Search Results (1,137)

Breast cancer is a significant cause of death worldwide. Recent research has focused on identifying natural compounds for developing effective cancer treatments. Resiniferatoxin, a transient receptor potential vanilloid 1 (TRPV1) agonist, is a common diterpene in Euphorbia bicolor Engelm. & A. Gray (Euphorbiaceae), a plant native to the southern United States that has not been studied before. We investigated the antiproliferative activities and mechanisms of action of E. bicolor xylene extract in estrogen receptor-positive T47D and triple-negative MDA-MB-231 cell lines. The extract significantly reduced the viability of T47D and MDA-MB-231 cells in a dose-dependent manner. In MDA-MB-231 cells, the extract induced apoptosis via intracellular calcium overload, triggered by TRPV1 activation. This effect was diminished by the TRPV1 antagonist capsazepine and the calcium chelator BAPTA-AM. Intracellular calcium influx was confirmed through Fura-2 AM staining, revealing that E. bicolor phytochemicals activated TRPV1 in MDA-MB-231 cells. Treatment of T47D cells with E. bicolor xylene extract resulted in apoptosis associated with reactive oxygen species (ROS) generation (10-fold higher in T47D cells than in MDA-MB-231 cells) and mitochondrial calcium overload. These effects were significantly blocked when cells were pretreated with N-acetyl-l-cysteine (NAC), a ROS inhibitor. Both cell lines underwent apoptosis via multiple mitochondrial- and endoplasmic reticulum stress–mediated pathways. This was supported by the activation of caspases 3, 8, and 9; increased expression of FAS, XBP1s, and CHOP; upregulation of BAX; and downregulation of BCL-2. In addition, PI3K, AKT, and pAKT protein expressions were also reduced in both cell lines, indicating downregulation of PI3K/Akt signaling pathway. Phytochemicals in E. bicolor xylene extract could become promising ingredients for developing breast cancer therapeutics. Full article

Gut microbial metabolites play a crucial role in modulating cognitive function. In a previous animal study, oral administration of Lactiplantibacillus plantarum MWFLp-182 (L. plantarum MWFLp-182) significantly increased inosine levels in both the serum and feces of D-galactose (D-gal)-induced mice, which was accompanied by improved cognitive performance. Building on this finding, we further investigated the neuroprotective mechanisms of inosine derived from L. plantarum MWFLp-182 in alleviating D-gal-induced neuronal damage in HT-22 cells. Reverse transcription-quantitative PCR (RT-qPCR) was used to analyze the addition of inosine (250 μg/mL, 500 μg/mL), which considerably reduces oxidative stress induced by D-gal (20 mg/mL), on the regulation of mRNA expression of the nuclear factor erythroid 2-related factor (Nrf2)/hemeoxygenase 1 (HO-1) signaling pathway factors. Compared to the D-gal group, the inosine-treated group exhibited a 4.3-fold and 8.7-fold increase in HO-1 and Nrf2 levels, respectively. Furthermore, inosine alleviates neuroinflammation by modulating the mRNA expression of the Toll-like receptor 4 (TLR4)/myeloid differentiation primary response protein 88 (MyD88)/nuclear factor kappa B (NF-κB) signaling pathway. Compared to the D-gal group, the inosine-treated group showed reductions of 41.75%, 28.29%, and 32.17% in TLR4, MyD88, and NF-κB levels, respectively. Moreover, immunofluorescence staining revealed that inosine exhibits anti-apoptotic properties by enhancing the levels of neurotrophic factors, including nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF), while simultaneously lowering the expression of the pro-apoptotic protein bcl-2-associated X (Bax). These findings suggest that inosine, a differentially expressed metabolite identified in a probiotic-intervention mouse model, alleviates D-gal-induced neuronal damage in HT-22 cells by modulating oxidative, inflammatory, and apoptotic pathways, providing mechanistic insights into the neuroprotective effects of this metabolite. Full article

Contrast-induced acute kidney injury (CIAKI) has emerged as the third most prevalent etiology of clinically acquired acute kidney injury, with a lack of specific preventive and therapeutic strategies. Rubia Cordifolia L. (madder root), a medicinal herb with a long-standing history and extensive clinical application, exhibits multiple pharmacological activities. This study aimed to clarify the renal protective effect of Rubia cordifolia L. dichloromethane extract (RCDE) on CIAKI modeling rats and investigate potential anti-apoptotic and autophagy-inducing effects molecular mechanisms. In this study, RCDE constituents were identified by UPLC-Q-TOF-MS. A CIAKI rat model was established to evaluate the nephroprotective effect of RCDE. The results showed that RCDE high-dose group significantly decreased serum SCr and BUN levels, attenuated renal histopathological damage, and modulated oxidative stress markers by decreasing MDA and CAT while increasing SOD, compared with the model group. It downregulated the expressions of Bcl-2, caspase-3 and p62, upregulated the expressions of Bax, Beclin1 and reduced the LC3B-II/LC3B-I ratio in renal tissues. Molecular docking indicates that anthraquinone compounds are probably the principal active constituents of RCDE. This study provides experimental evidence for the intervention efficacy of RCDE against CIAKI. Full article

Sharp declines in temperature pose a significant risk for mass mortality events in the Chinese soft-shelled turtle (Pelodiscus sinensis). To assess the effects of acute cold stress on intestinal health, turtles were exposed to temperatures of 28 °C (control), 14 °C, and 7 °C for 1, 2, 4, 8, and 16 days. The results showed that acute cold stress at 14 °C and 7 °C induced time-dependent alterations in intestinal morphology and histopathology. The damage was more severe at 7 °C, characterized by inflammatory cell infiltration, lymphoid hyperplasia, and extensive detachment and necrosis across the villi, muscle layer, and submucosa. 16S rDNA sequencing revealed significant shifts in intestinal microbiota composition in the 7 °C group, dominated by Helicobacter and Citrobacter. Transcriptomic analysis identified differentially expressed genes (DEGs) that respond to acute cold stress and are involved in the Toll-like receptor signaling pathway (Tlr2, Tlr4, Tlr5, Tlr7, and Tlr8), the NOD-like receptor signaling pathway (Traf6, Traf2, Casr, Rnasel, Pstpip1, Plcb2, Atg5, and Mfn2), apoptosis (Tuba1c, Ctsz, Ctsb, Kras, Hras, Pik3ca, Bcl2l11, Gadd45a, Pmaip1, Ddit3, and Fos), and the p53 signaling pathway (Serpine1, Sesn2, Ccng2, Igf1, Mdm2, Gadd45a, Pmaip1, and Cdkn1a). Metabolomic profiling highlighted differentially expressed metabolites (DEMs) that cope with acute cold stress, such as organic acids (oxoglutaric acid, L-aspartic acid, fumaric acid, DL-malic acid, and citric acid) and amino acids (including L-lysine, L-homoserine, and allysine). The integrated analysis of DEGs and DEMs underscored three key pathways modulated by acute cold stress: linoleic acid metabolism, neuroactive ligand–receptor interaction, and the FoxO signaling pathway. This study provides a comprehensive evaluation of intestinal health in Chinese soft-shelled turtles under acute cold stress and elucidates the underlying mechanisms. Full article

Background: β-site amyloid precursor protein cleaving enzyme 1 (BACE1) inhibitors demonstrated amyloid-lowering efficacy but failed in phase II/III clinical trials due to adverse effects and limited disease-modifying outcomes. This study employed an integrated network pharmacology and molecular docking approach to quantitatively elucidate the multitarget mechanisms of 4 (phase II/III) discontinued BACE1 inhibitors (Verubecestat, Lanabecestat, Elenbecestat, and Umibecestat) and the preclinical compound AM-6494 in Alzheimer’s disease (AD). Methods: Drug-associated targets were intersected with AD-related genes to construct a protein–protein interaction (PPI) network, followed by topological analysis to identify hub proteins. Gene Ontology (GO) and KEGG pathway enrichment analyses were performed using statistically significant thresholds (p < 0.05, FDR-adjusted). Molecular docking was conducted using AutoDock Vina to quantify binding affinities and interaction modes between the selected compounds and the identified hub proteins. Results: Network analysis identified 10 hub proteins (CASP3, STAT3, BCL2, AKT1, MTOR, BCL2L1, HSP90AA1, HSP90AB1, TNF, and MDM2). GO enrichment highlighted key biological processes, including the negative regulation of autophagy, regulation of apoptotic signalling, protein folding, and inflammatory responses. KEGG pathway analysis revealed significant enrichment in the PI3K–AKT–MTOR signalling, apoptosis, and TNF signalling pathways. Molecular docking demonstrated strong multitarget binding, with binding affinities ranging from approximately −6.6 to −11.4 kcal/mol across the hub proteins. Umibecestat exhibited the strongest binding toward AKT1 (−11.4 kcal/mol), HSP90AB1 (−9.5 kcal/mol), STAT3 (−8.9 kcal/mol), HSP90AA1 (−8.5 kcal/mol), and MTOR (−8.3 kcal/mol), while Lanabecestat showed high affinity for AKT1 (−10.6 kcal/mol), HSP90AA1 (−9.9 kcal/mol), BCL2L1 (−9.2 kcal/mol), and CASP3 (−8.5 kcal/mol), respectively. These interactions were stabilized by conserved hydrogen bonding, hydrophobic contacts, and π–alkyl interactions within key regulatory domains of the target proteins, supporting their multitarget engagement beyond BACE1 inhibition. Conclusions: This study demonstrates that clinically failed BACE1 inhibitors engage multiple non-structural regulatory proteins that are central to AD pathogenesis, particularly those governing autophagy, apoptosis, proteostasis, and neuroinflammation. The identified ligand–hub protein complexes provide a mechanistic rationale for repurposing and optimization strategies targeting network-level dysregulation in Alzheimer’s disease, warranting further in silico refinement and experimental validation. Full article

Background/Objectives: Plants of the Crassulaceae family have been utilized in traditional medicine because of their medicinal properties. Crassula capitella, an ornamental succulent plant, has not yet received significant attention from physiochemists or pharmacologists. The objective of this study was to investigate the in vitro phytochemical properties and biological activity of methanolic extracts obtained from the leaves (CCLE) and inflorescences (CCIE) of C. capitella. Methods: Phytochemical screening included GC/MS analysis. The in vitro investigation of biological properties includes the assessment of antibacterial activity, utilizing disk diffusion assays and measuring MIC and MBC values for Gram-positive and Gram-negative bacteria. Antioxidant properties were determined through IC50 values in DPPH and ABTS assays. Cytotoxicity properties were evaluated using the MTT assay in MCF-7 and HepG2 cells, along with an analysis of apoptosis gene expression. Additionally, the antidiabetic effects were examined through α-amylase or α-glucosidase inhibition assays. Results: GC/MS analysis revealed distinct differences. CCLE contained more terpenoids such as betulinaldehyde (30.53%) followed by lupeol (19%) and betulin (4.07%), whereas CCIE was rich in fatty acids. The TPC and TFC of CCIE (88.17 mg GAE/g and 57 mg QE/g) were significantly greater than those of CCLE. Compared with CCLE, CCIE exhibited greater antibacterial properties (MIC values of 6.25 µg/mL toward S. aureus), greater antioxidant properties (IC₅₀ values in the DPPH/ABTS assay), antitumor properties (IC₅₀ values of approximately 90–96 µg/mL), and antidiabetic properties (IC₅₀ values of 87–83 µg/mL in the α-amylase/α-glucosidase assay). Both bioactive extracts induced apoptosis in cancer cells by downregulating the expression of the tumorigenesis genes bcl-2 and bcl-xL. Conclusions: The findings provided the first evidence about the evaluated the potential antibacterial, antioxidant, anticancer, and antidiabetic activities of C. capitella, which is attributed to its robust chemical composition and position it as a compelling candidate for further in vivo and sub-clinical applications. Full article

Zanthoxylum piperitum is a food and culinary plant commonly used in East Asia. In traditional medicine, its fruits, seeds, and bark have been utilized to treat digestive disorders, pain, and stomachache. Prior research has demonstrated its health benefits, particularly its significant antioxidant properties. However, limited research has investigated the specific metabolites responsible for these pharmacological effects. In this study, the antioxidant activities (EC₅₀: 9.1–1084.5 μg/mL) and metabolite profiles of different organs (fruits, pericarps, and seeds) of Z. piperitum collected from different regions were comparatively analyzed. Chemical structures of 91 metabolites from different organs were identified using UHPLC-Orbitrap-MS/MS based on untargeted metabolomics. The LC-DPPH method was employed to screen antioxidants from the extracts of the most active organ (the pericarps). The potential effects of the active compounds on oxidation-related diseases were evaluated by integrating compound–target interaction network analysis. Protein–protein interaction (PPI) networks revealed EGFR, STAT3, AKT1, TNF, BCL2, CASP3, ESR1, PPARA, CYP19A1, and CDK2 as central hub genes. The significance of compound and target interactions was further supported by molecular docking studies, which demonstrated favorable binding affinities, with most proteins exhibiting docked scores below −4.27 kcal/mol. The extracts of Z. piperitum fruits and pericarps also exhibited antioxidative activity against ROS production in LPS-stimulated RAW264.7 cells. Our findings demonstrate the application of an optimized extraction process and underscore the medicinal value of this food-plant by characterizing its bioactive constituents. The results indicate that Z. piperitum may serve not only as a health-promoting food but also has the potential for prevention or treatment of oxidative-stress-related diseases. Future research should focus on in vivo studies by exploring the therapeutic mechanisms of actions of the active extracts. Full article

Cellular senescence is a stress-induced process that contributes to adipose tissue dysfunction by promoting inflammation, impaired adipogenesis, and insulin resistance, alterations that are closely associated with age-related cellular dysfunction and metabolic disorders. In this study, we evaluated the protective role of chlorogenic acid (CGA), a polyphenol with known antioxidant and anti-inflammatory properties, against oxidative stress-induced senescence in murine 3T3-L1 adipocytes. The results obtained showed that CGA treatment significantly alleviated the senescent phenotype by restoring Lamin B1 levels and the Bcl-2/Bax ratio. Additionally, CGA downregulated key senescence-related cell cycle progression markers, modulating p53, p21, and MAPK signaling. CGA also restored insulin signaling through the PI3K-AKT-GLUT4 axis and improved glucose uptake, while attenuating oxidative stress, inflammatory cytokine expression, and extracellular matrix remodeling factors associated with SASP. Collectively, these findings support the role of CGA as a promising senotherapeutic nutraceutical able to reduce adipocyte senescence and its metabolic consequences, offering novel insights for the development of dietary supplements targeting age-related cellular dysfunction. Full article

Background: Tumor-associated neutrophils (TANs) exhibit remarkable functional plasticity within tumor microenvironment (TME), with N1-like subtypes promoting anti-tumor immunity and N2-like subtypes facilitating tumor progression. Despite their critical role in cancer immunology, strategies to selectively modulate TAN polarization remain limited. Methods: We integrated transcriptomic analyses of TAN subtypes to identify potential hub molecules. Molecular docking and experimental assays were used to evaluate DHA’s effect on neutrophil-like cell polarization. Results: Hub genes (TNF, IL1B, PTGS2, BCL2A1, MSR1, ACOD1, CXCL16, CLEC10A, and SOCS3) were identified, with TNF serving as a potential core regulator. Molecular docking indicated that DHA forms stable interactions hub proteins. Experimentally, DHA treatment of neutrophil-like dNB4 cells promoted N1 polarization, evidenced by upregulation of TNF, IL1B, PTGS2, BCL2A1, MSR1, ACOD1, CXCL16, and N1 markers PD-L1 and NOX2, and downregulation of N2 marker CEACAM8 and hub genes CLEC10A and SOCS3. Functional assays demonstrated that DHA-treated cells exhibited increased secretion of TNF, IL1β, ROS, and PD-L1, accompanied by enhanced cytotoxic activity against hepatocellular carcinoma cells in a co-culture system. Conclusions: These findings reveal the molecular mechanisms underlying TAN polarization, and establish DHA as a potent immunomodulatory agent capable of reshaping TANs toward an anti-tumor phenotype. Full article

Cancer therapies frequently induce hepatotoxicity, complicating treatment courses and outcomes. Natural products, including polyphenol-rich extracts, have shown hepatoprotective activity via anti-oxidative and anti-inflammatory mechanisms, often linked to NF-κB and PI3K–Akt pathways. Apple-derived polyphenols (e.g., phlorizin/phloretin) also demonstrate liver-protective effects in experimental settings. In this study, we examined whether ASE mitigates cancer-related liver damage by rebalancing the apoptosis–survival axis and maintaining PI3K-Akt signaling in an endometrial cancer mouse model. Female Institute of Cancer Research mice with induced endometrial cancer received ASE (0–200 mg) over 13 days; liver tissues were analyzed for Caspase-3, p53, LC3, and SQSTM1 using histology stains, Western blot (e.g., Caspase-3/9, Bcl-xL, PI3K, Akt, PCNA, IGF-IR), ELISA, and qRT-PCR (GAPDH). ImageJ (version 1.54f; RRID: SCR_003070) quantification statistical analysis followed (mean ± SD; post-hoc tests). ASE exhibited dose-dependent modulation of apoptosis and survival readouts in liver tissue of cancer-bearing mice: (i) Caspase-9/3 and Bcl-xL showed differential regulation across doses; (ii) PI3K–Akt and IL-2 signals were preserved or restored toward baseline at specific doses; and (iii) histology indicated partial structural recovery. Thus, ASE may mitigate liver injury by re-balancing apoptosis–survival signaling and promoting structural recovery. Our interpretation emphasizes that dose, route, and formulation are critical for translational potential. Full article

To investigate the regulatory effects of miR-16-5p and miR-142-3p on inflammation and autophagy in human corneal epithelial cells (HCEpiCs) exposed to hyperosmotic stress, a key pathogenic condition in dry eye disease, HCEpiCs were cultured under NaCl-induced hyperosmotic conditions (450 mOsm, 24 h) and transfected with miR-16-5p or miR-142-3p mimics. Expression of inflammatory cytokines (IL-1β, IL-6, TNF-α, IRAK1), autophagy-related genes (ATG5, Beclin-1, ATG16L1, p62), and apoptotic markers (Bax, Bcl-2, caspase-3) was analyzed by qRT-PCR and Western blot. Reactive oxygen species (ROS), autophagic vesicles, and apoptosis were evaluated using DCFH-DA, DAPRed, and Annexin V assays. The expression levels of antioxidant proteins (SOD1, catalase, NRF2) were also measured. Hyperosmotic stress induces marked inflammatory activation and excessive autophagy in HCEpiCs, accompanied by increased ROS generation and apoptosis. Overexpression of miR-16-5p or miR-142-3p significantly attenuated these effects by suppressing NF-κB-mediated cytokine expression and downregulating ATG5 and ATG16L1 expression, while restoring p62 expression. Both miRNAs reduced oxidative stress and COX-2 expression, enhanced antioxidant defenses, and normalized the expression of apoptotic markers. miR-16-5p and miR-142-3p are important regulators of inflammation and autophagy under hyperosmotic stress. Our findings suggest that modulating intracellular miR-16-5p and miR-142-3p levels in corneal epithelial cells may represent a potential approach to protect the ocular surface under hyperosmotic stress, although their systemic roles in autoimmune dry eye require further clarification. Full article

Oocyte-granulosa cell complexes (OGCs) cultivation is crucial for advancing reproductive biotechnology but remains incomplete and needs further optimization. Insulin-like growth factor-I (IGF-I) regulates granulosa cell proliferation and apoptosis, and numerous studies have confirmed its role in promoting ovarian follicle development. Porcine follicular fluid (PFF) contains factors beneficial for oocyte growth, which may enhance oocyte development. To investigate whether IGF-I and PFF improve the in vitro culture efficiency of porcine OGCs, we cultured OGCs with IGF-I (0, 10, 50, 100 ng/mL) and PFF (from 3 to 6 mm follicles) at concentrations of 0, 2.5%, 5%, 10%, respectively. The results revealed that 50 and 100 ng/mL IGF-I significantly increased the antrum formation rate of OGCs (from 61.11 ± 7.35% to 88.89 ± 7.35%) and diameter growth of oocytes (from 108.77 ± 0.27 µm to 114.94 ± 0.58 and 113.29 ± 0.50 µm, respectively). However, only the 50 ng/mL group, but not the 100 ng/mL group, significantly improved the maturation rate (38.13 ± 3.77% vs. 25.00 ± 3.27%, p < 0.05) of oocytes. Additionally, 50 ng/mL IGF-I downregulated BAX (a pro-apoptotic gene) and upregulated BCL-2 (an anti-apoptotic factor) in granulosa cells, ultimately reducing apoptosis. In contrast, none of the PFF doses used in this study induced the formation of enclosed antrum-like structures in OGCs, nor did they significantly enhance their in vitro development. Our findings demonstrate that 50 ng/mL IGF-I effectively promotes the in vitro growth of porcine early antral follicle-derived OGCs by reducing apoptosis, whereas tested PFF concentrations had no beneficial effects and induced abnormal granulosa cell growth. How PFF modulates the adherent and spreading growth of granulosa cells has not been fully elucidated and requires further clarification. Full article

Background/Objectives: Uveal melanoma (UVM), the leading primary intraocular cancer in adults, is driven by GNAQ/GNA11 mutations, encoding the active forms of Gαq proteins. While local treatments like surgery or radiation can control primary tumors, nearly half of patients die from metastasis. Our aim was identifying potential pathways involved in resistance to targeted therapy in UVM. Methods: Here, we screened 100 pathway-activating mutant complementary DNAs by lentiviral overexpression to identify those that enhance the survival of cancer cells in the presence of clinically relevant targeted therapies, using BAP1 wild-type UVM cells and validated the most significant results in BAP1-mutant cells. Results: This revealed JAK/STAT activation, overexpression of anti-apoptotic BCL2/BCL-XL, and dysregulated PI3K/mTOR or Hippo pathways as escape routes under MEK-ERK or FAK inhibition. Bioinformatic analysis of UVM transcriptome in TCGA further showed that high expression of the hallmark PI3K/AKT/mTOR pathway and IL6/JAK/STAT signaling correlates with poor prognosis. A similar correlation was shown by YAP and anti-apoptotic signatures. The analysis of individual representative genes from these signatures revealed that MTOR, BCL2L1 (BCL-XL), and TEAD4 gene expression are linked to poorer survival, underscoring the potential clinical impact of these adaptive pathways. Proliferation and apoptosis assay demonstrated that aberrant activation of AKT and YAP promotes resistance to FAK and MEK inhibitors. Conclusions: These findings support the adaptability of UVM lesions and suggest rational combination therapies targeting both primary GNAQ/GNA11-driven oncogenic signals and their compensatory networks as a more effective, personalized treatment approach for advanced UVM. Full article

Background/Objectives: Astrocytic redox-inflammatory signaling has been implicated in Parkinson’s disease (PD) pathology and may constrain hippocampal neurogenesis. We previously identified an astrocytic NOX4–MPO–OPN axis associated with impaired neurogenic capacity. Here, we tested whether a saffron-derived antioxidant (SDA; Crocus sativus extract) and Passiflora incarnata L. extract (PI) modulate this pathway in an MPTP-induced PD mouse model. Methods: Male C57BL/6J mice were randomized to Sham, MPTP, and treatment groups (n = 9/group for behavior; n = 4–5/group for histology/immunoblotting). SDA or PI (50 mg/kg/day, oral, 5 weeks) was administered, with resveratrol as a positive control. Behavioral, histological, and molecular analyses were performed by investigators blinded to group allocation where feasible. Results: SDA and PI were associated with reduced NOX4/MPO/OPN signals, mainly in GFAP-positive astrocytes, along with recovery of neurogenesis markers (Ki67, DCX, BrdU/NeuN) and synaptic markers (PSD95, synaptophysin), and improved motor performance. Mitochondrial and oxidative injury markers (TIM23, TOM20, OXPHOS subunits; 4-HNE) and apoptotic markers (Bax, cleaved caspase-3, Bcl-2) also shifted toward Sham levels. Given previous reports of Passiflora extracts’ sedative effects, we note that metabolic measures (body weight, food intake, and water intake) were similar across groups; however, specific tests for sedation or arousal were not conducted. Conclusions: These findings offer preclinical evidence that SDA and PI modulate redox-inflammatory and mitochondrial stress signatures and are associated with neurogenic, synaptic, and behavioral improvements in an acute MPTP model. Further validation in chronic/genetic PD models and pharmacokinetic/brain exposure studies will be necessary to confirm their translational potential. Full article

This study aimed to evaluate the response mechanisms of zebrafish larvae to Norfloxacin nicotinate (NOR-N) exposure. Embryos were exposed to NOR-N from 4 h post-fertilization (hpf) until 96 hpf. The exposure concentrations included 0.002, 0.2, 1, and 5 mg/L (simulating both normal and exceptionally high environmentally relevant levels of NOR), as well as a high dose of 25 mg/L. Subsequent analyses focused on apoptosis, neurodevelopment, and DNA methylation in the resulting zebrafish larvae. The results showed that high-dose NOR-N (≥5 mg/L) induced obvious apoptotic cell death in zebrafish larvae, accompanied by increased activities of Cas3 and Cas9, up-regulated P53, Bax, Puma, Apaf1, Cas3 and Cas9 genes expression, and reduced Mdm2 levels and Bcl2/Bax ratio. Moreover, exposure to 5 and/or 25 mg/L NOR-N resulted in a significant up-regulation of neurodevelopment-related genes (Sox2, Sox3 and Sox19a), concomitantly with a marked decline in the transcription of DNA methylation genes, including Dnmt1, Dnmt3a1, Dnmt3b1, Dnmt3b2 and Dnmt3b4. Overall, our findings demonstrated that NOR-N exposure could induce apoptosis, developmental neurotoxicity and aberrant DNA methylation in zebrafish larvae. These findings provide insights to guide the safe application of NOR-N in aquaculture and support the assessment of its potential ecological risks to aquatic ecosystems. Full article