Search Results (30)

Background: Non-alcoholic steatohepatitis (NASH) lacks approved pharmacotherapies despite affecting approximately 25% of the global population. Agrimonia pilosa, a traditional herb with anti-inflammatory and antioxidant properties, remains unexplored for NASH treatment. Objective: This study investigated the hepatoprotective effects and mechanisms of Agrimonia pilosa extract (APE) in NASH models. Methods: HepG2 cells were treated with free fatty acids (0.125 mM) and APE (+12.5–50 μg/mL). C57BL/6J mice received a choline-deficient, L-amino acid-defined, high-fat diet (CDAHFD) for 12 weeks with APE (25–100 mg/kg/day), silymarin (100 mg/kg/day), or luteolin (20 mg/kg/day). Lipid accumulation, liver enzymes, histopathology, and molecular markers were assessed. Results: APE dose-dependently reduced lipid accumulation in FFA-treated cells, suppressed lipogenic factors (SREBF1, CEBPA, and PPARG), and upregulated fatty acid oxidation enzymes (CPT1A and PPARA) via AMPK/SIRT1 activation. In NASH mice, APE (100 mg/kg) significantly decreased serum ALT (160.0 ± 49.1 vs. 311.2 ± 66.7 U/L) and AST (96.0 ± 18.7 vs. 219.0 ± 55.7 U/L, p < 0.001), reduced hepatic macrophage infiltration by 68%, and substantially attenuated inflammatory markers (Ccl2, Tnf, and IL6), oxidative stress indicators (NRF2, HMOX1, and CYBB), and fibrogenic markers (ACTA2, COL1A1, and TGFB1) by 83–85% (p < 0.001). Collagen deposition decreased from 5.63 ± 0.39% to 1.54 ± 0.03% (p < 0.001). Conclusions: APE exerts potent hepatoprotective effects through multi-targeted modulation of lipid metabolism, inflammation, oxidative stress, and fibrosis via AMPK/SIRT1 pathway activation, supporting its potential as a natural therapeutic intervention for NASH. Full article

While Moringa oleifera Lam. (MO) extracts are known to have various bioactive properties, including anti-inflammatory properties, the components responsible still remain to be identified. This study explores the protective effects of the MO component, 7-octenoic acid (7OCT) in LPS-stimulated THP-1 macrophage inflammatory responses. The compound significantly downregulated the production of the pro-inflammatory cytokines TNF-α, IL-1β, and IL-6, as well as the expression of inflammation-related genes NFKB1, PTGS2, and NOS2. Additionally, it inhibited the nuclear translocation of NF-κB p65, a key transcription factor of inflammatory signaling cascade. Effects on oxidative stress showed that 7OCT inhibited LPS-induced NADPH oxidase 2 (NOX2) component genes including CYBB, CYBA, NCF1, NCF2, and NFE2L2, along with phosphorylated NOX2 and p47^phox proteins. The compound reduced the expression of TP53, BAX, CASP3, and CASP7, while enhancing BCL2 expression and Bcl-2 protein levels, suggesting an effect on apoptosis. Decreased levels of BAX, caspase-3, and cleaved caspase-3 proteins further confirmed its anti-apoptotic effect. Our findings suggest that 7OCT exhibits strong anti-inflammatory, antioxidant, and anti-apoptotic properties. Full article

This study designed a polyurethane core–shell fiber (PU CSF) wound dressing, which achieved unique redox catalytic function by loading nanoceria (n-CeO₂) nanozyme and effectively reduced potential side effects. The stability of ceria nanoparticles with superoxide dismutase (SOD) mimetic activity was optimized. Engineered PU CSFs with different doses of citrate-modified nanospheres (CeO₂@PU CSFs) were successfully fabricated via electrospinning and showed excellent SOD-mimetic activity in reducing oxidative stress both in vitro and in vivo. Notably, low-dose nanoceria PU CSFs demonstrated advantages in promoting wound healing and reducing scar formation compared to high-dose and SOD-loaded groups (p < 0.05), despite lower reactive oxygen species (ROS) scavenging capacity (p < 0.001). Transcriptome analysis revealed distinct mechanisms in rat skin studies: the CeO₂-loaded dressing systemically downregulated cell activation- and innate immunity-related genes (Fos, Trpm2, Cybb, and Nlrc4), while the SOD-loaded group specifically regulated inflammation mediated by oxidative stress (IL17a and Ccl20). The optimized core–shell structure and low-dose nanoceria provided balanced redox modulation, effectively protecting cells from oxidative damage while providing a multifunctional therapeutic platform for damaged wound healing. Full article

Background: Keratoconus (KTCN) is a multifactorial disease characterized by progressive corneal degeneration. Recent studies suggest that a gene expression analysis of corneas may uncover potential novel biomarkers involved in corneal matrix remodeling. However, identifying reliable combinations of biomarkers that are linked to disease risk or progression remains a significant challenge. Objective: This study employed multiple machine learning algorithms to analyze the transcriptomes of keratoconus patients, identifying feature gene combinations and their functional associations, with the aim of enhancing the understanding of keratoconus pathogenesis. Methods: We analyzed the GSE77938 (PRJNA312169) dataset for differential gene expression (DGE) and performed gene set enrichment analysis (GSEA) using Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways to identify enriched pathways in keratoconus (KTCN) versus controls. Machine learning algorithms were then used to analyze the gene sets, with SHapley Additive exPlanations (SHAP) applied to assess the contribution of key feature genes in the model’s predictions. Selected feature genes were further analyzed through Gene Ontology (GO) enrichment to explore their roles in biological processes and cellular functions. Results: Machine learning models, including XGBoost, Random Forest, Logistic Regression, and SVM, identified a set of important feature genes associated with keratoconus, with 15 notable genes appearing across multiple models, such as IL1R1, JUN, CYBB, CXCR4, KRT13, KRT14, S100A8, S100A9, and others. The under-expressed genes in KTCN were involved in the mechanical resistance of the epidermis (KRT14, KRT15) and in inflammation pathways (S100A8/A9, IL1R1, CYBB, JUN, and CXCR4), as compared to controls. The GO analysis highlighted that the S100A8/A9 complex and its associated genes were primarily involved in biological processes related to the cytoskeleton organization, inflammation, and immune response. Furthermore, we expanded our analysis by incorporating additional datasets from PRJNA636666 and PRJNA1184491, thereby offering a broader representation of gene features and increasing the generalizability of our results across diverse cohorts. Conclusions: The differing gene sets identified by XGBoost and SVM may reflect distinct but complementary aspects of keratoconus pathophysiology. Meanwhile, XGBoost captured key immune and chemotactic regulators (e.g., IL1R1, CXCR4), suggesting upstream inflammatory signaling pathways. SVM highlighted structural and epithelial differentiation markers (e.g., KRT14, S100A8/A9), possibly reflecting downstream tissue remodeling and stress responses. Our findings provide a novel research platform for the evaluation of keratoconus using machine learning-based approaches, offering valuable insights into its pathogenesis and potential therapeutic targets. Full article

Background: Metformin, a commonly prescribed medication for managing diabetes, has garnered increasing interest as a potential therapeutic option for combating cancer and aging. Methods: The current study investigated the effects of metformin treatment on human meibomian gland epithelial cells (hMGECs) at morphological, molecular, and electron microscopy levels. HMGECs were stimulated in vitro with 1 mM, 5 mM, and 10 mM metformin for 24, 48, and 72 h. The assessed outcomes were cell proliferation assays, lipid production, ultrastructural changes, levels of IGF-1, Nrf2, HO-1, apoptosis-inducing factor 1 (AIF1) at the protein level, and the expression of oxidative stress factors (matrix metallopeptidase 9, activating transcription factor 3, CYBB, or NADPH oxidase 2, xanthine dehydrogenase). Results: Morphological studies showed increased lipid production, the differentiation of hMGECs after stimulation with metformin, and the differentiation effects of undifferentiated hMGECs. Proliferation tests showed a reduction in cell proliferation with increasing concentrations over time. AIF1 apoptosis levels were not significantly regulated, but morphologically, the dying cells at a higher concentration of 5-10 mM showed a rupture and permeabilization of the plasma membrane, a swelling of the cytoplasm, and vacuolization after more than 48 h. The IGF-1 ELISA showed an irregular expression, which mostly decreased over time. Only at 72 h and 10 mM did we have a significant increase. Mitochondrial metabolic markers such as Nrf2 significantly increased over time, while HO-1 decreased partially. The RT-PCR showed a significant increase in MMP9, CYBB, XDH, and ATF with increasing time and metformin concentrations, indicating cell stress. Conclusions: Our results using a cell line suggest that metformin affects the cellular physiology of meibomian gland epithelial cells and induces cell stress in a dose- and duration-dependent manner, causing changes in their morphology and ultrastructure. Full article

Inflammatory Bowel Diseases (IBD), which encompass ulcerative colitis (UC) and Crohn’s disease (CD), are characterized by chronic inflammation and tissue damage of the gastrointestinal tract. This study aimed to uncover novel disease-gene signatures, dysregulated pathways, and the immune cell infiltration landscape of inflamed tissues. Eight publicly available transcriptomic datasets, including inflamed and non-inflamed tissues from CD and UC patients were analyzed. Common differentially expressed genes (DEGs) were identified through meta-analysis, revealing 180 DEGs. DEGs were implicated in leukocyte transendothelial migration, PI3K-Akt, chemokine, NOD-like receptors, TNF signaling pathways, and pathways in cancer. Protein–protein interaction network and cluster analysis identified 14 central IBD players, which were validated using eight external datasets. Disease module construction using the NeDRex platform identified nine out of 14 disease-associated genes (CYBB, RAC2, GNAI2, ITGA4, CYBA, NCF4, CPT1A, NCF2, and PCK1). Immune infiltration profile assessment revealed a significantly higher degree of infiltration of neutrophils, activated dendritic cells, plasma cells, mast cells (resting/activated), B cells (memory/naïve), regulatory T cells, and M0 and M1 macrophages in inflamed IBD tissue. Collectively, this study identified the immune infiltration profile and nine disease-associated genes as potential modulators of IBD pathogenesis, offering insights into disease molecular mechanisms, and highlighting potential disease modulators and immune cell dynamics. Full article

Chronic granulomatous disease (CGD) is an inherited immunodeficiency disease mainly caused by mutations in the X-linked CYBB gene that abrogate reactive oxygen species (ROS) production in phagocytes and microbial defense. Gene repair using the CRISPR/Cas9 system in hematopoietic stem and progenitor cells (HSPCs) is a promising technology for therapy for CGD. To support the establishment of efficient and safe gene therapies for CGD, we generated a mouse model harboring a patient-derived mutation in the CYBB gene. Our CybbC517del mouse line shows the hallmarks of CGD and provides a source for Cybb-deficient HSPCs that can be used to evaluate gene-therapy approaches in vitro and in vivo. In a setup using Cas9 RNPs and an AAV repair vector in HSPCs, we show that the mutation can be repaired in 19% of treated cells and that treatment restores ROS production by macrophages. In conclusion, our CybbC517del mouse line provides a new platform for refining and evaluating novel gene therapies and studying X-CGD pathophysiology. Full article

The liver plays a vital role in the defense against infections. Porphyromonas gingivalis (P. gingivalis), a dominant etiologic oral bacterium implicated in periodontal disease (PD), has been associated with various systemic diseases. This study aimed to investigate the influence of P. gingivalis on alcohol-associated liver diseases (ALD). Mice were fed a Lieber–DeCarli liquid diet containing 5% ethanol for 10 days after an initial adaptation period on a diet with lower ethanol content for 7 days. Two days before tissue sample collection, the mice were administered P. gingivalis strain W83 (Pg) through intraperitoneal injection (IP). Pair-fed mice with Pg infection (PF+Pg) exhibited an activated immune response to combat infections. However, alcohol-fed mice with Pg infection (AF+Pg) showed liver injury with noticeable abscess lesions and elevated serum alanine aminotransferase (ALT) levels. Additionally, these mice displayed liver infiltration of inflammatory monocytes and significant downregulation of proinflammatory cytokine gene expression levels; and AF+Pg mice also demonstrated increased intrahepatic neutrophil infiltration, as confirmed by chloroacetate esterase (CAE) staining, along with elevated gene expression levels of neutrophil cytosol factor 1 (Ncf1), neutrophilic inflammation driver lipocalin 2 (Lcn2), and complement component C5a receptor 1 (C5ar1), which are associated with neutrophilic inflammation. Interestingly, compared to PF+Pg mice, the livers of AF+Pg mice exhibited downregulation of gene expression levels of NADPH oxidase 2 (Cybb), the leukocyte adhesion molecule Cd18, and the Toll-like receptor adaptor Myd88. Consequently, impaired clearance of P. gingivalis and other bacteria in the liver, increased susceptibility to infections, and inflammation-associated hepatic necrotic cell death were observed in AF+Pg mice, which is likely to have facilitated immune cell infiltration and contributed to liver injury. Furthermore, in addition to the Srebf1/Fasn pathway induced by alcohol feeding, Pg infection also activated carbohydrate response element-binding protein (ChREBP) in AF+Pg mice. In summary, this study demonstrates that P. gingivalis infection, acting as a “second hit”, induces dysfunction of immune response and impairs the clearance of bacteria and infections in alcohol-sensitized livers. This process drives the development of liver injury. Full article

One of the hallmarks of microgravity-induced effects in several cellular models is represented by the alteration of oxidative balance with the consequent accumulation of reactive oxygen species (ROS). It is well known that male germ cells are sensitive to oxidative stress and to changes in gravitational force, even though published data on germ cell models are scarce. We previously studied the effects of simulated microgravity (s-microgravity) on a 2D cultured TCam-2 seminoma-derived cell line, considered the only human cell line available to study in vitro mitotically active human male germ cells. In this study, we used a corresponding TCam-2 3D cell culture model that mimics cell–cell contacts in organ tissue to test the possible effects induced by s-microgravity exposure. TCam-2 cell spheroids were cultured for 24 h under unitary gravity (Ctr) or s-microgravity conditions, the latter obtained using a random positioning machine (RPM). A significant increase in intracellular ROS and mitochondria superoxide anion levels was observed after RPM exposure. In line with these results, a trend of protein and lipid oxidation increase and increased pCAMKII expression levels were observed after RPM exposure. The ultrastructural analysis via transmission electron microscopy revealed that RPM-exposed mitochondria appeared enlarged and, even if seldom, disrupted. Notably, even the expression of the main enzymes involved in the redox homeostasis appears modulated by RPM exposure in a compensatory way, with GPX1, NCF1, and CYBB being downregulated, whereas NOX4 and HMOX1 are upregulated. Interestingly, HMOX1 is involved in the heme catabolism of mitochondria cytochromes, and therefore the positive modulation of this marker can be associated with the observed mitochondria alteration. Altogether, these data demonstrate TCam-2 spheroid sensitivity to acute s-microgravity exposure and indicate the capability of these cells to trigger compensatory mechanisms that allow them to overcome the exposure to altered gravitational force. Full article

Background and Objectives: This study aimed to investigate the causes of continuous deep fluctuations in the absolute lymphocyte count (ALC) in an untreated patient with Chronic Lymphocytic Leukemia (CLL), who has had a favorable prognosis since the time of diagnosis. Up until now, the patient has voluntarily chosen to adopt a predominantly vegetarian and fruitarian diet, along with prolonged periods of total fasting (ranging from 4 to 39 days) each year. Materials and Methods: For this purpose, we decided to analyze the whole transcriptome profiling of peripheral blood (PB) CD19+ cells from the patient (#1) at different time-points vs. the same cells of five other untreated CLL patients who followed a varied diet. Consequently, the CLL patients were categorized as follows: the 1st group comprised patient #1 at 20 different time-points (16 time-points during nutrition and 4 time-points during fasting), whereas the 2nd group included only one time point for each of the patients (#2, #3, #4, #5, and #6) as they followed a varied diet. We performed microarray experiments using a powerful tool, the Affymetrix Human Clariom™ D Pico Assay, to generate high-fidelity biomarker signatures. Statistical analysis was employed to identify differentially expressed genes and to perform sample clustering. Results: The lymphocytosis trend in patient #1 showed recurring fluctuations since the time of diagnosis. Interestingly, we observed that approximately 4–6 weeks after the conclusion of fasting periods, the absolute lymphocyte count was reduced by about half. The gene expression profiling analysis revealed that nine genes were statistically differently expressed between the 1st group and the 2nd group. Specifically, IGLC3, RPS26, CHPT1, and PCDH9 were under expressed in the 1st group compared to the 2nd group of CLL patients. Conversely, IGHV3-43, IGKV3D-20, PLEKHA1, CYBB, and GABRB2 were over-expressed in the 1st group when compared to the 2nd group of CLL patients. Furthermore, clustering analysis validated that all the samples from patient #1 clustered together, showing clear separation from the samples of the other CLL patients. Conclusions: This study unveiled a small gene expression signature consisting of nine genes that distinguished an untreated CLL patient who followed prolonged periods of total fasting, maintaining a gradual growth trend of lymphocytosis, compared to five untreated CLL patients with a varied diet. Future investigations focusing on patient #1 could potentially shed light on the role of prolonged periodic fasting and the implication of this specific gene signature in sustaining the lymphocytosis trend and the favorable course of the disease. Full article

Joint pain severity in arthritic diseases differs between sexes and is often more pronounced in women. This disparity is thought to stem from biological mechanisms, particularly innate immunity, yet the understanding of sex-specific differences in arthritic pain remains incomplete. This study aims to investigate these disparities using an innate immunity-driven inflammation model induced by intra-articular injections of Streptococcus Cell Wall fragments to mimic both acute and pre-sensitized joint conditions. Nociceptive behavior was evaluated via gait analysis and static weight-bearing, and inflammation was evaluated via joint histology and the synovial gene expression involved in immune response. Although acute inflammation and pain severity were comparable between sexes, distinct associations between synovial inflammatory gene expression and static nociceptive behavior emerged. These associations delineated sex-specific relationships with pain, highlighting differential gene interactions (Il6 versus Cybb on day 1 and Cyba/Gas6 versus Nos2 on day 8) between sexes. In conclusion, our study found that, despite similar pain severity between sexes, the association of inflammatory synovial genes revealed sex-specific differences in the molecular inflammatory mechanisms underlying pain. These findings suggest a path towards more personalized treatment strategies for pain management in arthritis and other inflammatory joint diseases. Full article

Chronic intermittent hypoxia (CIH)-induced redox alterations underlie diaphragm muscle dysfunction. We sought to establish if NADPH oxidase 2 (NOX2)-derived reactive oxygen species (ROS) underpin CIH-induced changes in diaphragm muscle, which manifest as impaired muscle performance. Adult male mice (C57BL/6J) were assigned to one of three groups: normoxic controls (sham); chronic intermittent hypoxia-exposed (CIH, 12 cycles/hour, 8 h/day for 14 days); and CIH + apocynin (NOX2 inhibitor, 2 mM) administered in the drinking water throughout exposure to CIH. In separate studies, we examined sham and CIH-exposed NOX2-null mice (B6.129S-Cybb^TM1Din/J). Apocynin co-treatment or NOX2 deletion proved efficacious in entirely preventing diaphragm muscle dysfunction following exposure to CIH. Exposure to CIH had no effect on NOX2 expression. However, NOX4 mRNA expression was increased following exposure to CIH in wild-type and NOX2 null mice. There was no evidence of overt CIH-induced oxidative stress. A NOX2-dependent increase in genes related to muscle regeneration, antioxidant capacity, and autophagy and atrophy was evident following exposure to CIH. We suggest that NOX-dependent CIH-induced diaphragm muscle weakness has the potential to affect ventilatory and non-ventilatory performance of the respiratory system. Therapeutic strategies employing NOX2 blockade may function as an adjunct therapy to improve diaphragm muscle performance and reduce disease burden in diseases characterised by exposure to CIH, such as obstructive sleep apnoea. Full article

Fibroepithelial lesions of the breast (FELs) are a heterogeneous group of neoplasms exhibiting a histologic spectrum ranging from fibroadenomas (FAs) to malignant phyllodes tumors (PTs). Despite published histologic criteria for their classification, it is common for such lesions to exhibit overlapping features, leading to subjective interpretation and interobserver disagreements in histologic diagnosis. Therefore, there is a need for a more objective diagnostic modality to aid in the accurate classification of these lesions and to guide appropriate clinical management. In this study, the expression of 750 tumor-related genes was measured in a cohort of 34 FELs (5 FAs, 9 cellular FAs, 9 benign PTs, 7 borderline PTs, and 4 malignant PTs). Differentially expressed gene analysis, gene set analysis, pathway analysis, and cell type analysis were performed. Genes involved in matrix remodeling and metastasis (e.g., MMP9, SPP1, COL11A1), angiogenesis (VEGFA, ITGAV, NFIL3, FDFR1, CCND2), hypoxia (ENO1, HK1, CYBB, HK2), metabolic stress (e.g., UBE2C, CDKN2A, FBP1), cell proliferation (e.g., CENPF, CCNB1), and the PI3K-Akt pathway (e.g., ITGB3, NRAS) were highly expressed in malignant PTs and less expressed in borderline PTs, benign PTs, cellular FAs, and FAs. The overall gene expression profiles of benign PTs, cellular FAs, and FAs were very similar. Although a slight difference was observed between borderline and benign PTs, a higher degree of difference was observed between borderline and malignant PTs. Additionally, the macrophage cell abundance scores and CCL5 were significantly higher in malignant PTs compared with all other groups. Our results suggest that the gene-expression-profiling-based approach could lead to further stratification of FELs and may provide clinically useful biological and pathophysiological information to improve the existing histologic diagnostic algorithm. Full article

Glioblastoma multiforme (GBM) is a highly heterogeneous disease with a mesenchymal subtype tending to exhibit more aggressive and multitherapy-resistant features. Glioblastoma stem-cells derived from mesenchymal cells are reliant on iron supply, accumulated with high reactive oxygen species (ROS), and susceptible to ferroptosis. Temozolomide (TMZ) treatment is the mainstay drug for GBM despite the rapid development of resistance in mesenchymal GBM. The main interconnection between mesenchymal features, TMZ resistance, and ferroptosis are poorly understood. Herein, we demonstrated that a subunit of NADPH oxidase, CYBB, orchestrated mesenchymal shift and promoted TMZ resistance by modulating the anti-ferroptosis circuitry Nrf2/SOD2 axis. Public transcriptomic data re-analysis found that CYBB and SOD2 were highly upregulated in the mesenchymal subtype of GBM. Accordingly, our GBM cohort confirmed a high expression of CYBB in the GBM tumor and was associated with mesenchymal features and poor clinical outcome. An in vitro study demonstrated that TMZ-resistant GBM cells displayed mesenchymal and stemness features while remaining resilient to erastin-mediated ferroptosis by activating the CYBB/Nrf2/SOD2 axis. The CYBB maintained a high ROS state to sustain the mesenchymal phenotype, TMZ resistance, and reduced erastin sensitivity. Mechanistically, CYBB interacted with Nrf2 and consequently regulated SOD2 transcription. Compensatory antioxidant SOD2 essentially protected against the deleterious effect of high ROS while attenuating ferroptosis in TMZ-resistant cells. An animal study highlighted the protective role of SOD2 to mitigate erastin-triggered ferroptosis and tolerate oxidative stress burden in mice harboring TMZ-resistant GBM cell xenografts. Therefore, CYBB captured ferroptosis resilience in mesenchymal GBM. The downstream compensatory activity of CYBB via the Nrf2/SOD2 axis is exploitable through erastin-induced ferroptosis to overcome TMZ resistance. Full article