Search Results (1,886)

Background/Objectives: The emergence of methicillin-resistant Staphylococcus aureus (MRSA) highlights the need for new antibacterial agents. Here, we report the identification and evaluation of a novel Gram-positive selective antibacterial: NDM-552. Methods: Antibacterial activity was determined by broth microdilution against a panel of Gram-positive and Gram-negative pathogens. Mechanistic studies included BOCILLIN-FL competition assays, microscale thermophoresis (MST), and antibiotic interaction with oxacillin and moenomycin. Additionally, time–kill analyses, frequency-of-resistance measurements, cytotoxicity assays, plasma stability studies, and a murine wound infection model were performed to characterize NDM-552. Results: NDM-552 exhibits activity against Gram-positive pathogens including S. aureus, Enterococcus faecium, and Staphylococcus epidermidis, with minimum inhibitory concentrations (MICs) of 1–2 µg/mL. NDM-552 displays no activity against Gram-negative bacteria. NDM-552 displays bacteriostatic activity against MRSA and additive interactions with oxacillin and moenomycin. The frequency of resistance in MRSA is low (2.39 × 10⁻⁹). BOCILLIN-FL competition assays and MST binding analysis identify the essential penicillin-binding protein 2 (PBP2) as the potential target of NDM-552. NDM-552 demonstrates acceptable cytotoxicity, favorable plasma stability, and reduces bacterial burden in a murine wound infection model. Conclusions: NDM-552 is a potent Gram-positive selective antibacterial agent that potentially targets PBP2-associated cell wall biosynthesis and exhibits in vivo efficacy against MRSA. Findings establish NDM-552 as a promising scaffold for the development of new antibacterial agents. Full article

Background: Streptococcus suis serotype 2 is a major zoonotic pathogen responsible for severe systemic infections in pigs and humans, including septicemia, meningitis, and high mortality outcomes. Cefquinome, a fourth-generation β-lactam antibiotic widely used in veterinary medicine, is commonly applied for the treatment of S. suis infections. However, optimized dosing strategies remain insufficiently defined, particularly under conditions of varying bacterial burden, inoculum size, and reduced susceptibility or resistance phenotypes. These factors may significantly alter pharmacodynamic responses and compromise the predictive value of conventional MIC-based approaches. Objectives: This study aimed to characterize the pharmacokinetics (PK) and pharmacodynamics (PD) of cefquinome against S. suis serotype 2 using an integrated ex vivo serum time-kill experiments and semi-mechanistic PK/PD modeling. A secondary objective was to evaluate optimized dosing regimens across different inoculum levels and susceptibility phenotypes, including a cefquinome-resistant mutant. Methods: Cefquinome pharmacokinetics following intramuscular administration at 2 and 4 mg/kg in piglets were described using a two-compartment model. Dose proportionality, exposure linearity, and clearance parameters were assessed. Ex vivo serum time-kill experiments were conducted using a parental strain and a cefquinome-resistant mutant (M1) under normal-inoculum (NI), high-inoculum (HI), and mutant/resistant (MS) conditions. A semi-mechanistic PK/PD model incorporating logistic bacterial growth, sigmoidal Emax killing, nutrient limitation, and a time-delay function was developed to describe dynamic bacterial responses. Model parameters (k₀, k_max, EC₅₀) were estimated using nonlinear least-squares regression (Scientist v2.0), and simulations were performed by integrating time-varying PK input functions. Results: Cefquinome demonstrated linear pharmacokinetics with dose-proportional increases in Cmax and AUC between 2 and 4 mg/kg, with comparable clearance across doses. Ex vivo studies revealed time-dependent antibacterial activity with a pronounced inoculum effect. Higher bacterial burdens significantly reduced bactericidal efficiency and promoted regrowth during declining drug exposure. No tested concentrations achieved ≥3-log₁₀ killing in HI or MS conditions, whereas the NI group achieved a maximal reduction of 3.5-log₁₀ CFU/mL. MIC values in serum and medium were consistent (0.03, 0.06, and 0.24 µg/mL for NI, HI, and MS, respectively), indicating minimal protein binding influence. The semi-mechanistic model accurately described observed bacterial dynamics (R² > 0.99; MSC > 1.5), capturing delayed drug effects, inoculum-dependent growth suppression, and regrowth phenomena. Growth rates were reduced under serum conditions, reflecting nutrient limitation. Importantly, inoculum size exerted a stronger impact on pharmacodynamic outcomes than resistance phenotype, as reflected by reductions in kmax and increases in EC₅₀ under HI conditions. Although %T>MIC exceeded conventional β-lactam targets (>40%) in most regimens, MIC-based indices poorly correlated with observed dynamic killing responses. Conclusions: Cefquinome exhibited time-dependent antibacterial activity against S. suis serotype 2, strongly modulated by inoculum size and reduced susceptibility. The developed semi-mechanistic PK/PD model provided robust prediction of bacterial time-kill behavior and outperformed MIC-based metrics in guiding dose optimization. Simulation results support 2 mg/kg every 24 h for normal infections and 2 mg/kg every 12 h for high-inoculum or less susceptible infections, emphasizing the value of model-informed dosing strategies for optimizing β-lactam therapy. Full article

Pseudomonas syringae pv. actinidiae (Psa) is the most destructive pathogen of kiwifruit, and the absence of curative measures makes the management of Psa-induced kiwifruit bacterial canker (KBC) particularly challenging. Elettaria cardamomum produces an essential oil (CAR) rich in bioactive compounds with demonstrated potential to act directly against Psa, but its mechanisms of action remain insufficiently explored. Here, we investigated CAR’s mode of action in plants with established mild KBC symptoms, and assessed its potential as a plant elicitor. In the in planta assay, CAR application (0.1% w/v, applied 7 days after inoculation) reduced KBC symptoms, with the strongest effect observed 14 days after treatment (DAT). However, CAR did not significantly affect oxidative stress biomarkers, antioxidant system, pigments and primary metabolism, or the expression of target genes related to systemic acquired resistance or salicylic acid and jasmonic acid pathways. For instance, Psa inoculation significantly upregulated PR1 (≈5.4-fold) and PR5 (≈4.3–5.5-fold), irrespective of CAR application. Complementary in vitro assays revealed a transient, phase-dependent antimicrobial activity of CAR: although the effect disappeared by 32 h in liquid-phase assay and no inhibition was observed under vapor-phase exposure, a strong reduction in Psa viable cells (79.7%) was observed after 8 h exposure in the liquid phase. This study demonstrates that CAR exerts a direct, albeit transient, antibacterial effect against Psa, conferring curative activity when applied to plants with mild KBC symptoms. Consequently, repeated applications may be required to maintain disease suppression, as CAR does not appear to induce a sustained preventive defense response in the host. Full article

Quinoline is a bicyclic aromatic heterocycle composed of a fused benzene and pyridine ring, and it has held an important place in medicinal chemistry for nearly two centuries. F. Runge first isolated it from coal tar in 1834, and it was later found to form the structural core of well-known natural products such as quinine, camptothecin, and γ-fagarine. Its asymmetric electron distribution, amphoteric character, and ability to undergo both electrophilic and nucleophilic substitution confer considerable pharmacophoric versatility on the quinoline scaffold. This review covers the chemistry and therapeutic relevance of quinoline derivatives, with an emphasis on their pharmacological significance rather than on their synthesis alone. It begins with a brief account of the scaffold’s structural features and main synthetic routes, then examines the biological activities reported for quinoline-based compounds, including antibacterial, anticancer, anti-inflammatory, antimalarial, antiparasitic, antitubercular, antioxidant, and antiviral activities, together with structure–activity relationships discussed at the level of specific ligand–target interactions where data allow. The last section covers more recent directions in quinoline-based drug discovery, including PROTAC degraders, kinase inhibitors, and multitarget-directed ligands, an area not extensively addressed in earlier reviews of this scaffold. The literature surveyed here shows that quinoline derivatives continue to serve as a versatile scaffold for generating new leads, linking established synthetic chemistry with current mechanistic and computational approaches. The continued diversification of quinoline-based chemotypes and their biological mechanisms reinforces the importance of this scaffold as a versatile platform for medicinal chemistry and drug discovery. Full article

Itaconic acid has been reported to possess anti-inflammatory and antibacterial properties. However, its specific mechanisms of action against pathogenic bacteria, especially in the context of purine metabolism, remain poorly understood. Here, we investigate the impact of itaconic acid on the purine metabolism of PCN033, a highly pathogenic porcine extraintestinal pathogenic Escherichia coli (ExPEC) strain. Our in vitro and in vivo experiments demonstrated that itaconic acid significantly inhibited the proliferation of PCN033. Multi-omics analyses, including transcriptome and metabolome sequencing, revealed that itaconic acid severely disrupted the purine metabolism pathway of PCN033. Further mechanistic studies identified PRPP amidotransferase (PurF), a key enzyme in de novo purine synthesis, as a direct target of itaconic acid. Molecular docking and click chemistry experiments provided compelling evidence that itaconic acid specifically binds to the second cysteine residue (2C) of PurF, leading to the inhibition of its enzymatic activity. These data reveal a novel and critical mechanism by which itaconic acid exerts its antibacterial effects in response to bacterial infection. Importantly, our in vivo data show that supplementation with itaconic acid alleviated weight loss, organ damage, and inflammatory responses induced by PCN033 infection in mice and nursery pigs. These novel findings enhance our understanding of the antibacterial mechanisms of itaconic acid. Supplementation with itaconic acid could serve as a therapeutic strategy for treating pathogenic bacterial infections in humans and other animals. Full article

Background/Objectives: Renal ischemia–reperfusion injury (IRI) and infection-associated renal damage are characterized by persistent inflammation, oxidative stress, microvascular dysfunction, and impaired tissue regeneration, creating a hostile microenvironment that limits effective repair. We developed an injectable, photocrosslinkable methacrylated chitosan (CSMA) hydrogel for the localized co-delivery of cerium oxide nanoparticles (CeO₂NPs) and vascular endothelial growth factor (VEGF), aiming to integrate redox modulation, antibacterial activity, and regenerative support. Methods: Gelation, microstructure, rheology, degradation, and CeO2NP/VEGF were characterized. Tubular epithelial and fibroblast migration and endothelial network formation, angiogenic gene expression, and antibacterial activity against Staphylococcus aureus and Escherichia coli were evaluated in vitro. Theraputic performance was assessed by renal surface application in a rat renal IRI model and catheter-mediated interavsical administration in an ascending urinary tract infection model. Systematic biocompatibility was evaluated separately in a 14-day subcutaneous implantation study. Renal response were further investigated using transcriptomic and targeted molecular analyses. Results: The CSMA/VEGF/CeO₂NPs hydrogel exhibited rapid in situ gelation, interconnected porous architecture, stable viscoelasticity, gradual degradation, and sustained release of both CeO₂NPs and VEGF. The formulation enhanced tubular epithelial and fibroblast migration, promoted endothelial network formation and angiogenic gene expression and effectively inhibited both S. aureus and E. coli. In a surgically controlled rat renal IRI model, direct renal-surface application of the hydrogel reduced tubular injury, inflammatory infiltration, and fibrotic remodeling. In a separate ascending urinary tract infection model, catheter-based intravesical administration reduced the ascending renal bacterial burden and infection-associated inflammatory injury. No detectable adverse systemic effects observed under the tested conditions over the 14-day observation period in the subcutaneous implantation. Transcriptomic analyses further revealed that CSMA/VEGF/CeO₂NPs treatment was associated with marked remodeling of the renal injury microenvironment, characterized by suppression of antigen presentation and immune activation pathways, alongside restoration of metabolic programs associated with amino acid, lipid, and purine metabolism. These molecular changes were accompanied by downregulation of CIITA/CD74/MHC-II signaling, recovery of metabolic regulators AGXT and ACOX1, modulation of Hippo/YAP- and ECM-associated pathways, and preservation of renal structural markers including nephrin and WT1. Conclusions: The localized CSMA-mediated co-delivery of CSMA/VEGF/CeO₂NPs hydrogel promotes renal repair through resolution of maladaptive immune activation, metabolic reprogramming, angiogenic enhancement, and preservation of renal structural integrity, providing a promising biomaterial strategy for the treatment of ischemic and infection-associated renal injuries. Full article

Implant-associated infections (IAIs) persist as a critical complication in biomaterial transplantation, driven by rapid microbial colonization, biofilm encapsulation, and escalating antibiotic resistance. Conventional antibiotic-based treatments and passive antimicrobial coatings often fail to achieve long-term infection control due to limited biofilm penetration, localized cytotoxicity, and active agent depletion. To overcome these limitations, piezoelectric nanocomposite coatings utilize a dynamic, stimulus-responsive framework that converts physiological mechanical forces or external ultrasound into localized electrical signals. These surface-bound electric fields systematically mitigate bacterial adhesion, eradicate mature biofilms via targeted reactive oxygen species (ROS) generation, disrupt microbial metabolic pathways, and favorably modulate the peri-implant immune microenvironment while supporting host tissue repair. This review evaluates the material design principles and classifications of inorganic, organic, and hybrid piezoelectric nanocoatings. We detail their multifaceted antibacterial mechanisms and trace their therapeutic potential in orthopedic and dental implants, as well as wound management. Lastly, we analyze current engineering bottlenecks to chart a clear trajectory for their clinical translation. Full article

Most translational work on bacteriophages has focused on antibacterial therapy or food biocontrol. A third use case is scientifically plausible but remains insufficiently defined: the intentional use of characterized phages to modulate gut microbial communities without infection-treatment claims. In this review, the term “phagobiotics” is used for defined, purified and process-controlled bacteriophages or phage cocktails intended for selective gut microbiota modulation. The concept is evaluated across natural human phage exposure, the gut phageome, mechanisms of phage-mediated community modulation, human intervention studies, preclinical models, manufacturing quality and regulatory boundaries. Current evidence supports biological plausibility and indicates that oral phage exposure can be well tolerated and, in some contexts, can selectively affect target bacterial groups without broad microbiota disruption. Generalized clinical efficacy and broad microbiome-support claims, however, remain insufficiently established. Microbiological-modulation claims require target-linked evidence, whereas claims to treat, prevent or cure disease or replace antibiotics fall outside the proposed non-therapeutic category. A proportionate framework is proposed in which natural exposure and food-use precedents inform, but do not determine, the safety rationale; product-specific controls focus on identity, purity, production-host control, manufacturing consistency, stability, genomic characterization and claim-linked evidence. Regulatory classification remains case-specific and depends on intended use, product format, target population and claims. Full article

Staphylococcus aureus (S. aureus) is a prominent foodborne pathogen that poses a continuous threat to global public health and food safety due to its possession of a variety of toxins and its multidrug resistance. Medium-chain fatty acids (MCFAs), notably lauric acid (LA), exhibit strong antimicrobial properties, but their application is heavily constrained by poor water solubility. In this study, optimized LA emulsions stabilized by chitosan (CS) and polyvinyl alcohol (PVA) were evaluated for their antibacterial activity and detailed mode of action against S. aureus ATCC 6538. The antibacterial activities were evaluated by the maximum inhibition zone, with the 20 CS-PVA/DLTA-LA formulation exhibiting stable dispersion and potent antibacterial activity at 1%. The underlying antibacterial mechanisms against S. aureus were specifically focused on cell membranes and peptidoglycan. Therein, the binding of emulsion droplets to the anionic bacterial surface was driven by electrostatic attraction. Membrane degradation was also observed with membrane dysfunctions involving membrane depolarization, increased permeability, and fluidity reduction triggered by their subsequent insertion into the lipid bilayer, which may cause cell dysmetabolism, disintegration, and eventual cell death. Overall, these findings substantiate that LA emulsions disrupt S. aureus by operating potentially multi-targeted effects involving cell membrane disruption and peptidoglycan interference, offering a promising alternative approach warranting further investigation for foodborne pathogen control. Full article

Fungal infections remain a major threat to human health, with non-albicans Candida (NAC) species causing more than half of all clinical cases and many strains gaining resistance to current treatments rapidly. Previously, N-arachidonoyl ethanolamine (anandamide, AEA) has been studied and shown to possess antibacterial and antifungal properties against various bacteria and Candida albicans. Given the previous findings, we aim here to expand the current preliminary research on AEA to investigate its antifungal activities against clinically relevant NAC species in vitro: Candida glabrata, Candida parapsilosis, and Candidaozyma auris. The minimum inhibitory concentration (MIC) and growth curve analysis determined planktonic inhibition. MTT metabolic assay and ATP production via BacTiter-Glo luminescence assay evaluated biofilm formation. Membrane fluidity, polarization and efflux pump activity were examined using fluorescence probes Laurdan, DiS-C3(3), and Rhodamine 6G, respectively. Reactive oxygen species (ROS) were assessed using DCFH-DA. Biofilm architecture and cell viability were analyzed by spinning disk confocal microscopy (SDCM). AEA reduced MIC values and slowed planktonic growth, while MTT and ATP assays demonstrated a pronounced dose-dependent reduction in biofilm metabolic activity. Membrane-targeted effects revealed increased fluidity and permeability at 125 µg/mL. Notably, AEA rapidly impaired efflux pump activity and induced intracellular ROS production. This effect was accompanied by reduced cell viability, increased proportions of PI-positive cells, and enhanced intracellular dye retention, as confirmed by SDCM. Together, these findings demonstrate that AEA exerts antifungal activity by disrupting membrane integrity and associated cellular functions and provide the first comparative characterization of species-specific membrane and oxidative stress responses to AEA across three major clinically relevant multidrug-resistant NAC species. Full article

Environmental concerns regarding the ecotoxicological effects of antifouling biocides have led to the development of products targeting biofilm-forming bacteria. However, the potential for sublethal biocide exposure to promote bacterial adaptation and increase the risk of resistance development poses a potential threat to marine ecosystems and human health, yet it remains insufficiently understood. Although this study focuses on conventional antifouling biocides, the findings are intended to inform the future development and evaluation of both conventional and environmentally friendly antifouling technologies by highlighting the importance of assessing resistance induction at sublethal concentrations. In this study, the effects of three representative antifouling biocides on marine bacterial growth and bacterial adhesion were investigated. Sea-Nine 211 (DCOIT), copper sulphate (CuSO₄), and tributyltin oxide (TBTO; included as a historical reference compound due to its environmental persistence) were tested at four concentrations (0.01, 0.1, 1.0, and 10 µg/mL) against six marine biofilm-forming bacteria: Vibrio proteolyticus, V. aestuarianus, V. harveyi, V. natriegens, Shewanella putrefaciens and Pseudoalteromonas elyakovii. The results showed that Sea-Nine 211 exhibited a strong antibacterial effect at 10 µg/mL against all tested species except V. harveyi, whereas at the lowest concentration it promoted bacterial adhesion in V. proteolyticus. In contrast, TBTO and CuSO₄ showed limited antibacterial activity and increased microbial adhesion at the three lowest concentrations tested. These findings demonstrate that antifouling biocides can induce distinct responses depending on the concentration, ranging from growth inhibition to enhanced bacterial adhesion. Given that reducing biocide release has been proposed as a strategy to mitigate environmental impacts, our results highlight two potential challenges: (i) reduced antifouling efficacy at sublethal concentrations and (ii) an increased risk of bacterial adaptation associated with enhanced adhesion. To support future monitoring and resistance risk assessment, we propose a conceptual Resistance Risk Index (RRI) framework that could contribute to the sustainable management of antifouling agents while accounting for local environmental conditions. Full article

Eucalyptus bacterial wilt in China, caused predominantly by Ralstonia pseudosolanacearum, severely threatens Eucalyptus plantation management in China and demands plant-derived alternatives to conventional bactericides. This study evaluated the in vitro antibacterial activities and underlying molecular mechanisms of two differentially substituted coumarin derivatives, daphnetin (DAP) and 6-methylcoumarin (MC), against a eucalyptus-derived R. pseudosolanacearum strain. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were determined using broth microdilution assays. Quantitative phenotypic assays were conducted to evaluate the effects of sub-lethal concentrations on biofilm formation, swimming motility, and extracellular polysaccharide (EPS) accumulation. Furthermore, high-throughput RNA sequencing integrated with GO and KEGG enrichment analyses was employed to characterize the genome-wide transcriptomic responses. Antimicrobial susceptibility testing demonstrated that DAP possessed superior bactericidal efficacy, yielding lower MIC (62.5 μg/mL) and MBC (250 μg/mL) values than MC. Conversely, sub-lethal MC exhibited a more pronounced, early-stage suppression of flagellum-dependent swimming motility and biofilm maturation. Both compounds consistently attenuated EPS production. Transcriptomic analysis revealed distinct yet partially convergent regulatory networks: DAP primarily exerted metabolic strangulation by downregulating genes governing aerobic respiratory chains and peripheral carbon/nitrogen pathways, whereas MC targeted collective behavior and active pathogenesis, systematically downregulating the expression of genes associated with flagellar assembly, quorum sensing, and Type III and VI secretion systems. Notably, both pathways converged downstream to repress the master virulence regulator xpsR and the epsA-P operon. These findings provide valuable insights into the potential molecular pathways affected by DAP and MC, offering a useful reference for future exploration of botanical formulations for ecologically responsible forest disease control. Full article

Global food spoilage precipitates monumental economic deficits and persistent public health risks, exposing the acute vulnerabilities of conventional passive preservation modalities reliant on energy-intensive refrigeration and synthetic additives. Intelligent responsive nanostructures (IRNs) offer a transformative paradigm for active food preservation. This review elucidates how IRNs transcend conventional approaches through dual modulation of targeted antibacterial activity and microenvironmental dynamics. Freshness visualization technologies based on colorimetric and fluorescent responses to spoilage biomarkers are delineated. Subsequently, the hierarchical antibacterial mechanisms of IRNs are elaborated, encompassing physical membrane disruption, controlled ion release, reactive oxygen species (ROS) generation, and genetic interference. Crucially, we emphasize the synergistic strategies where lethal mechanisms are coupled with environmental regulation. The construction of active prevention systems is presented, alongside critical biosafety challenges toward sustainable nanoplatforms that autonomously maintain food quality from farm to fork. Full article

Background/Objectives: Antimicrobial peptides (AMPs) have emerged as promising alternatives to conventional antibiotics in response to the escalating global threat of antimicrobial resistance (AMR), owing to their potent antimicrobial activity and low propensity for resistance development. However, their clinical application remains limited by poor selectivity and undesirable toxicity toward mammalian cells. Methods: In this study, the naturally occurring frog-derived AMP Dermaseptin-A4 (A4) was selected as a template for rational design. Guided by the principle that optimising the balance between peptide hydrophobicity and cationicity could improve bacterial membrane targeting while reducing interactions with mammalian membranes, three analogues were designed through the targeted modulation of these physicochemical properties. Results: Among the designed analogues, A4-3 exhibited the best overall biological profile. A4-3 maintained a stable α-helical conformation in membrane-mimicking environments and displayed potent antimicrobial activity against tested Gram-positive and Gram-negative bacteria while exhibiting lower haemolytic and cytotoxic effects than the parent peptide. As a result, A4-3 showed improved selectivity, achieving a selectivity index of up to 34.5. A4-3 rapidly eradicated bacterial cells through a membrane-targeting mechanism, leading to membrane disruption and the loss of cellular integrity, and exhibited a low propensity for resistance development following prolonged exposure. A4-3 also retained its antimicrobial activity under physiologically relevant conditions. Conclusions: Collectively, these findings demonstrate that achieving an optimal balance between peptide hydrophobicity and cationicity is an effective strategy for enhancing antimicrobial selectivity without compromising antibacterial activity, highlighting A4-3 as a promising lead candidate for the development of novel antimicrobial therapeutics against drug-resistant bacterial infections. Full article

Background/Objective: Cinnamaldehyde (CA) is a naturally occurring bioactive compound derived from the leaves, bark, roots, and flowers of the Chinese medicinal plant Cinnamomum cassia. It exhibits a broad spectrum of pharmacological properties, encompassing antioxidant, antibacterial, anti-diabetic, antifungal, and anticancer activities. Notably, it has shown potential therapeutic benefits in the management of type 2 diabetes mellitus (T2DM) and non-alcoholic fatty liver disease (NAFLD). Ochratoxin A (OTA), a common contaminant found in foods such as cereals, coffee, and raisins, is also present in traditional Chinese medicinal materials, including Astragalus and liquorice. T2DM and NAFLD share intertwined pathophysiological pathways, including insulin resistance, dyslipidaemia, chronic low-grade inflammation and oxidative stress, with insulin resistance serving as the common pathological hub for both conditions. Consequently, they frequently co-occur and exacerbate each other. OTA exerts dual-targeted toxicity to the pancreas and liver, which may synergistically drive the development of the comorbidity of T2DM and NAFLD. These two processes are mutually causal and together constitute the pathological basis of metabolic comorbidity. Methods: Network toxicology employs toxicological data, gene expression, and protein–protein interaction (PPI) networks to predict the targets of toxins, while network pharmacology, based on systems biology principles, reveals how drugs exert regulatory effects through multiple targets and pathways. In this study, we employed an integrated network toxicology and network pharmacology approach to jointly decipher the potential mechanisms by which CA intervenes in OTA-induced comorbid T2DM-NAFLD. First, a network toxicology approach was employed to preliminarily screen for core toxicological targets responsible for OTA’s pathogenicity. Subsequently, network pharmacology was used to identify potential targets of CA-mediated intervention in the disease. Finally, the common overlap among the CA intervention targets, OTA toxicity targets, and disease targets was defined as the final set of potential targets for CA-mediated intervention in OTA-induced T2DM-NAFLD comorbidity. A PPI network was constructed using the STRING database, and topological analysis was performed with Cytoscape. Core targets were selected using the median values of six parameters—betweenness centrality, closeness centrality, degree centrality, eigenvector centrality, LAC (local average connectivity) score, and network centrality—as cut-off thresholds, and the top 10 key genes were further identified using the cytoHubba plugin. Gene Ontology (GO) functional enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were conducted via the DAVID database, and the results were visualized on the CNSknowall platform. Lastly, molecular docking of the core targets was performed using the CB-DOCK2 platform to validate binding affinity. Results: Based on an integrated analysis of network toxicology, network pharmacology, and molecular docking, 10 key targets were systematically identified. These may serve as potential mediators of cinnamaldehyde in the treatment of OTA-induced T2DM-NAFLD comorbidity. Among these, six targets—albumin (ALB), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), interleukin-6 (IL-6), tumor necrosis factor (TNF), actin beta (ACTB), and estrogen receptor 1 (ESR1)—possess crystal structures amenable to molecular docking. KEGG enrichment analysis revealed that CA and OTA jointly participate in key pathological processes such as the cancer pathway, the lipid and atherosclerosis pathway, the advanced glycation end-products–receptor for advanced glycation end-products (AGE-RAGE) signaling pathway, the phosphatidylinositol 3-kinase–protein kinase B (PI3K-Akt) signaling pathway, the TNF signaling pathway, and the interleukin-17 (IL-17) signaling pathway. OTA exacerbates inflammatory responses, impairs insulin signaling, promotes hepatic steatosis, and disrupts systemic metabolic homeostasis, ultimately contributing to T2DM-NAFLD comorbidity. Conversely, cinnamaldehyde counteracts these pathological processes through multiple mechanisms, including antioxidant and anti-inflammatory effects as well as regulation of glucose and lipid metabolism, thereby restoring metabolic homeostasis. Conclusions: This study has preliminarily identified the toxicological targets of OTA and the potential intervention targets of CA, offering new avenues for preventing and intervening in OTA-induced metabolic toxicity. Furthermore, it provides a theoretical basis for CA as a potential multi-target therapeutic agent and presents novel insights worthy of further investigation into the prevention of T2DM-NAFLD comorbidity. Full article