Purpose Excessive conjunctival fibroblast proliferation is a major factor in postoperative scarring following ophthalmic surgery. While Rebamipide is clinically used for dry eye and has anti-inflammatory effects, its direct impact on the transcriptomic profiles and inflammatory signaling of conjunctival fibroblasts remains unclear. We aimed to elucidate the gene expression pathways by which Rebamipide modulates the inflammatory and fibrotic responses of Primary Human Conjunctival Fibroblasts (HCFCs). Methods HCFCs were treated with Rebamipide at 0.054 mM (0.002%) or 0.54 mM (0.02%), which represent 1/1000 and 1/100 of the clinical dose (2%), or control media for 24 hours. Global transcriptomic changes were analyzed using RNA-seq. Differential expression analysis (FDR < 0.05 and fold-change ≥ 2) was performed, followed by Ingenuity Pathway Analysis (IPA) to identify enriched pathways and upstream regulators. Key findings were validated at the protein level using immunofluorescence and Western blotting. Results In the functional assay, Rebamipide significantly and dose-dependently increased the metabolic activity and viability of conjunctival fibroblasts at concentrations between 0.054 mM and 1.08 mM, without inducing cytotoxicity. Concurrently, RNA-seq revealed significant dose-dependent gene alterations, with the 0.54 mM group showing the largest change. Rebamipide strongly inhibited pro-inflammatory pathways (TNF, IL1B, IL6) while activating cell cycle regulatory genes (TP53 at 0.054 mM and CDKN1A at 0.54 mM). Notably, the anti-fibrotic effect was associated with the marked downregulation of CSF3 (G-CSF) and other pro-inflammatory cytokines (CXCL6, CCL2, IL11). Mechanistically, the suppression of CSF3 is likely attributable to Rebamipide's modulation of NF-κB signaling, specifically through blockade of p65 nuclear translocation rather than a change in p65 phosphorylation status. Protein and immunofluorescence analyses supported the RNA-seq data, revealing a statistically significant reduction in Total NF-κB protein and a clear blockade of p65 nuclear translocation, which visually corresponded with the reduction in G-CSF expression. Conclusion Rebamipide modulates the gene expression profile of HCFCs toward an anti-inflammatory and anti-fibrotic state, primarily through the downregulation of CSF3 and associated pro-inflammatory cytokines. These findings suggest Rebamipide’s potential as a novel topical therapeutic strategy for reducing postoperative scarring and complications in transconjunctival surgery.
Plasma-activated water (PAW) has emerged as a promising indirect cold atmospheric plasma modality for cancer therapy due to its rich reactive oxygen and nitrogen species (RONS) content, yet its effects on malignant melanoma and the underlying mechanisms require further elucidation. In this study, deionized water was irradiated with a helium atmospheric pressure plasma jet (APPJ) for 1, 2, 3, 4, and 5 min to generate five PAWs (PAW1, PAW2, PAW3, PAW4, PAW5). Physicochemical analysis of PAWs revealed progressive increases in conductivity, decreases in pH, and accumulation of hydrogen peroxide (H2O2), nitrite (NO2-), and nitrate (NO3-) with prolonged APPJ irradiation. Compared to control group, PAWs treatment reduced malignant melanoma cell (B16) viability in a time-dependent manner, concomitant with increased apoptosis and elevated lactate dehydrogenase release. Oxidative stress markers demonstrated an obvious increase in malondialdehyde (MDA) content and a progressive decline in superoxide dismutase (SOD) activity. PAW5 treatment exerted the strongest cytotoxic effect on B16 cells. Western blotting analysis revealed significant downregulation of anti-apoptotic Bcl-2, accompanied by a sustained upregulation of pro-apoptotic Bax. This was followed by significant activation of cleaved caspase-9 and cleaved caspase-3, indicating the induction of the mitochondrial apoptotic pathway. These findings demonstrated that PAW-induced oxidative stress triggered the mitochondrial apoptotic pathway in melanoma cells, supporting the potential of PAW as a novel therapeutic strategy for malignant melanoma.
Thioredoxins are ubiquitous thiol-disulfide oxidoreductases that maintain intracellular redox homeostasis. In addition to its conserved catalytic domain, Escherichia coli thioredoxin 2 (EcTrx2) possesses a unique N-terminal zinc-binding domain whose physiological function remains largely unknown. Here, we identify a previously unrecognized DNA-binding activity of EcTrx2 and demonstrate its role in protecting DNA during oxidative stress. Electrophoretic mobility shift assays showed that EcTrx2 bound plasmid DNA in a concentration-dependent and GST-tag-independent manner, whereas EcTrx1 exhibited no detectable DNA-binding activity. DNA binding was abolished by deletion of the N-terminal zinc-binding domain and was blocked by zinc occupancy, indicating that this unique domain is essential for DNA interaction. Consistent with these findings, EcTrx2 significantly protected plasmid DNA from DNase I digestion and hydroxyl radical-mediated oxidative damage in vitro. Furthermore, EcTrx2 enhanced bacterial tolerance to the DNA-damaging agents zeocin and diamide, supporting the physiological relevance of its DNA-binding activity. Our results reveal a DNA-binding role for EcTrx2 and identify its N-terminal zinc-binding domain as a key determinant of DNA binding and protection against oxidative DNA damage.
Plant-mediated nanoparticles are being investigated for metabolic disorders, but therapeutic gains from full-dose co-administration should be distinguished from true pharmacological synergy. This study compared the antidiabetic effects of Cymbopogon citratus leaf ethanolic extract (LEE), biosynthesized silver nanoparticles (AgNPs), and their combination in alloxan-induced diabetic mice. AgNPs were synthesized using LEE as a reducing and capping agent and characterized by UV-Vis spectroscopy, dynamic light scattering, zeta potential analysis, transmission electron microscopy, Fourier-transform infrared spectroscopy, and X-ray diffraction. The AgNPs showed a surface plasmon resonance peak at 432 nm, mean hydrodynamic diameter of 38.2 ± 4.6 nm, zeta potential of -28.4 ± 3.1 mV, and predominantly spherical morphology. Diabetic mice received metformin, LEE, AgNPs, or LEE + AgNPs for 28 days, followed by evaluation of glycaemic, oxidative, lipid, hepatorenal, haematological, pancreatic histopathological, and integrated biomarker recovery responses. The combination group recorded fasting blood glucose of 128.0 ± 3.0 mg/dL and HbA1c of 6.5 ± 0.1%, compared with 141.0 ± 5.7 mg/dL and 7.3 ± 0.1%, respectively, in the AgNP group. Although combined administration produced the highest integrated biomarker recovery index (80.61 ± 0.61%), additional improvements over AgNP monotherapy were limited or nonsignificant across several endpoints. Pancreatic histopathology also showed improved islet area, cellular density, and lesion scores following treatment. Co-administration produced endpoint-specific additional benefits but did not demonstrate pharmacological synergy. Further route-matched dose-response studies, mechanistic validation, nanoparticle biodistribution, and long-term biosafety assessment are required.
Crosslinking of the high affinity IgE receptor (FcεRI) by multivalent antigen increases intracellular Ca2+ concentration ([Ca2+]i) through two sequential steps: (i) Ca2+ release from the endoplasmic reticulum (ER) and (ii) store-operated Ca2+ entry (SOCE) mediated by stromal interaction molecule 1 (STIM1)-Orai1 interaction. This process is essential for basophil and mast cell activation. We previously showed that FcεRI disaggregation by monomeric hapten rapidly and persistently inhibited [Ca2+]i increase although upstream signal disruption was transient in rat basophilic leukemia cells. However, the underlying inhibitory mechanisms remain unclear. In this study, we found that monomeric hapten dissociates STIM1-Orai1 interaction by promoting Orai1 re-phosphorylation. Although Ca2+ release from ER, the first step in [Ca2+]i increase, was blocked by monomeric hapten addition, phosphorylation of phospholipase Cγ1, a critical step for Ca2+ release from the ER, was inhibited only when the monomeric hapten was added immediately after multivalent antigen stimulation, but not when added several minutes later. However, STIM1-Orai1 interaction was dissociated by the monomeric hapten, regardless of the timing of its addition. Multivalent antigen induced Orai1 dephosphorylation, which is necessary for SOCE. In contrast, monomeric hapten addition promoted its re-phosphorylation. These results suggest that cessation of [Ca2+]i increase following FcεRI disaggregation primarily arises from immediate suppression of SOCE via STIM1-Orai1 dissociation.
Activated CD8+ T cells undergo metabolic reprogramming and shift to aerobic glycolysis to fulfill their energy and biosynthetic demands. However, the downstream pathways linking glycolytic flux to potent antitumor immunity remain unclear. In this study, we used a T-cell-specific phosphoglycerate mutase 1 (Pgam1)-deficient mouse model to demonstrate that accelerated lipid synthesis induced by glycolysis is necessary for CD8+ T cells to acquire antitumor activity. Pgam1 deficiency severely impaired antitumor activity and intratumoral infiltration in the MC38-OVA tumor model. Transcriptomic profiling of Pgam1-deficient CD8+ T cells activated in vitro revealed that Pgam1 deficiency caused a marked reduction in the lipid biosynthetic program and altered lipid composition. We found that in Pgam1-deficient CD8+ T cells, TCR stimulation dose not induce the upregulation of the Srebf1 and Srebf2 genes, which encode the master transcription factors Srebp1 and Srebp2, respectively, that regulate lipid synthesis. Pharmacological inhibition of SREBPs by fatostatin attenuated effector functions, such as TCR-induced proliferation, cytokine production, and cytotoxicity. These findings indicate that the activation of SREBP-dependent lipid synthesis pathways, which follow glycolysis, is important for the acquisition of antitumor activity by CD8+ T cells.
PTEN plays diverse tumor-suppressive roles, including inhibition of PI3K-AKT signaling and maintenance of genomic integrity in the nucleus. Elucidating the molecular mechanisms regulating its subcellular localization is therefore essential for understanding PTEN functions. PTEN350, a fragment comprising the N-terminal phosphatase and C2 domains of PTEN, preferentially localizes to the nucleus, although the residues responsible for this localization remain unclear. Previously, we demonstrated that Thr348 contributes to the prominent nuclear accumulation of the PTEN350 fragment and PTENA4 carrying alanine substitutions in the Ser380/Thr382/Thr383/Ser385 (STTS) motif. Since our previous findings suggested that Phe347 also contributes to PTEN nuclear localization, we investigated its role in the present study. Phe347 substitutions (F347A, F347Y, and F347L) were introduced into PTEN and its mutant or truncated derivatives, including PTEN350, PTEN, PTENA4, PTEN350,K13R, and PTENK13R,A4, either alone or in combination with T348D. The F347A substitution significantly attenuated the nuclear accumulation of PTEN350 and PTENA4, whereas F347L partially preserved nuclear accumulation and F347Y substitution exhibited an intermediate phenotype. Similar effects of the F347 substitutions were also observed in the T348D mutants, although the differences among the three substitutions were less pronounced. A similar pattern was observed for the plasma membrane localization of PTEN350,K13R and PTENK13R,A4, with F347A causing the greatest reduction, F347L retaining partial membrane localization, and F347Y exhibiting an intermediate phenotype. The effects of these substitutions were consistently observed in both HEK293T cells and HeLa cells. Collectively, these findings indicate that Phe347 is an important determinant of PTEN subcellular localization.
Nuclear pore complexes (NPCs) contribute to genome organization and cell identity, yet how post-mitotic NPC assembly is coordinated with chromatin architecture remains unclear. Here, we show that the nucleoporin ELYS preferentially associates with chromatin regions displaying distinct intrinsic DNA sequence features that are not explained by the repressive histone marks examined here. ELYS-bound regions are enriched for AT-rich sequences, whereas ELYS binding at super-enhancer-associated loci shift toward GC-rich sequence composition, revealing distinct sequence environments. These findings indicate that ELYS localization is associated with distinct intrinsic DNA sequence features and suggest a mechanism by which nuclear pore-associated architecture restores transcriptional programs after mitosis.
High-mobility group box 1 (HMGB1) is a chromatin-associated protein and a prototypical damage-associated molecular pattern whose dual intracellular and extracellular functions are increasingly implicated in cancer progression. Because viral proteins can harness HMGB1 to facilitate their own replication and remodel the microenvironment of transformed cells, human oncogenic viruses provide an instructive model for examining this duality. In this conceptual review, we organized the available evidence around two functional nodes. At the first node, intracellular HMGB1 supports viral replication, acting on viral chromatin in Kaposi's sarcoma-associated herpesvirus (KSHV) and Epstein-Barr virus, and on structured viral RNA in hepatitis C virus. At the second node, viral infection or specific viral oncoproteins induce HMGB1 secretion, which promotes infected-cell survival and remodels the tumor microenvironment, as reported for KSHV, hepatitis B virus, and human T-cell leukemia virus type 1. Human papillomavirus engage a receptor-level variant of this node through the HMGB1-TLR4 axis. Only KSHV currently supports both nodes in matched experimental systems. Therefore, we present a sequential two-node arrangement as a hypothesis, instead of an established property of oncogenic viruses. We further considered how viruses reverse the tumor-suppressive, genome-stabilizing functions of nuclear HMGB1, with conserved and divergent strategies apparent across viral families; why the absence of HMGB1 data for Merkel cell polyomavirus is a tractable and informative gap; and which HMGB1- and RAGE-directed agents are realistically positioned for evaluation in virus-associated cancers.
Perfluorooctane sulfonate (PFOS), a persistent member of the per- and polyfluoroalkyl substances (PFAS), has been associated with adverse neurodevelopmental outcomes. However, the direct effects of PFOS on the developing human hippocampus remain incompletely understood due to the limited availability of physiologically relevant human models. In the present study, we utilized human hippocampal organoids (hHOs) derived from human pluripotent stem cells to investigate the developmental effects of PFOS exposure. The organoids exhibited molecular characteristics consistent with hippocampal identity, including increased expression of the medial pallium-associated markers compared with human cortical organoids. Exposure to PFOS resulted in a dose-dependent reduction in MAP2 immunoreactivity together with increased cleaved caspase-3-positive cells, suggesting neuronal toxicity. Gene expression analyses further demonstrated increased expression of progenitor-associated genes accompanied by reduced expression of neuronal markers DCX and MAP2. Bulk RNA sequencing identified broad transcriptional alterations following PFOS exposure, including enrichment of proliferation-associated biological processes and downregulation of neuronal differentiation-related pathways. In addition, multiple genes involved in mitochondrial oxidative phosphorylation and the tricarboxylic acid cycle were reduced in PFOS-treated organoids. Collectively, these findings suggest that PFOS induces neuronal toxicity in human hippocampal organoids and is accompanied by alterations in developmental transcriptional programs and mitochondrial metabolism-related gene expression.
Mitochondrial calcium homeostasis is critical for bioenergetics, cell signaling, and cell survival and death, but its regulatory mechanism remains largely unknown. Here, a mitochondria-targeted genetically encoded calcium indicator has revealed that physiological concentrations of ascorbic acid (vitamin C) suppress mitochondrial calcium uptake in both intact living cells and permeabilized cells and enhance intracellular calcium signaling compared with ascorbate-deprived conditions. Mechanistic analyses indicate that this effect is mediated by a reduction in mitochondrial membrane potential, the primary driving force for mitochondrial calcium uptake. These findings uncover an unrecognized role of ascorbic acid in mitochondrial calcium homeostasis. Given the roles of mitochondrial calcium in neurodegeneration and cancer cell bioenergetics, our findings provide new insights into disease pathophysiology and potential therapeutic strategies.
