Astragalus Membranaceus: Exploring its Protective Role in Heat Stroke via Integrated Transcriptomic and Molecular Docking Approaches

Astragalus Membranaceus: Exploring its Protective Role in Heat Stroke via Integrated Transcriptomic and Molecular Docking Approaches | 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 Astragalus Membranaceus: Exploring its Protective Role in Heat Stroke via Integrated Transcriptomic and Molecular Docking Approaches Defeng Yin, Yandong Yao, Qin Guo, Hao Jiang, Yonglan Hu, Lu Liu, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5594393/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Objective: This study aims to explore the therapeutic potential of Astrolus Membranaceus in treating heatstroke and its potential therapeutic targets. Method: This study was conducted at the Affiliated Hospital of Southwest Medical University, and peripheral blood samples were collected from 10 heatstroke patients (HS=10) and 10 healthy individuals (NC=10) for RNA sequencing. Perform RNA differential analysis using the DESeq2 software package. In addition, the active ingredients and targets of Astragalus membranaceus were screened using the TCMSP database. Intersection the target and differential RNA to obtain the cross target. Then perform GO analysis and KEGG analysis on the intersection targets. Build a protein interaction network with cross targets using STRING website and Cytoscape software, and perform molecular docking between core targets and active molecules using AutoDock Tools. Results: RNA sequencing results showed that compared with the NC group, the HS group had a total of 2042 differentially expressed RNAs. After taking the intersection of Astrolus Membranaceus targets and differentially expressed RNA, 23 intersecting targets were obtained. GO analysis found that the enrichment of cellular components of cross genes is mainly in the extracellular region. The molecular functional enrichment of cross genes mainly involves molecular function regulators. The biological processes related to cross factors mainly include regulation of molecular function, regulation of multicellular organic processes, and response to stress. KEGG analysis showed that the enriched pathways in the crossover genes mainly include the MAPK signaling pathway. The key targets TP53, BCL2, and MMP9 in the protein-protein interaction network were identified using cytoHubba. The molecular docking results indicate that quercetin forms hydrogen bonds with TP53, BCL2, and MMP9, with low binding energies. Conclusion: This study reveals the potential molecular mechanism of Astrolus Membranaceus in treating heatstroke, providing a scientific basis for further drug development and clinical application. Heatstroke Astrolus Membranaceus RNA sequencing molecular docking Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Heat stroke is a serious and life-threatening illness characterized by a core body temperature exceeding 40 ° C, often accompanied by central nervous system dysfunction 1 .Heat stroke can lead to systemic inflammatory response and multiple organ dysfunction, with a very high mortality rate 2 .With climate change, abnormally high temperatures, and an increase in cases of heat stroke, understanding the pathogenesis of heat stroke and finding effective treatment measures have become increasingly important 3 . Astragalus Membranaceus is a traditional Chinese medicine that has been used by people for hundreds of years. It is said to have the effect of enhancing immune function and reducing inflammation 4 , 5 .Recent studies have found that Astragalus membranaceus can be used to treat ulcerative colitis. 6 ,Its extract can alleviate airway inflammation and remodeling in asthmatic mice 7 ,inhibit enterovirus replication 8 ,and is also an effective anti-cancer drug 9 .Although Astragalus membranaceus has been widely studied in other diseases, there is relatively little research on its role in heat stroke, and the specific molecular mechanism of its action in heat stroke is still unclear. The advancement of transcriptomics and molecular docking technologies provides valuable tools for exploring the molecular basis of diseases and evaluating the therapeutic potential of herbs. Transcriptome analysis is used to identify differential RNAs associated with Heat stroke, while molecular docking is used to predict the interactions between active molecules of traditional Chinese medicine and target proteins 10 , 11 .This comprehensive method can be used to predict the mechanism of action of herbs. In this study, transcriptome analysis was combined with molecular docking to investigate the protective effect of Astragalus membranaceus in Heat stroke. By analyzing the RNA sequencing data of peripheral blood samples from Heat stroke patients and healthy controls, differential RNAs related to Heat stroke were obtained. Obtain the intersection targets between differential RNA and Astragalus Membranaceus targets, as well as Astragalus Membranaceus active molecules acting on these intersection targets, through network pharmacology analysis. And predicted the pathways involved in intersecting genes through bioinformatics analysis. Then, molecular docking was used to predict the interactions between bioactive compounds of Astragalus membranaceus and potential target proteins involved in Heat stroke. Our research findings aim to provide new insights into the molecular basis of Heat stroke and highlight the therapeutic potential of Astragalus membranaceus. Materials and Methods Sample collection This research was conducted at the Affiliated Hospital of Southwest Medical University. Peripheral blood samples were collected from 10 patients with Heat stroke and 10 healthy individuals between June 28, 2023, and September 8, 2023. Peripheral blood from Heat stroke patients was collected within 24 hours of admission and was used for mRNA sequencing.Inclusion Criteria for heat stroke：(1)Clinical Diagnosis of Heat Stroke:Patients must have a core body temperature of >40°C and exhibit central nervous system dysfunction (2)Age:Adult patients aged 18 years and above 12 .(3)Informed Consent:Patients or their legal representatives must provide written informed consent to participate in the study.(4)Onset of Symptoms:Inclusion of patients presenting with heat stroke symptoms within the last 24 hours prior to hospital admission.Exclusion Criteria:(1)Pre-existing Severe Medical Conditions: Patients with severe cardiovascular diseases, end-stage liver or kidney diseases, or other life-threatening conditions that could confound the study outcomes.(2)Acute Central Nervous System Disorders:Patients with acute central nervous system infections (e.g., meningitis, encephalitis), acute stroke, or severe traumatic brain injury .(3)Pregnancy: Pregnant or lactating women to avoid potential risks to the mother and fetus.(4)Incomplete Medical Records:Patients whose medical records are incomplete or lack essential data for the study.(5)Prolonged Pre-hospital Treatment: Patients who have received extensive pre-hospital cooling treatment for more than 2 hours prior to admission, which might affect the study’s assessment of initial therapeutic interventions . The study protocol has received approval from the Ethics Committee of Southwest Medical University (Ethics Number:KY2023087). All procedures involving human participants conformed to the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki Declaration and its later amendments or comparable ethical standards 13 . And the study has passed the clinical trial registration.( registry: China Clinical Trial Registration Center； registration number: ChiCTR2400084775 ; date of registration: 24-05-2024). Blood sample preprocessing The collection of whole blood is a critical initial step for a variety of molecular assays designed to study intracellular RNA, particularly in conditions such as heat stroke. The PAXgene® Blood RNA System (BD Biosciences) includes a blood collection tube (PAXgene® Blood RNA Tube) and a nucleic acid purification kit (PAXgene® Blood RNA Kit). The PAXgene® Blood RNA Tube contains a unique additive formulated to maintain the native gene expression profile by preventing RNA degradation ex vivo and reducing gene induction. When used in combination with the PAXgene® Blood RNA Kit, this system ensures precise analysis and quantification of gene expression 14 . Before collecting samples, PAXgene ® blood RNA tubes were labeled with patient information and placed in a 18 ℃ freezer. After collecting peripheral blood into the tube, gently invert the tube to mix the liquid inside, and then store the tube in a 18 ℃ refrigerator. RNA sequencing In this study, we employed a precise method for the extraction and quality assessment of total RNA from peripheral blood samples. Initially, total RNA was isolated from the peripheral blood of patients with heat stroke, followed by quantitative analysis of RNA concentration and purity using the Nanodrop2000, and assessment of RNA integrity and RIN value using the Agilent2100 to ensure that the extracted RNA quality met the strict requirements for subsequent experiments. Next, mRNA was specifically enriched using the A-T base pairing technology of Oligo(dT) magnetic beads, laying the foundation for in-depth transcriptome analysis. A library construction kit compatible with the DNBSEQ platform was used, following the manufacturer's guidelines for library construction: first, the first strand cDNA was reverse transcribed and treated with UDG to avoid the incorporation of uracil. Then, the second strand cDNA is synthesized to form a stable double stranded cDNA, providing a high-quality template for subsequent sequencing library construction. Then we repaired the end of the double stranded cDNA and added an "A" base at the 3 'end to facilitate the connection of the Y-shaped linker, completing the library construction. Finally, connect the sequencing platform specific adapter for size selection and purification. Next, high-throughput sequencing specific biological analyzers or chips are used to detect the size distribution and concentration of the library. According to the operation process of the DNBSEQ platform, load the library onto the sequencing chip and set the sequencing parameters. Utilize the basecall software on the DNBSEQ platform to process raw image data, generate Cal files, and conduct preliminary data quality assessments, including signal strength, background noise, etc. This series of detailed operating procedures ensures the quality of the library and the accuracy of sequencing results, providing reliable data support for the molecular mechanism research of heat stroke. Differential RNA analysis The RNA-seq raw data were uploaded to the online analysis platform iDEP 2.0 15 (Integrated Differential Expression & Pathway Analysis) (http://bioinformatics.sdstate.edu/idep/ ） for standardization processing and Principal Component Analysis (PCA).Principal Component Analysis (PCA) can observe the level of separation between HS and NC groups and exclude abnormal samples. Subsequently, distribution density and box plot analysis were performed on the sample data that had undergone quality control and filtering to clarify the homogeneity and comparability of the data. The DESeq2 software package was used for differential analysis of raw RNA sequencing data, with screening criteria set at Q value1, to identify differential RNAs between the NC and HS groups. The ggplot2 R software package is used to visualize the results of differential analysis. Screening of active ingredients and targets of Astragalus Membranaceus The various active ingredients and targets of Astragalus Membranaceushave different mechanisms of action and therapeutic effects. Screening specific active ingredients is the key to understanding the mechanism of Astragalus Membranaceusin treating heat stroke.TCMSP（Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform ：http://tcmspw. com/tcmsp.php）an integrated traditional Chinese medicine system pharmacology database and analysis platform, provides an efficient screening method. By setting the criteria of bioavailability (OB value) ≥ 30% and drug similarity (DL) ≥ 0.18, active ingredients and their targets with high bioavailability and drug similarity were screened. Then, the gene names corresponding to the targets were obtained using the Uniprot database. Then, the targets of active ingredients in Astragalus membranaceus were plotted against differential RNA using a Venn diagram, and the intersection targets of active ingredient targets and differential RNA were screened. These intersecting targets may be potential targets for Astragalus Membranaceus in treating heat stroke, and a network diagram of the effective active ingredients of Astragalus Membranaceusn and intersecting targets was constructed using Cytoscape (version 3.10.2). Gene Ontology (GO) enrichment analysis Gene Ontology (GO) is an internationally standardized classification system used to describe the attributes of genes and gene products in organisms. It encompasses three ontologies: molecular function(MF), cellular component(CC), and biological process(BP). The fundamental unit of GO is the term, with each term representing a specific attribute. In GO functional analysis, differentially expressed genes are first mapped to corresponding GO terms in the database, and the number of genes associated with each term is calculated. Subsequently, a hypergeometric test is employed to identify GO terms that are significantly enriched among the differentially expressed genes. A p-value of 0.05 is used as the threshold for significance. GO terms meeting this criterion are considered significantly enriched in the differentially expressed genes, aiding in the identification of the primary biological functions these genes perform 16 . Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis In biological systems, genes work in concert to perform various biological functions, and pathway-based analysis provides deeper insights into these functions. The Kyoto Encyclopedia of Genes and Genomes (KEGG) is a primary public database for pathway information. Pathway enrichment analysis using KEGG pathways involves applying a hypergeometric test to identify pathways that are significantly enriched in differentially expressed genes compared to the entire genomic background 17 . A p-value of ≤0.05 is used as the significance threshold, and pathways meeting this criterion are considered significantly enriched in the differentially expressed genes. Construction of protein-protein interaction network To further screen potential core targets, submit the intersection targets to the STRING database（ https://cn.string-db.org/ ）The minimum interaction score is set to 0.7 and disconnected nodes hidden in the network are used to generate protein interaction networks and download protein interaction data. Export protein interaction data to Cytoscape (version 3.10.2), and then use the cytoHubba plugin to score and rank nodes using the Maximum Group Centrality (MCC) analysis method, and visualize the analysis results. Molecular docking Firstly, search for the corresponding drug small molecule in TCMSP and download the mol2 format file of the drug small molecule. Next, in the UniProt database（ https://www.uniprot.org/ ）Search for the protein corresponding to the core gene, select "X-ray" as the method, and select a single chain structure with "RESOLUTION" less than 3. Then in the RCSB-PDB database（ https://www.rcsb.org/ ）Download the PDB format file of the corresponding protein. Next, import the PDB format file of the protein into Pymol software to remove ions and ligands from the protein. Then use AutoDock Tools to perform hydrogenation treatment on the protein and set it as a receptor, exporting it as a pdbqt format file. Then, AutoDock Tools was used to perform hydrogenation treatment on the drug small molecule, and the drug small molecule was set as a ligand. The twisted bond was detected and set, and exported as a pdbqt format file. Next, import the pdbqt format files of protein and drug small molecules into AutoDock Tools software, set up the docking box, export as a gpf format file, then run Autogrid4, set the number of runs to 50, and set other parameters to default settings. Run autodock4, import the generated GPF format file, perform molecular docking, view the docking results, and export the result with the lowest binding energy. Finally, import the docking results into Pymol software for visualization processing. Results Differentially expressed RNA analysis PCA analysis was performed on the RNA sequencing results, which showed a good difference between NC samples and HS samples, and no abnormal samples were found (Figure 1A). The density distribution and box plot show that both groups of data have homogeneity and comparability (Figure 1BC). Perform differential analysis on the data of HS group and NC group, with screening criteria set as Q value1. Compared with the NC group, the HS group had a total of 2042 differentially expressed RNAs, of which 1640 were upregulated and 402 were downregulated. (Figure 1D). Screening of active ingredients and targets of Astragalus Membranaceus The TCMSP database was used, and a total of 17 active ingredients of Astragalus membranaceus were obtained by setting the standards of bioavailability (OB value) ≥ 30% and drug similarity (DL) ≥ 0.18, targeting 190 targets. Then the Uniprot database was used to obtain the gene names corresponding to the targets, with the species set as humans, and 188 targets were obtained. 188 targets were intersected with 2042 differentially expressed RNAs, resulting in a total of 23 intersecting targets, which are potential targets for Astragalus Membranaceusin the treatment of heat stroke. (Figure 2A) Heat map based on RNA sequencing showed that MMP9, FOS, SPP1, CXCL10, MAPK14, HSPB1, HMOX1, AHSA1, MET, 1GF2, PLAU, VCAM1, SLPI, and SULT1E1 were highly expressed in heat stroke, while TP53, BCL2, MYC, PRSS1, GSTM2, CD4OLG, DPP4, PKIA, and ERBB3 were lowly expressed in heat stroke. (Figure 2B) These 23 intersecting targets are the action targets of six active molecules of Astragalus membranaceus. (Figure 2C) GO analysis GO analysis revealed 18 biological processes, 1 cellular component, and 1 molecular functional category. The enrichment of cellular components of cross genes is mainly in the extracellular region. (Figure 3A-B) The molecular function enrichment of crossover genes mainly involves molecular function regulator. (Figure 3A-B) The biological processes related to the intersection factor mainly include regulation of molecular function, regulation of multicellular organic process, response to stress, response to hypoxia, response to reduced oxygen levels, regulation of cell population proliferation, negative regulation of response to stimulus, and response to biotic stimulus (Figure 3C). KEGG analysis KEGG analysis identified 20 noteworthy pathways, including 3 Environmental Information Processing, 2 Organic Systems, and 15 Human Diseases.(Figure 4A-B) The enriched pathways in the cross genes include Fluid sheet stress and atherosclerosis, TNF signaling pathway, IL-17 signaling pathway, Toll like receptor signaling pathway, and MAPK signaling pathway. (Figure 4C) Protein interaction network A protein interaction network diagram based on the STRING database, where the more connecting lines between proteins, the closer the protein interactions. (Figure 5A) Using the MCC analysis method in cytoHubba, the protein nodes in the PPI network were scored and ranked to obtain the top 10 most core proteins, including TP53, BCL2, MMP9, FOS, MYC, SPP1, VCAM1, HMOX1, CXCL10, PLAU. The redder the color, the higher the rating, indicating that the protein is more important. Among them, TP53, BCL2, and MMP9 have higher scores. (Figure 5B) Molecular docking The molecular docking results showed that quercetin forms hydrogen bonds with TP53, BCL2, and MMP9, and has a low binding energy. The docking results of quercetin with TP53 showed that hydrogen bonds were formed between quercetin and GLU-326, PHE-328, LEU-330, and GLU-349 of TP53, with a binding energy of -5.48 kcal/mol. (Figure 6A) The docking results of quercetin with BCL2 showed that hydrogen bonds were formed between quercetin and ASN-143, TRP-144, GLY-145, ASN-192, LEU-201, and TYR-202 of BCL2, with a binding energy of -6.14 kcal/mol. (Figure 6B) The docking results of quercetin with MMP9 showed that hydrogen bonds were formed between quercetin and GLU-227, ALA-242, and ARG-249 of MMP9, with a binding energy of -9.58 kcal/mol. (Figure 6C) Discussion Heat stroke is a serious acute disease caused by high temperature, mainly manifested as high fever, central nervous system dysfunction, and often accompanied by multiple organ dysfunction 18 – 20 .Current research indicates that its pathogenesis mainly involves inflammatory response, oxidative stress, coagulation abnormalities, etc 12 .The current treatment measures for heat stroke mainly include rapid physical cooling, anticoagulant therapy, and multi organ function support 21 , 22 .New treatment strategies are being explored, including the use of anti-inflammatory drugs and antioxidant drugs 23 , 24 .However, there is currently no treatment that can significantly improve the prognosis of patients with heat stroke, and precise treatment is the future trend of medical development 25 .Therefore, this study explored the potential therapeutic targets of active molecules of Astragalus membranaceus in heat stroke. Quercetin is a flavonoid compound found in Astragalus membranaceus, which has various biological activities such as anti-inflammatory, antioxidant, and cell protection. Studies have shown that quercetin can exert anti-inflammatory effects by inhibiting the nuclear factor kappa B (NF - κ B) pathway, reducing the production of inflammatory mediators, and thus exerting anti-inflammatory effects 26 .In addition, quercetin can also reduce cellular damage caused by oxidative stress by regulating ROS. Studies have also shown that quercetin can protect cells by inhibiting apoptosis 27 .Our research indicates that intersecting genes are mainly enriched in regulation of molecular function, regulation of multicellular organic processes, response to stress, response to biotic stimuli, and MAPK signaling pathways.Heat stroke can affect the normal molecular function regulation and physiological activity of cells by affecting enzyme activity. Systemic inflammation and organ dysfunction caused by heat stroke involve complex multicellular organism process regulation, which affects the overall stability of the body. Therefore, multiple organ dysfunction is often combined.Studies have shown that after experiencing heat stress, the body's DNA damage increases, which affects the normal physiological activities 28 .In addition, studies have shown that heat stress induces necrotic apoptosis of cells through the MAPK pathway, which plays an important role in the inflammatory response and tissue damage caused by heat stroke 29 . Our research indicates that quercetin has low binding energies with TP53, BCL2, and MMP9, with MMP9 upregulated in heat stroke and BCL2 and TP53 downregulated in heat stroke TP53 is a key regulatory factor of apoptosis and plays an important role in cellular stress response 30 .TP53 can regulate cell apoptosis caused by stress response by modulating autophagy, alleviate inflammatory response, and maintain cellular homeostasis in the nervous system 31 .Quercetin targeting TP53 may have a certain improvement effect on central nervous system dysfunction caused by heat stroke.BCL2 is an anti apoptotic protein that can protect cells from various stress-induced cell apoptosis 32 .Quercetin may regulate the apoptosis pathway by modulating the expression of BCL2, affecting the survival and functional status of cells under stress conditions, and playing a protective role in multicellular organism processes and stress response.Studies have shown that MMP9 (matrix metalloproteinase 9) plays a critical role in extracellular matrix degradation and remodeling, and plays an important role in tissue repair and inflammatory response 33 .Overexpression of MMP9 in heat stroke is associated with oxidative stress in cells. Studies have shown that MMP9 can increase the permeability of the blood-brain barrier, promote leukocyte infiltration into the central nervous system, damage neurons, and cause central nervous system disorders 34 , 35 .The expression of MMP9 may be related to the severity of heat stroke and can serve as a potential biomarker for the diagnosis of heat stroke. Studies have shown that downregulation of MMP9 expression can promote cellular autophagy, and quercetin may alleviate organ damage caused by inflammatory reactions by promoting leukocyte autophagy 36 . This study integrated transcriptomics, network pharmacology, and molecular docking analysis to explore the role of Astragalus membranaceus in heat stroke. It predicted the key pathways of Astragalus membranaceus in treating heat stroke and the interactions between Astragalus membranaceus bioactive compound quercetin and key targets such as TP53, BCL2, and MMP9, providing valuable insights into its mechanism of treating heat stroke.In the future, in vivo studies will be conducted to verify the therapeutic effect of Huangqi and explore its clinical applicability. In addition, in vitro experiments will be used to explore the specific mechanisms of Astragalus membranaceus and evaluate its potential side effects in clinical settings. Declarations Ethics approval The study protocol has received approval from the Ethics Committee of Southwest Medical University (Ethics Number:KY2023087). All procedures involving human participants conformed to the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki Declaration and its later amendments or comparable ethical standards 13 . Consent to participate All experimental participants have read and signed the informed consent form, and understand their rights and responsibilities as study participants. Consent for publication We promise that all published information is based on the voluntary consent of participants, and we respect their privacy rights and the protection of personal information. We further declare that the publication of this paper will not cause any form of harm or adverse impact to the participants. Availability of data and materials The study has passed the clinical trial registration.( registry: China Clinical Trial Registration Center； registration number: ChiCTR2400084775 ; date of registration: 24-05-2024). Competing interests I declare that the authors have no competing interests as defined by BMC, or other interests that might be perceived to influence the results and/or discussion reported in this paper. Funding This project is supported by the Economic, Commercial, Scientific and Technological Bureau of Xuyong County's project "Basic and Clinical Research on Heat Stroke (Project No.: 2023JSYF01). Authors' contributions 1. Kaiyu Jin and Yingchun Hu led the overall project and designed the experiments. 2. Defeng Yin,Yandong Yao and Qin Guo performed the majority of the experiments and data analysis, and contributed to the writing of the manuscript. 3. Hao Jiang and Yonglan Hu developed the theoretical framework and provided critical feedback on the manuscript. 4. Lu Liu collected and processed the data, and contributed to the statistical analysis. All authors have read and approved the final manuscript. Acknowledgements We express our sincere gratitude to the volunteers and participants who participated in this study, as their contributions were crucial for collecting data and analyzing results. 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The Roles of Matrix Metalloproteinases and Their Inhibitors in Human Diseases. Int. J. Mol. Sci. 21 , 9739 (2020). Kobayashi, H. et al. MMPs initiate Schwann cell-mediated MBP degradation and mechanical nociception after nerve damage. Mol. Cell. Neurosci. 39 , 619–627 (2008). Uemura, S. et al. Diabetes mellitus enhances vascular matrix metalloproteinase activity: role of oxidative stress. Circ. Res. 88 , 1291–1298 (2001). Yuan, Q. et al. Lycorine improves peripheral nerve function by promoting Schwann cell autophagy via AMPK pathway activation and MMP9 downregulation in diabetic peripheral neuropathy. Pharmacol. Res. 175 , 105985 (2022). Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted 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. 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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-5594393","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":394989961,"identity":"b08972b4-47f6-4269-b232-8f7881ea840a","order_by":0,"name":"Defeng Yin","email":"","orcid":"","institution":"The Affiliated Hospital of Southwest Medical University","correspondingAuthor":false,"prefix":"","firstName":"Defeng","middleName":"","lastName":"Yin","suffix":""},{"id":394989962,"identity":"51de5c77-0218-4f71-aa19-4c6619d601f6","order_by":1,"name":"Yandong Yao","email":"","orcid":"","institution":"The Affiliated Hospital of Southwest Medical University","correspondingAuthor":false,"prefix":"","firstName":"Yandong","middleName":"","lastName":"Yao","suffix":""},{"id":394989963,"identity":"773b8c71-056f-4ad9-b599-671982315d3b","order_by":2,"name":"Qin Guo","email":"","orcid":"","institution":"Xuyong People's Hospital","correspondingAuthor":false,"prefix":"","firstName":"Qin","middleName":"","lastName":"Guo","suffix":""},{"id":394989964,"identity":"fb66a6a6-f40d-4c2f-8b31-92b127b628ad","order_by":3,"name":"Hao Jiang","email":"","orcid":"","institution":"Xuyong People's Hospital","correspondingAuthor":false,"prefix":"","firstName":"Hao","middleName":"","lastName":"Jiang","suffix":""},{"id":394989965,"identity":"bb2c966b-8be3-4b8d-a5b5-dc725be0a575","order_by":4,"name":"Yonglan Hu","email":"","orcid":"","institution":"Xuyong People's Hospital","correspondingAuthor":false,"prefix":"","firstName":"Yonglan","middleName":"","lastName":"Hu","suffix":""},{"id":394989966,"identity":"d369298a-0b15-4e91-84a0-27e8374a2c35","order_by":5,"name":"Lu Liu","email":"","orcid":"","institution":"Xuyong People's Hospital","correspondingAuthor":false,"prefix":"","firstName":"Lu","middleName":"","lastName":"Liu","suffix":""},{"id":394989967,"identity":"7ef25ed9-487b-449c-b9c8-0da7a9379bc5","order_by":6,"name":"Kaiyu Jin","email":"","orcid":"","institution":"Xuyong People's Hospital","correspondingAuthor":false,"prefix":"","firstName":"Kaiyu","middleName":"","lastName":"Jin","suffix":""},{"id":394989968,"identity":"f3f0ed73-89dd-4fad-be7e-de61c4348f3c","order_by":7,"name":"Yingchun Hu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABCUlEQVRIiWNgGAWjYBACAyA+AMQJDAzMBw5+qIAK8xCnhS3xsMQZIrUwQLTwGB/gbSNCi7lEjuHhgl92eQa3ewwOSM6rS1w7I4Hxwds2BnlzHFosZ+QYHJ7Zl1xscOdYwYHCbYcTt91IYDac28ZguLMBh8NuALXw9hxI3HAjecMByW0HcoFa2KSBLkwwOEBQC1AN75w6kBb23wS18PwAaUkBamlgBtvCjFfLmWcFh3kbkhNn3khLOCxx7HD9tjMPmyXnnJMw3IBLy/HkzZ95/tgl9t1IPvzxQ02dsdnx5IMf3pTZyOOyhYGBw4CBsQ1FhLEBSEjgUg8E7A8YGP7gkR8Fo2AUjIJRAAB8F24g4ionpwAAAABJRU5ErkJggg==","orcid":"","institution":"The Affiliated Hospital of Southwest Medical University","correspondingAuthor":true,"prefix":"","firstName":"Yingchun","middleName":"","lastName":"Hu","suffix":""}],"badges":[],"createdAt":"2024-12-06 14:23:22","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5594393/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5594393/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":72612122,"identity":"1d034ca1-69f2-4ba6-94f3-5140c41aa35a","added_by":"auto","created_at":"2024-12-30 10:26:37","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":242322,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eData quality control and differential RNA screening.\u003c/strong\u003e(A) PCA analysis shows that HS group samples and NC group samples can be clearly distinguished without outlier samples. (B-C) The density distribution and box plot show that the data of each sample has uniformity and comparability. (D) The horizontal axis in the volcano plot is log2 (fc), and the vertical axis is - log10 (FDR). Red represents upregulated expression, blue represents downregulated expression, and black represents non differentially expressed RNA. Compared with the NC group, the HS group had a total of 2042 differentially expressed RNAs, 1640 up-regulated RNAs, and 402 down regulated RNAs. HS: heat stroke group; NC: normal control group.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-5594393/v1/0180694138311a8dc86ea987.png"},{"id":72612127,"identity":"0144b5f6-9d1b-42a5-9029-c13838a89658","added_by":"auto","created_at":"2024-12-30 10:26:37","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":212488,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTargeted screening of Astragalus Membranaceus for the treatment of heat stroke. \u003c/strong\u003e(A) The Venn diagram shows that the blue color represents 2042 differentially expressed RNAs in heat stroke, the pink color represents 188 targets of active molecules in Astragalus Membranaceus, and the middle represents 23 cross targets of the two, which are potential targets for Astragalus Membranaceusn in treating heat stroke. (B) The heatmap of 23 intersecting targets based on RNA sequencing shows the HS group in blue and the NC group in red. Red indicates upregulation of expression, while blue indicates downregulation of expression. MMP9, FOS, SPP1, CXCL10, MAPK14, HSPB1, HMOX1, AHSA1, MET, 1GF2, PLAU, VCAM1, SLPI, and SULT1E1 are highly expressed in heat stroke, while TP53, BCL2, MYC, PRSS1, GSTM2, CD4OLG, DPP4, PKIA, and ERBB3 are lowly expressed in heat stroke. (C) Component target network display, with yellow circles representing 23 intersecting targets and green hexagons representing 6 active ingredients acting on the targets. HS: heat stroke group; NC: normal control group.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-5594393/v1/13f9bdcb7accf071d6bb541c.png"},{"id":72612673,"identity":"bbb2d6fa-4377-49c1-a457-d9193d75b95f","added_by":"auto","created_at":"2024-12-30 10:34:37","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":389067,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGene Ontology Enrichment Analysis.\u003c/strong\u003e (A) Yellow represents molecular function, blue represents biological processes, and green represents cellular components. GO analysis revealed 18 biological processes, 1 cellular component, and 1 molecular functional category. (B) Yellow represents biological processes, blue represents molecular functions, and green represents biological components. The enrichment of cellular components of cross genes is mainly in the extracellular region. The molecular function enrichment of crossover genes mainly involves molecular function regulators. (C) The biological processes related to intersection factors mainly include regulation of molecular function, regulation of multicellular organic processes, response to stress, response to hypoxia, response to reduced oxygen levels, regulation of cell population reproduction, negative regulation of response to stimulus, and response to biotic stimulus.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-5594393/v1/de5a4043c9da7ad90c9d1026.png"},{"id":72612129,"identity":"dc7c8b76-6f7d-4903-9089-00793e661b31","added_by":"auto","created_at":"2024-12-30 10:26:37","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":343251,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eKEGG enrichment pathway.\u003c/strong\u003e (A-B) KEGG analysis identified 20 noteworthy pathways, including 3 Environmental Information Processing, 2 Organic Systems, and 15 Human Diseases. (C) The enriched pathways in cross genes include Fluid shear stress and atherosclerosis, TNF signaling pathway, IL-17 signaling pathway, Toll like receptor signaling pathway, and MAPK signaling pathway.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-5594393/v1/a6bea09740a11c348b1d6d5a.png"},{"id":72612130,"identity":"ebcf0592-5d66-4de0-9892-fb0050f20dd5","added_by":"auto","created_at":"2024-12-30 10:26:37","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":158850,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eprotein-protein interaction (PPI) network.\u003c/strong\u003e (A) A protein interaction network diagram based on the STRING database, where the more connecting lines between proteins, the closer the protein interactions. (B) The MCC analysis method in cytoHubba is used to score and rank protein nodes in the PPI network to obtain the top 10 most core proteins, including TP53, BCL2, MMP9, FOS, MYC, SPP1, VCAM1, HMOX1, CXCL10, PLAU. The redder the color, the higher the rating, indicating that the protein is more important. Among them, TP53, BCL2, and MMP9 have higher scores.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-5594393/v1/81300a5ff1ae7b6fbac06bd4.png"},{"id":72612132,"identity":"cea96ef5-f9d0-4b09-bbd5-79fb5ee4c764","added_by":"auto","created_at":"2024-12-30 10:26:37","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":256554,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMolecular docking.\u003c/strong\u003e (A) The docking results of quercetin with TP53 showed that hydrogen bonds were formed between quercetin and GLU-326, PHE-328, LEU-330, and GLU-349 of TP53, with a binding energy of -5.48 kcal/mol. (B) The docking results of quercetin with BCL2 show that hydrogen bonds are formed between quercetin and ASN-143, TRP-144, GLY-145, ASN-192, LEU-201, TYR-202 of BCL2, with a binding energy of -6.14Kcal/mol. (C) The docking results of quercetin with MMP9 showed that hydrogen bonds were formed between quercetin and GLU-227, ALA-242, and ARG-249 of MMP9, with a binding energy of -9.58 kcal/mol. GLU: Glutamic acid; PHE: Phenylalanine; LEU: Leucin; ASN: Asparagine; TRP: Tryptophane; GLY: Glycine; TYR :Tyrosine; ALA: Alanine; ARG: Arginine; TP53: Cellular tumor antigen p53; BCL2: Apoptosis regulator Bcl-2; MMP9: Matrix metalloproteinase-9.\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-5594393/v1/8d6ee1bb5e9b2b9d93951b7d.png"},{"id":73190522,"identity":"895d2ea4-6ce8-47f6-99c9-b8f08802a394","added_by":"auto","created_at":"2025-01-07 14:32:34","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2238165,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5594393/v1/cb408af1-2f95-4054-b4cf-84f22c9d61bf.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Astragalus Membranaceus: Exploring its Protective Role in Heat Stroke via Integrated Transcriptomic and Molecular Docking Approaches","fulltext":[{"header":"Introduction","content":"\u003cp\u003eHeat stroke is a serious and life-threatening illness characterized by a core body temperature exceeding 40 \u0026deg; C, often accompanied by central nervous system dysfunction\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e.Heat stroke can lead to systemic inflammatory response and multiple organ dysfunction, with a very high mortality rate\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e.With climate change, abnormally high temperatures, and an increase in cases of heat stroke, understanding the pathogenesis of heat stroke and finding effective treatment measures have become increasingly important\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eAstragalus Membranaceus is a traditional Chinese medicine that has been used by people for hundreds of years. It is said to have the effect of enhancing immune function and reducing inflammation\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e,\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e.Recent studies have found that Astragalus membranaceus can be used to treat ulcerative colitis.\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e,Its extract can alleviate airway inflammation and remodeling in asthmatic mice\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e,inhibit enterovirus replication\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e,and is also an effective anti-cancer drug\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e.Although Astragalus membranaceus has been widely studied in other diseases, there is relatively little research on its role in heat stroke, and the specific molecular mechanism of its action in heat stroke is still unclear.\u003c/p\u003e \u003cp\u003eThe advancement of transcriptomics and molecular docking technologies provides valuable tools for exploring the molecular basis of diseases and evaluating the therapeutic potential of herbs. Transcriptome analysis is used to identify differential RNAs associated with Heat stroke, while molecular docking is used to predict the interactions between active molecules of traditional Chinese medicine and target proteins\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e,\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e.This comprehensive method can be used to predict the mechanism of action of herbs. In this study, transcriptome analysis was combined with molecular docking to investigate the protective effect of Astragalus membranaceus in Heat stroke. By analyzing the RNA sequencing data of peripheral blood samples from Heat stroke patients and healthy controls, differential RNAs related to Heat stroke were obtained. Obtain the intersection targets between differential RNA and Astragalus Membranaceus targets, as well as Astragalus Membranaceus active molecules acting on these intersection targets, through network pharmacology analysis. And predicted the pathways involved in intersecting genes through bioinformatics analysis. Then, molecular docking was used to predict the interactions between bioactive compounds of Astragalus membranaceus and potential target proteins involved in Heat stroke. Our research findings aim to provide new insights into the molecular basis of Heat stroke and highlight the therapeutic potential of Astragalus membranaceus.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e\u003cstrong\u003eSample collection\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was conducted at the Affiliated Hospital of Southwest Medical University. Peripheral blood samples were collected from 10 patients with Heat stroke and 10 healthy individuals between June 28, 2023, and September 8, 2023. Peripheral blood from Heat stroke patients was collected within 24 hours of admission and was used for mRNA sequencing.Inclusion Criteria for heat stroke：(1)Clinical Diagnosis of Heat Stroke:Patients must have a core body temperature of \u0026gt;40\u0026deg;C \u0026nbsp;and exhibit central nervous system dysfunction (2)Age:Adult patients aged 18 years and above\u003csup\u003e12\u003c/sup\u003e.(3)Informed Consent:Patients or their legal representatives must provide written informed consent to participate in the study.(4)Onset of Symptoms:Inclusion of patients presenting with heat stroke symptoms within the last 24 hours prior to hospital admission.Exclusion Criteria:(1)Pre-existing Severe Medical Conditions: Patients with severe cardiovascular diseases, end-stage liver or kidney diseases, or other life-threatening conditions that could confound the study outcomes.(2)Acute Central Nervous System Disorders:Patients with acute central nervous system infections (e.g., meningitis, encephalitis), acute stroke, or severe traumatic brain injury .(3)Pregnancy: Pregnant or lactating women to avoid potential risks to the mother and fetus.(4)Incomplete Medical Records:Patients whose medical records are incomplete or lack essential data for the study.(5)Prolonged Pre-hospital Treatment: Patients who have received extensive pre-hospital cooling treatment for more than 2 hours prior to admission, which might affect the study\u0026rsquo;s assessment of initial therapeutic interventions . The study protocol has received approval from the Ethics Committee of Southwest Medical University (Ethics Number:KY2023087). All procedures involving human participants conformed to the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki Declaration and its later amendments or comparable ethical standards\u003csup\u003e13\u003c/sup\u003e. And the study has passed the clinical trial registration.(\u003cstrong\u003eregistry:\u003c/strong\u003eChina Clinical Trial Registration Center；\u003cstrong\u003eregistration number:\u003c/strong\u003eChiCTR2400084775 ;\u003cstrong\u003edate of registration:\u003c/strong\u003e24-05-2024).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBlood sample preprocessing\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe collection of whole blood is a critical initial step for a variety of molecular assays designed to study intracellular RNA, particularly in conditions such as heat stroke. The PAXgene\u0026reg; Blood RNA System (BD Biosciences) includes a blood collection tube (PAXgene\u0026reg; Blood RNA Tube) and a nucleic acid purification kit (PAXgene\u0026reg; Blood RNA Kit). The PAXgene\u0026reg; Blood RNA Tube contains a unique additive formulated to maintain the native gene expression profile by preventing RNA degradation ex vivo and reducing gene induction. When used in combination with the PAXgene\u0026reg; Blood RNA Kit, this system ensures precise analysis and quantification of gene expression\u0026nbsp;\u003csup\u003e14\u003c/sup\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eBefore collecting samples, PAXgene \u0026reg; blood RNA tubes were labeled with patient information and placed in a 18\u0026nbsp;℃\u0026nbsp;freezer. After collecting peripheral blood into the tube, gently invert the tube to mix the liquid inside, and then store the tube in a 18\u0026nbsp;℃\u0026nbsp;refrigerator.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRNA sequencing\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn this study, we employed a precise method for the extraction and quality assessment of total RNA from peripheral blood samples. Initially, total RNA was isolated from the peripheral blood of patients with heat stroke, followed by quantitative analysis of RNA concentration and purity using the Nanodrop2000, and assessment of RNA integrity and RIN value using the Agilent2100 to ensure that the extracted RNA quality met the strict requirements for subsequent experiments. Next, mRNA was specifically enriched using the A-T base pairing technology of Oligo(dT) magnetic beads, laying the foundation for in-depth transcriptome analysis.\u003c/p\u003e\n\u003cp\u003eA library construction kit compatible with the DNBSEQ platform was used, following the manufacturer\u0026apos;s guidelines for library construction: first, the first strand cDNA was reverse transcribed and treated with UDG to avoid the incorporation of uracil. Then, the second strand cDNA is synthesized to form a stable double stranded cDNA, providing a high-quality template for subsequent sequencing library construction. Then we repaired the end of the double stranded cDNA and added an \u0026quot;A\u0026quot; base at the 3 \u0026apos;end to facilitate the connection of the Y-shaped linker, completing the library construction. Finally, connect the sequencing platform specific adapter for size selection and purification. Next, high-throughput sequencing specific biological analyzers or chips are used to detect the size distribution and concentration of the library. According to the operation process of the DNBSEQ platform, load the library onto the sequencing chip and set the sequencing parameters. Utilize the basecall software on the DNBSEQ platform to process raw image data, generate Cal files, and conduct preliminary data quality assessments, including signal strength, background noise, etc. This series of detailed operating procedures ensures the quality of the library and the accuracy of sequencing results, providing reliable data support for the molecular mechanism research of heat stroke.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDifferential RNA analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe RNA-seq raw data were uploaded to the online analysis platform iDEP 2.0\u003csup\u003e15\u003c/sup\u003e (Integrated Differential Expression \u0026amp; Pathway Analysis) (http://bioinformatics.sdstate.edu/idep/ ） for standardization processing and Principal Component Analysis (PCA).Principal Component Analysis (PCA) can observe the level of separation between HS and NC groups and exclude abnormal samples. Subsequently, distribution density and box plot analysis were performed on the sample data that had undergone quality control and filtering to clarify the homogeneity and comparability of the data. The DESeq2 software package was used for differential analysis of raw RNA sequencing data, with screening criteria set at Q value\u0026lt;0.05 and |log2FC|\u0026gt;1, to identify differential RNAs between the NC and HS groups. The ggplot2 R software package is used to visualize the results of differential analysis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eScreening of active ingredients and targets of Astragalus Membranaceus\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe various active ingredients and targets of Astragalus Membranaceushave different mechanisms of action and therapeutic effects. Screening specific active ingredients is the key to understanding the mechanism of Astragalus Membranaceusin treating heat stroke.TCMSP（Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform\u0026nbsp;：http://tcmspw. com/tcmsp.php）an integrated traditional Chinese medicine system pharmacology database and analysis platform, provides an efficient screening method. By setting the criteria of bioavailability (OB value) \u0026ge; 30% and drug similarity (DL) \u0026ge; 0.18, active ingredients and their targets with high bioavailability and drug similarity were screened. Then, the gene names corresponding to the targets were obtained using the Uniprot database. Then, the targets of active ingredients in Astragalus membranaceus were plotted against differential RNA using a Venn diagram, and the intersection targets of active ingredient targets and differential RNA were screened. These intersecting targets may be potential targets for Astragalus Membranaceus in treating heat stroke, and a network diagram of the effective active ingredients of Astragalus Membranaceusn and intersecting targets was constructed using Cytoscape (version 3.10.2).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGene Ontology (GO) enrichment analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGene Ontology (GO) is an internationally standardized classification system used to describe the attributes of genes and gene products in organisms. It encompasses three ontologies: molecular function(MF), cellular component(CC), and biological process(BP). The fundamental unit of GO is the term, with each term representing a specific attribute. In GO functional analysis, differentially expressed genes are first mapped to corresponding GO terms in the database, and the number of genes associated with each term is calculated. Subsequently, a hypergeometric test is employed to identify GO terms that are significantly enriched among the differentially expressed genes. A p-value of 0.05 is used as the threshold for significance. GO terms meeting this criterion are considered significantly enriched in the differentially expressed genes, aiding in the identification of the primary biological functions these genes perform\u003csup\u003e16\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eKyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn biological systems, genes work in concert to perform various biological functions, and pathway-based analysis provides deeper insights into these functions. The Kyoto Encyclopedia of Genes and Genomes (KEGG) is a primary public database for pathway information. Pathway enrichment analysis using KEGG pathways involves applying a hypergeometric test to identify pathways that are significantly enriched in differentially expressed genes compared to the entire genomic background\u003csup\u003e17\u003c/sup\u003e. A p-value of\u0026nbsp;\u0026le;0.05 is used as the significance threshold, and pathways meeting this criterion are considered significantly enriched in the differentially expressed genes.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConstruction of protein-protein interaction network\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo further screen potential core targets, submit the intersection targets to the STRING database（\u0026nbsp;https://cn.string-db.org/\u0026nbsp;）The minimum interaction score is set to 0.7 and disconnected nodes hidden in the network are used to generate protein interaction networks and download protein interaction data. Export protein interaction data to Cytoscape (version 3.10.2), and then use the cytoHubba plugin to score and rank nodes using the Maximum Group Centrality (MCC) analysis method, and visualize the analysis results.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMolecular docking\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFirstly, search for the corresponding drug small molecule in TCMSP and download the mol2 format file of the drug small molecule. Next, in the UniProt database（ https://www.uniprot.org/ ）Search for the protein corresponding to the core gene, select \u0026quot;X-ray\u0026quot; as the method, and select a single chain structure with \u0026quot;RESOLUTION\u0026quot; less than 3. Then in the RCSB-PDB database（ https://www.rcsb.org/ ）Download the PDB format file of the corresponding protein. Next, import the PDB format file of the protein into Pymol software to remove ions and ligands from the protein. Then use AutoDock Tools to perform hydrogenation treatment on the protein and set it as a receptor, exporting it as a pdbqt format file. Then, AutoDock Tools was used to perform hydrogenation treatment on the drug small molecule, and the drug small molecule was set as a ligand. The twisted bond was detected and set, and exported as a pdbqt format file. Next, import the pdbqt format files of protein and drug small molecules into AutoDock Tools software, set up the docking box, export as a gpf format file, then run Autogrid4, set the number of runs to 50, and set other parameters to default settings. Run autodock4, import the generated GPF format file, perform molecular docking, view the docking results, and export the result with the lowest binding energy. Finally, import the docking results into Pymol software for visualization processing.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eDifferentially expressed RNA analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePCA analysis was performed on the RNA sequencing results, which showed a good difference between NC samples and HS samples, and no abnormal samples were found (Figure 1A). The density distribution and box plot show that both groups of data have homogeneity and comparability (Figure 1BC). Perform differential analysis on the data of HS group and NC group, with screening criteria set as Q value\u0026lt;0.05 and | log2FC |\u0026gt;1. Compared with the NC group, the HS group had a total of 2042 differentially expressed RNAs, of which 1640 were upregulated and 402 were downregulated. (Figure 1D).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eScreening of active ingredients and targets of Astragalus Membranaceus\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe TCMSP database was used, and a total of 17 active ingredients of Astragalus membranaceus were obtained by setting the standards of bioavailability (OB value) \u0026ge; 30% and drug similarity (DL) \u0026ge; 0.18, targeting 190 targets. Then the Uniprot database was used to obtain the gene names corresponding to the targets, with the species set as humans, and 188 targets were obtained. 188 targets were intersected with 2042 differentially expressed RNAs, resulting in a total of 23 intersecting targets, which are potential targets for Astragalus Membranaceusin the treatment of heat stroke. (Figure 2A) Heat map based on RNA sequencing showed that MMP9, FOS, SPP1, CXCL10, MAPK14, HSPB1, HMOX1, AHSA1, MET, 1GF2, PLAU, VCAM1, SLPI, and SULT1E1 were highly expressed in heat stroke, while TP53, BCL2, MYC, PRSS1, GSTM2, CD4OLG, DPP4, PKIA, and ERBB3 were lowly expressed in heat stroke. (Figure 2B) These 23 intersecting targets are the action targets of six active molecules of Astragalus membranaceus. (Figure 2C)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGO analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGO analysis revealed 18 biological processes, 1 cellular component, and 1 molecular functional category. The enrichment of cellular components of cross genes is mainly in the extracellular region. (Figure 3A-B) The molecular function enrichment of crossover genes mainly involves molecular function regulator. (Figure 3A-B) The biological processes related to the intersection factor mainly include regulation of molecular function, regulation of multicellular organic process, response to stress, response to hypoxia, response to reduced oxygen levels, regulation of cell population proliferation, negative regulation of response to stimulus, and response to biotic stimulus (Figure 3C).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eKEGG analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eKEGG analysis identified 20 noteworthy pathways, including 3 Environmental Information Processing, 2 Organic Systems, and 15 Human Diseases.(Figure 4A-B) \u0026nbsp;The enriched pathways in the cross genes include Fluid sheet stress and atherosclerosis, TNF signaling pathway, IL-17 signaling pathway, Toll like receptor signaling pathway, and MAPK signaling pathway. (Figure 4C)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eProtein interaction network\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA protein interaction network diagram based on the STRING database, where the more connecting lines between proteins, the closer the protein interactions. (Figure 5A) Using the MCC analysis method in cytoHubba, the protein nodes in the PPI network were scored and ranked to obtain the top 10 most core proteins, including TP53, BCL2, MMP9, FOS, MYC, SPP1, VCAM1, HMOX1, CXCL10, PLAU. The redder the color, the higher the rating, indicating that the protein is more important. Among them, TP53, BCL2, and MMP9 have higher scores. (Figure 5B)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMolecular docking\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe molecular docking results showed that quercetin forms hydrogen bonds with TP53, BCL2, and MMP9, and has a low binding energy. The docking results of quercetin with TP53 showed that hydrogen bonds were formed between quercetin and GLU-326, PHE-328, LEU-330, and GLU-349 of TP53, with a binding energy of -5.48 kcal/mol. (Figure 6A) The docking results of quercetin with BCL2 showed that hydrogen bonds were formed between quercetin and ASN-143, TRP-144, GLY-145, ASN-192, LEU-201, and TYR-202 of BCL2, with a binding energy of -6.14 kcal/mol. (Figure 6B) The docking results of quercetin with MMP9 showed that hydrogen bonds were formed between quercetin and GLU-227, ALA-242, and ARG-249 of MMP9, with a binding energy of -9.58 kcal/mol. (Figure 6C)\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eHeat stroke is a serious acute disease caused by high temperature, mainly manifested as high fever, central nervous system dysfunction, and often accompanied by multiple organ dysfunction\u003csup\u003e\u003cspan additionalcitationids=\"CR19\" citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e.Current research indicates that its pathogenesis mainly involves inflammatory response, oxidative stress, coagulation abnormalities, etc\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e.The current treatment measures for heat stroke mainly include rapid physical cooling, anticoagulant therapy, and multi organ function support\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e,\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e.New treatment strategies are being explored, including the use of anti-inflammatory drugs and antioxidant drugs\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e,\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e.However, there is currently no treatment that can significantly improve the prognosis of patients with heat stroke, and precise treatment is the future trend of medical development\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e.Therefore, this study explored the potential therapeutic targets of active molecules of Astragalus membranaceus in heat stroke.\u003c/p\u003e \u003cp\u003eQuercetin is a flavonoid compound found in Astragalus membranaceus, which has various biological activities such as anti-inflammatory, antioxidant, and cell protection. Studies have shown that quercetin can exert anti-inflammatory effects by inhibiting the nuclear factor kappa B (NF - κ B) pathway, reducing the production of inflammatory mediators, and thus exerting anti-inflammatory effects\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e .In addition, quercetin can also reduce cellular damage caused by oxidative stress by regulating ROS. Studies have also shown that quercetin can protect cells by inhibiting apoptosis\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e.Our research indicates that intersecting genes are mainly enriched in regulation of molecular function, regulation of multicellular organic processes, response to stress, response to biotic stimuli, and MAPK signaling pathways.Heat stroke can affect the normal molecular function regulation and physiological activity of cells by affecting enzyme activity. Systemic inflammation and organ dysfunction caused by heat stroke involve complex multicellular organism process regulation, which affects the overall stability of the body. Therefore, multiple organ dysfunction is often combined.Studies have shown that after experiencing heat stress, the body's DNA damage increases, which affects the normal physiological activities\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e.In addition, studies have shown that heat stress induces necrotic apoptosis of cells through the MAPK pathway, which plays an important role in the inflammatory response and tissue damage caused by heat stroke\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eOur research indicates that quercetin has low binding energies with TP53, BCL2, and MMP9, with MMP9 upregulated in heat stroke and BCL2 and TP53 downregulated in heat stroke TP53 is a key regulatory factor of apoptosis and plays an important role in cellular stress response\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e.TP53 can regulate cell apoptosis caused by stress response by modulating autophagy, alleviate inflammatory response, and maintain cellular homeostasis in the nervous system\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e.Quercetin targeting TP53 may have a certain improvement effect on central nervous system dysfunction caused by heat stroke.BCL2 is an anti apoptotic protein that can protect cells from various stress-induced cell apoptosis\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e.Quercetin may regulate the apoptosis pathway by modulating the expression of BCL2, affecting the survival and functional status of cells under stress conditions, and playing a protective role in multicellular organism processes and stress response.Studies have shown that MMP9 (matrix metalloproteinase 9) plays a critical role in extracellular matrix degradation and remodeling, and plays an important role in tissue repair and inflammatory response\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e.Overexpression of MMP9 in heat stroke is associated with oxidative stress in cells. Studies have shown that MMP9 can increase the permeability of the blood-brain barrier, promote leukocyte infiltration into the central nervous system, damage neurons, and cause central nervous system disorders\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e,\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e.The expression of MMP9 may be related to the severity of heat stroke and can serve as a potential biomarker for the diagnosis of heat stroke. Studies have shown that downregulation of MMP9 expression can promote cellular autophagy, and quercetin may alleviate organ damage caused by inflammatory reactions by promoting leukocyte autophagy\u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThis study integrated transcriptomics, network pharmacology, and molecular docking analysis to explore the role of Astragalus membranaceus in heat stroke. It predicted the key pathways of Astragalus membranaceus in treating heat stroke and the interactions between Astragalus membranaceus bioactive compound quercetin and key targets such as TP53, BCL2, and MMP9, providing valuable insights into its mechanism of treating heat stroke.In the future, in vivo studies will be conducted to verify the therapeutic effect of Huangqi and explore its clinical applicability. In addition, in vitro experiments will be used to explore the specific mechanisms of Astragalus membranaceus and evaluate its potential side effects in clinical settings.\u003c/p\u003e "},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe study protocol has received approval from the Ethics Committee of Southwest Medical University (Ethics Number:KY2023087). All procedures involving human participants conformed to the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki Declaration and its later amendments or comparable ethical standards\u003csup\u003e13\u003c/sup\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;Consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll experimental participants have read and signed the informed consent form, and understand their rights and responsibilities as study participants.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe promise that all published information is based on the voluntary consent of participants, and we respect their privacy rights and the protection of personal information. We further declare that the publication of this paper will not cause any form of harm or adverse impact to the participants.\u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe study has passed the clinical trial registration.(\u003cstrong\u003eregistry:\u003c/strong\u003eChina Clinical Trial Registration Center；\u003cstrong\u003eregistration number:\u003c/strong\u003eChiCTR2400084775 ;\u003cstrong\u003edate of registration:\u003c/strong\u003e24-05-2024).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eI declare that the authors have no competing interests as defined by BMC, or other interests that might be perceived to influence the results and/or discussion reported in this paper.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis project is supported by the Economic, Commercial, Scientific and Technological Bureau of Xuyong County\u0026apos;s project \u0026quot;Basic and Clinical Research on Heat Stroke (Project No.: 2023JSYF01).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; contributions\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e1. Kaiyu Jin and Yingchun Hu led the overall project and designed the experiments.\u003c/p\u003e\n\u003cp\u003e2. Defeng Yin,Yandong Yao and Qin Guo performed the majority of the experiments and data analysis, and contributed to the writing of the manuscript.\u003c/p\u003e\n\u003cp\u003e3. Hao Jiang and Yonglan Hu developed the theoretical framework and provided critical feedback on the manuscript.\u003c/p\u003e\n\u003cp\u003e4. Lu Liu collected and processed the data, and contributed to the statistical analysis.\u003c/p\u003e\n\u003cp\u003eAll authors have read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;Acknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe express our sincere gratitude to the volunteers and participants who participated in this study, as their contributions were crucial for collecting data and analyzing results. We promise that their participation and contributions will be fairly recorded and recognized.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eXia, R. \u003cem\u003eet al.\u003c/em\u003e The pathogenesis and therapeutic strategies of heat stroke-induced myocardial injury. \u003cem\u003eFront. Pharmacol.\u003c/em\u003e \u003cstrong\u003e14\u003c/strong\u003e, 1286556 (2023).\u003c/li\u003e\n\u003cli\u003eLeon, L. R. \u0026amp; Helwig, B. G. Heat stroke: role of the systemic inflammatory response. \u003cem\u003eJ. Appl. Physiol. Bethesda Md 1985\u003c/em\u003e \u003cstrong\u003e109\u003c/strong\u003e, 1980\u0026ndash;1988 (2010).\u003c/li\u003e\n\u003cli\u003eShi, L. \u003cem\u003eet al.\u003c/em\u003e Heatstroke: a multicenter study in Southwestern China. \u003cem\u003eFront. 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Res.\u003c/em\u003e \u003cstrong\u003e175\u003c/strong\u003e, 105985 (2022).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Heatstroke, Astrolus Membranaceus, RNA sequencing, molecular docking","lastPublishedDoi":"10.21203/rs.3.rs-5594393/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5594393/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eObjective:\u003c/strong\u003e This study aims to explore the therapeutic potential of Astrolus Membranaceus in treating heatstroke and its potential therapeutic targets.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethod:\u003c/strong\u003e This study was conducted at the Affiliated Hospital of Southwest Medical University, and peripheral blood samples were collected from 10 heatstroke patients (HS=10) and 10 healthy individuals (NC=10) for RNA sequencing. Perform RNA differential analysis using the DESeq2 software package. In addition, the active ingredients and targets of Astragalus membranaceus were screened using the TCMSP database. Intersection the target and differential RNA to obtain the cross target. Then perform GO analysis and KEGG analysis on the intersection targets. Build a protein interaction network with cross targets using STRING website and Cytoscape software, and perform molecular docking between core targets and active molecules using AutoDock Tools.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults: \u003c/strong\u003eRNA sequencing results showed that compared with the NC group, the HS group had a total of 2042 differentially expressed RNAs. After taking the intersection of Astrolus Membranaceus targets and differentially expressed RNA, 23 intersecting targets were obtained. GO analysis found that the enrichment of cellular components of cross genes is mainly in the extracellular region. The molecular functional enrichment of cross genes mainly involves molecular function regulators. The biological processes related to cross factors mainly include regulation of molecular function, regulation of multicellular organic processes, and response to stress. KEGG analysis showed that the enriched pathways in the crossover genes mainly include the MAPK signaling pathway. The key targets TP53, BCL2, and MMP9 in the protein-protein interaction network were identified using cytoHubba. The molecular docking results indicate that quercetin forms hydrogen bonds with TP53, BCL2, and MMP9, with low binding energies.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion:\u003c/strong\u003e This study reveals the potential molecular mechanism of Astrolus Membranaceus in treating heatstroke, providing a scientific basis for further drug development and clinical application.\u003c/p\u003e","manuscriptTitle":"Astragalus Membranaceus: Exploring its Protective Role in Heat Stroke via Integrated Transcriptomic and Molecular Docking Approaches","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-12-30 10:26:32","doi":"10.21203/rs.3.rs-5594393/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"c2f8c54c-a730-4612-8ba9-446132d37197","owner":[],"postedDate":"December 30th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-01-16T16:23:08+00:00","versionOfRecord":[],"versionCreatedAt":"2024-12-30 10:26:32","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5594393","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5594393","identity":"rs-5594393","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}