Background: Chronic thromboembolic pulmonary hypertension (CTEPH) is driven by unresolved pulmonary arterial thrombi and involves complex processes such as vascular remodeling and immune dysregulation. Early identification of molecular markers may support more accurate diagnosis and individualized therapy.
Methods: We integrated anthropometric and biochemical data with single-cell RNA sequencing (scRNA-seq), DNA methylation, and Mendelian randomization (MR) analyses. scRNA-seq data were analyzed to determine altered cell populations and pathways. MR and colocalization analyses were conducted to identify genetically supported candidates related to CTEPH.
Results: scRNA-seq analysis revealed altered immune composition, with a modest increase in NK cells and an angiogenesis-associated enrichment of monocytes and HSC-G-CSF cells in CTEPH patients, accompanied by activation of toll-like receptor and MAPK signaling pathways. MR and colocalization identified several genetically associated genes—CLEC7A, TNFSF13B, LRP1, ETS1, and FGR—of which ETS1 and FGR demonstrated good diagnostic performance. DNA methylation analysis indicated marked alterations in chromatin assembly and epigenetic regulation.
Conclusions: This multi-omics study highlights critical genes, epigenetic features, and immune-related cell populations associated with CTEPH. These findings improve understanding of disease mechanisms and offer potential biomarkers for early diagnosis and personalized management.
Background: Atherosclerosis (AS) is a chronic inflammatory vascular disease that can lead to severe cardiovascular events. Ferroptosis and autophagy have been increasingly recognized for their significant roles in AS; however, few clinically translatable hub genes that connect these processes in atherosclerotic lesions have been identified. There is an urgent need for novel diagnostic and therapeutic targets to improve the early detection and intervention of AS.
Methods: We downloaded AS-related datasets from the Gene Expression Omnibus (GEO) database. Common genes were identified using Limma and Weighted Gene Coexpression Network Analysis (WGCNA). We assessed ferroptosis and autophagy marker expression in AS mouse coronary tissues using immunohistochemistry (IHC) and immunofluorescence (IF). Hub genes were identified by intersecting common genes with known ferroptosis- and autophagy-related gene sets. Gene Set Enrichment Analysis (GSEA), receiver operating characteristic (ROC) curves, and 10-fold cross-validation repeated five times were performed to explore the potential roles and diagnostic capabilities of these hub genes. Immune cell infiltration analysis was performed using CIBERSORT, and Spearman's correlation analysis was subsequently performed to evaluate the associations between the identified hub genes and the relative abundance of immune cells. Finally, hub gene expression was validated in AS mouse coronary tissues using IHC and IF.
Results: Limma and WGCNA identified 104 common genes. IHC and IF analyses in AS mice confirmed the activation of ferroptosis and inhibition of autophagy in the coronary tissue. Intersecting common genes with pathway-specific genes identified three pivotal hub genes: CALCOCO2, TXNRD1, and SELENBP1. ROC analysis and 10-fold cross-validation repeated five times indicated excellent diagnostic efficacy for these genes. Immune infiltration analysis revealed significant alterations in immune cell populations in AS patients, and correlation analysis further demonstrated that CALCOCO2, TXNRD1, and SELENBP1 were significantly associated with multiple immune cell types, directly linking these hub genes to the atherosclerotic immune microenvironment. Consistent with patient data, IHC/IF validation showed significantly lower expression of the hub genes in AS mouse coronary tissues compared to controls.
Conclusions: CALCOCO2, TXNRD1, and SELENBP1 represent a novel panel of diagnostic biomarkers uniquely positioned at the intersection of ferroptosis and autophagy in AS. The robust diagnostic performance of these hub genes, as demonstrated by ROC analysis and cross‑validation, was further consolidated by IHC/IF validation showing their consistently downregulated expression in AS mouse models. Furthermore, these hub genes are significantly correlated with multiple immune cell populations, directly linking them to the atherosclerotic immune microenvironment and reinforcing their role as immune modulators in AS pathogenesis. Beyond diagnosis, these hub genes illuminate clinically actionable therapeutic targets: restoring their expression or function may simultaneously correct autophagy dysfunction and suppress ferroptosis, offering a dual-mechanism intervention strategy for atherosclerotic cardiovascular disease. Our findings provide a molecular framework for precision medicine approaches targeting ferroptosis–autophagy crosstalk and immune dysregulation in AS.
Background: Experimental animal models are essential for understanding the molecular mechanisms of human leukemia and testing potential therapies. Chemical induction using 7,12-dimethylbenz[a]anthracene (DMBA) has shown promise in recapitulating features of leukemogenesis in rodents, but its molecular fidelity to human disease remains underexplored. The aim of this study was to evaluate the transcriptomic landscape of a DMBA-induced leukemia rat model and assess its alignment with known gene expression signatures and oncogenic pathways in human leukemia.
Methods: Male Wistar rats were treated with DMBA to induce leukemia. RNA was extracted from whole blood and subjected to RNA sequencing. Differentially expressed genes (DEG) were identified using RNA-seq by expectation–maximization and NOISeq. Functional enrichment analysis, Gene Ontology mapping, and protein interaction networks were analyzed via STRING. Prognostic relevance of key DEGs was explored using data from the Human Protein Atlas.
Results: The DMBA-induced model showed significant upregulation of leukemia-associated genes, including FLT3, NRAS, KRAS, ABL1, BCR, and NPM1. The top 10 up- and downregulated genes were enriched in pathways related to cell fate, proliferation, apoptosis, and immune regulation. Particularly, genes such as MEIS2, CXCL9, and WIF1 were linked to hematopoietic dysregulation and poor prognosis in various cancers. Comparative analysis revealed strong transcriptomic overlap with human leukemia/carcinogenesis gene signatures, supporting the model's translational validity.
Conclusion: In conclusion, the DMBA-induced leukemia rat model exhibits a transcriptomic profile that closely mirrors key oncogenic and prognostic features of human leukemia. This model holds significant promise for preclinical studies targeting leukemogenesis and therapeutic intervention strategies.
Background: Atherosclerosis (AS), the leading cause of cardiovascular disease, involves complex molecular mechanisms that remain incompletely understood, particularly in the context of diet-induced vascular lesions.
Methods: We established an AS model in Bama miniature pigs using a high-cholesterol, high-fat diet (HCFD) and performed quantitative proteomic analysis on coronary artery tissues. Key proteins were identified using protein–protein interaction (PPI) network analysis and subsequently validated by histopathological evaluation in porcine and murine coronary arteries. The underlying molecular mechanisms were elucidated using Western blot analysis.
Results: The HCFD successfully induced an atherosclerotic phenotype characterized by significantly elevated serum lipid levels. Proteomic analysis identified 108 differentially expressed proteins (DEPs) between the AS and control groups. From four identified hub proteins, we focused on clusterin (CLU), which was markedly upregulated in atherosclerotic coronary tissues, particularly within endothelial cells (ECs) and smooth muscle cells (SMCs). Mechanistically, CLU upregulation activated the LRP1/AKT signaling pathway, thereby promoting atherogenesis.
Conclusion: Our study reveals that elevated CLU expression accelerates the process of AS by activating the LRP1/AKT pathway. These data elucidate a novel pro-atherogenic role for CLU and establish the CLU/LRP1/AKT axis as a promising therapeutic target for managing AS, particularly in pathologies driven by high-fat diets.
Nonclinical studies in animal models have often treated male animals as the default, based on the assumption that estrous-cycle hormonal variation in females increases variability and complicates interpretation. This convention is a structural limitation in study design, particularly in therapeutic areas where sex differences in pharmacokinetics (PK), pharmacodynamics (PD), and toxicity are substantial and clinically relevant. Clinical experience shows higher adverse drug reaction rates in women and recurring examples of sex-biased toxicities, underscoring the translational cost of limited sex consideration early in development. Sex differences in nonclinical PK and toxicity arise from defined mechanisms, including sex-dependent growth hormone secretion patterns that shape hepatic and renal enzyme/transporter expression, as well as sex-specific determinants of gastrointestinal absorption and cardiac electrophysiology. Omics analyses further show that sex effects are embedded in system-wide molecular networks across immune, metabolic, cardiovascular, renal, and neural systems, supporting a shift from descriptive to predictive and stratified animal study designs. Sex bias is also an animal welfare concern: male-only strategies can generate surpluses of unused females in breeding colonies, challenging the 3Rs—particularly Reduction—when evaluated at the project or facility level. Regulatory expectations are converging through International Council for Harmonization (ICH) principles, the National Institutes of Health (NIH) sex-as-a-biological-variable (SABV) policy, and reporting frameworks such as Sex and Gender Equity in Research (SAGER), with increasing emphasis on transparent sex reporting and analysis across nonclinical evidence streams. This review integrates scientific, welfare, and regulatory perspectives to provide a practical framework for sex-aware nonclinical study design, enhancing translational validity and ethical rigor.
Cochleovestibular impairment is a debilitating sequela of oncological intervention, resulting from multifaceted organic damage to the inner ear. Clinically manifesting as sensorineural hearing loss and vestibular dysfunction, ototoxicity significantly compromises cognitive health and long-term quality of life. Despite its prevalence, early detection and prophylactic intervention remain clinically suboptimal. This review provides a comprehensive analysis of the molecular pathogenesis and risk factors—including age, specific genetic polymorphisms, and cumulative dosage—associated with various cancer modalities such as platinum-based chemotherapy, radiotherapy, and emerging immunotherapies. We specifically evaluate the cellular mechanisms of cisplatin-induced irreversible damage to the stria vascularis and hair cells. Furthermore, this article critiques current clinical monitoring protocols, advocating for the integration of baseline audiological screening and high-frequency auditory assessments as mandatory standards of care. Finally, we synthesize the current therapeutic landscape, ranging from established otoprotective agents and cochlear implants to novel candidate drugs in the preclinical pipeline. By bridging mechanistic research with translational evidence, this review emphasizes a precision-medicine approach to balance therapeutic efficacy with the preservation of auditory integrity for cancer survivors.
Background: Cardiac structure and function undergo progressive age-related changes influenced by sex, yet systematic characterization of these physiological variations in preclinical models remains incomplete.
Methods: Using echocardiography and histopathology, we examined age- and sex-dependent cardiac phenotypes in healthy adult C57BL/6J mice (n = 40), stratified into eight groups by age (8, 12, 20, and 30 weeks) and sex (male/female, n = 5 per group). Longitudinal echocardiographic assessments quantified cardiac dimensions and function, whereas histopathological analyses (hematoxylin and eosin, wheat germ agglutinin, α-actinin and cTnI immunofluorescence, p16INK4a and ATP2B1 immunohistochemistry, and Masson's trichrome staining) evaluated myocardial architecture, fibrosis, and molecular expression characteristics.
Results: Female mice exhibited significant age-dependent left ventricular dilation, increased cardiac mass, and cardiomyocyte hypertrophy, whereas males exhibited greater structural stability. Conversely, males developed pronounced interventricular septal thickening and exacerbated myocardial fibrosis at later ages (20–30 weeks). Structural protein and molecular expression remodeling may represent the underlying mechanism.
Conclusion: These findings underscore the critical role of age and sex in cardiac remodeling and establish a normative reference dataset for cardiac parameters in healthy adult mice. By defining robust baseline metrics, this study enhances experimental design in cardiovascular research, improving reproducibility and translational relevance of preclinical studies.
Background: Intravaginal drug administration is the most promising strategy for treating vaginal infections to protect women's health, such as vulvovaginal candidiasis (VVC). However, conventional vaginal formulations in the clinic face challenges such as drug leakage and low bioavailability due to the mucus barrier and vaginal self-cleaning behavior. To address these limitations, we designed and optimized clotrimazole-loaded PLGA non-spherical microparticles (CPNMs) for vaginal drug delivery.
Methods: CPNMs and clotrimazole-loaded PLGA porous microspheres (CPMs) were prepared by the double emulsion-solvent evaporation method. The physicochemical features and in vitro drug release kinetics were characterized, and the effect of shape on drug delivery were also investigated. After intravaginal administration of CPMs and CPNMs, the pharmacokinetics and biocompatibility in the vagina of mice were studied.
Results: Both CPMs and CPNMs exhibited sustained release features in vitro. Initially, there was a rapid release within the first 24 h and the cumulative release of CPMs and CPNMs was 46.55% and 56.89% over 7 days, respectively. Compared to CPMs, CPNMs demonstrated an extended drug release in vivo, maintaining a residence duration of at least 72 h, which was beneficial for clotrimazole delivery at a high concentration in the vagina of mice. Histological evaluation demonstrated the low cytotoxicity and good biocompatibility of CPNMs, with no obvious damage to vaginal epithelial cells.
Conclusion: This vaginal drug delivery system offers potential applications for managing VVC, along with other health concerns in women.
Background: Adenostemma lavenia, a plant long used in traditional medicine to manage pain, inflammation, and circulatory disorders, has not been thoroughly validated using modern scientific methods. This study presents the first comprehensive investigation into the analgesic, anti-inflammatory, and thrombolytic potentials of the acetone extract of A. lavenia leaves (AEAL), employing an integrated strategy that combines in vivo, in vitro, and in silico methodologies.
Methods: The analgesic effects of the extract AEAL were evaluated in mice (five mice per group) using acetic acid-induced writhing, formalin-induced pain, and hot plate tests. Anti-inflammatory activity was assessed through xylene-induced ear edema in mice, heat-induced hemolysis of human red blood cells (HRBC), and protein denaturation assays. In vitro clot lysis was performed in triplicate to determine the thrombolytic activity of AEAL and a standard streptokinase. Molecular docking, density functional theory (DFT), and ADME/T profiling were conducted to identify and evaluate potential bioactive compounds.
Results: AEAL demonstrated significant, dose-dependent analgesic effects, reducing writhing responses by 53.74% (at 400 mg/kg), and inhibiting formalin-induced pain by up to 65.76% (p < 0.01). It also showed potent anti-inflammatory activity, with edema inhibition of 55.17% at a 400 mg/kg dose (p < 0.001), HRBC membrane stabilization (77.19%), and 85.96% suppression of protein denaturation. In thrombolytic testing, AEAL significantly (p < 0.001) achieved 68.1% clot dissolution compared with the control group. In silico analysis revealed that sesquiphellandrene strongly bound to COX-2 (−7.7 kcal/mol) and tissue plasminogen activator (tPA) (−6.9 kcal/mol), whereas derivatives of 2,3-hexadienoic acid exhibited notable COX-2 inhibition (−7.4 kcal/mol). ADMET predictions indicated favorable drug-like properties. In the DFT analysis, sesquiphellandrene and 2,3-hexadienoic acid, 2-methyl-4-phenyl-, methyl ester exhibited a narrow HOMO–LUMO energy gap, indicating high chemical reactivity and a greater propensity for electron-transfer processes.
Conclusions: The AEAL exhibits robust analgesic, anti-inflammatory, and thrombolytic activities, corroborated by computational analysis of its phytoconstituents. These results validate its ethnopharmacological use and highlight sesquiphellandrene as a promising candidate for future therapeutic development. Further isolation, mechanistic studies, and preclinical evaluation of sesquiphellandrene are warranted to harness its full pharmacological potential.
Background: The Chinese hamster (Cricetulus griseus) is not only an important model organism but also the source of Chinese hamster ovary cells, which play an indispensable role in biomedical and biopharmaceutical research. However, systematic investigations of the proteome and phosphoproteome across multiple organs of this species remain limited.
Methods: A comprehensive proteomic and phosphoproteomic analysis was performed across nine major organs of the Chinese hamster, including heart, liver, lung, kidney, spleen, cerebral cortex, skeletal muscle, stomach, and testis or ovary. High-throughput mass spectrometry–based approaches were used to identify and quantify proteins and phosphorylation sites across these tissues.
Results: In total, 14 219 proteins were identified in the proteome, with 11 828 phosphorylated proteins and 47 122 phosphorylation sites in the phosphoproteome. The comparative analysis revealed pronounced interorgan differences in protein composition and phosphorylation regulation, reflecting distinct physiological functions.
Conclusions: This work provides a systematic framework for understanding organ-specific functions of the Chinese hamster and establishes a theoretical basis for its use as a disease model, filling a critical gap in multiorgan proteomic and posttranslational modification datasets for this species.
Background: Stress urinary incontinence (SUI) is primarily caused by structural and functional damage to the external urethral sphincter (EUS). In previous studies, metformin, a known AMP-activated protein kinase (AMPK) activator, has shown repair effects after skeletal muscle injury. This study explored its therapeutic effect and molecular mechanisms in SUI associated with EUS injury.
Methods: The SUI model was established by double vaginal dilation. Rats were assigned to sham, vehicle-treated, or metformin-treated (200 mg/kg/day, oral) groups. Urodynamic testing was performed after 7 and 14 days. Subsequently, EUS tissues were isolated for histological (hematoxylin and eosin [H&E], Masson's), apoptotic (TUNEL), ultrastructural (transmission electron microscopy [TEM]), and molecular analyses. Finally, C2C12 myotubes cells were treated with varying concentrations of metformin to evaluate the expression of phosphorylated AMPK (p-AMPK) and MYH1/2, which are markers of skeletal muscle fiber type and regeneration.
Results: Abdominal leak point pressure (ALPP) and bladder leak point pressure (BLPP) decreased after double vaginal dilation, suggesting that SUI was established. After treatment for 7 and 14 days, respectively, metformin significantly increased ALPP and BLPP (p < 0.05) without affecting maximum bladder capacity (p > 0.05). Masson's staining and TEM revealed that metformin improved disorganized EUS architecture. Further, Western blot analysis demonstrated that metformin increased the expression of MYH1/2 and p-AMPK in vivo and in vitro. Meanwhile TUNEL assay showed that metformin treatment reduced apoptotic cells in the EUS from SUI rats.
Conclusions: These results indicate that metformin enhances EUS repair to improve SUI via activating the AMPK signaling pathway. These findings provide new insights into therapeutic strategies for SUI.
Background: Contusion spinal cord injury (SCI) models are extensively used in preclinical research because of their ability to mimic the pathophysiological characteristics observed in humans. Although various impact devices have been developed to establish graded contusion SCI models, few studies have systematically investigated the relationship between impact depth and injury severity. In this study, we aimed to establish and characterize a graded SCI model, with impact depth as the independent variable.
Methods: A precise impactor system was used to establish graded SCI models by adjusting the impact depth, with comprehensive evaluations of locomotor function, imaging, histology, and transcriptomic profiles of the injured spinal cord. Additionally, gene expression trend analysis and subsequent Gene Ontology enrichment analysis were performed to investigate the potential biological mechanisms associated with injury severity.
Results: The Basso, Beattie, Bresnahan (BBB) score and CatWalk gait analysis demonstrated a severity-dependent functional recovery pattern in different impact depth groups across multiple postinjury time points. Magnetic resonance imaging and histological results revealed a correlation between impact depth and lesion size. Principal component analysis and heat map clustering of the transcriptomic profile revealed intragroup clustering and intergroup separation, with different injury severities across different time points postinjury. Furthermore, biological mechanisms that correlated with injury severity were identified using gene expression trend analysis.
Conclusions: This study established a quantifiable, graded rat SCI model that was comprehensively evaluated using multiple approaches and may serve as a valuable platform for future SCI research.
Antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) is a systemic autoimmune disease characterized by small-vessel inflammation and the presence of ANCAs, which primarily target myeloperoxidase (MPO) and proteinase 3. Propylthiouracil (PTU), an antithyroid drug, is known as a causative drug of MPO-AAV. PTU induces the formation of DNase I-resistant neutrophil extracellular traps (NETs), which act as autoantigen sources and trigger the production of MPO-ANCAs. Although a rat model of PTU-induced AAV has been established, previous attempts to develop a corresponding mouse model have failed to replicate its histopathological features. In this study, we developed a novel murine model of PTU-induced AAV by enhancing neutrophil recruitment through intraperitoneal injection of thioglycolate, followed by intraperitoneal administration of phorbol 12-myristate 13-acetate and PTU to induce DNase I-resistant NETs. Freund's complete adjuvant was coadministered intradermally to amplify the immune response. This protocol resulted in abundant NET deposition in peritoneal tissues, elevated MPO-ANCA levels, and the development of pauci-immune glomerular lesions that closely resembled those observed in human AAV. Furthermore, a modified enzyme-linked immunosorbent assay (ELISA) reliably quantified murine MPO-ANCA levels. Thus, we established a novel murine model of PTU-induced AAV that recapitulates both the serological and histopathological features of the human disease. This model provides a valuable platform for investigating disease mechanisms, including NETs- and ANCA-mediated AAV pathogenesis.
Monkeypox virus (MPXV), a zoonotic orthopoxvirus, has emerged as a major public health concern following its global spread in 2022. Robust small animal models are urgently needed to investigate MPXV pathogenesis and evaluate medical countermeasures. Here, we systematically compared MPXV infection in Syrian hamsters, C57BL/6 mice, and BALB/c mice following intraperitoneal inoculation. Syrian hamsters displayed the highest susceptibility, with systemic viral dissemination peaking at 2 days post-infection (dpi) and clearance by 12 dpi. Viral burdens were particularly elevated in the spleen and kidneys, correlating with severe histopathological changes. In contrast, C57BL/6 and BALB/c mice exhibited more restricted viral replication, with the kidneys, liver, and spleen serving as major target organs. Across all models, no cutaneous lesions were observed, underscoring the influence of infection route on disease phenotype. Comparative analysis revealed species-specific tissue tropism and clearance kinetics, confirming the Syrian hamster as a permissive model for systemic MPXV disease, while C57BL/6 and BALB/c mice offer complementary value for mechanistic immunology studies. These findings establish a framework for rational selection of small animal models to study MPXV pathogenesis and to evaluate antiviral and vaccine strategies.
Intraocular pressure (IOP) is the only modifiable risk factor for glaucoma, making animal models of ocular hypertension (OHT) foundational for research. Steroid-induced OHT is a strong candidate model, yet published steroid models rarely provide quantitative guidance on how route, dose, and schedule shape the pressure–time course, leaving laboratories to pilot protocols and hindering cross-study comparisons. We examined an OHT model using depot dexamethasone acetate injections, and systematically characterized IOP profiles in C57BL/6J mice across two injection routes (subconjunctival or periorbital), two dosing schedules (3 doses/1 week; 5 doses/4 weeks), and two concentrations (20 μL, 10 or 20 mg/mL). Steroid exposure produced OHT in every treated mouse, occurring rapidly by day 2, peaking on days 27–31, and declining to baseline by day 98. Maximum IOP increased ~90%–100% above baseline (from ~11–12 mmHg to ~22–23 mmHg). Increasing the dosing regimen from three to five doses modestly raised mean IOP over the study period without changing maximum IOP and peak timing. Increasing dexamethasone concentration increased maximum and mean IOP, without prolonging the duration of IOP elevation. Subconjunctival and periorbital injections were performed similarly, with consistent responses across sexes and minimal diurnal variation. These results provide researchers with clear benchmarks for selecting steroid regimens, guide interpretation of therapeutic effects, and offer a reproducible, practical platform for glaucoma research.
Chemical castration offers a noninvasive way to sterilize male animals without surgical complications. An ideal chemical castration agent should be affordable, effective with a single injection, free of side effects, and without negative impacts on animal welfare. Because there is limited research on chemical castration in rabbits, we aimed to investigate the histopathological and hormonal effects of intra-testicular injections of dextrose, mannitol, and saline solutions in male rabbits. Nine adult rabbits were divided into three equal groups. Under anesthesia, 50% dextrose, 20% mannitol, or 0.9% saline was injected bilaterally into the testicles, with the volume of each solution determined by the width of each testicle. Sixty days after the intra-testicular injections, the rabbits underwent orchiectomy. All testicular tissues were examined histopathologically. Serum testosterone levels were assessed on days 0 and 60. Histopathological examination showed a significant decrease in the quality and maturity of the seminiferous tubules and spermiogenesis index, as well as an increase in histopathological changes in the testis of the saline-receiving group compared to other groups (p < 0.05). Except for the percentage of mature testicular seminiferous tubules, there was no significant difference between the mannitol- and dextrose-receiving groups (p > 0.05). Intra-testicular injections of any of the solutions did not significantly affect testosterone levels (p > 0.05). Our study showed that intra-testicular injection of saline, unlike dextrose and mannitol, can cause significant testicular tissue damage without side effects. Consequently, 0.9% saline may have potential as a chemical sterilization agent in rabbits.