Apurinic/apyrimidinic endonuclease 1/redox factor-1 (APE1/Ref-1) is a multifunctional stress-response regulator that coordinates genome maintenance, redox signaling, RNA biology, and cellular metabolism. Its expression and subcellular localization further determine disease states and severity. The growing appreciation of its biological complexity and clinical relevance makes APE1/Ref-1 an increasingly attractive therapeutic target for redox-stress-related diseases. In this review, we aim to consolidate information on the structural and mechanistic basis of APE1/Ref-1 redox and repair functions, while recognizing emerging evidence in DNA/RNA-forming G-quadruplex (rG4) biology, RNA metabolism, protein homeostasis, and mitochondrial function. We discuss mechanisms regulating APE1/Ref-1 expression, activity, and trafficking, which dynamically influence function in physiological and disease contexts. We specifically emphasize therapeutic strategies including redox-specific inhibition, endonuclease-targeted approaches, and genetic perturbations that result in distinct effects across disease models. Evolving understanding of APE1/Ref-1 biology has accelerated therapeutic development, particularly through redox-selective targeting strategies. Small-molecule inhibitors such as APX3330 and new-generation analogs like APX2009 and APX2014 have advanced into therapeutic applications spanning cancer, inflammatory disorders, and ocular diseases. Continued investigation into the context-dependent and multifunctional roles of APE1/Ref-1, together with the progression of mechanism-informed therapeutic design, is steadily strengthening the translational potential of APE1/Ref-1-directed therapies. APE1/Ref-1 is a protein central to how cells respond to stress. It assists in repairing damaged DNA and governs key signaling pathways that promote inflammation, survival, and adaptation to low-oxygen environments. These same pathways are often overactive in diseases such as cancer, eye conditions like diabetic retinopathy, inflammatory bowel disease, and other disorders linked to chronic inflammation. Because of this, APE1/Ref-1 has been studied for many years as a potential drug target. However, its high abundance in disease states and its multiple functions make targeting this protein challenging. Moreover, since this protein is vital for normal cell function, completely inhibiting it can damage healthy tissues and hinder the development of safe, effective treatments. Recent research has shifted how APE1/Ref-1 can be targeted: newer strategies aim to selectively interfere with specific functions that are more active in disease states, especially those involved in the cellular response to stress. Several drugs designed with this approach have shown promising results in laboratory studies and early clinical trials, particularly for eye diseases and cancer. In this review, we compile decades of research to explain how APE1/Ref-1 works, why it has been difficult to target, and the emerging strategies. We highlight both progress and ongoing challenges and discuss how more precise targeting of this protein could lead to safer, more effective therapies in the future.
Alzheimer's disease (AD) is the most common neurodegenerative disorder and a leading cause of dementia worldwide, characterized by progressive cognitive decline, memory impairment, and neuronal loss. The pathological hallmarks of AD include extracellular accumulation of amyloid-β (Aβ) plaques, intracellular neurofibrillary tangles composed of hyperphosphorylated tau protein, chronic neuroinflammation, oxidative stress, mitochondrial dysfunction, and synaptic degeneration. Current symptomatic therapies provide modest clinical benefits, while recently approved amyloid-targeting monoclonal antibodies, such as lecanemab and donanemab, can slow decline in selected early-stage AD patients but do not cure the disease and are associated with safety, access, and cost concerns. This narrative review summarizes mechanistic evidence from in vitro and in vivo studies and distinguishes preclinical promise from validated clinical utility. Phytochemicals, including polyphenols, flavonoids, alkaloids, terpenoids, and carotenoids, demonstrate neuroprotective effects through antioxidant activity, anti-inflammatory modulation, inhibition of amyloid aggregation, regulation of tau phosphorylation, and support of mitochondria and synapses. Evidence from experimental models suggests that several phytochemicals may help slow AD pathology and improve cognitive function, but clinical translation remains limited due to poor bioavailability, inadequate blood-brain barrier (BBB) penetration, and a lack of large-scale clinical trials. This review highlights critical research gaps and emerging strategies to facilitate phytochemical-based preventive and therapeutic approaches in AD.
The Developmental Origins of Health and Disease (DOHaD) theory proposes that adverse conditions during critical developmental windows can increase disease susceptibility throughout life. Maternal protein restriction (MPR) is a well-established experimental model that impairs offspring lung development, affecting pulmonary structure, metabolism, and molecular pathways from early life to aging. However, little is known about its effects on maternal pulmonary health and female offspring, representing an important gap in the literature. Therefore, this study investigated the effects of MPR on lung structure and metabolism in dams at the end of lactation and in female offspring at weaning. Sprague Dawley rats were fed either a control diet (17% protein) or a low-protein diet (6% protein) throughout gestation and lactation. Dams and female offspring were euthanized at weaning, and pulmonary alterations were assessed through histological, biochemical, molecular, zymographic, and in silico analyses. MPR increased collagen deposition, mast cell density, and Acta expression, while reducing reticular fibers in maternal lungs, accompanied by a trend toward increased lipid peroxidation. In female offspring, MPR reduced alveolar diameter, mast cell density, and reticular fibers, while increasing collagen deposition, MMP-2 activity, PI3K and MyoD expression, and decreasing mTOR expression as well as Nduf transcript levels. No significant alterations in antioxidant parameters were observed in either dams or offspring. Integrative analyses indicate that MPR promotes structural remodeling in maternal lungs, potentially compromising ventilatory function. In female offspring, MPR induces delayed pulmonary development, extracellular matrix remodeling, alterations in smooth muscle-related pathways, and disturbances in energy metabolism. Together, these findings demonstrate that maternal protein resQ1triction adversely affects pulmonary health in both dams and female offspring, revealing potential mechanisms linking early-life nutritional adversity to the developmental origins of respiratory disease.
Diabetic kidney disease (DKD) is a major microvascular complication of diabetes. The glucagon-like peptide-1 receptor agonist (GLP-1RA) liraglutide exerts renoprotective effects beyond glucose control; however, the underlying mechanisms remain incompletely understood. The protective effects and mechanisms of liraglutide were investigated using in vitro (HK-2 cells under glucolipotoxic conditions) and in vivo (DKD rat) models. Key molecular and functional assessments included the evaluation of oxidative stress, apoptosis, mitochondrial function (dynamics (MFN1, MFN2, FIS1, and DRP1), mitophagy (PINK1, PARKIN, LC3II/I, and P62), and ultrastructure), and histone acetylation (H3K9/14/18/27ac). The critical role of the NQO1/SIRT3 pathway was validated using pharmacological inhibition and genetic silencing (shRNA/AAV). Both in vitro and in vivo, a multifaceted injury phenotype, including oxidative stress, apoptosis, mitochondrial dysfunction, and histone hyperacetylation, was induced, accompanied by downregulation of the NQO1/SIRT3 pathway. Disruption of this pathway further exacerbated these injuries. Conversely, liraglutide treatment effectively counteracted this phenotype, improving metabolic parameters (blood glucose, blood insulin, and blood lipids), renal function (blood urea nitrogen, serum creatinine, and urine albumin-to-creatinine ratio) and histopathology. Liraglutide attenuated oxidative stress and apoptosis, restored mitochondrial function and mitophagy, and reduced histone hyperacetylation. These protective effects were consistently associated with the restoration of NQO1/SIRT3 expression. Genetic or pharmacological disruption of the NQO1/SIRT3 axis significantly attenuated the efficacy of liraglutide, and combined inhibition completely abolished its effects. Our findings demonstrate that liraglutide attenuates DKD by activating the NQO1/SIRT3 pathway, which coordinates the enhancement of mitochondrial function and the restoration of epigenetic homeostasis. This study revealed that the NQO1/SIRT3 pathway is a critical mechanistic mediator of the renoprotective effect of liraglutide.
Glutamate excitotoxicity is a key contributor to the pathogenesis of glaucoma, a leading cause of irreversible blindness worldwide; however, the molecular events driving progressive retinal ganglion cell (RGC) loss and axonal degeneration remain incompletely understood, and effective neuroprotective therapies are lacking. Here, we evaluated the preventive potential of K9-C-peptide, a biopolymer-conjugated human C-peptide, in a mouse model of N-methyl-D-aspartate (NMDA)-induced retinal neurodegeneration and optic nerve axonal transport impairment, and examined potential mechanisms underlying its protective effects. In NMDA-induced excitotoxic mouse retinas, intracellular Ca2+ elevation mediated NMDA-induced oxidative stress, including both intracellular and mitochondrial reactive oxygen species (ROS) generation and lipid peroxidation. NMDA exposure induced activation of Müller glia and microglia and upregulation of inflammatory cytokines, ultimately leading to RGC death; these effects were attenuated by prolonged intraocular delivery of ROS scavengers. K9-C-peptide significantly reduced NMDA-induced retinal degeneration, including RGC loss and retinal thinning, and preserved optic nerve axonal transport function in both whole-mount retinas and optic nerve longitudinal sections. These protective effects were associated with suppression of NMDA-induced oxidative stress, mitochondrial dysfunction, and inflammation and reactive gliosis, without altering intracellular Ca2+ levels. Notably, sustained intraocular delivery of human C-peptide conferred robust neuroprotection for at least 3 weeks against NMDA-induced retinal degeneration and optic nerve axonal transport impairment. These findings suggest that K9-C-peptide acts as a long-acting neuroprotective agent that mitigates oxidative stress-driven retinal damage and axonal dysfunction, highlighting its translational potential as a C-peptide-based neuroprotective strategy for retinal glutamate excitotoxicity.
Background and Objectives: Acute myeloid leukemia (AML) is characterized not only by its heterogeneity but also by its high relapse rate. This results in limited treatment options, especially in elderly or therapy-refractory patients. It is known that inhibiting anti-apoptotic BCL-2 family proteins can be effective; however, cellular resistance mechanisms often limit the efficacy of this treatment. We studied the effects of the BCL-2 inhibitor ABT-737, the MCL-1 inhibitor S63845, and their combination on AML cell lines and primary AML patient cells. Materials and Methods: To analyze the effects of ABT-737 and S63845 treatment on cells, cell energy phenotype, apoptosis, and cell cycle were assessed, and gene expression by RT-qPCR and protein levels by Western blot analysis were measured. Results: Treatment with the BCL-2 inhibitor ABT-737, the MCL-1 inhibitor S63845, and their combination reduced AML cell viability and induced apoptosis. Dual treatment also altered the expression of epigenetic regulators, as the levels of DNMT1, EZH2, SUZ12, and HDAC1 were reduced, while histone acetylation was increased. An increase in pro-apoptotic markers (PARP cleavage, caspase-9) was observed, and the expression of oncogenes (MYC, WT1) was reduced in model cell lines and primary AML patient cells. Conclusions: BCL-2 and MCL-1 inhibition, alone or in combination, induced apoptosis and altered the expression of epigenetic regulators and oncogenes in AML cell lines and primary patient cells, with no consistent advantage of combined treatment over single agents. BCL-2/MCL-1 inhibition remains a promising approach for AML, and further work should clarify which patients or disease subtypes are most likely to benefit from combined versus single-agent treatment.
This study investigated the effects of dietary lemongrass essential oil nanoemulsion (LEONE) on growth performance, feed efficiency, physiological responses, and overall health in heat-stressed New Zealand White rabbits. Eighty healthy male rabbits, aged five weeks, were randomly assigned to four groups (20 rabbits per group) and fed diets containing 0, 150, 300, or 600 mg LEONE/kg for eight consecutive weeks. Supplementation with LEONE at 300 mg/kg significantly improved average daily weight gain by 11.73%, dressing percentage by 11.59%, while concurrently reducing the feed conversion ratio by 10.81%, respiration rate by 1.82%, and rectal temperature by 6.25% compared to the control group. Regression analysis indicated that the optimal dietary dose for maximizing average daily gain and minimizing feed conversion ratio was approximately 450 mg/kg diet. Hematological profiles were enhanced, with increased red blood cell counts and decreased white blood cell counts following LEONE supplementation. Additionally, supplementation with 300 or 600 mg LEONE/kg diet increased total protein, globulin, thyroid hormones, and immunoglobulins A, G, and M. At the same time, levels of cholesterol, triglycerides, liver enzyme activities, urea, creatinine, and cortisol were significantly reduced. At the micro-mechanism level, LEONE enhanced antioxidant defenses by increasing total antioxidant capacity, elevating key antioxidant enzyme activities (SOD, CAT, GSH-Px), and reducing oxidative stress markers such as malondialdehyde, protein carbonyls, and 8-hydroxy-2'-deoxyguanosine. Moreover, LEONE modulated inflammatory responses by significantly lowering tumor necrosis factor-α, interferon-γ, serum amyloid A, and Toll-like receptor levels, indicating a protective effect on cellular and molecular pathways under heat stress. Importantly, no significant differences were observed between the groups receiving 300 or 600 mg LEONE/kg for any of the blood chemistry assays. Histological assessments demonstrated that LEONE provided protection against heat stress-induced liver and kidney damage. In conclusion, LEONE supplementation improved growth, immunity, antioxidant capacity, and inflammatory status under heat stress, with an optimal dose of ~ 450 mg/kg diet specifically for maximizing growth performance and enhancing feed conversion efficiency.
Early detection of diabetic atherosclerosis (DAS) remains challenging, and the mechanisms underlying endothelial barrier dysfunction in this condition are not fully understood. Extracellular Hsp90α (eHsp90α) and the ER stress marker GRP78 have been implicated in vascular injury; however, their roles in DAS remain unclear. Therefore, this study aimed to investigate the association of eHsp90α and GRP78 with DAS and explore the underlying mechanisms. We recruited patients with diabetes mellitus (DM) and patients with DAS. We then compared the levels and potential efficacies of serum eHsp90α and the ER stress marker GRP78 between the two groups. We subsequently conducted cytological experiments and experiments with ApoE-/- mice to further explore the underlying mechanisms involved. The serological analysis revealed that the levels of serum eHsp90α and the ER stress marker GRP78 were significantly higher in the DAS group than in the DM group and that the eHsp90α level was correlated with GRP78 level. The association and preliminary discriminative performance of the combination of eHsp90α and GRP78 levels were appeared higher than that of either marker alone. In addition, GRP78 plays a mediating role in the relationship between eHsp90α and DAS. Furthermore, the expression of GRP78 was higher in both diabetic ApoE-/- mice and DAS patients than in control ApoE-/- mice and AS patients. Cytological experiments revealed that eHsp90α induced endothelial barrier dysfunction mediated by ER stress via the LRP1 receptor. Our findings suggest that eHsp90α and GRP78 are associated with diabetic atherosclerosis and may be associated biomarkers with potential discriminative value, although further validation is required. The eHsp90α-LRP1-ER stress pathway may contribute to endothelial barrier dysfunction. However, further large-scale and longitudinal studies are required to validate these findings.
Immunohistochemistry is a tissue-based technique that enables in situ visualization of specific molecules, most commonly proteins or peptides, within preserved cellular and tissue architecture through antigen-antibody specificity. By using labeled antibodies, immunohistochemistry allows spatially resolved and cell type-specific detection of target molecules and maintains the morphological context, thereby providing both localization and relative expression information at the light or electron microscopy level. Owing to this integrative capacity, immunohistochemistry has become an essential tool in biological research and diagnostic pathology for characterizing molecular distribution and tissue organization across animal and plant systems. The detection techniques based on fluorescence and enzymes (chromogenic) are compared in terms of their repeatability, multiplexing capability, sensitivity, and specificity. With a focus on protocol standardization and methodological rigor, important procedural processes are covered, including fixation, antigen extraction, antibody optimization, signal detection, and quality control. Additionally, the immunohistochemistry technique remains essential for locating and identifying immunogenic substances in a variety of biological settings. Therefore, drawing on more than two decades of laboratory experience, this narrative review summarizes the history of the immunohistochemistry assay, its underlying principles and procedures, common immunohistochemistry protocol, methodological process, essential reagents, infrastructure, and best-practice principles required for the establishment and optimization of a high-quality immunohistochemistry laboratory.
Sarcopenia is characterized by progressive loss of skeletal muscle mass and function and is a major contributor to frailty, disability, and mortality in older adults. Store-operated calcium entry (SOCE) is a crucial regulator of skeletal muscle calcium homeostasis, and impaired SOCE has been linked to age-related muscle weakness. Here, we identify the synaptophysin family member synaptophysin-like protein 2, also known as mitsugumin 29 (MG29; encoded by the human gene SYPL2 and the mouse ortholog Mg29), as a key organizer of triad membrane cholesterol and lipid signaling required for normal SOCE during aging. Using Mg29-/- mice as a model of accelerated sarcopenia, together with RNA interference against Mg29 in adult muscle and primary myotubes, we quantified changes in muscle morphology, contractile function, SOCE activity, and targeted lipidomic profiles. Reduced MG29 expression led to decreased muscle fiber cross-sectional area, reduced specific force, blunted SOCE, and marked alterations in membrane cholesterol content and fatty acid-derived lipid mediators. Cholesterol depletion by methyl-β-cyclodextrin in wild-type myotubes produced SOCE defects similar to those observed in aged wild-type and young Mg29-/- muscles, indicating that MG29-dependent maintenance of membrane cholesterol is required for normal SOCE. Acute Mg29 knockdown also altered myogenic differentiation, the expression of calcium-handling and stress-response genes, and the release and consumption of specific polyunsaturated fatty acid-derived lipid mediators. Together, these findings identify MG29 as a critical regulator of SOCE and lipid signaling in skeletal muscle and suggest that its age-related decline contributes to sarcopenia by disrupting triad membrane organization and excitation-contraction coupling.
Trimethylamine N-oxide (TMAO), a gut microbiota-derived metabolite, has been linked to cardiovascular diseases. This study aimed to explore the role of TMAO in cardiac fibrosis by examining its effects on the NLRP3 inflammasome, endoplasmic reticulum stress (ERS), mitochondria-associated membranes (MAMs), and mitochondrial dynamics in cardiac fibroblasts (CFs), alongside clinical data from acute myocardial infarction (AMI) patients and unstable angina (UA) patients and AMI animal model data. Plasma TMAO levels were measured in AMI patients and healthy controls. In vitro, CFs were treated with TMAO to assess cellular activation and fibrosis markers. Western blot, immunofluorescence, and RNA sequencing identified key pathways and proteins related to ERS, NLRP3 inflammasome activation, and mitochondrial dynamics. In vivo, Masson's trichrome staining, Hematoxylin-Eosin (HE) staining and Immunohistochemical were used to evaluate the effects of TMAO on AMI mice. Plasma TMAO levels were significantly higher in the AMI group. TMAO promoted cardiac fibroblast activation and fibrosis by increasing α-SMA and Collagen I expression. It induced ERS, marked by elevated GRP78, p-PERK, and CHOP, and upregulated Sigma-1R, enhancing MAM formation. TMAO also altered mitochondrial dynamics via DRP1 phosphorylation and Mfn2 expression. RNA sequencing identified macrophage migration inhibitory factor (MIF) as a key mediator linking TMAO to NLRP3 inflammasome activation. TMAO exacerbates myocardial injury and fibrotic remodeling in AMI mice. TMAO exacerbates cardiac fibrosis via ERS and NLRP3 activation, with implications for mitochondrial dynamics and MAM formation. Elevated TMAO levels in AMI patients underscore its potential as a therapeutic target for ventricular remodeling fibrosis.
Given the limitations of the existing monoclonal antibody (mAb)-based therapies, more efficient and safer small-molecule-based checkpoint therapies targeting the programmed cell death-1 (PD-1) / programmed cell death ligand-1 (PD-L1) axis are gaining growing attention and urgently required. To identify a novel PD-L1 small-molecule inhibitor from natural products and systematically evaluate its antitumor activity, mechanism of action, and potential biomarkers. Based on the co-crystal structure of PD-L1 with BMS-202 (PDB ID: 5J89), molecular docking was employed for high-throughput virtual screening of 16,563 natural products. The binding affinity of candidate compounds to PD-L1 protein was validated through microscale thermophoresis (MST), cellular thermal shift assay (CETSA), drug affinity responsive target stability (DARTS) assays and nuclear magnetic resonance (NMR). The blockade of PD-1/PD-L1 interaction was assessed using homogeneous time-resolved fluorescence (HTRF), NFAT-Luc luciferase reporter gene system, and cell membrane PD-1 binding assays. In vivo antitumor efficacy was systematically assessed in humanized PD-L1 knock-in B16F10 and MC38 tumor-bearing mouse models. The mechanism of action was investigated using RNA-seq transcriptomics, flow cytometry, and immunofluorescence staining. Finally, response mechanisms and potential biomarkers were systematically analyzed by comparing differential responses across distinct tumor models. Through high-throughput virtual screening, we identified rosavin as a small molecule with a novel scaffold that targets PD-L1, exhibiting the unusual small-molecule property of inhibiting PD-L1 without inducing its dimerization. Rosavin demonstrated significant antitumor activity in vivo by promoting antitumor immunity through enhancing CD8+ T cell activation, consistent with the effects of PD-L1/PD-1 blockade. Notably, rosavin was particularly effective for fighting against tumor progression in microsatellite instability-high (MSI-H) solid tumors and robustly strengthened the expression levels of CXCL9 and CXCL10 in tumors, which may serve as potential biomarkers for predicting responsiveness to rosavin-mediated PD-1/PD-L1 blockade. Rosavin serves as a privileged novel and unexpected scaffold for designing potent PD-1/PD-L1 modulators, offering promising candidates for cancer immunotherapy.
Hexavalent chromium [Cr(VI)] is a highly toxic environmental pollutant that induces severe renal injury primarily through oxidative stress, inflammation, DNA damage, and dysregulation of cell death pathways. Thymoquinone (Tq) has been reported to exert antioxidant and anti-inflammatory effects; however, its potential protective role against Cr(VI)-induced nephrotoxicity remains unclear. This study aimed to investigate the protective effects of Tq against Cr(VI)-induced renal injury, focusing on oxidative stress, inflammation, DNA damage, apoptosis, and ferroptosis-related markers. Forty male Wistar albino rats were randomly assigned into five groups (n = 8): Control, vehicle, Cr(VI) (15 mg/kg), Tq (30 mg/kg), and Cr(VI) + Tq. All treatments were administered orally for 10 days. Biochemical analyses included malondialdehyde (MDA), glutathione (GSH), superoxide dismutase (SOD), catalase (CAT), tumor necrosis factor-alpha (TNF-α), nuclear factor kappa B (NF-κB), transforming growth factor-beta 1 (TGF-β1), and 8-hydroxy-2'-deoxyguanosine (8-OHdG) levels. Histopathological, histometric, and immunohistochemical evaluations (GPX4, ACSL4, Caspase-3) were also performed. Cr(VI) exposure significantly increased MDA, TNF-α, NF-κB, TGF-β1, and 8-OHdG levels, while decreasing GSH, SOD, and CAT activities (p < 0.01). These changes were associated with marked histopathological damage, renal dysfunction reflected by elevated serum BUN and creatinine levels, decreased GPX4 expression, and increased ACSL4 and Caspase-3 levels in renal tissue, suggesting the involvement of ferroptotic and apoptotic pathways. Tq treatment significantly ameliorated these alterations by restoring antioxidant defenses, suppressing inflammation and fibrosis, reducing DNA damage, and modulating markers of ferroptosis and apoptosis (p < 0.01). In conclusion, Tq attenuates Cr(VI)-induced nephrotoxicity, as evidenced by improvements in oxidative stress, inflammation, DNA damage, ferroptosis- and apoptosis-related markers, together with preservation of renal structure and function.
Therapeutic resistance to trastuzumab remains a critical limitation in the management of HER2-positive (HER2+) breast cancer. Emerging evidence implicates epigenetic regulators, including microRNAs and histone demethylases, in modulating HER2 signaling and drug responsiveness. This study aimed to investigate the mechanistic role of miR-770-5p and its interplay with KDM5B in the context of trastuzumab resistance. An integrative approach combining in silico analyses with in vitro functional assays was employed in trastuzumab-sensitive and -resistant HER2 + breast cancer cell lines. Molecular, subcellular, and phenotypic effects of miR-770-5p modulation were assessed using qRT-PCR, Western blotting, immunofluorescence, RNA-FISH, luciferase reporter assays, and live-cell imaging. KDM5B was significantly overexpressed in HER2 + tumors and inversely correlated with HER2 expression. miR-770-5p directly targeted KDM5B and was associated with coordinated regulation of HER2 signaling at both transcription-associated and post-transcriptional levels. Overexpression of miR-770-5p was associated with reduced expression of KDM5B, TCF12, and GRB7, supporting the involvement of a KDM5B-TCF12-GRB7 regulatory network in HER2 modulation. Functionally, miR-770-5p reversed EMT-associated phenotypic features and enhanced trastuzumab sensitivity. Clinically, reduced KDM5B expression was associated with improved distant metastasis-free survival in ER+/HER2-high breast cancer patients. Collectively, miR-770-5p functions as a nuclear-associated regulatory microRNA that modulates HER2 signaling through epigenetic regulation of KDM5B and associated downstream networks. These findings suggest a previously unrecognized regulatory mechanism contributing to trastuzumab response and provide potential therapeutic avenues for HER2-positive breast cancer.
Gastric precancerous lesions (GPLs) are a pivotal stage in the gastritis-gastric cancer sequence, and the absence of effective treatments presents a clinical challenge. Veratramine is a natural anti-inflammatory and analgesic steroid alkaloid; however, its effects on GPLs and the mechanisms have remained unexplored. This study investigated the effects of veratramine on GPLs using 1-Methyl-3-nitro-1-nitrosoguanidine (MNNG)-induced GES-1 cells and rat models, utilizing RNA-seq to elucidate the underlying mechanisms. In vitro, veratramine inhibited malignant cells (MC) proliferation, induced apoptosis, and triggered G0/G1 cell cycle arrest. It also suppressed epithelial-mesenchymal transition (EMT), migration, and invasion by upregulating E‑cadherin and downregulating Slug and vimentin. Transcriptomic and molecular docking analyses highlighted the Wnt/β-catenin pathway as a key target, regulated via DDX60, MUC1, APOL1, and MSH5. Veratramine reduced Wnt10B, β-catenin, and cyclin D1 expression and blocked β‑catenin nuclear translocation; these effects were reversed by the Wnt activator BML-284. In vivo, treatment with veratramine ameliorated the GPLs-induced pathological changes in rats. It restored body weight and preserved gastric mucosal integrity, as evidenced by intact glandular and cellular morphology, reduced hyperplasia, and attenuated intestinal metaplasia. These improvements were associated with a modulation of key molecular markers, specifically a decrease in the expression of N-cadherin, Wnt10B, and β-catenin, alongside an increase in E-cadherin expression in gastric tissues. These results collectively indicate that veratramine exerts its therapeutic effects against GPLs primarily by suppressing the Wnt/β-catenin signaling pathway. Taken together, our findings suggest that veratramine is a promising candidate small-molecule drug for the treatment of GPLs.
Triple-negative breast cancer (TNBC) has a poor prognosis due to the lack of targeted treatment. Previous studies have shown that the deubiquitinase UCHL1 is significantly upregulated in TNBC tissues and positively correlated with shorter overall survival in patients, suggesting that UCHL1 may drive TNBC progression. The latest research suggests that ferroptosis deficiency can promote tumor metastasis, but it is still unclear whether UCHL1 affects TNBC by regulating ferroptosis. Based on the potential role of UCHL1 in ferroptosis, we propose the hypothesis that UCHL1 enhances the survival and invasion ability of TNBC cells by inhibiting ferroptosis. We downloaded the single-cell RNA sequencing dataset for breast cancer (GSE176078) from the Gene Expression Omnibus database and integrated it with The Cancer Genome Atlas data to conduct bioinformatics analysis, focusing on identifying key genes related to TNBC. We performed gene set enrichment analysis (GSEA) to explore the pathways associated with UCHL1. After that, we validated the expression of UCHL1 in TNBC tissues and cell lines and examined its influence on cell proliferation, migration, and invasion through functional experiments. Finally, we explored the downstream targets of UCHL1, utilizing co-immunoprecipitation, western blot, immunofluorescence colocalization, and establishing TNBC xenograft models in nude mice to elucidate its mechanisms in TNBC progression. Public database analysis revealed high levels of UCHL1 in TNBC. Additional research confirmed its overexpression in TNBC tissues and cells, along with significantly increased TNBC cell proliferation, migration, and invasion associated with high UCHL1 expression. GSEA further identified UCHL1 as being predominantly enriched in pathways related to ferroptosis. UCHL1 interacted with Parkinson's disease-related glycosylation enzyme PARK7 and reduced the degradation of PARK7 protein, which was mediated through the ubiquitin-proteasome pathway. Lastly, both in vitro and in vivo studies revealed that the UCHL1-PARK7 axis fostered tumor progression in TNBC by inhibiting ferroptosis. UCHL1 deubiquitinates and stabilizes PARK7, thereby inhibiting ferroptosis and boosting tumor progression in TNBC, indicating the potential of UCHL1 and PARK7 as therapeutic targets for TNBC patients.
Infants exposed to opioids in utero are at risk of developing Neonatal Opioid Withdrawal Syndrome (NOWS). Rodent models of perinatal opioid exposure can reliably recapitulate the acute withdrawal and developmental deficits exhibited in clinical NOWS, but few persisting phenotypes are consistently observed between studies, limiting mechanistic insight into the long-lasting effects of early-life opioid exposure. To investigate the enduring impact of perinatal opioid exposure, we employed a multi-region, multi-omic approach. We combined RNA sequencing (RNA-seq) and H3K27ac chromatin immunoprecipitation sequencing (ChIP-seq) from NeuN+ neuronal nuclei in a mouse model of NOWS. Additionally, cytokine levels were measured in the brain and spleen, and physiological responses were assessed under both basal and immune-challenged conditions. Analysis revealed differentially expressed genes and H3K27ac modifications were enriched for immune and metabolic pathways in hypothalamic neurons. Transcription factor network inference identified state-dependent rewiring of immune and metabolic regulatory circuits, with Transcription factor 4 (TCF4) emerging as a convergent epigenomic and transcriptional hub under immune-challenged conditions. Consistent with molecular signatures, cytokine levels were suppressed in morphine-exposed mice both in adulthood, both at baseline and under immune-challenged conditions. Several metabolic properties, including changes in weight and basal body temperature, were also altered. Our findings suggest that perinatal opioid exposure creates a lasting enhancer imprint in hypothalamic neurons, leading to suppressed baseline immune gene expression and altered regulatory responses to subsequent inflammatory challenges. These epigenomic and transcriptional changes may underlie the long-term physiological impacts of early-life opioid exposure, offering new insights into the enduring consequences of NOWS.
Caveolin-1 (CAV1), a principal structural component of caveolae, plays a pivotal role in the regulation of lipid metabolism, signal transduction, and cellular homeostasis. Dysregulation of CAV1 has been implicated in the pathogenesis of metabolic diseases, particularly non-alcoholic fatty liver disease (NAFLD). However, the precise molecular mechanisms responsible for CAV1 in NAFLD remain largely unclear. In vitro experiments were performed using THLE-3 or HepG2 cells treated with palmitic acid (PA) to establish a lipotoxic model. Quantitative real-time polymerase chain reaction was used to detect mRNA levels, whereas western blotting was performed to analyze protein expression. Cell viability, proliferation, apoptosis, and lipid deposition were assessed using Cell Counting Kit-8 (CCK-8), 5-ethynyl-2'-deoxyuridine (EdU), flow cytometry, and Oil Red O staining, respectively. Ferroptosis was evaluated by measuring Fe2+ levels, malondialdehyde (MDA), superoxide dismutase (SOD), and lipid peroxidation. Molecular interactions, including ubiquitination, co-immunoprecipitation (Co-IP), methylated RNA immunoprecipitation (MeRIP), RNA immunoprecipitation (RIP), and dual-luciferase reporter assays, were used to analyze the association among m6A methyltransferase-like 3 (METTL3), tripartite motif containing 37 (TRIM37) and CAV1. An in vivo NAFLD model was generated in mice fed a high-fat diet (HFD). In the animal study, liver injury and steatosis were visualized by hematoxylin and eosin (H&E) and Oil Red O staining. Serum levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT), as well as hepatic triglyceride (TG) and total cholesterol (TC) levels, were quantified to assess liver function and lipid accumulation. CAV1 expression was downregulated in blood samples of NAFLD patients. PA treatment significantly downregulated CAV1 expression, inhibited cell proliferation, increased apoptosis, and enhanced ferroptosis and lipid deposition; however, all of which were significantly reversed by CAV1 overexpression. Mechanistically, the E3 ubiquitin ligase TRIM37 was identified as a negative regulator of CAV1; TRIM37 was found to interact with CAV1 and promote its ubiquitination and degradation. Furthermore, METTL3 upregulated TRIM37 expression by binding to its mRNA transcript in an IGF2BP1-dependent manner, thereby enhancing TRIM37 mRNA stability. Knockdown of TRIM37 or METTL3 mitigated PA-induced cellular damage, but these protective effects were abolished by CAV1 silencing or TRIM37 overexpression, respectively. Consistent with the in vitro findings, in vivo experiments confirmed that CAV1 overexpression attenuated HFD-induced liver injury. This study unveils a novel METTL3/TRIM37/CAV1 regulatory axis that represents an important pathway contributing to NAFLD exacerbation. Restoring CAV1 expression represents a promising therapeutic strategy for NAFLD.
Cryo-electron tomography (Cryo-ET) has emerged as a transformative tool for visualizing viral components within their native cellular environment, enabling structural interrogation of viral life cycle events at nanometer resolution without chemical fixation or heavy metal staining. However, a persistent challenge in applying Cryo-ET to virus research is the unambiguous identification of specific viral components within densely crowded tomographic volumes. Electron density encodes mass and shape but not molecular identity, and as the cellular environment grows more complex, the assumption that a given density has no plausible alternative assignment becomes increasingly difficult to defend. This review surveys labeling and localization strategies for in situ Cryo-ET of viral components, encompassing label-free exploitation of native electron density, Cryo-immunogold labeling, genetically encoded and synthetic molecular tags, and correlative Cryo-light/electron microscopy (Cryo-CLEM) combined with Cryo-focused ion beam (Cryo-FIB) milling. We first summarize the landmark structural discoveries that in situ Cryo-ET has delivered across virus families, and then evaluate each labeling strategy against the structural and functional constraints that viral proteins impose, providing a practical framework for matching a labeling approach to a specific viral component and life-cycle stage.
The mechanisms linking chronic hyperglycemia to intestinal inflammation and epithelial dysfunction remain incompletely understood, highlighting an important gap in our understanding of diabetes-associated gastrointestinal pathology. In this study, we investigated the effects of sustained hyperglycemia on intestinal inflammation, endoplasmic reticulum (ER) stress, and autophagy in a translational porcine model of diabetes. Diabetes was induced in Yucatan mini pigs using a high-fat, high-carbohydrate/fructose diet (HFHFD) followed by streptozotocin administration. Intestinal tissues from the terminal ileum and sigmoid colon were analyzed using histological evaluation, quantitative real-time PCR, and immunohistochemistry. Histological analysis revealed structural alterations in diabetic animals, including villous degeneration, crypt depletion, goblet-cell loss, and increased inflammatory-cell infiltration. Gene expression analysis revealed significant upregulation of inflammatory mediators (NF-κB, TNF-α, IL-6, IL-1β), inflammasome components (NLRP3), and macrophage markers (CD68, CD86, CD163). In parallel, ER stress-related genes (ORMDL3, ATF6) and autophagy-associated genes (NOD2, ULK1, ATG4a) were significantly elevated in diabetic pigs. At the protein level, increased expression of ER stress markers was confirmed in both intestinal regions, while autophagy-related proteins showed less consistent changes and did not fully reflect the observed transcriptional patterns, suggesting a potential disconnect between transcriptional activation and downstream autophagy-related protein expression under diabetic conditions. Chronic hyperglycemia is associated with intestinal inflammation and disruption of cellular stress pathways, including ER stress and autophagy, in a porcine model. These findings provide mechanistic insight into how chronic hyperglycemia contributes to intestinal dysfunction through coordinated alterations in inflammatory signaling, ER stress, and autophagy pathways, identifying these processes as potential targets for therapeutic intervention in diabetes-associated gastrointestinal disease.