Aspartame and Ischemic Stroke: Unraveling the Molecular Link through Network Toxicology and Molecular Docking Analysis

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While epidemiological studies link artificial sweeteners to cerebrovascular disease, the molecular mechanisms connecting aspartame to ischemic stroke are unclear. This study integrates network toxicology and molecular docking to identify key targets and pathways. Potential aspartame targets were predicted using STITCH, SwissTargetPrediction, and SEA databases, while ischemic stroke-related genes were retrieved from GeneCards, OMIM, and TTD. Venn analysis identified 201 overlapping genes, with IL1B, MMP9, SRC, AGT, and TNF as core targets. GO/KEGG enrichment revealed their roles in the renin-angiotensin system, complement/coagulation cascades, and inflammatory pathways. Molecular docking demonstrated strong binding affinities between aspartame and these targets, suggesting direct modulation. Our findings indicate that aspartame exacerbates ischemic brain injury by disrupting inflammatory responses, vascular homeostasis, and coagulation via multi-target interactions. This study provides the first systematic evidence of aspartame’s neurotoxicity mechanisms, offering insights for food additive safety evaluation and stroke prevention. Further validation is required to clarify metabolite synergies and dose-response relationships. Biological sciences/Computational biology and bioinformatics Health sciences/Health care Health sciences/Risk factors Health sciences/Neurology/Neurological disorders Aspartame Ischemic Stroke Network Toxicology Molecular Docking Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Aspartame, chemically known as L-α-aspartyl-L-phenylalanine methyl ester, is a high-intensity artificial sweetener that has been widely used in the global food industry since its discovery in 1965. With a sweetness approximately 200 times that of sucrose and minimal caloric contribution, aspartame quickly became one of the most extensively utilized sugar substitutes worldwide. 1 Currently, aspartame has been approved for use in over 90 countries and is present in thousands of products, including sugar-free beverages, low-calorie desserts, chewing gum, pharmaceuticals, and energy drinks. 2 Its primary advantage lies in its ability to provide the desired sweetness with minimal consumption, making it particularly favored by individuals with diabetes and those managing their weight. However, debates over its safety have persisted for decades. Although the U.S. Food and Drug Administration (FDA) has established an acceptable daily intake (ADI) of 40 mg/kg body weight and deemed aspartame safe at recommended doses, the recent classification of aspartame as a potential carcinogen by the World Health Organization (WHO) has intensified scientific controversy. The core of this debate stems from the limitations of early animal studies, where high-dose exposure results are difficult to extrapolate to long-term, low-dose human consumption. 3 Moreover, aspartame undergoes complex metabolic pathways, breaking down into phenylalanine, aspartic acid, and methanol, which may interfere with neurotransmitter balance, induce oxidative stress, and trigger inflammatory responses—mechanisms potentially linked to cerebrovascular diseases, depression, and autism. 4 Notably, several studies suggest that aspartame may increase the risk of neurodegeneration by disrupting the blood-brain barrier (BBB) and causing neuronal damage through its metabolic byproducts, 5 a mechanism that may intersect with the pathological processes of ischemic stroke. Ischemic stroke, the second leading cause of death globally, accounts for approximately 87% of all stroke cases. Its pathophysiological hallmark is the obstruction of cerebral arteries, leading to local hypoxia and blood flow disruption, followed by BBB breakdown, cerebral edema, and neuronal death. 6 According to the Global Burden of Disease (GBD) study, 13.7 million new stroke cases were reported worldwide in 2016, with ischemic strokes comprising 84.4% of cases, and East Asia exhibiting the highest age-standardized incidence rates. 7 The loss of BBB integrity is a critical determinant of poor prognosis in ischemic stroke, with mechanisms involving tight junction protein degradation, endothelial cell injury, and inflammatory mediator release, ultimately resulting in irreversible neurological deficits. Although reperfusion therapies, such as thrombolysis or thrombectomy, can partially restore cerebral blood flow, reperfusion injury-induced oxidative stress and inflammatory cascades remain major challenges in clinical management. 8 In recent years, increasing attention has been directed toward the chronic impact of exogenous chemicals (e.g., environmental toxins and food additives) on BBB function, with aspartame metabolites emerging as potential modulators of stroke risk by disrupting cerebrovascular homeostasis. However, while existing research primarily focuses on the carcinogenicity of aspartame, its mechanistic link to cerebrovascular diseases remains largely unexplored. Against this backdrop, integrating network toxicology and molecular docking approaches provides a novel paradigm for investigating the potential association between aspartame and ischemic stroke. Network toxicology, an interdisciplinary approach combining bioinformatics, systems biology, and cheminformatics, offers a systematic framework for understanding how chemical compounds disrupt biomolecular networks and cellular functions, ultimately contributing to disease pathogenesis. 9 Molecular docking further enables atomic-level simulations of aspartame and its metabolites binding to target proteins, allowing the prediction of structure-activity relationships and toxicity mechanisms. 10 By integrating these interdisciplinary methodologies, this study aims to elucidate how aspartame influences ischemic stroke pathogenesis, providing a theoretical basis for risk assessment and intervention strategies, while also establishing a methodological framework for the systematic study of food additive neurotoxicity. Materials and Methods Toxicity Identification of Aspartame The chemical structure and standardized SMILES sequence of aspartame were retrieved from the PubChem database 11 ( https://pubchem.ncbi.nlm.nih.gov/ ) using its unique identifier (CID: 134601). To comprehensively assess its toxicity profile, a dual-platform cross-validation approach was employed, utilizing ProTox-3.0 ( https://tox.charite.de/protox3/ ) and ADMETLab 2.0 ( https://admetmesh.scbdd.com/ ). ProTox-3.0 employs machine learning models to predict compound toxicity categories and median lethal dose (LD50), whereas ADMETLab 2.0 evaluates its drug-likeness and potential risks based on ADMET (absorption, distribution, metabolism, excretion, and toxicity) parameters. This dual validation strategy was adopted to minimize biases associated with a single database and enhance the reliability of toxicity assessment. Collection of Aspartame Target Genes Potential targets of aspartame were predicted from the STITCH 12 ( http://stitch.embl.de/ ), SwissTargetPrediction 13 ( http://www.swisstargetprediction.ch/ ), and SEA 14 ( https://sea.bkslab.org/ ) databases, using “Aspartame” as the keyword and restricting the species to Homo sapiens . The retrieved data were analyzed for structural consistency, and overlapping targets were integrated and de-duplicated using Venn diagrams to establish a comprehensive aspartame target library. Collection of Ischemic Stroke-Related Genes The disease-related target library was constructed based on data retrieved from the GeneCards 15 ( https://www.genecards.org/ ), OMIM ( https://www.omim.org/ ), and TTD ( https://db.idrblab.net/ttd/ ) databases using the keyword “Ischemic Stroke.” To ensure strong relevance between the identified genes and both ischemic stroke and aspartame exposure, a “Relevance score” threshold of 1.0 was applied in GeneCards, and only genes with a score exceeding 1.0 were selected. After merging and removing duplicates from the three databases using a Venn diagram, a final target library for ischemic stroke was established. The database search for this study was completed as of March 3, 2025. Intersection of Aspartame and Ischemic Stroke Targets The overlapping genes between aspartame targets and ischemic stroke-related genes were identified using the “ggvenn” package in R. The results were visualized using Cytoscape 3.10.3 ( https://cytoscape.org/ ). GO/KEGG Enrichment Analysis To elucidate the biological significance of the overlapping genes, Gene Ontology (GO) functional annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis were conducted using the “ClusterProfiler,” “Enrichplot,” and “Org.Hs.eg.db” packages in R. This analysis aimed to reveal the potential roles of key genes in biological processes, molecular functions, and cellular components, as well as their involvement in signaling pathways. Screening of Core Targets and Construction of PPI Network Protein-protein interaction (PPI) analysis was performed on the overlapping genes using the STRING database 16 ( https://cn.string-db.org/ ), with a confidence score threshold set at ≥ 0.7 and species limited to Homo sapiens . Isolated nodes were excluded to ensure biologically meaningful interactions. The PPI network was visualized using Cytoscape 3.10.3 17 ( https://cytoscape.org/ ), and key hub genes were identified based on degree centrality using the cytoHubba plugin. Molecular Docking Analysis To assess the binding affinity and interaction patterns between aspartame and its core targets, protein structures in PDB format were obtained from the RCSB Protein Data Bank 18 ( https://www.rcsb.org/ ), while the 3D molecular structure of aspartame was downloaded from the PubChem database. Molecular docking analysis was conducted using the CB-Dock2 web server 19 ( https://cadd.labshare.cn/cb-dock2/php/blinddock.php#job_list_load ). Binding energy values were used to evaluate ligand-receptor interactions: a binding energy of less than 0 kcal/mol indicated spontaneous binding, while a value below − 5 kcal/mol suggested a relatively stable interaction. The workflow of this study is illustrated in Fig. 1. Results Collection of Aspartame Target Genes By integrating target prediction data from the STITCH, SwissTargetPrediction, and SEA databases, we identified potential target genes of aspartame. After removing duplicates, we constructed an aspartame target gene database comprising 352 targets (Supplementary Table S1 ). Collection of Ischemic Stroke-Associated Genes We searched the GeneCards ( https://www.genecards.org/ ), OMIM ( https://www.omim.org/ ), and TTD ( https://db.idrblab.net/ttd/ ) databases using the keyword “Ischemic Stroke.” To ensure the relevance of the retrieved genes to both ischemic stroke and aspartame, we set the “Relevance Score” threshold to 1.0 in the GeneCards database and selected genes with scores above this threshold. After removing duplicate targets from the three databases, we established an ischemic stroke target gene database containing 6,833 targets (Supplementary Table S2 ). Identification of Core Targets and Interaction Network Between Aspartame and Ischemic Stroke Using the “ggvenn” package in R, we identified 201 overlapping target genes between aspartame and ischemic stroke. These were then visualized using Cytoscape 3.10.3 ( https://cytoscape.org/ ) (Fig. 2 ). We performed a protein–protein interaction (PPI) analysis on the overlapping genes using the STRING database ( https://cn.string-db.org/ ), setting the confidence score threshold to ≥ 0.7, restricting the species to Homo sapiens , and excluding isolated nodes. This process yielded 200 biologically significant interaction targets (Fig. 3 A). The PPI network was visualized in Cytoscape 3.10.3, and core hub genes were identified using the cytoHubba plugin based on degree centrality. The five key hub genes— IL1B, MMP9, SRC, AGT , and TNF —were selected, with darker colors and larger circles indicating stronger interactions with other proteins (Fig. 3 B). This visualization provides a comprehensive overview of the interactions among key targets. GO and KEGG Enrichment Analysis By analyzing the intersection of aspartame-related genes and ischemic stroke-associated genes, we identified 201 overlapping genes. Gene Ontology (GO) functional enrichment analysis revealed that these genes are primarily involved in biological processes such as blood pressure regulation, coagulation/fibrinolysis balance, extracellular matrix (ECM) remodeling, hypoxia adaptation, inflammation regulation, apoptosis, and neurotransmitter homeostasis (Fig. 3 C). Additionally, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis demonstrated that these genes are significantly enriched in multiple critical pathways, including the renin-angiotensin system, neuroactive ligand-receptor interaction, apoptosis, proteasome, Alzheimer’s disease, complement and coagulation cascades, lipid metabolism and atherosclerosis, prostate cancer, proteoglycans in cancer, and fluid shear stress and atherosclerosis (Fig. 3 D). These findings suggest that aspartame may influence the occurrence and progression of ischemic stroke through pathways related to neuroprotection, inflammatory response, apoptosis, vascular regulation, and tissue repair. Molecular Docking of Aspartame with Core Target Genes Molecular docking analysis demonstrated that all five key target genes could spontaneously bind to aspartame (binding energy < 0 kcal/mol) (Table 1 ). Among them, IL1B, MMP9, AGT , and TNF exhibited binding energies lower than − 5 kcal/mol, indicating stable interactions with aspartame (Fig. 4 A-E). These results suggest that aspartame may exert its effects on ischemic stroke by directly interacting with these key target genes, thereby modulating relevant biological processes. Table 1 Binding energies between the core targets and aspartame. Target Degree PDB number Hydrone Binding energy (kcal/mol) TNF 39 4V46 Aspartame -6.4 IL1B 30 1T4Q Aspartame -5.8 SRC 28 8BQ3 Aspartame -4.9 AGT 25 1T38 Aspartame -6.6 MMP9 24 1ITV Aspartame -6.3 Discussion Aspartame, an artificially synthesized high-intensity sweetener, has been widely utilized in the food industry since the 1980s. Its metabolites—phenylalanine, aspartic acid, and methanol—have raised persistent scientific debates regarding their potential toxicity. 2 Although the U.S. Food and Drug Administration (FDA) has established an acceptable daily intake (ADI) of 40 mg/kg body weight, 4 the World Health Organization (WHO) has recently classified it as a potential carcinogen, further underscoring the complexity of its safety concerns. Existing studies have primarily focused on the carcinogenicity and metabolic disturbances associated with aspartame. Notably, recent work by the team led by Yihai Cao demonstrated that aspartame can accelerate the formation of atherosclerotic plaques through an insulin-mediated inflammatory response, suggesting a link with cardiovascular disease. 20 However, ischemic stroke, the world’s second leading cause of death, is closely associated with blood–brain barrier disruption, inflammatory cascades, and oxidative stress, 6 and the chronic impact of aspartame metabolites on cerebrovascular homeostasis remains underexplored. Consequently, the potential mechanisms by which aspartame may influence stroke risk—whether via modulation of inflammatory mediators, vascular tone, or coagulation balance—warrant further investigation. Traditional risk factors for ischemic stroke include atherosclerosis, hypertension, and atrial fibrillation, 21 yet the continuously rising incidence suggests that emerging environmental factors should not be overlooked. Recent studies have gradually elucidated the prothrombotic effects of exogenous chemicals, such as erythritol, 22 Given that aspartame is one of the most pervasive artificial sweeteners used in the global food industry, its cumulative effects from long-term, low-dose exposure may indirectly affect cerebrovascular health through metabolic interference. Although epidemiological evidence implies a potential association between artificial sweetener consumption and stroke risk, the underlying molecular mechanisms remain unclear. This study is the first to integrate network toxicology and molecular docking techniques to systematically explore the target interaction network between aspartame and ischemic stroke. The aim is to fill the gap in understanding the neurotoxic mechanisms of food additives and to provide a new paradigm for risk assessment. Through the intersection analysis of genes related to aspartame and ischemic stroke, we identified 201 overlapping genes and further screened five core targets: IL1B, MMP9, SRC, AGT, and TNF. These targets were significantly enriched in pathways including the renin–angiotensin system (RAS), the complement and coagulation cascades, and inflammatory regulatory pathways. Overactivation of the RAS can induce vasoconstriction and oxidative stress, and AGT, as a key component of this system, may exacerbate post-ischemic microcirculatory dysfunction by promoting angiotensin II production. Moreover, the enrichment of the complement and coagulation cascades suggests that aspartame may interfere with the coagulation–fibrinolysis balance, thereby increasing the propensity for thrombosis—a mechanism analogous to the recently reported prothrombotic effect of erythritol. These findings indicate that aspartame may compromise cerebrovascular homeostasis and exacerbate ischemic injury through multi-target interactions, providing a theoretical basis for the neurotoxicity evaluation of artificial sweeteners. TNF-α, a central proinflammatory cytokine, 23 exhibits a complex dual role in ischemic stroke. On one hand, TNF-α promotes neutrophil infiltration and blood–brain barrier permeability via activation of the NF-κB signaling pathway, thereby aggravating cerebral edema and neuronal death; 24 on the other hand, animal studies suggest that moderate expression of TNF-α can induce ischemic tolerance by modulating apoptosis-related proteins, such as members of the Bcl-2 family, thereby mitigating reperfusion injury. 25 Our molecular docking results confirmed a stable binding between aspartame and TNF (binding energy − 6.2 kcal/mol), suggesting that aspartame may directly interfere with the conformation or signal transduction of TNF-α. However, the team led by Yihai Cao found that aspartame mediates inflammatory responses via the chemokine CX3CL1, with TNF-α potentially acting as a downstream effector. This hierarchical regulatory network implies that the intervention of aspartame on TNF may be dose-dependent, necessitating further in vivo studies to determine its critical threshold for either proinflammatory or neuroprotective effects. IL1B is a key driver of the inflammatory cascade in ischemic stroke, promoting the maturation and release of IL-18 and IL-1β through the activation of the NLRP3 inflammasome, 26 thereby amplifying neuroinflammation following cerebral ischemia. 27 The high affinity binding between IL1B and aspartame observed in this study suggests that aspartame may directly activate Toll-like receptor (TLR) signaling by mimicking pathogen-associated molecular patterns (PAMPs), leading to the overexpression of IL1B. 28 Preclinical studies have demonstrated that IL1B inhibitors, such as anakinra, can significantly reduce infarct volume and improve neurological deficits. 29 Nevertheless, complete inhibition of IL1B may impair host defense mechanisms and elevate infection risk. Therefore, targeting upstream regulators of IL1B (such as NLRP3 or ASC) 30 or developing small-molecule allosteric inhibitors may offer safer therapeutic strategies. The interaction between aspartame and IL1B provides a potential target for the development of novel anti-inflammatory agents, although its clinical translation requires careful evaluation of the risk–benefit ratio. MMP9, a member of the matrix metalloproteinase family, plays a pivotal role in ischemia–reperfusion injury by degrading type IV collagen and laminin, thereby disrupting the blood–brain barrier and promoting inflammatory cell infiltration and cerebral edema. 31 Our study found that the binding energy between aspartame and MMP9 is as low as − 6.3 kcal/mol, suggesting that aspartame may competitively inhibit MMP9 activity or interfere with its substrate binding, thereby mitigating blood–brain barrier leakage. This hypothesis contrasts with findings from the team led by Yihai Cao, who reported that MMP9 plays a key role in plaque instability during atherosclerosis. 20 The apparent discrepancy may arise from tissue-specific differences: in brain vascular endothelial cells, aspartame’s inhibitory effect on MMP9 might be protective, whereas in atherosclerotic plaques, it may promote MMP9 expression through alternative pathways such as CX3CL1. AGT, the rate-limiting component of the renin–angiotensin system (RAS), is significantly associated with stroke risk due to its genetic polymorphisms. 32 This study is the first to propose that aspartame may upregulate AGT expression, thereby promoting the production of angiotensin II, which induces vasoconstriction, oxidative stress, and endothelial dysfunction. This mechanism is consistent with recent epidemiological evidence suggesting a positive correlation between artificial sweetener consumption and the incidence of hypertension, 33 a core risk factor for ischemic stroke. Furthermore, angiotensin II can exacerbate free radical generation via NADPH oxidase activation, synergistically contributing to the oxidative stress effects of methanol, a metabolite of aspartame. Interventions targeting AGT—such as ACE inhibitors or angiotensin receptor blockers—have been widely applied in stroke prevention. The interaction between aspartame and AGT suggests that reducing the intake of artificial sweeteners may enhance the efficacy of existing antihypertensive therapies. SRC, a member of the non-receptor tyrosine kinase family, influences angiogenesis and neuronal survival in ischemic stroke through the regulation of VEGF and the PI3K/Akt signaling pathways. 34 Although the binding energy between aspartame and SRC (− 4.9 kcal/mol) is lower than that of the other core targets, the pivotal role of SRC in cellular migration and inflammatory responses cannot be overlooked. Overactivation of SRC may increase blood–brain barrier permeability and activate microglia, whereas its specific inhibition might interfere with physiological repair processes. 35 For instance, dasatinib, an SRC inhibitor, has been shown to alleviate cerebral ischemic injury, yet its long-term use may suppress neural stem cell proliferation. (36) Further in vitro studies are required to elucidate whether aspartame regulates SRC activity through direct binding or via epigenetic modifications. Molecular docking analysis revealed that aspartame binds to IL1B, MMP9, TNF, and AGT with binding energies lower than − 5 kcal/mol, with the interactions being most stable for AGT (− 6.6 kcal/mol) and TNF (− 6.4 kcal/mol). The binding energy analysis indicates that aspartame may interact with the active pockets of these targets via hydrogen bonds and hydrophobic interactions. Notably, the docking results for the aspartame prototype suggest that it may directly interfere with target protein functions; however, the indirect effects of metabolites such as methanol require further elucidation through dynamic simulations and in vitro experiments. This study innovatively integrates network toxicology with molecular docking techniques to systematically elucidate how aspartame may influence the progression of ischemic stroke through a multi-target interaction network, thereby addressing a significant gap in the research on the neurotoxic mechanisms of food additives. Nonetheless, certain limitations must be acknowledged. First, target prediction is dependent on algorithmic databases, and the in vivo relevance of these targets requires validation in animal models. Second, the molecular docking analysis did not account for the effects of metabolites or the cumulative impact of long-term exposure. Finally, the absence of clinical cohort data limits the ability to support the predicted pathways with epidemiological correlations. Future research should focus on developing transgenic animal models for chronic aspartame exposure to dynamically monitor blood–brain barrier permeability and inflammatory cytokine expression, as well as conducting large-scale human studies to analyze the relationship between aspartame intake and stroke incidence, and to explore the mediating effects of biomarkers such as CX3CL1. Such efforts will deepen our understanding of the neurotoxicity of artificial sweeteners and provide a scientific basis for the formulation of precise food safety policies. Conclusion This study systematically elucidated the potential molecular associations between the artificial sweetener aspartame and ischemic stroke by integrating network toxicology with molecular docking techniques. Based on cross-validation across multiple databases, five core targets—IL1B, MMP9, SRC, AGT, and TNF—were identified, with functional enrichment in the renin–angiotensin system (RAS), complement and coagulation cascades, and inflammatory regulatory pathways. These findings suggest that aspartame may exacerbate ischemic brain injury by modulating vascular tone, coagulation balance, and neuroinflammation. Molecular docking analyses further confirmed a high-affinity binding between aspartame and the aforementioned targets, indicating its potential to directly interfere with target protein conformation or signal transduction. Notably, this study is the first to propose that aspartame may activate the RAS by upregulating AGT expression, thereby inducing oxidative stress and endothelial dysfunction. This mechanism aligns with epidemiological evidence linking artificial sweetener consumption to hypertension, offering a novel perspective on its cerebrovascular toxicity. The results provide a theoretical basis for assessing the neurotoxicity of aspartame and serve as a methodological reference for the re-evaluation of food additive safety. Future research should validate target functions using animal models, delineate the synergistic effects of metabolites, and combine epidemiological cohort studies to clarify the dose–response relationship, thereby advancing the refinement of food safety guidelines and the optimization of stroke prevention strategies. Declarations Conflict of interest disclosure: The authors report there are no competing interests to declare. Funding No funding was received for conducting this study. Ethical statement The data used in this study were obtained from publicly available databases, Since these databases consist of anonymized data that is freely accessible to the public, no ethical approval or informed consent was required for this study. The data were used solely for research purposes and were in compliance with the relevant ethical guidelines for the use of publicly available data. CRediT authorship contribution statement Tao Wang: Methodology, Writing – original draft, Writing – review & editing. Tianyu Zhang: Data curation, Methodology, Software, Writing original draft, Writing – review & editing. Kunyang Bao: Conceptualization, Funding acquisition, Methodology, Writing – review & editing. Kuangyang Yu: Conceptualization, Methodology, Writing – original draft, Writing – review & editing. Changren Huang: Formal analysis, Writing original draft. Zhu Xinyao: Data curation, Writing – original draft. Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgements We would like to express our sincere gratitude to all the staff members of the databases utilized in this study for their valuable contributions. Data Availability The data supporting the findings of this study are available from the following public databases: PubChem (https://pubchem.ncbi.nlm.nih.gov/) STITCH (http://stitch.embl.de/) SwissTargetPrediction (http://www.swisstargetprediction.ch/) SEA (https://sea.bkslab.org/) GeneCards (https://www.genecards.org/) OMIM (https://www.omim.org/) Therapeutic Target Database (TTD, https://db.idrblab.net/ttd/) STRING (https://cn.string-db.org/) RCSB Protein Data Bank (https://www.rcsb.org/) CB-Dock2 (https://cadd.labshare.cn/cb-dock2/php/blinddock.php#job_list_load) References Soffritti, M. et al. The carcinogenic effects of aspartame: The urgent need for regulatory re-evaluation. Am. J. Ind. Med. 57 , 383–397 (2014). Shaher, S. A. A., Mihailescu, D. F. & Amuzescu, B. Aspartame safety as a food sweetener and related health hazards. Nutrients 15 , 3627 (2023). Chen, D. & Hou, X. Aspartame carcinogenic potential revealed through network toxicology and molecular docking insights. Sci. 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Supplementary Files SupplementaryTableS1.xlsx SupplementaryTableS2.xlsx Cite Share Download PDF Status: Published Journal Publication published 04 Jul, 2025 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Revision requested 01 Apr, 2025 Reviews received at journal 30 Mar, 2025 Reviews received at journal 27 Mar, 2025 Reviews received at journal 22 Mar, 2025 Reviewers agreed at journal 19 Mar, 2025 Reviewers agreed at journal 18 Mar, 2025 Reviewers agreed at journal 18 Mar, 2025 Reviewers invited by journal 18 Mar, 2025 Editor assigned by journal 18 Mar, 2025 Editor invited by journal 17 Mar, 2025 Submission checks completed at journal 14 Mar, 2025 First submitted to journal 08 Mar, 2025 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-6183078","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":432405301,"identity":"020665ea-8189-4d82-bf88-6844e979b6aa","order_by":0,"name":"Tianyu Zhang","email":"","orcid":"","institution":"The Affiliated Hospital of Southwest Medical University","correspondingAuthor":false,"prefix":"","firstName":"Tianyu","middleName":"","lastName":"Zhang","suffix":""},{"id":432405303,"identity":"caab1995-bbbb-47dc-9b45-5fd76e26f082","order_by":1,"name":"Tao Wang","email":"","orcid":"","institution":"The Affiliated Hospital of Southwest Medical University","correspondingAuthor":false,"prefix":"","firstName":"Tao","middleName":"","lastName":"Wang","suffix":""},{"id":432405306,"identity":"3a595ef3-af36-4539-9056-f96d7318f78c","order_by":2,"name":"Kuangyang Yu","email":"","orcid":"","institution":"The Affiliated Hospital of Southwest Medical University","correspondingAuthor":false,"prefix":"","firstName":"Kuangyang","middleName":"","lastName":"Yu","suffix":""},{"id":432405307,"identity":"bfa3e1c1-5d29-49bd-9033-52a2e5203f8d","order_by":3,"name":"Changren huang","email":"","orcid":"","institution":"The Affiliated Hospital of Southwest Medical University","correspondingAuthor":false,"prefix":"","firstName":"Changren","middleName":"","lastName":"huang","suffix":""},{"id":432405308,"identity":"a8c6e736-bc80-4cc2-937d-2c2f04e57f8f","order_by":4,"name":"Kunyang Bao","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA1ElEQVRIiWNgGAWjYBACAx4GxgMQJvOBAx9+EKeFAaqFLfHgzB7StPAYH+ZgI0KLOc8Zg4M//tyLNriR8+EwAw+DPL/YAfxaLHt7DA5IthXnbriRu+FwgQWD4czZCQQcdp7H4IBhQwJEywwehgSD28RoSfgD0pLz4DAPGzFazgIddoANrIWBOC2WPccKDja2JeTOPPPMABjIEoT9Ys6TvPHhD6DD+o4nP/7w4YeNPL80AS0MDBwGYErhAlilBCHlIMD+AEzJ9x8gRvUoGAWjYBSMRAAA561SVHEgmBQAAAAASUVORK5CYII=","orcid":"","institution":"The Affiliated Hospital of Southwest Medical University","correspondingAuthor":true,"prefix":"","firstName":"Kunyang","middleName":"","lastName":"Bao","suffix":""}],"badges":[],"createdAt":"2025-03-08 09:08:02","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6183078/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6183078/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-025-08898-z","type":"published","date":"2025-07-04T15:58:15+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":79356393,"identity":"83193662-cc15-4d31-abf2-e766c3d3f571","added_by":"auto","created_at":"2025-03-27 11:26:38","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":544177,"visible":true,"origin":"","legend":"\u003cp\u003eFlowchart of the study design.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-6183078/v1/5f33b8ea96bc6a3df8ceb64c.png"},{"id":79356399,"identity":"622859ad-f1fd-4773-890a-d9b7624a2dab","added_by":"auto","created_at":"2025-03-27 11:26:38","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":185818,"visible":true,"origin":"","legend":"\u003cp\u003e(A) Venn diagram of targets associated with aspartame and ischemic stroke. (B) Network diagram depicting the potential targets shared between aspartame and ischemic stroke.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-6183078/v1/bc3fced4d461144e4c7c447a.png"},{"id":79356396,"identity":"401f32e6-8fdd-4164-a527-b382a76febfd","added_by":"auto","created_at":"2025-03-27 11:26:38","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":313079,"visible":true,"origin":"","legend":"\u003cp\u003e(A) Protein–protein interaction (PPI) network of potential targets, with a confidence score threshold set at ≥0.7; nodes represent proteins and edges indicate their interactions. (B) Visualization of the PPI network using Cytoscape, with node color and size adjusted based on degree values—darker colors and larger circles represent stronger interactions. (C) Gene Ontology (GO) enrichment analysis of aspartame–ischemic stroke targets. (D) Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of aspartame–ischemic stroke targets.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-6183078/v1/93d2ded3d5d9d5680560eef2.png"},{"id":79356398,"identity":"3255f48a-6832-4d4f-bbcd-b100c57bb4e6","added_by":"auto","created_at":"2025-03-27 11:26:38","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":330460,"visible":true,"origin":"","legend":"\u003cp\u003eMolecular docking visualizations: (A) AGT with aspartame; (B) IL1B with aspartame; (C) SRC with aspartame; (D) TNF with aspartame; (E) MMP9 with aspartame.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-6183078/v1/dec89f945ec144f293a2d211.png"},{"id":86179883,"identity":"6e7576e7-947d-4ffe-8751-64c10a0ffbb1","added_by":"auto","created_at":"2025-07-07 16:20:18","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2225269,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6183078/v1/cc148715-5589-4eea-a68b-72ed9351f611.pdf"},{"id":79356675,"identity":"2e2ecb9b-9cb7-4dfa-8235-b87258baf12f","added_by":"auto","created_at":"2025-03-27 11:34:38","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":13319,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTableS1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-6183078/v1/34fe23ff1d6b86bb62c99ac9.xlsx"},{"id":79356402,"identity":"b5bcddcc-1c8e-4f40-86fb-96da43419f06","added_by":"auto","created_at":"2025-03-27 11:26:38","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":95192,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTableS2.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-6183078/v1/8a12681b8ac328b5d60ac475.xlsx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Aspartame and Ischemic Stroke: Unraveling the Molecular Link through Network Toxicology and Molecular Docking Analysis","fulltext":[{"header":"Introduction","content":"\u003cp\u003eAspartame, chemically known as L-α-aspartyl-L-phenylalanine methyl ester, is a high-intensity artificial sweetener that has been widely used in the global food industry since its discovery in 1965. With a sweetness approximately 200 times that of sucrose and minimal caloric contribution, aspartame quickly became one of the most extensively utilized sugar substitutes worldwide. \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e Currently, aspartame has been approved for use in over 90 countries and is present in thousands of products, including sugar-free beverages, low-calorie desserts, chewing gum, pharmaceuticals, and energy drinks. \u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e Its primary advantage lies in its ability to provide the desired sweetness with minimal consumption, making it particularly favored by individuals with diabetes and those managing their weight. However, debates over its safety have persisted for decades. Although the U.S. Food and Drug Administration (FDA) has established an acceptable daily intake (ADI) of 40 mg/kg body weight and deemed aspartame safe at recommended doses, the recent classification of aspartame as a potential carcinogen by the World Health Organization (WHO) has intensified scientific controversy. The core of this debate stems from the limitations of early animal studies, where high-dose exposure results are difficult to extrapolate to long-term, low-dose human consumption. \u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e Moreover, aspartame undergoes complex metabolic pathways, breaking down into phenylalanine, aspartic acid, and methanol, which may interfere with neurotransmitter balance, induce oxidative stress, and trigger inflammatory responses\u0026mdash;mechanisms potentially linked to cerebrovascular diseases, depression, and autism. \u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e Notably, several studies suggest that aspartame may increase the risk of neurodegeneration by disrupting the blood-brain barrier (BBB) and causing neuronal damage through its metabolic byproducts, \u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e a mechanism that may intersect with the pathological processes of ischemic stroke.\u003c/p\u003e \u003cp\u003eIschemic stroke, the second leading cause of death globally, accounts for approximately 87% of all stroke cases. Its pathophysiological hallmark is the obstruction of cerebral arteries, leading to local hypoxia and blood flow disruption, followed by BBB breakdown, cerebral edema, and neuronal death. \u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e According to the Global Burden of Disease (GBD) study, 13.7\u0026nbsp;million new stroke cases were reported worldwide in 2016, with ischemic strokes comprising 84.4% of cases, and East Asia exhibiting the highest age-standardized incidence rates. \u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e The loss of BBB integrity is a critical determinant of poor prognosis in ischemic stroke, with mechanisms involving tight junction protein degradation, endothelial cell injury, and inflammatory mediator release, ultimately resulting in irreversible neurological deficits. Although reperfusion therapies, such as thrombolysis or thrombectomy, can partially restore cerebral blood flow, reperfusion injury-induced oxidative stress and inflammatory cascades remain major challenges in clinical management. \u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e In recent years, increasing attention has been directed toward the chronic impact of exogenous chemicals (e.g., environmental toxins and food additives) on BBB function, with aspartame metabolites emerging as potential modulators of stroke risk by disrupting cerebrovascular homeostasis. However, while existing research primarily focuses on the carcinogenicity of aspartame, its mechanistic link to cerebrovascular diseases remains largely unexplored.\u003c/p\u003e \u003cp\u003eAgainst this backdrop, integrating network toxicology and molecular docking approaches provides a novel paradigm for investigating the potential association between aspartame and ischemic stroke. Network toxicology, an interdisciplinary approach combining bioinformatics, systems biology, and cheminformatics, offers a systematic framework for understanding how chemical compounds disrupt biomolecular networks and cellular functions, ultimately contributing to disease pathogenesis. \u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e Molecular docking further enables atomic-level simulations of aspartame and its metabolites binding to target proteins, allowing the prediction of structure-activity relationships and toxicity mechanisms. \u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e By integrating these interdisciplinary methodologies, this study aims to elucidate how aspartame influences ischemic stroke pathogenesis, providing a theoretical basis for risk assessment and intervention strategies, while also establishing a methodological framework for the systematic study of food additive neurotoxicity.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eToxicity Identification of Aspartame\u003c/h2\u003e \u003cp\u003eThe chemical structure and standardized SMILES sequence of aspartame were retrieved from the PubChem database\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e (\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) using its unique identifier (CID: 134601). To comprehensively assess its toxicity profile, a dual-platform cross-validation approach was employed, utilizing ProTox-3.0 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://tox.charite.de/protox3/\u003c/span\u003e\u003cspan address=\"https://tox.charite.de/protox3/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and ADMETLab 2.0 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://admetmesh.scbdd.com/\u003c/span\u003e\u003cspan address=\"https://admetmesh.scbdd.com/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). ProTox-3.0 employs machine learning models to predict compound toxicity categories and median lethal dose (LD50), whereas ADMETLab 2.0 evaluates its drug-likeness and potential risks based on ADMET (absorption, distribution, metabolism, excretion, and toxicity) parameters. This dual validation strategy was adopted to minimize biases associated with a single database and enhance the reliability of toxicity assessment.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eCollection of Aspartame Target Genes\u003c/h3\u003e\n\u003cp\u003ePotential targets of aspartame were predicted from the STITCH \u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://stitch.embl.de/\u003c/span\u003e\u003cspan address=\"http://stitch.embl.de/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), SwissTargetPrediction\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e (\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), and SEA\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e (\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) databases, using \u0026ldquo;Aspartame\u0026rdquo; as the keyword and restricting the species to \u003cem\u003eHomo sapiens\u003c/em\u003e. The retrieved data were analyzed for structural consistency, and overlapping targets were integrated and de-duplicated using Venn diagrams to establish a comprehensive aspartame target library.\u003c/p\u003e\n\u003ch3\u003eCollection of Ischemic Stroke-Related Genes\u003c/h3\u003e\n\u003cp\u003eThe disease-related target library was constructed based on data retrieved from the GeneCards \u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e (\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://www.omim.org/\u003c/span\u003e\u003cspan address=\"https://www.omim.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), and TTD (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://db.idrblab.net/ttd/\u003c/span\u003e\u003cspan address=\"https://db.idrblab.net/ttd/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) databases using the keyword \u0026ldquo;Ischemic Stroke.\u0026rdquo; To ensure strong relevance between the identified genes and both ischemic stroke and aspartame exposure, a \u0026ldquo;Relevance score\u0026rdquo; threshold of 1.0 was applied in GeneCards, and only genes with a score exceeding 1.0 were selected. After merging and removing duplicates from the three databases using a Venn diagram, a final target library for ischemic stroke was established. The database search for this study was completed as of March 3, 2025.\u003c/p\u003e\n\u003ch3\u003eIntersection of Aspartame and Ischemic Stroke Targets\u003c/h3\u003e\n\u003cp\u003eThe overlapping genes between aspartame targets and ischemic stroke-related genes were identified using the \u0026ldquo;ggvenn\u0026rdquo; package in R. The results were visualized using Cytoscape 3.10.3 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://cytoscape.org/\u003c/span\u003e\u003cspan address=\"https://cytoscape.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e\n\u003ch3\u003eGO/KEGG Enrichment Analysis\u003c/h3\u003e\n\u003cp\u003eTo elucidate the biological significance of the overlapping genes, Gene Ontology (GO) functional annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis were conducted using the \u0026ldquo;ClusterProfiler,\u0026rdquo; \u0026ldquo;Enrichplot,\u0026rdquo; and \u0026ldquo;Org.Hs.eg.db\u0026rdquo; packages in R. This analysis aimed to reveal the potential roles of key genes in biological processes, molecular functions, and cellular components, as well as their involvement in signaling pathways.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eScreening of Core Targets and Construction of PPI Network\u003c/h2\u003e \u003cp\u003eProtein-protein interaction (PPI) analysis was performed on the overlapping genes using the STRING database \u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e (\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 a confidence score threshold set at \u0026ge;\u0026thinsp;0.7 and species limited to \u003cem\u003eHomo sapiens\u003c/em\u003e. Isolated nodes were excluded to ensure biologically meaningful interactions. The PPI network was visualized using Cytoscape 3.10.3 \u003csup\u003e17\u003c/sup\u003e (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://cytoscape.org/\u003c/span\u003e\u003cspan address=\"https://cytoscape.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), and key hub genes were identified based on degree centrality using the cytoHubba plugin.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eMolecular Docking Analysis\u003c/h3\u003e\n\u003cp\u003eTo assess the binding affinity and interaction patterns between aspartame and its core targets, protein structures in PDB format were obtained from the RCSB Protein Data Bank\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e (\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), while the 3D molecular structure of aspartame was downloaded from the PubChem database. Molecular docking analysis was conducted using the CB-Dock2 web server\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://cadd.labshare.cn/cb-dock2/php/blinddock.php#job_list_load\u003c/span\u003e\u003cspan address=\"https://cadd.labshare.cn/cb-dock2/php/blinddock.php#job_list_load\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Binding energy values were used to evaluate ligand-receptor interactions: a binding energy of less than 0 kcal/mol indicated spontaneous binding, while a value below \u0026minus;\u0026thinsp;5 kcal/mol suggested a relatively stable interaction. The workflow of this study is illustrated in Fig.\u0026nbsp;1.\u003c/p\u003e "},{"header":"Results","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eCollection of Aspartame Target Genes\u003c/h2\u003e \u003cp\u003eBy integrating target prediction data from the STITCH, SwissTargetPrediction, and SEA databases, we identified potential target genes of aspartame. After removing duplicates, we constructed an aspartame target gene database comprising 352 targets (Supplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eCollection of Ischemic Stroke-Associated Genes\u003c/h2\u003e \u003cp\u003eWe searched the GeneCards (\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://www.omim.org/\u003c/span\u003e\u003cspan address=\"https://www.omim.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), and TTD (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://db.idrblab.net/ttd/\u003c/span\u003e\u003cspan address=\"https://db.idrblab.net/ttd/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) databases using the keyword \u0026ldquo;Ischemic Stroke.\u0026rdquo; To ensure the relevance of the retrieved genes to both ischemic stroke and aspartame, we set the \u0026ldquo;Relevance Score\u0026rdquo; threshold to 1.0 in the GeneCards database and selected genes with scores above this threshold. After removing duplicate targets from the three databases, we established an ischemic stroke target gene database containing 6,833 targets (Supplementary Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eIdentification of Core Targets and Interaction Network Between Aspartame and Ischemic Stroke\u003c/h2\u003e \u003cp\u003eUsing the \u0026ldquo;ggvenn\u0026rdquo; package in R, we identified 201 overlapping target genes between aspartame and ischemic stroke. These were then visualized using Cytoscape 3.10.3 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://cytoscape.org/\u003c/span\u003e\u003cspan address=\"https://cytoscape.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003e). We performed a protein\u0026ndash;protein interaction (PPI) analysis on the overlapping genes using the STRING 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), setting the confidence score threshold to \u0026ge;\u0026thinsp;0.7, restricting the species to \u003cem\u003eHomo sapiens\u003c/em\u003e, and excluding isolated nodes. This process yielded 200 biologically significant interaction targets (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). The PPI network was visualized in Cytoscape 3.10.3, and core hub genes were identified using the cytoHubba plugin based on degree centrality. The five key hub genes\u0026mdash;\u003cem\u003eIL1B, MMP9, SRC, AGT\u003c/em\u003e, and \u003cem\u003eTNF\u003c/em\u003e\u0026mdash;were selected, with darker colors and larger circles indicating stronger interactions with other proteins (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). This visualization provides a comprehensive overview of the interactions among key targets.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eGO and KEGG Enrichment Analysis\u003c/h2\u003e \u003cp\u003eBy analyzing the intersection of aspartame-related genes and ischemic stroke-associated genes, we identified 201 overlapping genes. Gene Ontology (GO) functional enrichment analysis revealed that these genes are primarily involved in biological processes such as blood pressure regulation, coagulation/fibrinolysis balance, extracellular matrix (ECM) remodeling, hypoxia adaptation, inflammation regulation, apoptosis, and neurotransmitter homeostasis (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). Additionally, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis demonstrated that these genes are significantly enriched in multiple critical pathways, including the renin-angiotensin system, neuroactive ligand-receptor interaction, apoptosis, proteasome, Alzheimer\u0026rsquo;s disease, complement and coagulation cascades, lipid metabolism and atherosclerosis, prostate cancer, proteoglycans in cancer, and fluid shear stress and atherosclerosis (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). These findings suggest that aspartame may influence the occurrence and progression of ischemic stroke through pathways related to neuroprotection, inflammatory response, apoptosis, vascular regulation, and tissue repair.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eMolecular Docking of Aspartame with Core Target Genes\u003c/h2\u003e \u003cp\u003eMolecular docking analysis demonstrated that all five key target genes could spontaneously bind to aspartame (binding energy\u0026thinsp;\u0026lt;\u0026thinsp;0 kcal/mol) (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Among them, \u003cem\u003eIL1B, MMP9, AGT\u003c/em\u003e, and \u003cem\u003eTNF\u003c/em\u003e exhibited binding energies lower than \u0026minus;\u0026thinsp;5 kcal/mol, indicating stable interactions with aspartame (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003eA-E). These results suggest that aspartame may exert its effects on ischemic stroke by directly interacting with these key target genes, thereby modulating relevant biological processes.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eBinding energies between the core targets and aspartame.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTarget\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eDegree\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePDB number\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eHydrone\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eBinding energy (kcal/mol)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTNF\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e39\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4V46\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eAspartame\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e-6.4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIL1B\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1T4Q\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eAspartame\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e-5.8\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSRC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e28\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e8BQ3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eAspartame\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e-4.9\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAGT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1T38\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eAspartame\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e-6.6\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMMP9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1ITV\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eAspartame\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e-6.3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eAspartame, an artificially synthesized high-intensity sweetener, has been widely utilized in the food industry since the 1980s. Its metabolites\u0026mdash;phenylalanine, aspartic acid, and methanol\u0026mdash;have raised persistent scientific debates regarding their potential toxicity.\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e Although the U.S. Food and Drug Administration (FDA) has established an acceptable daily intake (ADI) of 40 mg/kg body weight,\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e the World Health Organization (WHO) has recently classified it as a potential carcinogen, further underscoring the complexity of its safety concerns. Existing studies have primarily focused on the carcinogenicity and metabolic disturbances associated with aspartame. Notably, recent work by the team led by Yihai Cao demonstrated that aspartame can accelerate the formation of atherosclerotic plaques through an insulin-mediated inflammatory response, suggesting a link with cardiovascular disease.\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e However, ischemic stroke, the world\u0026rsquo;s second leading cause of death, is closely associated with blood\u0026ndash;brain barrier disruption, inflammatory cascades, and oxidative stress,\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e and the chronic impact of aspartame metabolites on cerebrovascular homeostasis remains underexplored. Consequently, the potential mechanisms by which aspartame may influence stroke risk\u0026mdash;whether via modulation of inflammatory mediators, vascular tone, or coagulation balance\u0026mdash;warrant further investigation.\u003c/p\u003e \u003cp\u003eTraditional risk factors for ischemic stroke include atherosclerosis, hypertension, and atrial fibrillation,\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e yet the continuously rising incidence suggests that emerging environmental factors should not be overlooked. Recent studies have gradually elucidated the prothrombotic effects of exogenous chemicals, such as erythritol,\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e Given that aspartame is one of the most pervasive artificial sweeteners used in the global food industry, its cumulative effects from long-term, low-dose exposure may indirectly affect cerebrovascular health through metabolic interference. Although epidemiological evidence implies a potential association between artificial sweetener consumption and stroke risk, the underlying molecular mechanisms remain unclear. This study is the first to integrate network toxicology and molecular docking techniques to systematically explore the target interaction network between aspartame and ischemic stroke. The aim is to fill the gap in understanding the neurotoxic mechanisms of food additives and to provide a new paradigm for risk assessment.\u003c/p\u003e \u003cp\u003eThrough the intersection analysis of genes related to aspartame and ischemic stroke, we identified 201 overlapping genes and further screened five core targets: IL1B, MMP9, SRC, AGT, and TNF. These targets were significantly enriched in pathways including the renin\u0026ndash;angiotensin system (RAS), the complement and coagulation cascades, and inflammatory regulatory pathways. Overactivation of the RAS can induce vasoconstriction and oxidative stress, and AGT, as a key component of this system, may exacerbate post-ischemic microcirculatory dysfunction by promoting angiotensin II production. Moreover, the enrichment of the complement and coagulation cascades suggests that aspartame may interfere with the coagulation\u0026ndash;fibrinolysis balance, thereby increasing the propensity for thrombosis\u0026mdash;a mechanism analogous to the recently reported prothrombotic effect of erythritol. These findings indicate that aspartame may compromise cerebrovascular homeostasis and exacerbate ischemic injury through multi-target interactions, providing a theoretical basis for the neurotoxicity evaluation of artificial sweeteners.\u003c/p\u003e \u003cp\u003eTNF-α, a central proinflammatory cytokine,\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e exhibits a complex dual role in ischemic stroke. On one hand, TNF-α promotes neutrophil infiltration and blood\u0026ndash;brain barrier permeability via activation of the NF-κB signaling pathway, thereby aggravating cerebral edema and neuronal death;\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e on the other hand, animal studies suggest that moderate expression of TNF-α can induce ischemic tolerance by modulating apoptosis-related proteins, such as members of the Bcl-2 family, thereby mitigating reperfusion injury.\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e Our molecular docking results confirmed a stable binding between aspartame and TNF (binding energy \u0026minus;\u0026thinsp;6.2 kcal/mol), suggesting that aspartame may directly interfere with the conformation or signal transduction of TNF-α. However, the team led by Yihai Cao found that aspartame mediates inflammatory responses via the chemokine CX3CL1, with TNF-α potentially acting as a downstream effector. This hierarchical regulatory network implies that the intervention of aspartame on TNF may be dose-dependent, necessitating further in vivo studies to determine its critical threshold for either proinflammatory or neuroprotective effects.\u003c/p\u003e \u003cp\u003eIL1B is a key driver of the inflammatory cascade in ischemic stroke, promoting the maturation and release of IL-18 and IL-1β through the activation of the NLRP3 inflammasome,\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e thereby amplifying neuroinflammation following cerebral ischemia.\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e The high affinity binding between IL1B and aspartame observed in this study suggests that aspartame may directly activate Toll-like receptor (TLR) signaling by mimicking pathogen-associated molecular patterns (PAMPs), leading to the overexpression of IL1B.\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e Preclinical studies have demonstrated that IL1B inhibitors, such as anakinra, can significantly reduce infarct volume and improve neurological deficits.\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e Nevertheless, complete inhibition of IL1B may impair host defense mechanisms and elevate infection risk. Therefore, targeting upstream regulators of IL1B (such as NLRP3 or ASC)\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e or developing small-molecule allosteric inhibitors may offer safer therapeutic strategies. The interaction between aspartame and IL1B provides a potential target for the development of novel anti-inflammatory agents, although its clinical translation requires careful evaluation of the risk\u0026ndash;benefit ratio.\u003c/p\u003e \u003cp\u003eMMP9, a member of the matrix metalloproteinase family, plays a pivotal role in ischemia\u0026ndash;reperfusion injury by degrading type IV collagen and laminin, thereby disrupting the blood\u0026ndash;brain barrier and promoting inflammatory cell infiltration and cerebral edema.\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e Our study found that the binding energy between aspartame and MMP9 is as low as \u0026minus;\u0026thinsp;6.3 kcal/mol, suggesting that aspartame may competitively inhibit MMP9 activity or interfere with its substrate binding, thereby mitigating blood\u0026ndash;brain barrier leakage. This hypothesis contrasts with findings from the team led by Yihai Cao, who reported that MMP9 plays a key role in plaque instability during atherosclerosis. \u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e The apparent discrepancy may arise from tissue-specific differences: in brain vascular endothelial cells, aspartame\u0026rsquo;s inhibitory effect on MMP9 might be protective, whereas in atherosclerotic plaques, it may promote MMP9 expression through alternative pathways such as CX3CL1.\u003c/p\u003e \u003cp\u003eAGT, the rate-limiting component of the renin\u0026ndash;angiotensin system (RAS), is significantly associated with stroke risk due to its genetic polymorphisms.\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e This study is the first to propose that aspartame may upregulate AGT expression, thereby promoting the production of angiotensin II, which induces vasoconstriction, oxidative stress, and endothelial dysfunction. This mechanism is consistent with recent epidemiological evidence suggesting a positive correlation between artificial sweetener consumption and the incidence of hypertension,\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e a core risk factor for ischemic stroke. Furthermore, angiotensin II can exacerbate free radical generation via NADPH oxidase activation, synergistically contributing to the oxidative stress effects of methanol, a metabolite of aspartame. Interventions targeting AGT\u0026mdash;such as ACE inhibitors or angiotensin receptor blockers\u0026mdash;have been widely applied in stroke prevention. The interaction between aspartame and AGT suggests that reducing the intake of artificial sweeteners may enhance the efficacy of existing antihypertensive therapies.\u003c/p\u003e \u003cp\u003eSRC, a member of the non-receptor tyrosine kinase family, influences angiogenesis and neuronal survival in ischemic stroke through the regulation of VEGF and the PI3K/Akt signaling pathways.\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e Although the binding energy between aspartame and SRC (\u0026minus;\u0026thinsp;4.9 kcal/mol) is lower than that of the other core targets, the pivotal role of SRC in cellular migration and inflammatory responses cannot be overlooked. Overactivation of SRC may increase blood\u0026ndash;brain barrier permeability and activate microglia, whereas its specific inhibition might interfere with physiological repair processes.\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e For instance, dasatinib, an SRC inhibitor, has been shown to alleviate cerebral ischemic injury, yet its long-term use may suppress neural stem cell proliferation. (36) Further in vitro studies are required to elucidate whether aspartame regulates SRC activity through direct binding or via epigenetic modifications.\u003c/p\u003e \u003cp\u003eMolecular docking analysis revealed that aspartame binds to IL1B, MMP9, TNF, and AGT with binding energies lower than \u0026minus;\u0026thinsp;5 kcal/mol, with the interactions being most stable for AGT (\u0026minus;\u0026thinsp;6.6 kcal/mol) and TNF (\u0026minus;\u0026thinsp;6.4 kcal/mol). The binding energy analysis indicates that aspartame may interact with the active pockets of these targets via hydrogen bonds and hydrophobic interactions. Notably, the docking results for the aspartame prototype suggest that it may directly interfere with target protein functions; however, the indirect effects of metabolites such as methanol require further elucidation through dynamic simulations and in vitro experiments.\u003c/p\u003e \u003cp\u003eThis study innovatively integrates network toxicology with molecular docking techniques to systematically elucidate how aspartame may influence the progression of ischemic stroke through a multi-target interaction network, thereby addressing a significant gap in the research on the neurotoxic mechanisms of food additives. Nonetheless, certain limitations must be acknowledged. First, target prediction is dependent on algorithmic databases, and the in vivo relevance of these targets requires validation in animal models. Second, the molecular docking analysis did not account for the effects of metabolites or the cumulative impact of long-term exposure. Finally, the absence of clinical cohort data limits the ability to support the predicted pathways with epidemiological correlations. Future research should focus on developing transgenic animal models for chronic aspartame exposure to dynamically monitor blood\u0026ndash;brain barrier permeability and inflammatory cytokine expression, as well as conducting large-scale human studies to analyze the relationship between aspartame intake and stroke incidence, and to explore the mediating effects of biomarkers such as CX3CL1. Such efforts will deepen our understanding of the neurotoxicity of artificial sweeteners and provide a scientific basis for the formulation of precise food safety policies.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis study systematically elucidated the potential molecular associations between the artificial sweetener aspartame and ischemic stroke by integrating network toxicology with molecular docking techniques. Based on cross-validation across multiple databases, five core targets\u0026mdash;IL1B, MMP9, SRC, AGT, and TNF\u0026mdash;were identified, with functional enrichment in the renin\u0026ndash;angiotensin system (RAS), complement and coagulation cascades, and inflammatory regulatory pathways. These findings suggest that aspartame may exacerbate ischemic brain injury by modulating vascular tone, coagulation balance, and neuroinflammation. Molecular docking analyses further confirmed a high-affinity binding between aspartame and the aforementioned targets, indicating its potential to directly interfere with target protein conformation or signal transduction. Notably, this study is the first to propose that aspartame may activate the RAS by upregulating AGT expression, thereby inducing oxidative stress and endothelial dysfunction. This mechanism aligns with epidemiological evidence linking artificial sweetener consumption to hypertension, offering a novel perspective on its cerebrovascular toxicity. The results provide a theoretical basis for assessing the neurotoxicity of aspartame and serve as a methodological reference for the re-evaluation of food additive safety. Future research should validate target functions using animal models, delineate the synergistic effects of metabolites, and combine epidemiological cohort studies to clarify the dose\u0026ndash;response relationship, thereby advancing the refinement of food safety guidelines and the optimization of stroke prevention strategies.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eConflict of interest disclosure:\u0026nbsp;\u003c/strong\u003eThe authors report there are no competing interests to declare.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNo funding was received for conducting this study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data used in this study were obtained from publicly available databases, Since these databases consist of anonymized data that is freely accessible to the public, no ethical approval or informed consent was required for this study. The data were used solely for research purposes and were in compliance with the relevant ethical guidelines for the use of publicly available data.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCRediT authorship contribution statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTao Wang:\u003c/strong\u003e Methodology, Writing \u0026ndash; original draft, Writing \u0026ndash; review \u0026amp; editing.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTianyu Zhang:\u0026nbsp;\u003c/strong\u003eData curation, Methodology, Software, Writing original draft, Writing \u0026ndash; review \u0026amp; editing.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eKunyang Bao:\u003c/strong\u003e Conceptualization, Funding acquisition, Methodology, Writing \u0026ndash; review \u0026amp; editing.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eKuangyang Yu:\u0026nbsp;\u003c/strong\u003eConceptualization, Methodology, Writing \u0026ndash; original draft, Writing \u0026ndash; review \u0026amp; editing.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eChangren Huang:\u0026nbsp;\u003c/strong\u003eFormal analysis, Writing original draft. Zhu Xinyao: Data curation, Writing \u0026ndash; original draft.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of Competing Interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe would like to express our sincere gratitude to all the staff members of the databases utilized in this study for their valuable contributions.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability\u0026nbsp;\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe data supporting the findings of this study are available from the following public databases: \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003ePubChem (https://pubchem.ncbi.nlm.nih.gov/) \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eSTITCH (http://stitch.embl.de/) \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eSwissTargetPrediction (http://www.swisstargetprediction.ch/) \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eSEA (https://sea.bkslab.org/) \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eGeneCards (https://www.genecards.org/) \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eOMIM (https://www.omim.org/) \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTherapeutic Target Database (TTD, https://db.idrblab.net/ttd/) \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eSTRING (https://cn.string-db.org/) \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eRCSB Protein Data Bank (https://www.rcsb.org/) \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eCB-Dock2 (https://cadd.labshare.cn/cb-dock2/php/blinddock.php#job_list_load) \u0026nbsp;\u003cbr\u003e\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eSoffritti, M. \u003cem\u003eet al.\u003c/em\u003e The carcinogenic effects of aspartame: The urgent need for regulatory re-evaluation. \u003cem\u003eAm. 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Ethnopharmacol.\u003c/em\u003e \u003cstrong\u003e317\u003c/strong\u003e, 116771 (2023).\u003c/li\u003e\n\u003cli\u003eYan, J. \u003cem\u003eet al.\u003c/em\u003e Met-RANTES preserves the blood-brain barrier through inhibiting CCR1/SRC/Rac1 pathway after intracerebral hemorrhage in mice. \u003cem\u003eFluids Barriers CNS\u003c/em\u003e \u003cstrong\u003e19\u003c/strong\u003e, 7 (2022).\u003c/li\u003e\n\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":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Aspartame, Ischemic Stroke, Network Toxicology, Molecular Docking","lastPublishedDoi":"10.21203/rs.3.rs-6183078/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6183078/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eAspartame, a widely used artificial sweetener, remains controversial due to neurotoxic risks from its metabolites\u0026mdash;phenylalanine, aspartic acid, and methanol. While epidemiological studies link artificial sweeteners to cerebrovascular disease, the molecular mechanisms connecting aspartame to ischemic stroke are unclear. This study integrates network toxicology and molecular docking to identify key targets and pathways. Potential aspartame targets were predicted using STITCH, SwissTargetPrediction, and SEA databases, while ischemic stroke-related genes were retrieved from GeneCards, OMIM, and TTD. Venn analysis identified 201 overlapping genes, with IL1B, MMP9, SRC, AGT, and TNF as core targets. GO/KEGG enrichment revealed their roles in the renin-angiotensin system, complement/coagulation cascades, and inflammatory pathways. Molecular docking demonstrated strong binding affinities between aspartame and these targets, suggesting direct modulation. Our findings indicate that aspartame exacerbates ischemic brain injury by disrupting inflammatory responses, vascular homeostasis, and coagulation via multi-target interactions. This study provides the first systematic evidence of aspartame\u0026rsquo;s neurotoxicity mechanisms, offering insights for food additive safety evaluation and stroke prevention. Further validation is required to clarify metabolite synergies and dose-response relationships.\u003c/p\u003e","manuscriptTitle":"Aspartame and Ischemic Stroke: Unraveling the Molecular Link through Network Toxicology and Molecular Docking Analysis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-03-27 11:26:34","doi":"10.21203/rs.3.rs-6183078/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-04-01T08:39:50+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-03-30T06:22:21+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-03-27T20:14:21+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-03-22T08:34:44+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"187411380207568015105984653254457929745","date":"2025-03-20T03:43:38+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"180705266603438817843110809651119532463","date":"2025-03-18T22:39:25+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"329486365313879349152197186081570390898","date":"2025-03-18T14:27:38+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-03-18T14:23:29+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-03-18T14:19:04+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-03-18T03:00:25+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-03-14T08:56:02+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-03-08T08:52:02+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"57fa9c63-b231-4a85-9354-38ac8feee54a","owner":[],"postedDate":"March 27th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":46057489,"name":"Biological sciences/Computational biology and bioinformatics"},{"id":46057490,"name":"Health sciences/Health care"},{"id":46057491,"name":"Health sciences/Risk factors"},{"id":46057492,"name":"Health sciences/Neurology/Neurological disorders"}],"tags":[],"updatedAt":"2025-07-07T16:12:08+00:00","versionOfRecord":{"articleIdentity":"rs-6183078","link":"https://doi.org/10.1038/s41598-025-08898-z","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2025-07-04 15:58:15","publishedOnDateReadable":"July 4th, 2025"},"versionCreatedAt":"2025-03-27 11:26:34","video":"","vorDoi":"10.1038/s41598-025-08898-z","vorDoiUrl":"https://doi.org/10.1038/s41598-025-08898-z","workflowStages":[]},"version":"v1","identity":"rs-6183078","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6183078","identity":"rs-6183078","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}