Abstract Background Wound healing is an environmentally adaptive process. It requires a tightly coordinated transition from an inflammatory to a proliferative state, which occurs in response to oxidative and inflammatory stress. Molecular hydrogen (H2) is a widely recognized medical gas, and it alleviates oxidative stress and selectively eliminates highly cytotoxic reactive oxygen species. However, it remains unclear whether redox-active gases such as H2 can actively orchestrate tissue state transitions, rather than merely mitigating oxidative damage. Methods Bulk RNA sequencing, single-cell RNA sequencing, and spatial transcriptomic analyses were integrated to investigate the cellular and molecular mechanisms underlying H2-mediated wound healing. Functional studies were performed in hydrogen-mediated mouse skin injury models and in self-assembled skin organoid models. Immunofluorescence staining, Masson’s trichrome staining, and qRT-PCR were used to validate key signaling pathways and assess tissue regeneration, angiogenesis, and wound healing outcomes. Results H2 significantly accelerated wound healing, especially during the proliferative phase, where it acted as a critical regulator of the inflammatory-to-proliferative transition. Multi-omics analyses identified a hydrogen-associated macrophage program and revealed a CXCL2-ACKR1 signaling axis mediating macrophage-endothelial communication. Functional validation confirmed that activation of this axis promoted endothelial migration, angiogenesis, and granulation tissue formation. As a result, the transition from inflammation to proliferation was facilitated, and wound closure was accelerated. Conclusions H2 spatiotemporally coordinates adaptive wound healing by regulating a macrophage-endothelial CXCL2-ACKR1 signaling axis that drives the inflammatory-to-proliferative transition. These findings provide mechanistic insight into how H2 spatiotemporally coordinates adaptive wound healing and highlight the potential of hydrogen-mediated redox modulation as a mechanism for regulating tissue repair.
Abstract Background Nitrogen mustard (HN2) is a bifunctional alkylating vesicant that produces blistering and ulcerative skin lesions, yet the molecular events responsible for this tissue damage remain incompletely defined. This study aimed to identify the direct protein targets of HN2 in human skin and elucidate how target alkylation affects mitochondrial function and thereby causes skin injury. Methods An activity based probe, HNP-1, was designed for fluorescence imaging and chemical proteomic target mapping. Biochemical assays and site-directed mutagenesis were employed to confirm HN2 binding to electron-transferring flavoprotein dehydrogenase (ETFDH) and to identify the modified residue. Additionally, transcriptomic profiling and a murine cutaneous injury model were used to characterize downstream events and evaluate the effects of vitamin B2 (VB2). Results HNP-1 enabled direct visualization of HN2-derived labeling in skin cells and tissues and showed that the signal was predominantly extranuclear. Unbiased proteomic analysis then identified ETFDH as a high-affinity mitochondrial target. HN2 alkylated ETFDH at His112, impairing its interaction with ubiquinol–cytochrome c reductase core protein 2 (UQCRC2) and triggering mitochondrial oxidative stress, voltage-dependent anion channel 1 (VDAC1) oligomerization, and mitochondrial DNA (mtDNA) release. These events promoted cGAS-STING signaling and pyroptosis, whereas the H112A substitution blunted the response. Intraperitoneal VB2 administration restored the epidermal flavin adenine dinucleotide (FAD) level and attenuated HN2-induced skin injury in mice. Conclusions These findings link ETFDH alkylation to pyroptotic signaling in acute HN2 skin injury and show that VB2 mitigates skin damage in severely exposed mice.
Abstract In recent years, the role of mechanosensation in lymphatic system development, functional homeostasis, and disease progression has received increasing attention. As key mechanosensitive receptors in lymphatic endothelial cells, Piezo1/2 ion channels respond to biomechanical stimuli such as fluid shear stress and interstitial pressure, thereby regulating calcium influx and downstream signaling pathways. These channels play pivotal roles in physiological processes, including lymphatic network formation, valve development, and the maintenance of meningeal lymphatic function. This review systematically describes the structural features and dynamic mechanisms of Piezo1/2 channels, with a particular focus on their biological functions within the lymphatic system. Piezo1 integrates multiple signaling pathways, including the Orai1/Notch, ANGPT/TIE/FOXO1, and EPHB4/RASA1 signaling pathways, to regulate lymphatic sprouting, valve morphogenesis, and meningeal lymphatic development. Dysregulation of Piezo1 function has been demonstrated to be closely associated with congenital lymphoedema, tumor lymphatic metastasis, and neurological disorders such as hydrocephalus and craniosynostosis. In addition, the potential role of Piezo2 in the sensory impairments associated with lymphoedema warrants attention. With respect to therapeutic strategies, this review comprehensively summarizes approaches targeting Piezo1/2, including the development of small-molecule agonists/inhibitors, gene-editing technologies, physical modulation methods, and advanced drug delivery systems, as well as their research progress and clinical prospects. Despite persistent challenges such as insufficient tissue specificity and limited spatiotemporal precision, the integration of multiscale biomechanical models, organoid platforms, and multidisciplinary research strategies holds promise for translational breakthroughs in Piezo1/2 ion channel research, providing a new paradigm for precision therapy of lymphatic system-related diseases.
Background Diabetic foot ulcer (DFU) is a severe diabetic complication characterized by impaired healing, often involving fibroblast senescence and the senescence-associated secretory phenotype (SASP). The role of ribonucleic acid (RNA)-binding proteins (RBPs) in this process remains undefined. This study investigates the function and mechanism of the RBP interleukin enhancer-binding factor 2 (ILF2) in DFU pathogenesis. Methods Differentially expressed RBPs were identified via bioinformatics analysis of public single-cell and bulk transcriptomic datasets. ILF2 downregulation was subsequently validated in clinical DFU samples and diabetic mouse models. Functional assays in high-glucose (HG)-treated fibroblasts evaluated proliferation, migration, and SASP. Mechanistically, RNA sequencing, RNA-binding protein immunoprecipitation, and RNA pull-down assays identified downstream targets, while co-IP and rescue experiments verified the NPM1/NF-u03BAB axis. Finally, a diabetic mouse model was used to study the effects of ILF2 overexpression/knockdown and NPM1 knockdown on wound healing. Results Bioinformatics analysis identified ILF2 as significantly downregulated in DFU. This reduction was consistently validated in DFU patient tissues, diabetic mouse wounds, and HG-treated fibroblasts. Functionally, ILF2 overexpression promoted fibroblast proliferation and migration while suppressing SASP, whereas knockdown exacerbated senescence. Mechanistically, ILF2 directly bound to nucleophosmin (NPM1) mRNA to promote its degradation. ILF2 deficiency led to aberrant NPM1 accumulation, enhancing the NPM1u2013phospho-p65 interaction and NF-u03BAB pathway activation. Rescue experiments confirmed that NPM1 knockdown reversed ILF2 deficiency-induced cellular dysfunction. Crucially, these findings were validated in primary fibroblasts isolated from DFU patients. In vivo, ILF2 overexpression accelerated wound healing, while knockdown delayed the process. Furthermore, NPM1 knockdown effectively ameliorated the impaired healing phenotype and reduced SASP levels. Conclusions This study elucidates a novel ILF2u2013NPM1u2013NF-u03BAB regulatory axis. ILF2 acts as a critical suppressor of inflammatory senescence by destabilizing NPM1 mRNA, highlighting its potential as a therapeutic target for DFU treatment.
Background Hypertrophic scars are a major clinical challenge with limited treatments. Adipose-derived stem cells (ADSCs) play an important role in inhibiting pathological scar formation. However, the underlying mechanisms remain unclear. In this study, we aimed to investigate the function, mechanism, and therapeutic potential of adipose-derived stem cell peptide 5 (ADSCP5), a novel peptide from adipose-derived stem cell-conditioned medium. Methods We used in vitro hypertrophic scar fibroblast, macrophageu2013fibroblast coculture and human umbilical vein endothelial cell (HUVEC) assays, RNA sequencing, pathway analysis, peptide pull-down, molecular docking, cellular thermal shift assays, surface plasmon resonance, immunofluorescence colocalization analysis, western blotting, rescue experiments, reactive oxygen species (ROS), autophagy tests, and mitochondrial membrane potential assays and untargeted metabolomics. The efficacy of ADSCP5 was validated in rabbit and porcine scar models. Results In hypertrophic scar fibroblasts, ADSCP5 significantly downregulated the expression of collagen genes (COL1A1, COL1A2, and COL3A1) and actin alpha 2, smooth muscle, without affecting cell proliferation, apoptosis, or migration. Transcriptomic, enrichment, and western blot analyses confirmed that ADSCP5 reduced the protein levels of phosphorylated p65 (p-p65, NF-u03BAB subunit), p-PI3K, p-AKT, and p-mTOR. Moreover, ADSCP5 decreased IL-6 transcription. Mechanistically, ADSCP5 bound directly to pyruvate carboxylase or the NF-u03BAB subunit p50. This interaction resulted in the downregulation of PC or the upregulation of p50, ultimately inhibiting collagen expression, a finding confirmed by rescue assays. Furthermore, ADSCP5 induced ROS generation and autophagy, altered global metabolism, and modulated macrophageu2013fibroblast crosstalk to suppress fibrosis. It also exhibited antiangiogenic effects in HUVECs. In both the rabbit and porcine scar models, ADSCP5 treatment effectively attenuated collagen deposition and scar hyperplasia, increased the number of macrophages (increased CD68), reduced angiogenesis (decreased VEGFA and CD34), and promoted autophagy (reduced p62). Conclusions Overall, this study demonstrate that ADSCP5 alleviates hypertrophic scarring by directly binding to PC and p50, suppressing the PI3K/AKT/mTOR and NF-u03BAB pathways, reducing IL-6 and collagen production, promoting ROS and autophagy, and modulating metabolic, macrophage and angiogenic responses. These findings position ADSCP5 as a promising therapeutic agent for hypertrophic scar prevention and treatment.
