal injection of 1% pentobarbital sodium (50 mg/kg), then fixed in a supine position. After shaving and disinfecting the abdominal area with alcohol, 1 cm incision was made along the midline. The incision was deepened layer by layer until the abdominal cavity was exposed. The mesentery and cecum were freed, and the cecum was ligated at the junction of the mid- and outer thirds of its antimesenteric border. A sterile 1 mL syringe needle was used to puncture the ligated tip of the cecum, and intestinal contents the size of a mung bean were extruded. The cecum was then returned to the abdominal cavity, and the incision was closed. Mice in the sham group underwent the same surgical procedure except for the ligation and puncture of the cecum. Successful modeling was indicated by lethargy, hypersomnia, reduced activity, cold intolerance, piloerection, and tachypnea in the mice. Animal Grouping and Drug Administration All mice used in this experiment were purchased from SPF (Beijing) Biotechnology Co., Ltd. (Animal Use License No. SCXK (Jing) 2019-0010), and the animal studies complied with the ethical standards set by the Experimental Animal Ethics Committee of Wannan Medical College. After one week of acclimatization, mice were randomly divided into four groups of 10 mice each: Sham group, CLP group, CGA low-dose (Low) group, and CGA high-dose (High) group. Twelve hours before CLP modeling, mice in the Sham and CLP groups received an intraperitoneal injection of saline (20 mg/kg), while mice in the Low group received CGA (20 mg/kg) and mice in the High group received CGA (100 mg/kg). Eight hours after CLP modeling, blood samples were collected from the eyeball and liver tissues were harvested. Main reagents and Reagents BCA Protein Assay Kit (Batch No. P0010), NF-κBp65 antibody (Catalog No. AF0246), TLR4 antibody (Batch No. AF8187), and α-tubulin (Catalog No. AF2827) were obtained from Beyotime Biotechnology (Shanghai). Histological Analysis After modeling, the right superior lobe of the mouse liver was harvested and fixed in 4% paraformaldehyde for 24 hours. The liver tissue was then dehydrated through a graded ethanol series, embedded in paraffin, and sectioned into 5 µm thick slices. Sections were stained with hematoxylin and eosin (HE) and examined under a microscope. Three random fields were selected from each slide, and a liver injury pathology score was assigned based on the following criteria: 1) Spotty necrosis (scored 0–4, with 0 indicating no injury and 4 indicating severe injury); 2) Fatty degeneration (scored 0–3); 3) Portal inflammation (scored 0–3); 4) Ballooning degeneration (scored 0–3); 5) Leukocyte infiltration and fibrin exudation (scored 0–3). The scores were summed to determine the severity of liver injury, ranging from 0 (no injury) to 16 (severe injury). Western Blotting Analysis Proteins were extracted from liver tissue using RIPA lysis buffer. The extracted proteins were then quantified using an enhanced BCA protein assay kit (Beyotime), heated for denaturation, separated by SDS-PAGE electrophoresis, and transferred onto a PVDF membrane. After blocking the membrane with a 1% bovine serum albumin (BSA) solution for 1 hour, the membrane was incubated with primary antibodies (TLR4 at 1:1000 dilution; NF-κB p65 at 1:1000 dilution) overnight at 4°C. Subsequently, the membrane was incubated with a secondary antibody conjugated with HRP (1:10000 dilution) for 1 hour at room temperature. The membrane was exposed using a Fluor Chem M imaging system, and the relative densities were analyzed with Image J2x analysis software (NIH). Statistical Analysis Data are presented as mean ± standard deviation ( ‾x ± s). For comparisons among multiple groups, one-way ANOVA was performed, followed by Fisher's least significant difference (LSD) test. Values of p < 0.05 were considered statistically significant. All statistical analyses were conducted using GraphPad Prism 9 program software. Results Septic Acute Liver Injury Target Prediction Results A total of 97 targets were obtained from the CGA database. Additionally, 2852 relevant targets related to septic acute liver injury were retrieved from the Gene Cards, OMIM, TTD, and PharmGKB databases. By using Venn diagram software to intersect the CGA targets with the relevant targets of septic acute liver injury, 60 common targets were identified (Fig. 1 ). PPI Network Construction and Topological Analysis The 60 intersecting targets were imported into the STRING website to conduct a Protein-Protein Interaction (PPI) analysis of the potential therapeutic targets of CGA for septic acute liver injury (Fig. 2B). The protein interaction information was visualized in a graphical format. Topological analysis was performed using Cytoscape 3.9.1 software with the CytoNCA plugin, which identified and placed nine core targets at the center of the network: EGFR, ESR1, GSK3B, PTGS2, TLR4, PPARA, HSP90AA1, ACE, and MMP9 (Fig. 2C). GO and KEGG Analysis Enrichment Analysis The key targets obtained were subjected to GO enrichment analysis, and a bubble plot (Fig. 2D) was generated using a bioinformatics platform. The results revealed 150 molecular functions (MF), 92 cellular components (CC) enriched in various cell types, and 1375 biological processes (BP) affected. In terms of MF, the primary enrichments were in protein kinase activity, kinase binding, and protein domain-specific binding. For BP, significant enrichments were observed in platelet activation, regulation of inflammatory response, blood coagulation, hemostasis, coagulation, reactive oxygen species metabolic process, and peptide-serine phosphorylation. Regarding CC, high enrichment percentages were found in membrane rafts, membrane microdomains, and membrane regions. The key targets were further subjected to KEGG enrichment analysis, which showed that the target genes were significantly enriched in 116 signaling pathways (Count > 2, P < 0.05). The top 20 pathways were selected for visual analysis in a bubble plot (Fig. 2E)(where the y-axis represents the enriched terms, the x-axis indicates the proportion of genes, larger bubbles represent a higher number of enriched genes, and redder colors indicate more significant enrichment). These pathways were mainly concentrated in cancer, insulin resistance, inflammatory diseases, and signaling pathways such as IL-17 and NF-kB. Figure 2 Network Pharmacology Diagram of CGA Treatment for Septic Acute Liver Injury A: Chemical structural formula of CGA. B: Targets intersection PPI network diagram between CGA and septic acute liver injury. C: Topological analysis diagram of the targets intersection. D: GO enrichment analysis diagram for targets intersection. E: Analyzed targets intersection KEGG pathway map. Molecular Docking verfication To further validate the predictive capabilities of bioinformatics, molecular docking techniques were employed to explore the potential of CGA in treating septic acute liver injury. Following selection through CytoNCA analysis, the most crucial SALI targets (EGFR, ESR1, GSK3B, PTGS2, TLR4, PPARA, HSP90AA1, ACE, and MMP9) were subjected to molecular docking with CGA (Fig. 3A-I). The binding free energies of CGA with EGFR, ESR1, GSK3B, PTGS2, TLR4, PPARA, HSP90AA1, ACE, MMP9 were − 7.2, -6.8, -7.7, -8.7, -6.1, -6.8, -7.3, -8.4, and − 8.6 kcal/mol, respectively. The free energies of docking binding of the above molecules were all not greater than − 5 kcal/mol, indicating that CGA had good binding ability to the core targets of septic acute liver injury. The cartoon structure diagrams provide an enlarged view of the ligand-protein binding residues. Figure 3 CGA docking with receptor protein molecules CGA binds to A: EGFR, B: ESR1, C: GSK3B, D: PTGS2, E: MMP9, F: TLR4, G: PPAR, H: HSP90A1, I: ACE, respectively. The Effect of CGA on Liver Injury in Septic Mice Macroscopic examination of the liver from mice in each group revealed (Fig. 4A) that the sham group exhibited no apparent abnormalities, while the CLP group showed visibly darker, swollen, and congested livers. In contrast, the CGA group (20 mg/kg) displayed significant improvement compared to the CLP group. These findings suggest that CGA can alleviate liver injury in mice. HE staining results (Fig. 4B) demonstrated that the liver tissue of the sham group had normal cellular morphology and structure, with intact central veins, clear lobular architecture, and neatly arranged cells, without significant cellular edema or necrosis. In contrast, the CLP group showed disrupted hepatocyte structure, hydropic degeneration, massive inflammatory cell infiltration, punctate necrosis, and prominent bridging necrosis between central veins, indicating liver damage and successful induction of sepsis through cecal ligation and puncture. Treatment with both low and high doses of CGA significantly reduced inflammatory cell infiltration, cellular edema, and necrosis in the liver tissue, with the high-dose group exhibiting lower pathological damage scores compared to the low-dose group. The liver pathological scores (Fig. 4D) indicated a marked increase in SALI pathological damage scores, which were significantly improved by CGA pretreatment, as evidenced by reduced scores for cellular inflammation and edema (P < 0.0001). These results suggest that CGA can effectively ameliorate CLP-induced septic liver injury in mice. TUNEL staining results (Fig. 4C, E) showed a significant increase in the apoptotic rate of hepatocytes in the CLP group. However, pretreatment with both low and high doses of CGA reduced the apoptotic rate in the kidney tissue of septic mice (P < 0.05), indicating a protective effect against cell death. Figure 4 Experimental validation of CGA treatment in a mouse model of SALI. A: General liver of mice in each group. B: HE staining of liver tissues in each group (Original magnification, 400x. Scale bar = 40µm); C: TUNNEL of liver cells in SALI mice. D: Pathological score of liver tissue in each group. E: The proportion of apoptotic cells n = 10, ± s. ### P < 0.001, *** P < 0.001, ### P < 0.001 vs the sham group; *** P < 0.001 vs the CLP group. sham: sham group; CLP: cecum ligation and puncture. Low: CGA low-dose group, High: CGA high-dose group. The Effect of CGA on Serum Inflammatory Factors and TLR4/NF-κB Pathway in Mice with Septic Acute Liver Injury ELISA analysis of serum samples from mice in each group (Fig. 5A-B) showed that compared to the sham group, the CLP group had significantly elevated serum levels of the inflammatory cytokines TNF-α and IL-1β (p < 0.001). However, both low and high doses of CGA treatment significantly reduced the levels of TNF-α and IL-1β compared to the CLP group (p < 0.05). These results indicate that CGA can decrease the expression of inflammatory factors in the liver tissue of SALI mice, thereby alleviating the inflammatory response in the liver during sepsis. Further validation of TLR4 and NF-κB protein expression was performed using Western blot analysis (Fig. 5C-E). Compared to the sham group, the CLP group exhibited significantly increased expression of TLR4 and NF-κB proteins (P < 0.05). In contrast, both the low and high dose CGA treatment groups showed significantly reduced expression of TLR4 and NF-κB proteins compared to the CLP group (P < 0.05). These findings suggest that CGA may mitigate hepatic cellular damage by regulating the TLR4/NF-κB pathway. Figure 5 Effect of CGA on Inflammatory Factors and TLR4/NF-κB Signaling Pathway in SALI A: The expression of TNF-αin serum of each group. B: The expression of IL-1βin serum of each group. C:The Western blot strip of TLR4 and NF-κB;D: Gray level analysis of TLR4 relative to internal reference; E: Gray level analysis of NF-κB relative to internal reference. n = 10. ###P < 0.001 vs the sham group; *P < 0.05, ***P < 0.001 vs the CGA group. # P < 0.05 vs the sham group; * P < 0.05 vs the CLP group. sham: sham group; CLP: cecum ligation and puncture. Low: CGA low-dose group, High: CGA high-dose group. Discussion Septic Acute Liver Injury, a high-morbidity condition in ICUs, is associated with a significant mortality rate, and currently, there are no definitive and effective therapeutic agents with minimal side effects available in clinical practice [ 11 , 12 ]. Consequently, many research efforts have focused on exploring potential treatments for SALI from traditional Chinese medicine (TCM) [ 13 , 14 ]. Chlorogenic acid (CGA), primarily found in plants such as Lonicera, Artemisia, Eucommia, honeysuckle, coffee, and chrysanthemum, has been extensively studied for its antioxidant, anti-inflammatory, antibacterial, and antiviral properties [ 15 ]. Bagdas' study revealed that CGA accelerates wound healing in diabetic rats by increasing hydroxyproline content and reducing malondialdehyde, nitric oxide, and glutathione levels, without affecting the expression of superoxide dismutase and catalase [ 16 ]. Wang L et al. discovered that CGA enhances the permeability of both the plasma membrane and outer membrane of bacterial cells, leading to damage in barrier function, release of cytoplasmic macromolecules, and depletion of intracellular potential, thereby exerting an antibacterial effect [ 17 ]. Karar et al. found that CGA inhibits the activity of ceramidase and Clostridium perfringens, demonstrating its antiviral potential [ 18 ]. Furthermore, CGA downregulates lipopolysaccharide-induced cyclooxygenase, NF-κB activity, and cytokine expression, contributing to ROS clearance and anti-inflammatory effects [ 19 ]. In our study, the CLP model successfully induced septic acute liver injury, as evidenced by gross structural changes such as liver swelling, increased volume, and pronounced congestion, accompanied by significantly elevated serum TNF-α,IL-1β levels. Similar to previous studies by Arunachalam AR et al. using LPS/GalN (lipopolysaccharide/galactosamine) induce septic acute liver injury respectively [ 20 ]. Pretreatment with CGA significantly alleviated liver damage and improved hepatic pathological injury score, indicating its protective role in septic acute liver injury. This protection may be attributed to CGA's systemic distribution following intraperitoneal absorption, with accumulation in the liver to mitigate injury upon occurrence. To investigate the underlying mechanism, we measured inflammatory factor levels in serum and liver tissue. Our results showed that CGA pretreatment significantly reduced TNF-α and IL-1β levels compared to the sepsis-induced liver injury group. IL-1β, an inflammatory mediator, plays a crucial role in mediating liver injury [ 21 ], while TNF-α, apart from regulating tumor cell growth, also promotes inflammation by activating TNFR1 and TNFR2, leading to apoptosis or necrosis and augmenting the inflammatory response [ 22 ]. Our findings demonstrate that CGA intervention significantly reduced the number of TUNEL-stained apoptotic hepatocytes, indicating its ability to inhibit cell death. Consistent with this, Ranjbary AG et al. reported that CGA can treat colon cancer by inducing cytotoxicity, cell cycle arrest, and apoptosis in colon cancer cell lines. TLR4, a transmembrane receptor belonging to the Toll-like receptor family, is primarily expressed on immune cells and endothelial cells. TLR4 recognizes various pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs), triggering immune and inflammatory responses[ 23 ]. NF-κB, a protein complex present in almost all animal cell types, regulates cellular responses to stimuli such as stress, cytokines, and free radicals. It plays a pivotal role in immune responses to infection and is closely associated with the development of inflammation, autoimmune diseases, and cancer[ 24 ]. Studies have shown that CGA inhibits the activation of the TLR4/NF-κB signaling pathway, thereby reducing the production and release of inflammatory mediators[ 25 ]. This mechanism has potential therapeutic applications in various diseases, including atherosclerosis and inflammatory bowel disease. In atherosclerosis, CGA inhibits the TLR4/MAPK/NF-κB pathway, activates autophagy, and suppresses inflammation and oxidative stress, protecting vascular endothelial cells from damage. In inflammatory bowel disease, excessive TLR4 activation is considered a key factor in intestinal inflammation. Network pharmacology research and molecular docking technology explore the key proteins and potential signaling pathways through which chlorogenic acid (CGA) exerts its therapeutic effects in acute septic liver injury (SALI) by leveraging large databases. This study's comprehensive analysis of CGA targets, pathways, and molecular docking results provides invaluable clues. Firstly, the molecular docking results indicate that CGA exhibits good binding affinity with key proteins such as EGFR, ESR1, GSK3B, PTGS2, TLR4, PPARA, HSP90AA1, ACE, MMP9, and itself (CGA). Relevant studies have also confirmed that CGA can reduce EGFR expression and phosphorylation levels, thereby inhibiting EGFR-mediated cell migration and invasion. The possible mechanism involves CGA binding to the ligand-binding domain of EGFR, preventing the binding of ligands like EGF, or it may interfere with receptor dimerization, blocking the transmission of phosphorylation signals and thus inhibiting EGFR activation. Furthermore, by inhibiting the TLR4/NF-κB signaling pathway, CGA can alleviate intestinal inflammatory responses and improve intestinal barrier function. CGA is capable of suppressing the expression of various inflammatory factors downstream of the TLR4/NF-κB pathway, including TNF-α, IL-6, VCAM-1, and ICAM-1. These inflammatory factors play pivotal roles in inflammatory reactions and immune responses, and their reduced expression levels contribute to mitigating the severity of inflammatory reactions. The study, however, has a few shortcomings. First, more research is required to determine the role of core targets in disease. The second limitation is that we did not examine the cellular effect of CGA on SALI. In our subsequent studies, we will explore the other molecular mechanisms by which CGA regulates SALI. In conclusion, chlorogenic acid can inhibit the release of inflammatory factors in mouse livers, reduce inflammatory reactions, improve liver function, alleviate hepatocyte edema, and mitigate sepsis-induced acute liver injury. Its mechanism of action likely involves inhibiting the TLR4/NF-κB pathway and suppressing the expression of TNF-α and IL-1β. This study provides novel insights and robust evidence for the clinical application of chlorogenic acid in the treatment of SALI. Declarations Ethical Approval: Our studies did not include human participates, human data or human tissues. All animal experiments conducted were compliant were approved by the Institutional Animal Care and Ethics Committee of Wannan Medical College (LLSC-2024–073). Funding: This work was supported by the Key Project Research Fund of Wannan Medical College (grant number: WK2022Z10); National College Student Innovation and Entrepreneurship Project (grant number:202310368016). Author Contribution Shangping Fang and Kecheng Zhai provided research materials and statistics.Huixian Cheng and Jiameng Liu provided the article design and data analysis. Hui Su and Huan Li provided administrative support and article design. Yangmengna Gao and Renke Sun provide the Network pharmacology and molecular docking.All the authors contributed to the manuscript writing and final review. The writers are accountable for the whole of the work, including making sure that any questions regarding the precision or integrity of any individual section are carefully investigated and resolved. The authors confirm that all data were generated in house and that no paper mill was used. Availability of data and materials: The datasets used and/or analyzed during the present study are available from the corresponding author on reasonable request. References Rello J, Valenzuela-Sánchez F, Ruiz-Rodriguez M et al (2017) Sepsis: A Review of Advances in Management[J]. 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Supplementary Files NFKB.tif TLR4.tif tubulin.tif Cite Share Download PDF Status: Published Journal Publication published 02 Jan, 2025 Read the published version in Naunyn-Schmiedeberg's Archives of Pharmacology → Version 1 posted Editorial decision: Revision requested 01 Oct, 2024 Reviewers agreed at journal 17 Sep, 2024 Reviews received at journal 16 Aug, 2024 Reviewers agreed at journal 31 Jul, 2024 Reviewers agreed at journal 26 Jul, 2024 Reviewers invited by journal 26 Jul, 2024 Editor assigned by journal 25 Jul, 2024 Submission checks completed at journal 25 Jul, 2024 First submitted to journal 22 Jul, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4780298","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":341313660,"identity":"82b53fa0-0c82-40b9-85c7-d0d9e0729f03","order_by":0,"name":"shangping fang","email":"","orcid":"","institution":"Wannan Medical College, Wuhu, Anhui Province, China","correspondingAuthor":false,"prefix":"","firstName":"shangping","middleName":"","lastName":"fang","suffix":""},{"id":341313661,"identity":"50937cea-0e09-4877-b763-335b6389427a","order_by":1,"name":"Hui Su","email":"","orcid":"","institution":"Wannan Medical College, Wuhu, Anhui Province, China","correspondingAuthor":false,"prefix":"","firstName":"Hui","middleName":"","lastName":"Su","suffix":""},{"id":341313662,"identity":"5ba11ace-08e6-4195-94c3-b97fbc2b5aea","order_by":2,"name":"Jiameng Liu","email":"","orcid":"","institution":"Wannan Medical College, Wuhu, Anhui Province, China","correspondingAuthor":false,"prefix":"","firstName":"Jiameng","middleName":"","lastName":"Liu","suffix":""},{"id":341313663,"identity":"5dc06498-2252-4794-82e2-db184f3f5235","order_by":3,"name":"Kecheng Zhai","email":"","orcid":"","institution":"Wannan Medical College, Wuhu, Anhui Province, China","correspondingAuthor":false,"prefix":"","firstName":"Kecheng","middleName":"","lastName":"Zhai","suffix":""},{"id":341313664,"identity":"9a872157-dec1-4cd9-82b0-903100a8a7fb","order_by":4,"name":"Yangmengna Gao","email":"","orcid":"","institution":"Wannan Medical College, Wuhu, Anhui Province, China","correspondingAuthor":false,"prefix":"","firstName":"Yangmengna","middleName":"","lastName":"Gao","suffix":""},{"id":341313665,"identity":"47b3bb9d-d6ff-4ac1-90c9-835872220b65","order_by":5,"name":"Huan Li","email":"","orcid":"","institution":"Wannan Medical College, Wuhu, Anhui Province, China","correspondingAuthor":false,"prefix":"","firstName":"Huan","middleName":"","lastName":"Li","suffix":""},{"id":341313666,"identity":"3bf86784-a54e-4899-bcd9-4d9fd01106af","order_by":6,"name":"Renke Sun","email":"","orcid":"","institution":"Wannan Medical College, Wuhu, Anhui Province, China","correspondingAuthor":false,"prefix":"","firstName":"Renke","middleName":"","lastName":"Sun","suffix":""},{"id":341313667,"identity":"b0e015b9-c392-4acb-ba44-29188d74369a","order_by":7,"name":"Huixian Cheng","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA5klEQVRIiWNgGAWjYBACfmbGxgcJBmw8/FABxgZCWiTbm5sNPlTwyUg2EKvF4MzxNskZZ+RsDA4Qq4XhRmKzMW+bGY/xjdyjG34w2MhuOMD87AE+HYwzEhsf87al8ZjdyEu72cOQZrzhAJu5AT4tzBJgW47xmN3OMbvNwHA4ccMBHjYJfFrYJBLbpHnb/vMYzwZr+U9YCw/PQZD32XgMpMFaDhDWIsHeCApkNh6J+2/MbvYYJBvPPMxmhleL/WH2h6CotOfvOWN240eFnWzf8eZneLWgAVBQMZOgfhSMglEwCkYBdgAACEFLFRJMKiYAAAAASUVORK5CYII=","orcid":"","institution":"Wannan Medical College, Wuhu, Anhui Province, China","correspondingAuthor":true,"prefix":"","firstName":"Huixian","middleName":"","lastName":"Cheng","suffix":""}],"badges":[],"createdAt":"2024-07-22 08:30:38","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4780298/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4780298/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s00210-024-03712-5","type":"published","date":"2025-01-02T15:57:34+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":63007437,"identity":"c7eee2d6-51dd-4519-9ee8-60e34f4b8830","added_by":"auto","created_at":"2024-08-22 04:53:06","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":39943,"visible":true,"origin":"","legend":"\u003cp\u003eVenn Diagram of CGA and Septic Acute Liver Injury Targets\u003c/p\u003e","description":"","filename":"Figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4780298/v1/f90ccd320348913c09dc9014.jpg"},{"id":63007957,"identity":"aac326ac-2d5d-45ec-bb40-f1d274efbfe9","added_by":"auto","created_at":"2024-08-22 05:01:07","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":510931,"visible":true,"origin":"","legend":"\u003cp\u003eNetwork Pharmacology Diagram of CGA Treatment for Septic Acute Liver Injury\u003c/p\u003e\n\u003cp\u003eA: Chemical structural formula of CGA. B: Targets intersection PPI network diagram between CGA and septic acute liver injury. C: Topological analysis diagram of the targets intersection. D: GO enrichment analysis diagram for targets intersection. E: Analyzed targets intersection KEGG pathway map.\u003c/p\u003e","description":"","filename":"figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4780298/v1/5073b653c72255572a4583ed.jpg"},{"id":63009381,"identity":"fe663b9e-2f58-4617-b279-3bc784f9fa3f","added_by":"auto","created_at":"2024-08-22 05:25:07","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1143265,"visible":true,"origin":"","legend":"\u003cp\u003eCGA docking with receptor protein molecules\u003c/p\u003e\n\u003cp\u003eCGA binds to A: EGFR, B: ESR1, C: GSK3B, D: PTGS2, E: MMP9, F: TLR4, G: PPAR, H: HSP90A1, I: ACE, respectively.\u003c/p\u003e","description":"","filename":"figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4780298/v1/aac9f72645134fff87fb1f4f.jpg"},{"id":63007955,"identity":"130b164a-ce01-4ee4-91b6-4990ea19bd08","added_by":"auto","created_at":"2024-08-22 05:01:07","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1144275,"visible":true,"origin":"","legend":"\u003cp\u003eExperimental validation of CGA treatment in a mouse model of SALI. A: General liver of mice in each group. B: HE staining of liver tissues in each group (Original magnification, 400x. Scale bar=40μm); C: TUNNEL of liver cells in SALI mice. D: Pathological score of liver tissue in each group. E: The proportion of apoptotic cells n=10, x̄± s.\u003csup\u003e ###\u003c/sup\u003eP\u0026lt;0.001，\u003csup\u003e***\u003c/sup\u003eP\u0026lt;0.001，\u003csup\u003e###\u003c/sup\u003eP\u0026lt;0.001 vs the sham group;\u003csup\u003e ***\u003c/sup\u003eP\u0026lt;0.001 vs the CLP group. sham: sham group; CLP: cecum ligation and puncture. Low: CGA low-dose group, High: CGA high-dose group.\u003c/p\u003e","description":"","filename":"figure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4780298/v1/b30e996d4cfc7b18ab80b1f9.jpg"},{"id":63007438,"identity":"c45ed550-32c0-4a68-9b8c-c4fa3dde0c3f","added_by":"auto","created_at":"2024-08-22 04:53:07","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":224540,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of CGA on Inflammatory Factors and TLR4/NF-κB Signaling Pathway in SALI\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA: The expression of TNF-αin serum of each group. B: The expression of IL-1βin serum of each group. C:The Western blot strip of TLR4 and NF-κB；D: Gray level analysis of TLR4 relative to internal reference; E: Gray level analysis of NF-κB relative to internal reference. n=10. ###P\u0026lt;0.001 vs the sham group; *P\u0026lt;0.05, ***P\u0026lt;0.001 vs the CGA group. \u003csup\u003e#\u003c/sup\u003eP\u0026lt;0.05 vs the sham group;\u003csup\u003e *\u003c/sup\u003eP\u0026lt;0.05 vs the CLP group. sham: sham group; CLP: cecum ligation and puncture. Low: CGA low-dose group, High: CGA high-dose group.\u003c/p\u003e","description":"","filename":"figure5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4780298/v1/be6a3b169cc36e288f1b56e2.jpg"},{"id":73093329,"identity":"23c7bbfb-f8aa-40fa-860f-bb56541aca52","added_by":"auto","created_at":"2025-01-06 16:13:50","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3617031,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4780298/v1/a759e4ea-b346-412c-a5bc-e78b46393997.pdf"},{"id":63007443,"identity":"890f166f-6396-4876-8b30-b9afd67e928c","added_by":"auto","created_at":"2024-08-22 04:53:07","extension":"tif","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1473456,"visible":true,"origin":"","legend":"","description":"","filename":"NFKB.tif","url":"https://assets-eu.researchsquare.com/files/rs-4780298/v1/b48ecf0cc0c428c37f738d6f.tif"},{"id":63007442,"identity":"d638c06b-552b-4cb0-a6f1-debfc2040a9c","added_by":"auto","created_at":"2024-08-22 04:53:07","extension":"tif","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":1473456,"visible":true,"origin":"","legend":"","description":"","filename":"TLR4.tif","url":"https://assets-eu.researchsquare.com/files/rs-4780298/v1/61ce1b444c4e5a9a62120452.tif"},{"id":63007439,"identity":"7f06a8b1-689c-444f-bedf-2e73dd668a8e","added_by":"auto","created_at":"2024-08-22 04:53:07","extension":"tif","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":1473456,"visible":true,"origin":"","legend":"","description":"","filename":"tubulin.tif","url":"https://assets-eu.researchsquare.com/files/rs-4780298/v1/2b44226e5e74f2e2e88b8b57.tif"}],"financialInterests":"No competing interests reported.","formattedTitle":"Network pharmacology and molecular docking to explore the potential mechanism of Chlorogenic acid in combined septic acute liver injury","fulltext":[{"header":"Introduction","content":"\u003cp\u003eSepsis is a life-threatening condition characterized by a dysregulated host response to infection or injury, leading to systemic inflammation and multiple organ dysfunction, which can progress to septic shock and organ failure [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. According to the Global Sepsis Alliance, sepsis affects 47 to 50\u0026nbsp;million people worldwide annually, with at least 11\u0026nbsp;million deaths, resulting in one death every 2.8 seconds, and mortality rates ranging from 15% to over 50% annually [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Sepsis acute liver injury (SALI) is a common complication that directly contributes to disease progression and mortality, posing a significant challenge in ICUs due to its poor prognosis and high mortality rates [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Currently, there is no definitive therapeutic drug available in clinical practice, highlighting the importance of exploring novel treatment options.\u003c/p\u003e \u003cp\u003eChlorogenic acid (CGA), extracted from traditional Chinese medicines such as honeysuckle and eucommia bark, possesses a wide range of biological activities. Modern pharmacological studies have shown that CGA exhibits antibacterial, antiviral, hepatoprotective, cholagogic, antitumor, hypotensive, hypolipidemic, free radical scavenging, and central nervous system stimulating effects, making it a promising therapeutic agent for sepsis [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. The Chemical structural formula of CGA in Fig.\u0026nbsp;2A.Research indicates that CGA can mitigate LPS-induced septic liver injury [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e], but the underlying molecular mechanisms remain unclear. Therefore, this study aims to investigate whether CGA can improve CLP-induced septic acute liver injury in mice and delve into its specific mechanisms of action, providing experimental and theoretical foundations for the clinical application of CGA. Additionally, network pharmacology and molecular docking techniques were employed to further elucidate the therapeutic mechanisms of CGA.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eCGA Target Gene Analysis of CGA\u003c/h2\u003e \u003cp\u003eThe molecular formula of CGA was obtained from the PubChem database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://pubchem.ncbi.nlm.nih.gov/\u003c/span\u003e\u003cspan address=\"https://pubchem.ncbi.nlm.nih.gov/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). This compound was then imported into the Swiss Target Prediction database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.swisstargetprediction.ch/\u003c/span\u003e\u003cspan address=\"http://www.swisstargetprediction.ch/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), with screening conditions set to target genes of the species Homo sapiens with Prob\u0026thinsp;\u0026gt;\u0026thinsp;0, to identify potential targets of CGA. Based on the UniProt database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.uniprot.org/\u003c/span\u003e\u003cspan address=\"https://www.uniprot.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), the target names of the genes were standardized along with their corresponding protein IDs. By searching the Similarity Ensemble Approach database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://sea.bkslab.org/\u003c/span\u003e\u003cspan address=\"https://sea.bkslab.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), the target genes of CGA were identified, with duplicates removed and merged based on the criterion of MaxTC\u0026thinsp;\u0026gt;\u0026thinsp;0.4. Ultimately, the potential targets of CGA were identified.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eTarget Prediction for Septic Acute Liver Injury\u003c/h2\u003e \u003cp\u003eDatabase Search: Using the keyword \"septic acute liver injury,\" searches were conducted in Gene Cards (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.genecards.org/\u003c/span\u003e\u003cspan address=\"https://www.genecards.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), OMIM (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://omim.org/\u003c/span\u003e\u003cspan address=\"https://omim.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), TTD (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://db.idrblab.net/ttd/\u003c/span\u003e\u003cspan address=\"http://db.idrblab.net/ttd/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), Drug Bank (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://go.drugbank.com/\u003c/span\u003e\u003cspan address=\"https://go.drugbank.com/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and Pharm GKB databases (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.pharmgkb.org/\u003c/span\u003e\u003cspan address=\"https://www.pharmgkb.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). The target information from these five databases was combined, and duplicate targets were removed.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eConstruction of Overlapping Targets and Protein\u0026ndash;protein interaction network\u003c/h2\u003e \u003cp\u003eThe overlapping targets between CGA and acute septic liver injury were identified using the jvenn online tool (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://jvenn.toulouse.inrae.fr/app/example.html)t\u003c/span\u003e\u003cspan address=\"https://jvenn.toulouse.inrae.fr/app/example.html)t\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003eo create a Venn diagram, yielding the intersection targets representing CGA's potential targets in treating acute septic liver injury. These intersecting targets were imported into the String 12.0 database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://cn.string-db.org/\u003c/span\u003e\u003cspan address=\"https://cn.string-db.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), with the species set to \"Homo sapiens\" and a minimum required interaction score\u0026thinsp;\u0026gt;\u0026thinsp;0.9, to construct a PPI (protein-protein interaction) network. The top 10 core potential targets were selected using the CytoNCA tool in Cytoscape 3.9.1.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eGO and KEGG pathway enrichment analysis\u003c/h2\u003e \u003cp\u003eThe biological processes (BP), molecular functions (MF), and cellular components (CC) of the core potential targets were enriched through GO analysis, while KEGG pathway enrichment analysis was also conducted using the bioinformatics platform (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.bioinformatics.com.cn\u003c/span\u003e\u003cspan address=\"http://www.bioinformatics.com.cn\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). This process aimed to identify the potential biological processes and pathways involved in CGA's treatment of septic acute liver injury.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eMolecular Docking\u003c/h2\u003e \u003cp\u003eProtein crystal structures of the 10 core targets identified in Section of Protein\u0026ndash;protein interaction network (EGFR(2xkn), ESR1(1xpc), GSK3B(1q5k), PTGS2(3nt1), LCK(1qpc), TLR4(2z62), PPARA(2p54), HSP90AA1(2qfo), ACE(3nxq), MMP9(4xct)) were retrieved from the RCSB PDB database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.rcsb.org/\u003c/span\u003e\u003cspan address=\"https://www.rcsb.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Using PyMol software, water molecules were removed, ligands were detached, and duplicate sequences were deleted, saving the files in PDB format. AutoDock Tools was then used to add hydrogen atoms, calculate charges, and assign AD4 atom types to the protein receptors. Finally, PyMol was employed for visualization.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eAnimal experiment\u003c/h2\u003e \u003cp\u003eAnimal Model Establishment\u003c/p\u003e \u003cp\u003eThe septic acute liver injury mouse model was established following the cecal ligation and puncture (CLP) method described in reference [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Mice were acclimatized for one week prior to the procedure, with food withheld for 12 hours before surgery but water allowed ad libitum. Mice were anesthetized with an intraperitoneal injection of 1% pentobarbital sodium (50 mg/kg), then fixed in a supine position. After shaving and disinfecting the abdominal area with alcohol, 1 cm incision was made along the midline. The incision was deepened layer by layer until the abdominal cavity was exposed. The mesentery and cecum were freed, and the cecum was ligated at the junction of the mid- and outer thirds of its antimesenteric border. A sterile 1 mL syringe needle was used to puncture the ligated tip of the cecum, and intestinal contents the size of a mung bean were extruded. The cecum was then returned to the abdominal cavity, and the incision was closed. Mice in the sham group underwent the same surgical procedure except for the ligation and puncture of the cecum. Successful modeling was indicated by lethargy, hypersomnia, reduced activity, cold intolerance, piloerection, and tachypnea in the mice.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eAnimal Grouping and Drug Administration\u003c/h2\u003e \u003cp\u003eAll mice used in this experiment were purchased from SPF (Beijing) Biotechnology Co., Ltd. (Animal Use License No. SCXK (Jing) 2019-0010), and the animal studies complied with the ethical standards set by the Experimental Animal Ethics Committee of Wannan Medical College. After one week of acclimatization, mice were randomly divided into four groups of 10 mice each: Sham group, CLP group, CGA low-dose (Low) group, and CGA high-dose (High) group. Twelve hours before CLP modeling, mice in the Sham and CLP groups received an intraperitoneal injection of saline (20 mg/kg), while mice in the Low group received CGA (20 mg/kg) and mice in the High group received CGA (100 mg/kg). Eight hours after CLP modeling, blood samples were collected from the eyeball and liver tissues were harvested.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eMain reagents and Reagents\u003c/h2\u003e \u003cp\u003eBCA Protein Assay Kit (Batch No. P0010), NF-κBp65 antibody (Catalog No. AF0246), TLR4 antibody (Batch No. AF8187), and α-tubulin (Catalog No. AF2827) were obtained from Beyotime Biotechnology (Shanghai).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eHistological Analysis\u003c/h2\u003e \u003cp\u003eAfter modeling, the right superior lobe of the mouse liver was harvested and fixed in 4% paraformaldehyde for 24 hours. The liver tissue was then dehydrated through a graded ethanol series, embedded in paraffin, and sectioned into 5 \u0026micro;m thick slices. Sections were stained with hematoxylin and eosin (HE) and examined under a microscope. Three random fields were selected from each slide, and a liver injury pathology score was assigned based on the following criteria: 1) Spotty necrosis (scored 0\u0026ndash;4, with 0 indicating no injury and 4 indicating severe injury); 2) Fatty degeneration (scored 0\u0026ndash;3); 3) Portal inflammation (scored 0\u0026ndash;3); 4) Ballooning degeneration (scored 0\u0026ndash;3); 5) Leukocyte infiltration and fibrin exudation (scored 0\u0026ndash;3). The scores were summed to determine the severity of liver injury, ranging from 0 (no injury) to 16 (severe injury).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eWestern Blotting Analysis\u003c/h2\u003e \u003cp\u003eProteins were extracted from liver tissue using RIPA lysis buffer. The extracted proteins were then quantified using an enhanced BCA protein assay kit (Beyotime), heated for denaturation, separated by SDS-PAGE electrophoresis, and transferred onto a PVDF membrane. After blocking the membrane with a 1% bovine serum albumin (BSA) solution for 1 hour, the membrane was incubated with primary antibodies (TLR4 at 1:1000 dilution; NF-κB p65 at 1:1000 dilution) overnight at 4\u0026deg;C. Subsequently, the membrane was incubated with a secondary antibody conjugated with HRP (1:10000 dilution) for 1 hour at room temperature. The membrane was exposed using a Fluor Chem M imaging system, and the relative densities were analyzed with Image J2x analysis software (NIH).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analysis\u003c/h2\u003e \u003cp\u003eData are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation ( \u0026oline;x\u0026thinsp;\u0026plusmn;\u0026thinsp;s). For comparisons among multiple groups, one-way ANOVA was performed, followed by Fisher's least significant difference (LSD) test. Values of p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 were considered statistically significant. All statistical analyses were conducted using GraphPad Prism 9 program software.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eSeptic Acute Liver Injury Target Prediction Results\u003c/h2\u003e \u003cp\u003eA total of 97 targets were obtained from the CGA database. Additionally, 2852 relevant targets related to septic acute liver injury were retrieved from the Gene Cards, OMIM, TTD, and PharmGKB databases. By using Venn diagram software to intersect the CGA targets with the relevant targets of septic acute liver injury, 60 common targets were identified (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003ePPI Network Construction and Topological Analysis\u003c/h2\u003e \u003cp\u003eThe 60 intersecting targets were imported into the STRING website to conduct a Protein-Protein Interaction (PPI) analysis of the potential therapeutic targets of CGA for septic acute liver injury (Fig.\u0026nbsp;2B). The protein interaction information was visualized in a graphical format. Topological analysis was performed using Cytoscape 3.9.1 software with the CytoNCA plugin, which identified and placed nine core targets at the center of the network: EGFR, ESR1, GSK3B, PTGS2, TLR4, PPARA, HSP90AA1, ACE, and MMP9 (Fig.\u0026nbsp;2C).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eGO and KEGG Analysis Enrichment Analysis\u003c/h2\u003e \u003cp\u003eThe key targets obtained were subjected to GO enrichment analysis, and a bubble plot (Fig.\u0026nbsp;2D) was generated using a bioinformatics platform. The results revealed 150 molecular functions (MF), 92 cellular components (CC) enriched in various cell types, and 1375 biological processes (BP) affected. In terms of MF, the primary enrichments were in protein kinase activity, kinase binding, and protein domain-specific binding. For BP, significant enrichments were observed in platelet activation, regulation of inflammatory response, blood coagulation, hemostasis, coagulation, reactive oxygen species metabolic process, and peptide-serine phosphorylation. Regarding CC, high enrichment percentages were found in membrane rafts, membrane microdomains, and membrane regions.\u003c/p\u003e \u003cp\u003eThe key targets were further subjected to KEGG enrichment analysis, which showed that the target genes were significantly enriched in 116 signaling pathways (Count\u0026thinsp;\u0026gt;\u0026thinsp;2, P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). The top 20 pathways were selected for visual analysis in a bubble plot (Fig.\u0026nbsp;2E)(where the y-axis represents the enriched terms, the x-axis indicates the proportion of genes, larger bubbles represent a higher number of enriched genes, and redder colors indicate more significant enrichment). These pathways were mainly concentrated in cancer, insulin resistance, inflammatory diseases, and signaling pathways such as IL-17 and NF-kB.\u003c/p\u003e \u003cp\u003eFigure\u0026nbsp;2 Network Pharmacology Diagram of CGA Treatment for Septic Acute Liver Injury\u003c/p\u003e \u003cp\u003eA: Chemical structural formula of CGA. B: Targets intersection PPI network diagram between CGA and septic acute liver injury. C: Topological analysis diagram of the targets intersection. D: GO enrichment analysis diagram for targets intersection. E: Analyzed targets intersection KEGG pathway map.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eMolecular Docking verfication\u003c/h2\u003e \u003cp\u003eTo further validate the predictive capabilities of bioinformatics, molecular docking techniques were employed to explore the potential of CGA in treating septic acute liver injury. Following selection through CytoNCA analysis, the most crucial SALI targets (EGFR, ESR1, GSK3B, PTGS2, TLR4, PPARA, HSP90AA1, ACE, and MMP9) were subjected to molecular docking with CGA (Fig.\u0026nbsp;3A-I). The binding free energies of CGA with EGFR, ESR1, GSK3B, PTGS2, TLR4, PPARA, HSP90AA1, ACE, MMP9 were \u0026minus;\u0026thinsp;7.2, -6.8, -7.7, -8.7, -6.1, -6.8, -7.3, -8.4, and \u0026minus;\u0026thinsp;8.6 kcal/mol, respectively. The free energies of docking binding of the above molecules were all not greater than \u0026minus;\u0026thinsp;5 kcal/mol, indicating that CGA had good binding ability to the core targets of septic acute liver injury. The cartoon structure diagrams provide an enlarged view of the ligand-protein binding residues.\u003c/p\u003e \u003cp\u003eFigure\u0026nbsp;3 CGA docking with receptor protein molecules\u003c/p\u003e \u003cp\u003eCGA binds to A: EGFR, B: ESR1, C: GSK3B, D: PTGS2, E: MMP9, F: TLR4, G: PPAR, H: HSP90A1, I: ACE, respectively.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eThe Effect of CGA on Liver Injury in Septic Mice\u003c/h2\u003e \u003cp\u003eMacroscopic examination of the liver from mice in each group revealed (Fig.\u0026nbsp;4A) that the sham group exhibited no apparent abnormalities, while the CLP group showed visibly darker, swollen, and congested livers. In contrast, the CGA group (20 mg/kg) displayed significant improvement compared to the CLP group. These findings suggest that CGA can alleviate liver injury in mice.\u003c/p\u003e \u003cp\u003eHE staining results (Fig.\u0026nbsp;4B) demonstrated that the liver tissue of the sham group had normal cellular morphology and structure, with intact central veins, clear lobular architecture, and neatly arranged cells, without significant cellular edema or necrosis. In contrast, the CLP group showed disrupted hepatocyte structure, hydropic degeneration, massive inflammatory cell infiltration, punctate necrosis, and prominent bridging necrosis between central veins, indicating liver damage and successful induction of sepsis through cecal ligation and puncture. Treatment with both low and high doses of CGA significantly reduced inflammatory cell infiltration, cellular edema, and necrosis in the liver tissue, with the high-dose group exhibiting lower pathological damage scores compared to the low-dose group.\u003c/p\u003e \u003cp\u003eThe liver pathological scores (Fig.\u0026nbsp;4D) indicated a marked increase in SALI pathological damage scores, which were significantly improved by CGA pretreatment, as evidenced by reduced scores for cellular inflammation and edema (P\u0026thinsp;\u0026lt;\u0026thinsp;0.0001). These results suggest that CGA can effectively ameliorate CLP-induced septic liver injury in mice.\u003c/p\u003e \u003cp\u003eTUNEL staining results (Fig.\u0026nbsp;4C, E) showed a significant increase in the apoptotic rate of hepatocytes in the CLP group. However, pretreatment with both low and high doses of CGA reduced the apoptotic rate in the kidney tissue of septic mice (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05), indicating a protective effect against cell death. \u003c/p\u003e \u003cp\u003eFigure\u0026nbsp;4 Experimental validation of CGA treatment in a mouse model of SALI. A: General liver of mice in each group. B: HE staining of liver tissues in each group (Original magnification, 400x. Scale bar =\u0026thinsp;40\u0026micro;m); C: TUNNEL of liver cells in SALI mice. D: Pathological score of liver tissue in each group. E: The proportion of apoptotic cells n\u0026thinsp;=\u0026thinsp;10, \u003cspan class=\"InlineEquation\"\u003e\u003c/span\u003e\u0026plusmn; s. \u003csup\u003e###\u003c/sup\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.001, \u003csup\u003e***\u003c/sup\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.001, \u003csup\u003e###\u003c/sup\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.001 vs the sham group; \u003csup\u003e***\u003c/sup\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.001 vs the CLP group. sham: sham group; CLP: cecum ligation and puncture. Low: CGA low-dose group, High: CGA high-dose group.\u003c/p\u003e \u003cp\u003e \u003cb\u003eThe Effect of CGA on Serum Inflammatory Factors and TLR4/NF-κB Pathway in Mice with Septic Acute Liver Injury\u003c/b\u003e \u003c/p\u003e \u003cp\u003eELISA analysis of serum samples from mice in each group (Fig.\u0026nbsp;5A-B) showed that compared to the sham group, the CLP group had significantly elevated serum levels of the inflammatory cytokines TNF-α and IL-1β (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). However, both low and high doses of CGA treatment significantly reduced the levels of TNF-α and IL-1β compared to the CLP group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). These results indicate that CGA can decrease the expression of inflammatory factors in the liver tissue of SALI mice, thereby alleviating the inflammatory response in the liver during sepsis.\u003c/p\u003e \u003cp\u003eFurther validation of TLR4 and NF-κB protein expression was performed using Western blot analysis (Fig.\u0026nbsp;5C-E). Compared to the sham group, the CLP group exhibited significantly increased expression of TLR4 and NF-κB proteins (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). In contrast, both the low and high dose CGA treatment groups showed significantly reduced expression of TLR4 and NF-κB proteins compared to the CLP group (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). These findings suggest that CGA may mitigate hepatic cellular damage by regulating the TLR4/NF-κB pathway.\u003c/p\u003e \u003cp\u003e \u003cb\u003eFigure\u0026nbsp;5 Effect of CGA on Inflammatory Factors and TLR4/NF-κB Signaling Pathway in SALI\u003c/b\u003e \u003c/p\u003e \u003cp\u003eA: The expression of TNF-αin serum of each group. B: The expression of IL-1βin serum of each group. C:The Western blot strip of TLR4 and NF-κB;D: Gray level analysis of TLR4 relative to internal reference; E: Gray level analysis of NF-κB relative to internal reference. n\u0026thinsp;=\u0026thinsp;10. ###P\u0026thinsp;\u0026lt;\u0026thinsp;0.001 vs the sham group; *P\u0026thinsp;\u0026lt;\u0026thinsp;0.05, ***P\u0026thinsp;\u0026lt;\u0026thinsp;0.001 vs the CGA group. \u003csup\u003e#\u003c/sup\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.05 vs the sham group; \u003csup\u003e*\u003c/sup\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.05 vs the CLP group. sham: sham group; CLP: cecum ligation and puncture. Low: CGA low-dose group, High: CGA high-dose group.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eSeptic Acute Liver Injury, a high-morbidity condition in ICUs, is associated with a significant mortality rate, and currently, there are no definitive and effective therapeutic agents with minimal side effects available in clinical practice [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Consequently, many research efforts have focused on exploring potential treatments for SALI from traditional Chinese medicine (TCM) [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Chlorogenic acid (CGA), primarily found in plants such as Lonicera, Artemisia, Eucommia, honeysuckle, coffee, and chrysanthemum, has been extensively studied for its antioxidant, anti-inflammatory, antibacterial, and antiviral properties [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eBagdas' study revealed that CGA accelerates wound healing in diabetic rats by increasing hydroxyproline content and reducing malondialdehyde, nitric oxide, and glutathione levels, without affecting the expression of superoxide dismutase and catalase [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Wang L et al. discovered that CGA enhances the permeability of both the plasma membrane and outer membrane of bacterial cells, leading to damage in barrier function, release of cytoplasmic macromolecules, and depletion of intracellular potential, thereby exerting an antibacterial effect [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Karar et al. found that CGA inhibits the activity of ceramidase and Clostridium perfringens, demonstrating its antiviral potential [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Furthermore, CGA downregulates lipopolysaccharide-induced cyclooxygenase, NF-κB activity, and cytokine expression, contributing to ROS clearance and anti-inflammatory effects [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn our study, the CLP model successfully induced septic acute liver injury, as evidenced by gross structural changes such as liver swelling, increased volume, and pronounced congestion, accompanied by significantly elevated serum TNF-α,IL-1β levels. Similar to previous studies by Arunachalam AR et al. using LPS/GalN (lipopolysaccharide/galactosamine) induce septic acute liver injury respectively [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Pretreatment with CGA significantly alleviated liver damage and improved hepatic pathological injury score, indicating its protective role in septic acute liver injury. This protection may be attributed to CGA's systemic distribution following intraperitoneal absorption, with accumulation in the liver to mitigate injury upon occurrence.\u003c/p\u003e \u003cp\u003eTo investigate the underlying mechanism, we measured inflammatory factor levels in serum and liver tissue. Our results showed that CGA pretreatment significantly reduced TNF-α and IL-1β levels compared to the sepsis-induced liver injury group. IL-1β, an inflammatory mediator, plays a crucial role in mediating liver injury [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e], while TNF-α, apart from regulating tumor cell growth, also promotes inflammation by activating TNFR1 and TNFR2, leading to apoptosis or necrosis and augmenting the inflammatory response [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Our findings demonstrate that CGA intervention significantly reduced the number of TUNEL-stained apoptotic hepatocytes, indicating its ability to inhibit cell death. Consistent with this, Ranjbary AG et al. reported that CGA can treat colon cancer by inducing cytotoxicity, cell cycle arrest, and apoptosis in colon cancer cell lines.\u003c/p\u003e \u003cp\u003eTLR4, a transmembrane receptor belonging to the Toll-like receptor family, is primarily expressed on immune cells and endothelial cells. TLR4 recognizes various pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs), triggering immune and inflammatory responses[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. NF-κB, a protein complex present in almost all animal cell types, regulates cellular responses to stimuli such as stress, cytokines, and free radicals. It plays a pivotal role in immune responses to infection and is closely associated with the development of inflammation, autoimmune diseases, and cancer[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Studies have shown that CGA inhibits the activation of the TLR4/NF-κB signaling pathway, thereby reducing the production and release of inflammatory mediators[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. This mechanism has potential therapeutic applications in various diseases, including atherosclerosis and inflammatory bowel disease. In atherosclerosis, CGA inhibits the TLR4/MAPK/NF-κB pathway, activates autophagy, and suppresses inflammation and oxidative stress, protecting vascular endothelial cells from damage. In inflammatory bowel disease, excessive TLR4 activation is considered a key factor in intestinal inflammation.\u003c/p\u003e \u003cp\u003eNetwork pharmacology research and molecular docking technology explore the key proteins and potential signaling pathways through which chlorogenic acid (CGA) exerts its therapeutic effects in acute septic liver injury (SALI) by leveraging large databases. This study's comprehensive analysis of CGA targets, pathways, and molecular docking results provides invaluable clues. Firstly, the molecular docking results indicate that CGA exhibits good binding affinity with key proteins such as EGFR, ESR1, GSK3B, PTGS2, TLR4, PPARA, HSP90AA1, ACE, MMP9, and itself (CGA). Relevant studies have also confirmed that CGA can reduce EGFR expression and phosphorylation levels, thereby inhibiting EGFR-mediated cell migration and invasion. The possible mechanism involves CGA binding to the ligand-binding domain of EGFR, preventing the binding of ligands like EGF, or it may interfere with receptor dimerization, blocking the transmission of phosphorylation signals and thus inhibiting EGFR activation. Furthermore, by inhibiting the TLR4/NF-κB signaling pathway, CGA can alleviate intestinal inflammatory responses and improve intestinal barrier function. CGA is capable of suppressing the expression of various inflammatory factors downstream of the TLR4/NF-κB pathway, including TNF-α, IL-6, VCAM-1, and ICAM-1. These inflammatory factors play pivotal roles in inflammatory reactions and immune responses, and their reduced expression levels contribute to mitigating the severity of inflammatory reactions.\u003c/p\u003e \u003cp\u003eThe study, however, has a few shortcomings. First, more research is required to determine the role of core targets in disease. The second limitation is that we did not examine the cellular effect of CGA on SALI. In our subsequent studies, we will explore the other molecular mechanisms by which CGA regulates SALI.\u003c/p\u003e \u003cp\u003eIn conclusion, chlorogenic acid can inhibit the release of inflammatory factors in mouse livers, reduce inflammatory reactions, improve liver function, alleviate hepatocyte edema, and mitigate sepsis-induced acute liver injury. Its mechanism of action likely involves inhibiting the TLR4/NF-κB pathway and suppressing the expression of TNF-α and IL-1β. This study provides novel insights and robust evidence for the clinical application of chlorogenic acid in the treatment of SALI.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003cstrong\u003eEthical Approval:\u003c/strong\u003e \u003cp\u003eOur studies did not include human participates, human data or human tissues. All animal experiments conducted were compliant were approved by the Institutional Animal Care and Ethics Committee of Wannan Medical College (LLSC-2024\u0026ndash;073).\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding:\u003c/h2\u003e \u003cp\u003eThis work was supported by the Key Project Research Fund of Wannan Medical College (grant number: WK2022Z10); National College Student Innovation and Entrepreneurship Project (grant number:202310368016).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eShangping Fang and Kecheng Zhai provided research materials and statistics.Huixian Cheng and Jiameng Liu provided the article design and data analysis. Hui Su and Huan Li provided administrative support and article design. Yangmengna Gao and Renke Sun provide the Network pharmacology and molecular docking.All the authors contributed to the manuscript writing and final review. The writers are accountable for the whole of the work, including making sure that any questions regarding the precision or integrity of any individual section are carefully investigated and resolved. The authors confirm that all data were generated in house and that no paper mill was used.\u003c/p\u003e\u003ch2\u003eAvailability of data and materials:\u003c/h2\u003e \u003cp\u003eThe datasets used and/or analyzed during the present study are available from the corresponding author on reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eRello J, Valenzuela-S\u0026aacute;nchez F, Ruiz-Rodriguez M et al (2017) Sepsis: A Review of Advances in Management[J]. Adv Ther 34(11):2393\u0026ndash;2411\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNapolitano LM, Esposito S, De Simone G et al (2018) Sepsis 2018: Definitions and Guideline Changes[J]. 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Food Chem Toxicol 81:54\u0026ndash;61\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang L, Pan X, Jiang L et al (2022) The Biological Activity Mechanism of Chlorogenic Acid and Its Applications in Food Industry: A Review[J]. Front Nutr 9:943911\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGamaleldin Elsadig Karar M, Matei MF, Jaiswal R et al (2016) Neuraminidase inhibition of Dietary chlorogenic acids and derivatives - potential antivirals from dietary sources[J]. Food Funct 7(4):2052\u0026ndash;2059\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZheng Y, Li L, Chen B et al (2022) Chlorogenic acid exerts neuroprotective effect against hypoxia-ischemia brain injury in neonatal rats by activating Sirt1 to regulate the Nrf2-NF-κB signaling pathway[J]. Cell Commun Signal 20(1):84\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eArunachalam AR, Samuel SS, Mani A et al (2023) P2Y2 purinergic receptor gene deletion protects mice from bacterial endotoxin and sepsis-associated liver injury and mortality[J]. Am J Physiol Gastrointest Liver Physiol 25(5):G471\u0026ndash;G91\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBusch K, Kny M, Huang N et al (2021) Inhibition of the NLRP3/IL-1β axis protects against sepsis-induced cardiomyopathy[J]. J Cachexia Sarcopenia Muscle 12(6):1653\u0026ndash;1668\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePuri VK (1998) Of mice and MODS, TNF-alpha, and sepsis[J]. Crit Care Med 26(7):1160\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang Y et al (2022) Toll-like receptor 4 (TLR4) inhibitors: Current research and prospective. Eur J Med Chem 235:114291\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eQuagliariello V et al (2024) Effect of sodium-glucose cotransporter 2 inhibitor dapagliflozin on ejection fraction reduction, myocardial and renal NF-κB expression and systemic pro-inflammatory biomarkers in models of short-term doxorubicin cardiotoxicity. J Clin Oncol _suppl 42(16):e24013\u0026ndash;e24013\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJain, Siddhi PS, Syamprasad N, ParambilPanda (2023) Samir RajanRajdev, BishalJannu, Arun KumarSharma, PawanNaidu, Vegi Ganga Modi. Targeting TLR4/3 using chlorogenic acid ameliorates LPS plus POLY I:C-induced acute respiratory distress syndrome via alleviating oxidative stress-mediated NLRP3/NF-kappa B axis. Clin Sci 137(10):785\u0026ndash;805\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"naunyn-schmiedebergs-archives-of-pharmacology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"nsap","sideBox":"Learn more about [Naunyn-Schmiedeberg's Archives of Pharmacology](https://www.springer.com/journal/210)","snPcode":"210","submissionUrl":"https://submission.nature.com/new-submission/210/3","title":"Naunyn-Schmiedeberg's Archives of Pharmacology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"chlorogenic acid, septic acute liver injury, network pharmacology, molecular docking, TLR4","lastPublishedDoi":"10.21203/rs.3.rs-4780298/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4780298/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eObjective\u003c/h2\u003e \u003cp\u003eTo investigate the biological activities and mechanisms of chlorogenic acid (CGA) in treating septic acute liver injury (SALI) using network pharmacology, molecular docking, and in vivo studies.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003ePotential targets related to both chlorogenic acid and septic acute liver injury were searched from public databases. Protein-protein interaction (PPI), Gene Ontology (GO), and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were conducted. Molecular docking was performed to predict the binding affinity between the active compounds and core targets. Finally, in vitro and in vivo experiments were carried out for further validation.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eA total of 60 common targets were identified between acute septic liver injury and chlorogenic acid, among which 10 shared core targets were screened using Cytoscape. Molecular docking results indicated that these core targets had good binding activity with chlorogenic acid. In the SALI mouse model, chlorogenic acid demonstrated significant protective effects on the liver and anti-inflammatory properties, acting through the TLR4/NF-κB pathway.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eCGA not only improves pathological damage in acute septic liver injury but also exerts its effects potentially through multiple pathways including TLR4.\u003c/p\u003e","manuscriptTitle":"Network pharmacology and molecular docking to explore the potential mechanism of Chlorogenic acid in combined septic acute liver injury","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-08-22 04:53:02","doi":"10.21203/rs.3.rs-4780298/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-10-01T19:44:29+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"175350789460595368431245600069004039511","date":"2024-09-18T02:20:51+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-08-17T00:37:09+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"336256863908012606569618853556557700797","date":"2024-07-31T13:49:48+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"161904461851136665635389739338530884833","date":"2024-07-26T08:09:43+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-07-26T06:32:29+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-07-26T03:04:00+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-07-26T03:03:35+00:00","index":"","fulltext":""},{"type":"submitted","content":"Naunyn-Schmiedeberg's Archives of Pharmacology","date":"2024-07-22T08:29:08+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"naunyn-schmiedebergs-archives-of-pharmacology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"nsap","sideBox":"Learn more about [Naunyn-Schmiedeberg's Archives of Pharmacology](https://www.springer.com/journal/210)","snPcode":"210","submissionUrl":"https://submission.nature.com/new-submission/210/3","title":"Naunyn-Schmiedeberg's Archives of Pharmacology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"2f35f1fe-9994-4bdd-9c70-dc9c6d67cc75","owner":[],"postedDate":"August 22nd, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-01-06T16:02:19+00:00","versionOfRecord":{"articleIdentity":"rs-4780298","link":"https://doi.org/10.1007/s00210-024-03712-5","journal":{"identity":"naunyn-schmiedebergs-archives-of-pharmacology","isVorOnly":false,"title":"Naunyn-Schmiedeberg's Archives of Pharmacology"},"publishedOn":"2025-01-02 15:57:34","publishedOnDateReadable":"January 2nd, 2025"},"versionCreatedAt":"2024-08-22 04:53:02","video":"","vorDoi":"10.1007/s00210-024-03712-5","vorDoiUrl":"https://doi.org/10.1007/s00210-024-03712-5","workflowStages":[]},"version":"v1","identity":"rs-4780298","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4780298","identity":"rs-4780298","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}