Metabolic support protects oral mucosa from ferroptosis in radiation-induced mucositis

Metabolic support protects oral mucosa from ferroptosis in radiation-induced mucositis | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Metabolic support protects oral mucosa from ferroptosis in radiation-induced mucositis Li-na Niu, Weiwei Yu, Kai Jiao, Kaiyan Wang, Qianqian Wan, Xiang Li, and 7 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5617929/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 08 Dec, 2025 Read the published version in Nature Communications → Version 1 posted You are reading this latest preprint version Abstract Ionizing radiation is effective in combating cancer but inflicts severe damage on the oral mucosa. The mechanisms behind this damage remain unclear, and current treatment modalities are primarily palliative. This study revealed that ferroptosis is the predominant reason for oral-radiation depletion of oral mucosal epithelial cells. More importantly, compensatory mechanisms are activated in the organism during the early stage after radiation exposure. These compensatory mechanisms arise from the metabolic support provided by fibroblasts. In the early post-radiation stage, fibroblasts supply polyamines, which are readily absorbed by basal epithelial cells, protecting them from ferroptosis. Local supplementation of polyamines effectively mitigates mucosal damage. This study emphasizes the important role of fibroblast-mediated metabolic support in protecting the oral mucosa from radiation-induced damage. Results of the study provide new insights into combating radiation-related diseases by enhancing the self-protective responses of living organisms. Health sciences/Medical research/Drug development Biological sciences/Cell biology/Cell death Compensation ferroptosis fibroblasts-epithelium interaction metabolic milieu polyamine metabolism microenvironment radiation-induced oral mucositis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Highlights 1. Basal epithelial cell ferroptosis is crucial for radiation-induced epithelial ablation. 2. Fibroblast-epithelial cell interactions promote compensatory protection by inhibiting ferroptosis. 3. Fibroblast-derived polyamines drive the compensatory mechanisms. 4. Polyamine supplementation inhibits ferroptosis and prevents radiation-induced mucosal injury. Introduction Radiation-induced oral mucositis (RIOM) is a common side effect of radiotherapy for head and neck cancer 1 . It is usually manifested as mucosal atrophy or peeling, and is accompanied by severe pain and loss of mucosal function. This condition results from the death of epithelial cells after their exposure to ionizing radiation 2–4 . Current research suggests that ionizing radiation can directly cause DNA breaks, and activate reactive oxygen species (ROS) that indirectly damage biomolecules 5 . These damages result in the upregulation of apoptosis-related cytokines and activation of downstream signaling pathways, ultimately triggering the loss of mucosal continuity 1,2 . The limited effectiveness of anti-apoptotic drugs in treating RIOM 3–5 suggests the involvement of other forms of cell death during disease progression. Thus, exploring the specific mechanisms of epithelial cell death may offer new insights for maintaining the structure and function of mucosa during radiotherapy. In this study, the authors resorted to single-cell RNA sequencing (scRNA-seq) and confirmed that ferroptosis is the primary mode of epithelial cell death. Currently, ferroptosis is primarily inhibited by blocking the propagation of lipid peroxyl radicals and chelating intracellular iron pools 6,7 . We found that using the specific ferroptosis inhibitor Liproxstatin-1 (LIP-1) effectively inhibits radiation-induced epithelial cell death and alleviates mucosal damage. However, this approach has limitations from a safety perspective, and may not be the optimal solution for treating RIOM 8–10 . Therefore, further investigations of epithelial cell ferroptosis are required to identify new pathways for mucosal protection. Surprisingly, at the early stage post-radiation, we observed that epithelial cell death was not significant, with even enhanced proliferation causing increase in the thickness of the oral mucosa. This suggests that the body might be in a compensatory and adaptive state to respond to radiation stimulation. Based on this discovery, we propose to amplify the inherent early-stage protective mechanism to maintain epithelial cells in an adaptive state long-term post-radiation, thereby preventing cell death. The objective of this study was to elucidate the specific mechanisms underlying mucosal damage in RIOM by investigating the death modes of mucosal epithelial cells under ionizing radiation. Specifically, it was discovered that early after radiation, the body can leverage intercellular communication within the microenvironment to provide metabolic support for the integrity of mucosal epithelia. Further, the utilization of metabolic supporters can effectively prevent RIOM. This discovery sheds light on the self-protective mechanisms of organisms under radiation stress via intercellular communication. Based on this discovery, it has significant potential to prevent radiation-related diseases and improve the prognosis of radiotherapy through amplifying the inherent self-protective mechanisms. Results The effect of ionizing radiation on epithelial thinning and basal cell count The impact of radiation on oral mucosa was investigated using biopsies of human tongues affected by RIOM (Fig. 1 a). Significant epithelial thinning and discontinuity were observed (Fig. 1 b, c). Reduced basal cell proliferation was also observed (Fig. 1 d). For in vivo modeling, C57BL/6 mice received 18 Gy cranial radiation. Onset of oral ulceration occurred on the 9th day after radiation (Fig. 1 e). Histological examination confirmed the manifestation of mucositis in the murine specimens that was similar to the human tongue mucosa specimens (Fig. 1 f, g). Suppressed basal cell proliferation was also evident (Fig. 1 h). Subsequently, scRNA-seq was performed on murine tongue mucosa to analyze cellular changes before and after radiation. After quality control to remove low-quality cells, the dataset included ~ 28,189 cells. Graph-based clustering and cell marker annotation identified 9 major cell types: epithelial cells, fibroblasts, endothelial cells, immune cells, Schwann cells, smooth muscle cells, pericytes, endo-pericytes, and myocytes (Fig. 1 i and Supplementary Fig. 1a, b). Transcriptomic analysis documented differential expression of cell-defining genes for the cell compartments, which were generally conserved across mucosal sites (Supplementary Fig. 1c). There was alteration in cell composition after radiation, with a sharp decline in the number of epithelial cells (Fig. 1 j). Because of the heterogeneity of oral epithelial cells, they were classified into 4 subpopulations: basal, spinous, granular cells, and corneocytes (Fig. 1 k) 11 . The basal epithelial cells exhibit stem cell traits, and were the most prevalent both before and after radiation (Fig. 1 l, Supplementary Fig. 1d, e). Immunohistochemistry confirmed a drastic reduction in basal cells after radiation (Supplementary Fig. 1f). Finally, histological imaging confirmed the presence of the 4 major compartments-epithelial cells, fibroblasts, endothelial cells, and immune cells within the oral mucosal tissues. This observation provided insight into the local tissue architecture (Fig. 1 m). Taken together, the findings demonstrate that ionizing radiation significantly thins the oral mucosa. Epithelial cells, especially basal cells, are severely depleted. The impact of ionizing radiation on death in basal epithelial cells Different cell death pathways were examined using Add Module Score to evaluate the cause of epithelial cell depletion. Ferroptosis was markedly increased after radiation exposure. Apoptosis was also detected in the epithelial cells (Fig. 2 a). Transmission electron microscopy of irradiated tongue tissue revealed ferroptotic features such as mitochondrial shrinkage and increased membrane density (Supplementary Fig. 2a). Gene set variation analysis (GSVA) identified altered iron ion homeostasis in the irradiated cells (Supplementary Fig. 2b). The ferroptosis marker prostaglandin-endoperoxide synthase 2 (PTGS2) was significantly upregulated in epithelial cells after irradiation (Fig. 2 b). The ferroptosis inhibitor LIP-1 suppressed PTGS2 expression and increased mucosal thickness (Fig. 2 b, c). Lipid peroxide buildup, a hallmark of ferroptosis, was evident and notably reduced by the ferroptosis inhibitor ferrostatin-1 (Supplementary Fig. 2c). These results indicate that ferroptosis is the primary cause of oral mucosal epithelial cell depletion after ionizing radiation. Analysis of ferroptosis gene set scores was subsequently conducted for different epithelial cell subtypes. Basal cells, the predominant epithelial subtype with stem cell characteristics, had the highest ferroptosis gene set scores (Fig. 2 d and Supplementary Fig. 2d). Immunofluorescence for acyl-CoA synthetase long chain family member 4 (ACSL4), the major enzyme in polyunsaturated fatty acid activation and ferroptosis induction 12 , showed elevated expression levels in basal cells from murine and human oral mucositis lesions (Fig. 2 e, f). Similarly, LIP-1 inhibited the expression of ACSL4 in mouse basal cells (Fig. 2 e). In vitro experiments confirmed reduced proliferation and increased mortality rates of post-irradiated basal cells (Supplementary Fig. 2d-f). The effects of ferroptosis inhibitor ferrostatin-1, apoptosis inhibitor Z-Val-Ala-Asp fluoromethylketone (Z-VAD-fmk), necroptosis inhibitor necrostatin-1, and pyroptosis inhibitor Belnacasan (VX-765) on the survival of post-irradiated basal cells were further evaluated. Treatment with ferrostatin-1 or Z-VAD-fmk partially restored the clonogenic survival rate of basal cells that was reduced by radiation exposure. The ferroptosis inhibitors demonstrated a more pronounced restorative effect than other cell death inhibitors (Supplementary Fig. 2g). There was a significant increase in labile iron and ROS levels within the basal cells; these features were substantially reduced by ferrostatin-1 treatment (Supplementary Fig. 2h, i). Collectively, these data support ferroptosis in basal cells as a primary component of radiation-induced epithelial cell death. Potential early-stage protective phenomenon in RIOM Murine tongue mucosal specimens were analyzed at sequential time-points to elucidate post-radiation basal cell ferroptosis. Expression of PTGS2, a ferroptosis biomarker 13 , did not rise markedly in basal cells during early radiation phase, but increased in the late phase (Fig. 2 g). Importantly, enhanced mucosal thickness and robust basal cell proliferation without DNA damage suggested early phase hyperactivity in RIOM (Fig. 2 h and Supplementary Fig. 3a, b). Single-cell sequencing of oral mucosal tissues was conducted at different time-points after radiation (Supplementary Fig. 3c, d). There was an increase in basal cell numbers in the early-phase, followed by a decline in the late-phase (Supplementary Fig. 3e). In agreement with these results, GSVA of basal cells indicated active enzymatic and protein folding functions in the early-phase of post-radiation. There was also regulation of the cell cycle to enhance proliferative and suppress apoptosis (Fig. 2 i and Supplementary Fig. 3f). In the late stage, the decline in protein folding, wound healing processes, and basement membrane functions was observed (Fig. 2 i and Supplementary Fig. 3g). These results suggest that a protective mechanism may be activated during the early phase of RIOM. This protective mechanism promotes compensatory proliferation of basal cells to counteract adverse stimuli. Effect of microenvironment factors on basal cell ferroptosis Basal cells are crucial for epithelium renewal and injury response. They are controlled by localized cues within their microenvironment 14 . Understanding the cellular and molecular environment that signals post-injury repair is essential for appreciating basal cell homeostasis and mucosal regeneration after injury 15 . Accordingly, the cellular components of the microenvironment were analyzed to investigate the protective mechanisms for basal cells in the early phase of RIOM. Notably, in irradiated mouse and human tongue mucosal specimens, fibroblasts were closely located around basal cells, with numbers peaking in the early phase and fibrosis enhanced in the late phase (Fig. 3 a, b and Supplementary Fig. 4a). There was sustained high-level fibroblast-basal cell interaction during the early phase. This activity declined markedly in the late phase (Fig. 3 e). When basal cells were co-cultured with fibroblasts under irradiation exposure, the proliferative activity of the basal cells was significantly higher compared to that of basal cells cultured alone (Fig. 3 f, g). Indeed, when basal cells were co-cultured with fibroblasts and subsequently irradiated, there was a significant reduction in iron content and lipid peroxidation levels in the basal cells (Fig. 3 h, i). These data demonstrate that post-radiation crosstalk between fibroblasts and basal cells plays a critical role in controlling compensatory protection against oral mucosal injury. Functional analysis of fibroblasts was performed to elucidate their protective mechanism. The analysis revealed distinct patterns of functional clustering at different post-radiation time-points. Apart from their role in secreting collagen and extracellular matrix components for connective tissue integrity, fibroblasts displayed active metabolic functions at the early phase after exposure to ionizing radiation. They transitioned to an immunomodulatory role, particularly T-cell regulation, in the late phase (Fig. 3 c, d and Supplementary Fig. 4b). A trajectory of fibroblast state transitions at different post-radiation times was used for gene enrichment analysis (Supplementary Fig. 4c). Differential gene expression was noted along the fibroblast differentiation trajectory. Metabolism and epithelial protection genes (e.g., ODC1, CRCT1, SLPI) were upregulated in the early phase, while inflammation-related genes (e.g., CCL4, CXCL13, LCN2) were enriched in the late phase (Supplementary Fig. 4d). This indicates that fibroblasts exert protective effects on basal cells in the early phase of RIOM. These protective effects may be associated with metabolic adjustments. Mechanism by which fibroblasts inhibit ferroptosis The mechanism by which fibroblasts inhibit ferroptosis was subsequently investigated. On the ninth day after irradiation, single-cell sequencing identified ferroptosis-related differentially expressed genes in the basal cells. This finding indicated a reduced protective effect of fibroblasts on the basal cells and increased ferroptosis. Among these genes, the spermidine/spermine N1-acetyltranferase 1 (SAT1), ACSL4, and solute carrier family 39 member 14 (SLC39A14) demonstrated the most significant upregulation (Fig. 4 a). Further validation using basal cells co-cultured with fibroblasts revealed that fibroblasts had the most pronounced inhibitory effect on SAT1 expression (Fig. 4 b). Flow cytometry cell sorting of epithelial cells also identified a significant increase in SAT1 expression (Supplementary Fig. 5a, b). Likewise, there was pronounced elevation of SAT1 protein levels in the basal cell lines after exposure to ionizing radiation (Supplementary Fig. 5c). Co-culturing basal epithelial cells with fibroblasts resulted in a rapid, transient decline in SAT1 expression, followed by gradual recovery (Fig. 4 c). Interference with SAT1 reduced lipid peroxidation levels in the basal cells (Supplementary Fig. 5d). This finding suggests that fibroblasts suppress SAT1 expression to inhibit ferroptosis. Mice deficient in SAT1 were used to study the role of SAT1 in radiation-induced ferroptosis in vivo . The mucosal epithelium of SAT1-deficient mice maintained its thickness and resilience against radiation. The basal cells in these mice preserved a high proliferative capacity during the late phase (Fig. 4 d, e). This finding validates that SAT1 contributes to the development of radiation injury. The downstream targets of SAT1 were subsequently examined to identify the mediator responsible for fibroblast-induced suppression of ferroptosis. Studies have shown that arachidonate 15-lipoxygenase (ALOX15) is a downstream molecule of SAT1 that promotes lipid peroxidation of cell membranes and ferroptosis 16, 17 . However, single-cell sequencing and immunofluorescence failed to detect ALOX15 protein expression in the basal cells (Fig. 4 f and Supplementary Fig. 5e). This prompted re-evaluation of the SAT1 downstream pathway in regulating ferroptosis. Protein interaction analysis was performed on all ferroptosis-relevant differentially expressed genes within the basal cells (Fig. 4 g). Among these genes, ACSL4 was identified as the most strongly interacting partner of SAT1. Concurrently, levels of ACSL4 were markedly elevated after radiation exposure (Supplementary Fig. 5f). This finding highlights the important role of ACSL4 in the ferroptosis cascade in basal cells. The relationship between SAT1 and ACSL4 was further elucidated using small interfering RNA (siRNA) to silence their endogenous expressions in the basal cells. Silencing SAT1 significantly suppressed ACSL4 expression, whereas silencing ACSL4 had negligible effects on SAT1 levels (Fig. 4 h). In SAT1-deficient mice exposed to radiation, a marginal increase in ACSL4 expression was identified during the late post-radiation phase (Fig. 4 i). These observations substantiate that SAT1 exerts regulatory control over its downstream target, ACSL4. Finally, the protein expression levels of ACSL4 in basal cells co-cultured with fibroblasts were monitored to examine if fibroblasts exert their protective influence by targeting ACSL4. Basal cells that were co-cultured with fibroblasts displayed a marked decrease in ACSL4 expression. However, this inhibitory effect could not be sustained over time, with ACSL4 expression recovering after an initial decline after radiation exposure (Fig. 4 j). These in vitro and in vivo experiments consistently demonstrate that fibroblasts alleviate ferroptosis in basal cells through suppression of the SAT1-ACSL4 axis in the early phase of RIOM. However, this protective mechanism appears to weaken in the late phase, exacerbating ferroptosis in the basal cells. The role of fibroblast-derived polyamines and JunD in regulating basal cell ferroptosis Fibroblasts exhibit increased metabolic activity in the early phase. They also regulate SAT1, a key enzyme in polyamine metabolism (Fig. 5 a). Hence, it is hypothesized that this regulation may occur through the secretion of polyamines by fibroblasts. Accordingly, polyamine metabolism in fibroblasts was quantified using the scMetabolism analytical tool. There was a relatively high level during the early phase after radiation exposure. This activity decreased over time (Supplementary Fig. 6a). The polyamine content between fibroblasts and basal cells was compared after radiation exposure. When cultivated independently, radiation led to an increase in polyamines within fibroblasts, and a decrease in basal cells and cellular supernatant. However, basal cells that were co-cultured with fibroblasts displayed an initial elevation in spermidine/spermine content, followed by a reduction at later stages (Fig. 5 b and Supplementary Fig. 6b). Similarly, spermidine/spermine was detected in the supernatant of co-cultured cells, with elevated levels at the early stage and significantly-reduce levels at the later stage (Fig. 5 b and Supplementary Fig. 6c). Putrescine levels within the basal cells remained high during the early phase after radiation exposure; these levels decreased in the later stage. Compared to culturing alone, the levels of polyamines within the basal cells were higher when they were co-cultured with fibroblasts (Supplementary Fig. 6d). Ornithine decarboxylase 1 (ODC1), a major gene in the synthesis pathway 18, 19 , was substantially up-regulated after radiation (Fig. 5 e). This was confirmed at the protein level by immunofluorescence imaging. A pronounced reduction in ODC1 expression was observed in fibroblasts derived from late-stage RIOM mice (Fig. 5 e). ODC1 gene knockdown was performed in fibroblasts, followed by co-culture with basal cells before radiation (Supplementary Fig. 6f). The supernatant showed higher levels of spermidine/spermine when ODC1 was expressed in fibroblasts. In contrast, spermidine levels significantly decreased when ODC1 expression was reduced (Fig. 5 f). Immunofluorescence staining for spermidine/spermine in basal cells supported these findings, showing high polyamine concentrations during the early post-radiation phase. The polyamine concentrations were notably reduced in human mucositis specimens and in mice in the later phase, confirming the observed decline in spermidine/spermine levels (Fig. 5 c and Supplementary Fig. 6e). These results validate that fibroblasts regulate the polyamine level in basal cells in a paracrine manner. In agreement with these results, pre-treatment of basal cells with different concentrations of polyamines before radiation exposure showed that spermidine and spermine effectively suppressed the expression of SAT1 and ACSL4 (Fig. 5 g). In addition, when ODC1 expression in fibroblasts was inhibited, the expression of SAT1 and ACSL4 in the basal cells increased following irradiation (Fig. 5 h). This treatment also reduced lipid peroxidation (Supplementary Fig. 6g), and enhanced cell proliferation. Spermidine and spermine demonstrated better efficacy than putrescine (Supplementary Fig. 6h). These findings confirm the function of fibroblast-derived polyamines in regulating the SAT1-ACSL4 axis to protect basal cells against ferroptosis. Immunofluorescence observations further indicated that spermidine and spermine accumulated in both the cytoplasm and the nucleus of basal cells (Fig. 5 c, d). As highly-charged aliphatic polycations, polyamines are capable of binding to negatively-charged nucleic acids. This interaction potentially modulates gene expression by altering transcription factor specificity 20,21 . Because of the observed nuclear localization, it was further hypothesized that polyamines regulate the upstream transcription factors of SAT1. Analysis of single cell RNA-sequencing data through the Single-Cell Regulatory Network Inference and Clustering (Scenic) platform identified JunD and JunB as potential transcription factors that control SAT1. In particular, JunD had a higher regulon activity score (RAS) compared to JunB (Supplementary Fig. 7a). Expression of JunD was upregulated in murine basal cells during the late stage of radiation exposure (Supplementary Fig. 7b). Small interfering RNA-mediated knockdown was subsequently used to investigate the role of JunD in basal cell ferroptosis. Knockdown of JunD resulted in the reduction of lipid peroxidation levels (Supplementary Fig. 7c). Transcription factor binding analysis of the SAT1 promoter using Joint Accessible Sites of Promoters and Regulators (JASPER) identified JunD as binding sequences of SAT1 promoter (Fig. 6a, b). The ChIP analysis identified increased enrichment of JUND at the indicated binding sites in the SAT1 gene promoter, which was further increased upon radiation treatment (Fig. 6c). The protein levels of both SAT1 and ACSL4 were substantially diminished after JunD disruption (Fig. 6d, e). These findings clarify that JunD modulates SAT1 expression. Activation of JUND dictates the onset of ferroptosis in basal cells. Taken together, the results identified sophisticated crosstalk among fibroblast-secreted polyamines, JunD-directed transcriptional regulation, and the regulation of the SAT1-ACSL4 pathway. Polyamines in mucosal protection against ionizing radiation-induced damage To further validate the role of polyamines in mucosal injury, difluoromethylornithine (DFMO), an ODC1 inhibitor, was used to see if polyamines inhibit mucosal injury by regulating basal cell ferroptosis. Administration of DFMO reduced mucosal thickness and weakened basal cell proliferation in the early phase after ionizing radiation exposure (Fig. 6f and Supplementary Fig. 7d). In addition, JunD was prematurely activated in basal cells treated with DFMO after radiation exposure. This was accompanied by a significant up-regulation of ACSL4 (Fig. 6g, h). The potential of spermidine and spermine as a preventive strategy against mucositis was investigated due to their ability to block ferroptosis in basal cells during the early phase of RIOM. Local injections of polyamines were performed prior to the onset of ionizing radiation exposure and continued until day 9 after radiation. Tongue tissues were examined 3 days and 9 days post-radiation (Fig. 6i). Polyamine supplementation suppressed JunD activation in the basal cells until the late phase of radiation (Fig. 6j). This inhibition reduced ACSL4 expression and markedly lowered ferroptosis levels (Fig. 6k and Supplementary Fig. 7e). In order to validate the therapeutic efficacy of polyamines compared to commonly used clinical drugs, two additional groups received KGF-1 (6.25mg/kg) intraperitoneal injection and 0.15% benzydamine (BDM) mouthwash treatment daily from 7 days before radiation to 9 days after the last dose of radiation (Fig. 6I). Spermidine treatment exhibited the best therapeutic effect, surpassing that of the ferroptosis inhibitor LIP-1, spermine, KGF-1 and BDM mouthwash. Immunofluorescence staining revealed that spermidine-treated mucosa exhibited significantly greater proliferation 9 days after radiation (Fig. 6l and Supplementary Fig. 7f). What’s more, spermidine treatment resulted in thicker mucosa, increased basal layer cell structure, and improved continuity in the late post-radiation phase (Fig. 6m). These findings demonstrate that spermidine supplementation effectively inhibits ferroptosis and promotes mucosal proliferation. This therapeutic strategy is potentially promising for mitigating RIOM. Further, we explored the therapeutic potential of polyamines in mitigating damage to other tissues during abdominal radiation. Mice were intraperitoneally injected with saline, spermidine (10mg/kg), and amifostine (AM) (10mg/kg) and subsequently subjected to a single 10Gy abdominal radiation. On the eighth day post-radiation, we observed changes in the colon. We found that the colon length of mice supplemented with spermidine was significantly longer (Supplementary Fig. 8a), indicating reduced inflammatory damage. H&E staining confirmed the protective effect of spermidine against radiation-induced colon injury. Spermidine treatment notably safeguarded the colonic mucosa, reducing the shedding of intestinal villi (Supplementary Fig. 8b). Concurrently, crypt base columnar stem cells maintained a higher proliferative capacity (Supplementary Fig. 8c). To confirm the impact of spermidine on inhibiting ferroptosis, we assessed the protein expression levels of key biomarkers involved in the ferroptosis pathway in radiated colon tissues. We observed a significant decrease in ACSL4 expression levels in radiated colon tissues following spermidine treatment (Supplementary Fig. 8d, e), suggesting that polyamines can suppress ferroptosis to inhibit radiation-induced colon injury. Additionally, the protective effects of spermidine on liver and lung tissues post-irradiation were observed (Supplementary Fig. 8f). The results indicated that spermidine inhibited vacuolar degeneration in the liver following radiation and reduced the degree of alveolar collapse, demonstrating a promising protective effect. In summary, polyamines, through the inhibition of ferroptosis, also provide good protection against radiation-induced injuries to the colon as well as the liver and lungs. Overall, treatment targeting ferroptosis with polyamines holds promise in minimizing normal tissue damage caused by radiation therapy. Discussion Prevention and treatment of RIOM must address the problem of epithelial ablation caused by massive basal cell death 22 . However, the mechanisms and processes behind basal cell death caused by ionizing radiation remain largely unknown. In this study, scRNA-seq, functional analysis, and in vitro and in vivo experiments were used to demonstrate that ferroptosis is the major cell death pathway of oral epithelial basal cells. More importantly, the study focused on the crosstalk between fibroblasts and basal cells. The study identified that polyamines produced by ODC1 activation in fibroblasts are absorbed by basal cells during the early radiation stage. These polyamines bind to JUND in the basal cells and inhibit expression of the SAT1-ACSL4 signaling pathway. This mechanism protects basal cells from ferroptosis. The focus on prevention in RIOM treatment led to the use of polyamines to prevent mucosal injury. This strategy produced promising results, and highlights the potential of polyamines for wide-ranging clinical applications. Previous studies have suggested that strong ionizing radiation induces DNA double-strand breaks. This causes cell cycle arrest, senescence, and different modes of cell death, including apoptosis, necrosis, autophagy, and mitotic catastrophe. Apart from direct DNA damage, ionizing radiation can produce ROS such as hydroxyl radicals and hydrogen peroxide through radiolysis of cellular water and stimulation of oxidases. These ROS species may damage nucleic acids, proteins, and lipids. Indirect damage is the primary mode of cell death 4,23 . Through single-cell sequencing and in vitro and in vivo experiments, ferroptosis was identified as the primary form of cell death in the basal layer of oral epithelial cells after their exposure to ionizing radiation. Ferroptosis is a unique form of iron-dependent cell death driven by the accumulation of lipid peroxides, due to the imbalance of the cellular antioxidant system 24,25 . Ferroptosis is distinct from apoptosis, necroptosis, and pyroptosis, which have specific functions during development and immune processes and respond to particular physiological stimuli 26,27 . Results of the present study provide a possible explanation for the occurrence of mucosal damage following massive cell death events induced by ionizing radiation. Radiation may cause a widespread elevation of ROS, and ferroptosis can propagate through ROS waves at a constant rate over long distances among human cells. This makes cell populations a medium for ROS propagation 28,29 . The rupture of cell membranes following ferroptosis further disrupts the barrier formed by epithelial cells, allowing invasion of microorganisms and infiltration of harmful substance. This subsequently triggers an inflammatory response of monocytes and the release of pro-inflammatory cytokines that exacerbates tissue damage 30,31 . However, the specific mechanisms by which ionizing radiation induces ferroptosis in oral mucosal epithelial cells remain largely unknown. Single-cell RNA-sequencing analysis performed in the present work revealed the important roles of ferroptosis-related molecular targets (such as SAT1, ACSL4, JunD, and PTGS2) in promoting basal cell death. These findings establish a foundation for understanding the pathogenesis of RIOM, and offer novel molecular biomarkers for prognostic assessment in individuals who are exposed to ionizing radiation. Radiation-induced cell death is a complex biological process involving a dynamic sequence of interrelated events throughout the mucosa. These events ultimately target highly proliferative epithelial basal cells 2 . Time-series analysis using sc-RNA seq was used to track the dynamic process of ferroptosis in basal cells following radiation stress over time. Unexpectedly, ferroptosis in basal cells was not prominently associated with mucosal thickening during the early stages of radiation exposure. Concurrently, a substantial increase in the number of fibroblasts within the microenvironment was observed. This finding indicates that the organism is in a compensatory phase. This compensatory mechanism appears to be crucial for inhibiting ferroptosis in basal cells. Understanding this mechanism may aid in developing early intervention strategies against RIOM. Fibroblasts in the epithelial microenvironment play a significant role during the compensatory phase by secreting metabolites to inhibit ferroptosis in the basal cells. Recent single-cell analyses have shown that fibroblasts undergo transcriptional changes that resemble the processes seen in cell differentiation. These changes help promote tissue repair during injury, suggesting that fibroblasts adapt their gene expression to support healing 32,33 . Because the functional state of fibroblasts is dynamic during tissue repair, these cells can interact with the epithelium to promote successful resistance to injury 34 . For example, viral transduction with keratinocyte lineage-related transcription factors such as Dnp63a, Grhl2, Tfap2a, and Myc can reprogram fibroblasts in situ within skin wounds. This process generates fibroblast-derived epidermis and enhances re-epithelialization of the wound 35 . This complex cell-to-cell communication includes not only traditional protein-ligand-receptor pairs, but also small metabolites that diffuse within the microenvironment. Metabolic interactions are important regulators in tissue repair 36 . Specifically, ferroptosis is regulated by multiple metabolic pathways, including redox balance, iron handling, mitochondrial activity, and metabolism of amino acids, lipids, and sugars 10 . Metabolic regulation is a key mechanism for modulating cell susceptibility to ferroptosis 37–39 . Fibroblasts act as signaling contributors to the epithelial microenvironment by secreting metabolites that regulate epithelial cell function 33 . In this work, fibroblasts were found to secrete polyamines that regulate basal cells after radiation damage. This discovery uncovers a new mechanism by which fibroblasts maintain the compensatory capacity of basal cells through metabolic support. In this work, ionizing radiation was found to activate ODC1 in fibroblasts, causing them to produce polyamines. These polyamines are then absorbed and accumulated in basal cells. Ornithine decarboxylase 1 is a key enzyme in polyamine synthesis. For example, the aryl hydrocarbon receptor binds to the xenobiotic response element sequence in the ODC1 promoter region. This binding promotes the transcription of ODC1. As a result, downstream metabolites such as putrescine, spermidine, and spermine are produced. These metabolites can inhibit pyroptosis in macrophages 40 . Polyamines regulate arginine metabolism and enhance the antioxidant capacity of glutathione 41–44 . This study identified a direct link between polyamine metabolism and ferroptosis and suggested that using polyamines to inhibit ferroptosis may be a new way to protect against radiation damage. This discovery has important implications for treating not only RIOM, but also radiation-induced damage to the skin, intestines, and other tissues. Polyamines have strong affinity for nucleic acids, enabling them to regulate fundamental cellular processes such as DNA replication, gene transcription regulation, and control of translation elongation and termination 45 . Consistent with the findings of those studies, JUND was identified in the present work as a transcription factor upstream of the SAT1-ACSL4 signaling pathway. Polyamines can regulate JUND expression at the transcriptional level and mediate interactions between fibroblasts and basal cells. This helps to inhibit ferroptosis in basal cells. These findings reveal a new compensatory function of polyamines produced by fibroblasts and emphasize the importance of the inhibited JUND-SAT1-ACSL4 signaling axis in promoting radiation-induced oral mucosal injury. The present study identified that under radiation stress, fibroblasts in the microenvironment inhibit ferroptosis in basal cells by producing polyamines. This discovery introduces a safer and more effective therapeutic approach for RIOM. In the context of RIOM, polyamine supplementation may support the natural healing process. Overall, these results increase the potential for leveraging the body’s own protective mechanisms for stress protection in clinical settings. These insights may also contribute to the design and development of treatment for radiation-related diseases. Despite the promising findings, there are limitations to this study. One such limitation is the unclear mechanism by which polyamines are transported from fibroblasts to basal cells, whether through passive diffusion or the release of extracellular vesicles 46 . Understanding this process may result in improved strategies for delivering polyamines to enhance mucosal protection. Another area for future research is the interaction between polyamine metabolism and the oral microbiome. This has not been investigated in the present work. Future experiments should examine how specific microorganisms influence polyamine production and their impact on radiation-induced mucosal injury. This may result in novel treatment regimens that combine microbiome modulation with polyamine supplementation to improve therapeutic outcomes. Addressing these limitations in future research will be essential for optimizing polyamine-based therapy and fully understanding fibroblast-basal cell interactions during radiation-induced injury. Methods The methods in the present study are provided in Supplementary Information. Declarations Data availability The main data supporting the results of this study are available within the paper and its Supplementary Information. The raw and analyzed datasets generated during the study are available for research purposes from the corresponding author on reasonable request. Acknowledgment This work was supported by the National Natural Science Foundation of China (no. 82325012 to Lina Niu). Author contributions W.Y., K.J., K.W., X.L., Y.M., and L.N. designed and organized experiments. W.Y., K.W., X.L., and X. H. performed experiments and analyzed data. W.Y., K.J., K.W., Q.W., M.W., J.W., F.T., and L.N. prepared figures and edited manuscript. W.Y., Q.W., J.W., Q.L., M.S., and L.N. wrote the paper. L.N. conceived, supervised, and directed the study. Declare of interest The authors do not declare competing financial interests. 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Supplementary Files GA.png ExtendedDataFig.docx SupplenmentalinformationNaturemedicine.docx Metabolic support protects oral mucosa from ferroptosis in radiation-induced mucositis Cite Share Download PDF Status: Published Journal Publication published 08 Dec, 2025 Read the published version in Nature Communications → Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5617929","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":389041634,"identity":"ef6785f5-4cf5-45fe-ba78-bf9c0a6425af","order_by":0,"name":"Li-na 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15:11:02","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5617929/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5617929/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41467-025-67214-5","type":"published","date":"2025-12-08T05:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":75147000,"identity":"3e3dea5f-2531-4803-8e77-9f86c5bd9538","added_by":"auto","created_at":"2025-01-31 07:13:50","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":10588927,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIonizing radiation causes mucosal thinning and marked basal cell reduction\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(a) Glossal tissue specimens were collected from patients with head and neck malignant tumors undergoing surgery after radiotherapy.\u003c/p\u003e\n\u003cp\u003e(b) Changes in the glossal mucosa observed with hematoxylin and eosin (H\u0026amp;E) staining. ▲ represents the boundary between the epithelium and stroma, * represents disruption of epithelial continuity. Scale bar, 25 mm.\u003c/p\u003e\n\u003cp\u003e(c) Immunohistochemistry images using Keratin 19 (KRT19) show changes in epithelial thickness and morphology. Five random images per mouse were analyzed. Representative images are shown. Scale bar, 25 mm.\u003c/p\u003e\n\u003cp\u003e(d) Quantitative analysis of proliferating cell nuclear antigen (PCNA)-positive basal cells. Scale bar, 25 mm. (e) Schematic diagram for the construction of an animal model of radiation-induced oral mucositis. (f) H\u0026amp;E staining of mouse tongues before and after radiation. The dotted line indicates the basal membrane. The boxed area represents disruption of mucosal continuity (n = 10). Three random images per mouse were analyzed. Representative images are shown. Scale bar, top, 100 μm; bottom, 50 μm.\u003c/p\u003e\n\u003cp\u003e(g) Sections from normal murine glossal mucosa and ulcerated areas near irradiated glossal tissues (n = 6). Scale bar, 100 μm.\u003c/p\u003e\n\u003cp\u003e(h) PCNA immunofluorescence staining of murine glossae before and after exposure to ionizing radiation (n = 6). Five random images per mouse were analyzed. Representative images are shown. Scale bar, 100 μm. (i) Uniform manifold approximation and projection (UMAP) plots and annotations by major cell type from single-cell transcriptome analysis of murine glossal mucosa before and after radiation.\u003c/p\u003e\n\u003cp\u003e(j) Pie charts showing the percentages of different cell types before and after radiation.\u003c/p\u003e\n\u003cp\u003e(k) Structure of the oral mucosal epithelium and major cell types. Scale bar, 50 μm.\u003c/p\u003e\n\u003cp\u003e(l) Pie charts showing the percentages of different cell types before and after radiation.\u003c/p\u003e\n\u003cp\u003e(m) Immunofluorescence depicts major cell types in murine glossal mucosa before and after radiation (n = 3). Three random images per mouse were analyzed. Representative images are shown. Scale bar, 50 μm. Data represent means ± standard deviations. ****p \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-5617929/v1/268d72b0ba52027a6be7219b.png"},{"id":75147001,"identity":"cd3a9043-a59a-44be-bc26-3a9219abd02e","added_by":"auto","created_at":"2025-01-31 07:13:50","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":12309989,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIonizing radiation induces ferroptosis in basal epithelial cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(a) Heat map showing Add Module Scores for apoptosis, ferroptosis, pyroptosis, and necrosis in epithelial cells.\u003c/p\u003e\n\u003cp\u003e(b-c) C57BL/6 mice were treated with Liproxstatin-1 (LIP-1) (5 mg/kg) locally by multipoint injection into the tongue, starting 7 days before radiation and continuing for 9 days post-radiation. (b) Immunofluorescence showing expression of prostaglandin-endoperoxide synthase 2 (PTGS2) in murine epithelial cells (n = 6). Five random images per mouse were analyzed. Representative images are shown. Scale bar, 100 μm. (c) H\u0026amp;E staining images were taken before radiation and 9 days post-radiation to display changes in mucosal thickness and morphology (n = 10). ▲ represents the boundary between the epithelium and stroma, * represents disruption of epithelial continuity. Three random images per mouse were analyzed. Representative images are shown. Scale bar, 100 μm.\u003c/p\u003e\n\u003cp\u003e(d) Heat map showing Add module scores for ferroptosis in basal cell, keratinized cell, granular cell, and spinous cell subtypes of epithelial cells.\u003c/p\u003e\n\u003cp\u003e(e) C57BL/6 mice were treated with LIP-1 (5 mg/kg) locally by multipoint injection into the tongue, starting 7 days before radiation and continuing for 9 days post-radiation. Representative images of ACSL4 expression in basal cells of murine glossal tissues before radiation and 9 days post-radiation (n = 6). Five random images per mouse were analyzed. Representative images are shown. Scale bar, 100 μm.\u003c/p\u003e\n\u003cp\u003e(f) Representative images of ACSL4 expression in basal cells of normal human tissue and mucositis tissue (n = 6). Scale bar, 25 mm.\u003c/p\u003e\n\u003cp\u003e(g) Representative images of PTGS2 expression in basal cells of murine glossal tissues at different time points after radiation (n = 6). Five random images per mouse were analyzed. Representative images are shown. Scale bar, 100 μm.\u003c/p\u003e\n\u003cp\u003e(h) Representative H\u0026amp;E staining of the morphology and thickness of murine epithelial mucosa at different time-points after radiation (n = 10). ▲ represents the boundary between the epithelium and stroma, * represents disruption of epithelial continuity. Three random images per mouse were analyzed. Representative images are shown. Scale bar, left, 100 μm; right, 50 μm.\u003c/p\u003e\n\u003cp\u003e(i) Chord diagram showing up-regulated genes and gene ontology pathways in basal cells when comparing normal tissue with tissue 3 days post-radiation, and comparing tissue 3 days post-radiation with tissue 9 days post-radiation.\u003c/p\u003e\n\u003cp\u003eData represent the means ± standard deviations. \u003csup\u003ens\u003c/sup\u003ep \u0026gt; 0.05, ****p \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-5617929/v1/77011769e4d05abb848d5c24.png"},{"id":75147082,"identity":"85abfe3b-2376-4f49-a90e-8f5530e78375","added_by":"auto","created_at":"2025-01-31 07:21:50","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":7085307,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFibroblasts provide protective support for basal cells in the early phase of radiation-induced mucositis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(a) Representative images showing changes in the number of fibroblasts within the murine mucosa and their relationship with basal cells via immunofluorescence staining (n = 6). Five random images per mouse were analyzed. Scale bar, 100 μm.\u003c/p\u003e\n\u003cp\u003e(b) Representative images showing changes in the number of fibroblasts within normal human glossal tissue and glossal mucositis tissue via immunofluorescence staining. Scale bar, 25 mm.\u003c/p\u003e\n\u003cp\u003e(c) UMAP (left) and bar graphs (right) for different fibroblast subtypes. The bar graph shows the proportion of different fibroblast subpopulations.\u003c/p\u003e\n\u003cp\u003e(d) Heat map showing Standardized Gene Set Enrichment analysis scores for selected gene ontology pathways in each fibroblast subtype.\u003c/p\u003e\n\u003cp\u003e(e) Circos plot showing the analysis of directed interaction intensity between fibroblasts and epithelial cell subgroups at different time points before and after radiation.\u003c/p\u003e\n\u003cp\u003e(f) Schematic of co-culture between fibroblasts (bottom) and basal cells (top).\u003c/p\u003e\n\u003cp\u003e(g) Western blot analysis of PCNA expression in basal cell lines 24 hours after exposure to 8 Gy radiation with or without co-culture with fibroblasts (n = 3).\u003c/p\u003e\n\u003cp\u003e(h) Total iron concentration in basal cells 24 hours after exposure to 8 Gy radiation, with or without co-culture with fibroblasts (n = 6).\u003c/p\u003e\n\u003cp\u003e(i) Assessment of lipid peroxidation in basal cell lines 24 hours after exposure to 8 Gy radiation with or without co-culture with fibroblasts. Bar graph showing the relative fold change in radiation-induced lipid peroxidation in cells stained with C11-BODIPY (n = 6).\u003c/p\u003e\n\u003cp\u003eData represent the mean ± SD. \u003csup\u003ens\u003c/sup\u003ep \u0026gt; 0.05; *p \u0026lt; 0.05; **p \u0026lt; 0.01; ***p \u0026lt; 0.001; ****p \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-5617929/v1/37fd77be85de84c1e61f0cde.png"},{"id":75147009,"identity":"b0202f41-bffa-4cd4-a10e-50501f9eb2df","added_by":"auto","created_at":"2025-01-31 07:13:50","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":9291113,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFibroblasts inhibit ferroptosis in basal cells through suppression of the SAT1-ACSL4 pathway\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(a) Table showing changes in ferroptosis gene expression in basal cells before and 9 days after radiation.\u003c/p\u003e\n\u003cp\u003e(b) qRT-PCR analysis of SAT1, ACSL4, and SLC39A14 inhibition rate in basal cell lines with or without fibroblasts for 24 hours after exposure to 8 Gy radiation.\u003c/p\u003e\n\u003cp\u003e(c) Western blot analysis of SAT1 expression in basal cells cultured with or without fibroblasts for 24 hours and 48 hours after exposure to 8 Gy radiation. (n = 3).\u003c/p\u003e\n\u003cp\u003e(d) Representative H\u0026amp;E staining of the oral mucosal epithelium of SAT1\u003csup\u003e+/+ \u003c/sup\u003eand SAT1\u003csup\u003e-/- \u003c/sup\u003emice before and 9 days after radiation (n = 6). ▲ represents the boundary between the epithelium and stroma, * represents disruption of epithelial continuity. Scale bar, top, 100 μm; bottom, 50 μm.\u003c/p\u003e\n\u003cp\u003e(e) Immunofluorescence staining for PCNA expression in basal cells of SAT1\u003csup\u003e+/+\u003c/sup\u003e and SAT1\u003csup\u003e-/- \u003c/sup\u003emurine tongues before and 9 days after radiation (n = 6). Five random images per mouse were analyzed. Representative images are shown. Scale bar, 50 μm.\u003c/p\u003e\n\u003cp\u003e(f) Immunofluorescence showing the location and expression of ALOX15 in wild-type mice at different time-points before and after radiation. Scale bar, 50 μm.\u003c/p\u003e\n\u003cp\u003e(g) String network analysis of interactions among ferroptosis-related genes in basal cells.\u003c/p\u003e\n\u003cp\u003e(h) Western blot analysis of SAT1 and ACSL4 expression in basal cell lines with or without interference of the ACSL4 gene (n = 3).\u003c/p\u003e\n\u003cp\u003e(i) Immunofluorescence staining for ACSL4 expression in basal cells of SAT1\u003csup\u003e+/+\u003c/sup\u003e and SAT\u003csup\u003e1-/- \u003c/sup\u003emice before and 9 days after radiation (n = 3). Five random images per mouse were analyzed. Representative images are shown. Scale bar, 50 μm.\u003c/p\u003e\n\u003cp\u003e(j) Western blot analysis of ACSL4 expression in basal cells cultured with or without fibroblasts for 24 hours and 48 hours after exposure to 8 Gy radiation (n = 3).\u003c/p\u003e\n\u003cp\u003eData represent the mean ± SD. \u003csup\u003ens\u003c/sup\u003ep \u0026gt; 0.05; *p \u0026lt; 0.05; **p \u0026lt; 0.01; ***p \u0026lt; 0.001; ****p \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-5617929/v1/bb9d108d947fb8c8e6a6e1af.png"},{"id":75147007,"identity":"45a36ead-e2c6-484e-ba98-b26a4f356d20","added_by":"auto","created_at":"2025-01-31 07:13:50","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":4742067,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFibroblasts produce polyamines that inhibit ferroptosis in basal cells in the early phase of post-ionizing radiation exposure\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(a) Schematic of the polyamine metabolic pathway.\u003c/p\u003e\n\u003cp\u003e(b) Levels of spermidine produced by basal cells, fibroblasts, and cellular supernatant before and after radiation, measured by mass spectrometry when cultured with or without fibroblasts (n = 6).\u003c/p\u003e\n\u003cp\u003e(c) Representative immunofluorescence images showing spermidine and spermine content in basal cells of murine glossal tissues before and after radiation (n = 6). Five random images per mouse were analyzed. Scale bar, 100 μm.\u003c/p\u003e\n\u003cp\u003e(d) Heat map from genes regulating polyamines in fibroblasts at different time-points before and after radiation.\u003c/p\u003e\n\u003cp\u003e(e) Quantification of ODC1 expression levels in fibroblasts of mice at different time-points before and after radiation (n = 6). Five random images per mouse were analyzed. Representative images are shown. Scale bar, 100 μm.\u003c/p\u003e\n\u003cp\u003e(f) Levels of spermidine and spermine produced by cellular supernatant measured by mass spectrometry when cultured without radiation, or 24 hours after exposure to 8 Gy radiation, with or without ODC1 siRNA fragments (n = 6).\u003c/p\u003e\n\u003cp\u003e(g) Western blot analysis of SAT1/ACSL4 expression in basal cell lines without radiation, or 24 hours after exposure to 8 Gy radiation, with or without supplementation of spermidine and spermine (n = 3).\u003c/p\u003e\n\u003cp\u003e(h) Western blot analysis of SAT1/ACSL4 expression in basal cell lines without radiation, or 24 hours after exposure to 8 Gy radiation, with or without ODC1 siRNA fragments (n = 3).\u003c/p\u003e\n\u003cp\u003eData represent the mean ± standard deviation.\u003csup\u003e ns\u003c/sup\u003ep \u0026gt; 0.05; *p \u0026lt; 0.05; **p \u0026lt; 0.01; ***p \u0026lt; 0.001; ****p \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-5617929/v1/5fa9d20c45fbf0b25eb27efb.png"},{"id":75147008,"identity":"c3423c04-7518-4305-b9aa-1e04ce77d78e","added_by":"auto","created_at":"2025-01-31 07:13:50","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":10857271,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePolyamines inhibit ferroptosis and reverse mucosal damage in basal cells through activation of the nuclear transcription factor JunD\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(a) Consensus sequence for the JunD binding motif.\u003c/p\u003e\n\u003cp\u003e(b) Presumed binding site of JunD in the promoter region of the SAT1 gene.\u003c/p\u003e\n\u003cp\u003e(c) Fold enrichment of JUND binding in SAT1 gene promoter upon radiation treatment normalized to IgG control (n = 3).\u003c/p\u003e\n\u003cp\u003e(e-f) Western blot analysis of JunD, SAT1, and ACSL4 expression in basal cell lines, with or without interference of the JunD gene (n = 3).\u003c/p\u003e\n\u003cp\u003e(g-h) C57BL/6 mice received vehicle or DFMO (300 mg/kg/day) locally by multipoint injection into the tongue, with continuous treatment starting 7 days prior to radiation and continuing for 9 days post-radiation. (g)H\u0026amp;E staining images were taken 3 days post-radiation to display changes in mucosal thickness and morphology (n = 10). ▲ represents the boundary between the epithelium and stroma, * represents disruption of epithelial continuity. Three random images per mouse were analyzed. Representative images are shown. Scale bar, 100 μm. (g) Immunofluorescence shows the expression of JunD in basal cells without radiation and basal cells 3 days post-radiation (n = 6). Five random images per mouse were analyzed. Representative images are shown. Scale bar, 100 μm. (h) Immunofluorescence showing expression of ACSL4 in basal cells without radiation and basal cells 3 days post-radiation (n = 6). Five random images per mouse were analyzed. Representative images are shown. Scale bar, 100 μm.\u003c/p\u003e\n\u003cp\u003e(i) Schematic diagram of the timing of Vehicle, LIP-1 (5mg/kg), KGF-1 (6.25 mg/kg), spermidine, and spermine (8.4mg/kg) injections and 0.15% benzdamine mouthwash in mice.\u003c/p\u003e\n\u003cp\u003e(j-m) C57BL/6 mice were treated with Vehicle, LIP-1, KGF-1, spermidine, and spermine by intraperitoneal injection and 0.15% benzydamine mouthwash for 10 consecutive days, starting 7 days before radiation and continuing for 9 days post-radiation. (j) Immunofluorescence shows the expression of JunD in basal cells 9 days after radiation. Five random images per mouse were analyzed. Representative images are shown. Scale bar, 100 μm (n = 6). (k) Immunofluorescence showed the expression of ACSL4 in basal cells 9 days after radiation (n = 6). Representative images are shown. Scale bar, 100 μm. (l) Quantification of PCNA+ basal cells 9 days after radiation (n = 6). Five random images per mouse were analyzed. Representative images are shown. Scale bar, 100 μm. (m) Comparison of H\u0026amp;E staining and epithelial thickness of mouse mucosa 9 days after radiation (n = 10). ▲ represents the boundary between the epithelium and stroma, * represents disruption of epithelial continuity. Three random images per mouse were analyzed. Representative images are shown. Scale bar, 100 μm. Data represent the mean ± standard deviation. *p \u0026lt; 0.05; **p \u0026lt; 0.01; ***p \u0026lt; 0.001; ****p \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"image6.png","url":"https://assets-eu.researchsquare.com/files/rs-5617929/v1/f94fccc7b43c356c74c8c52e.png"},{"id":100295560,"identity":"d22bf786-1d0c-4139-af4f-080ab9fde390","added_by":"auto","created_at":"2026-01-15 08:06:36","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":55480428,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5617929/v1/d3671b74-5453-4c18-a362-3ddd88b6b0b6.pdf"},{"id":75147081,"identity":"c741799a-19b6-42f6-8093-5b54772de71d","added_by":"auto","created_at":"2025-01-31 07:21:50","extension":"png","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1247546,"visible":true,"origin":"","legend":"","description":"","filename":"GA.png","url":"https://assets-eu.researchsquare.com/files/rs-5617929/v1/f7785d82a76d6423479ac6e0.png"},{"id":75147003,"identity":"217cc9fb-5c47-4311-8004-0016031f4f54","added_by":"auto","created_at":"2025-01-31 07:13:50","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":8788059,"visible":true,"origin":"","legend":"","description":"","filename":"ExtendedDataFig.docx","url":"https://assets-eu.researchsquare.com/files/rs-5617929/v1/2e1215b96bdf7b50ac1c0869.docx"},{"id":75147005,"identity":"d4367eb6-f107-4057-96c1-a05168d47598","added_by":"auto","created_at":"2025-01-31 07:13:50","extension":"docx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":136591,"visible":true,"origin":"","legend":"Metabolic support protects oral mucosa from ferroptosis in radiation-induced mucositis","description":"","filename":"SupplenmentalinformationNaturemedicine.docx","url":"https://assets-eu.researchsquare.com/files/rs-5617929/v1/e927faf49a72ab7cec9b7e07.docx"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Metabolic support protects oral mucosa from ferroptosis in radiation-induced mucositis","fulltext":[{"header":"Highlights","content":"\u003cp\u003e1. Basal epithelial cell ferroptosis is crucial for radiation-induced epithelial ablation.\u003c/p\u003e\u003cp\u003e2. Fibroblast-epithelial cell interactions promote compensatory protection by inhibiting ferroptosis.\u003c/p\u003e\u003cp\u003e3. Fibroblast-derived polyamines drive the compensatory mechanisms.\u003c/p\u003e\u003cp\u003e4. Polyamine supplementation inhibits ferroptosis and prevents radiation-induced mucosal injury.\u003c/p\u003e"},{"header":"Introduction","content":"\u003cp\u003eRadiation-induced oral mucositis (RIOM) is a common side effect of radiotherapy for head and neck cancer\u003csup\u003e1\u003c/sup\u003e. It is usually manifested as mucosal atrophy or peeling, and is accompanied by severe pain and loss of mucosal function. This condition results from the death of epithelial cells after their exposure to ionizing radiation\u003csup\u003e2\u0026ndash;4\u003c/sup\u003e. Current research suggests that ionizing radiation can directly cause DNA breaks, and activate reactive oxygen species (ROS) that indirectly damage biomolecules\u003csup\u003e5\u003c/sup\u003e. These damages result in the upregulation of apoptosis-related cytokines and activation of downstream signaling pathways, ultimately triggering the loss of mucosal continuity\u003csup\u003e1,2\u003c/sup\u003e. The limited effectiveness of anti-apoptotic drugs in treating RIOM\u003csup\u003e3\u0026ndash;5\u003c/sup\u003e suggests the involvement of other forms of cell death during disease progression. Thus, exploring the specific mechanisms of epithelial cell death may offer new insights for maintaining the structure and function of mucosa during radiotherapy.\u003c/p\u003e \u003cp\u003eIn this study, the authors resorted to single-cell RNA sequencing (scRNA-seq) and confirmed that ferroptosis is the primary mode of epithelial cell death. Currently, ferroptosis is primarily inhibited by blocking the propagation of lipid peroxyl radicals and chelating intracellular iron pools\u003csup\u003e6,7\u003c/sup\u003e. We found that using the specific ferroptosis inhibitor Liproxstatin-1 (LIP-1) effectively inhibits radiation-induced epithelial cell death and alleviates mucosal damage. However, this approach has limitations from a safety perspective, and may not be the optimal solution for treating RIOM\u003csup\u003e8\u0026ndash;10\u003c/sup\u003e. Therefore, further investigations of epithelial cell ferroptosis are required to identify new pathways for mucosal protection. Surprisingly, at the early stage post-radiation, we observed that epithelial cell death was not significant, with even enhanced proliferation causing increase in the thickness of the oral mucosa. This suggests that the body might be in a compensatory and adaptive state to respond to radiation stimulation. Based on this discovery, we propose to amplify the inherent early-stage protective mechanism to maintain epithelial cells in an adaptive state long-term post-radiation, thereby preventing cell death.\u003c/p\u003e \u003cp\u003eThe objective of this study was to elucidate the specific mechanisms underlying mucosal damage in RIOM by investigating the death modes of mucosal epithelial cells under ionizing radiation. Specifically, it was discovered that early after radiation, the body can leverage intercellular communication within the microenvironment to provide metabolic support for the integrity of mucosal epithelia. Further, the utilization of metabolic supporters can effectively prevent RIOM. This discovery sheds light on the self-protective mechanisms of organisms under radiation stress via intercellular communication. Based on this discovery, it has significant potential to prevent radiation-related diseases and improve the prognosis of radiotherapy through amplifying the inherent self-protective mechanisms.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eThe effect of ionizing radiation on epithelial thinning and basal cell count\u003c/h2\u003e \u003cp\u003eThe impact of radiation on oral mucosa was investigated using biopsies of human tongues affected by RIOM (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). Significant epithelial thinning and discontinuity were observed (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb, c). Reduced basal cell proliferation was also observed (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed). For \u003cem\u003ein vivo\u003c/em\u003e modeling, C57BL/6 mice received 18 Gy cranial radiation. Onset of oral ulceration occurred on the 9th day after radiation (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ee). Histological examination confirmed the manifestation of mucositis in the murine specimens that was similar to the human tongue mucosa specimens (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ef, g). Suppressed basal cell proliferation was also evident (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eh).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eSubsequently, scRNA-seq was performed on murine tongue mucosa to analyze cellular changes before and after radiation. After quality control to remove low-quality cells, the dataset included\u0026thinsp;~\u0026thinsp;28,189 cells. Graph-based clustering and cell marker annotation identified 9 major cell types: epithelial cells, fibroblasts, endothelial cells, immune cells, Schwann cells, smooth muscle cells, pericytes, endo-pericytes, and myocytes (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ei and Supplementary Fig.\u0026nbsp;1a, b). Transcriptomic analysis documented differential expression of cell-defining genes for the cell compartments, which were generally conserved across mucosal sites (Supplementary Fig.\u0026nbsp;1c). There was alteration in cell composition after radiation, with a sharp decline in the number of epithelial cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ej). Because of the heterogeneity of oral epithelial cells, they were classified into 4 subpopulations: basal, spinous, granular cells, and corneocytes (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ek) \u003csup\u003e11\u003c/sup\u003e. The basal epithelial cells exhibit stem cell traits, and were the most prevalent both before and after radiation (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003el, Supplementary Fig.\u0026nbsp;1d, e). Immunohistochemistry confirmed a drastic reduction in basal cells after radiation (Supplementary Fig.\u0026nbsp;1f). Finally, histological imaging confirmed the presence of the 4 major compartments-epithelial cells, fibroblasts, endothelial cells, and immune cells within the oral mucosal tissues. This observation provided insight into the local tissue architecture (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003em). Taken together, the findings demonstrate that ionizing radiation significantly thins the oral mucosa. Epithelial cells, especially basal cells, are severely depleted.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eThe impact of ionizing radiation on death in basal epithelial cells\u003c/h3\u003e\n\u003cp\u003eDifferent cell death pathways were examined using Add Module Score to evaluate the cause of epithelial cell depletion. Ferroptosis was markedly increased after radiation exposure. Apoptosis was also detected in the epithelial cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). Transmission electron microscopy of irradiated tongue tissue revealed ferroptotic features such as mitochondrial shrinkage and increased membrane density (Supplementary Fig.\u0026nbsp;2a). Gene set variation analysis (GSVA) identified altered iron ion homeostasis in the irradiated cells (Supplementary Fig.\u0026nbsp;2b). The ferroptosis marker prostaglandin-endoperoxide synthase 2 (PTGS2) was significantly upregulated in epithelial cells after irradiation (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb). The ferroptosis inhibitor LIP-1 suppressed PTGS2 expression and increased mucosal thickness (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb, c). Lipid peroxide buildup, a hallmark of ferroptosis, was evident and notably reduced by the ferroptosis inhibitor ferrostatin-1 (Supplementary Fig.\u0026nbsp;2c). These results indicate that ferroptosis is the primary cause of oral mucosal epithelial cell depletion after ionizing radiation.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAnalysis of ferroptosis gene set scores was subsequently conducted for different epithelial cell subtypes. Basal cells, the predominant epithelial subtype with stem cell characteristics, had the highest ferroptosis gene set scores (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed and Supplementary Fig.\u0026nbsp;2d). Immunofluorescence for acyl-CoA synthetase long chain family member 4 (ACSL4), the major enzyme in polyunsaturated fatty acid activation and ferroptosis induction\u003csup\u003e12\u003c/sup\u003e, showed elevated expression levels in basal cells from murine and human oral mucositis lesions (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ee, f). Similarly, LIP-1 inhibited the expression of ACSL4 in mouse basal cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ee). \u003cem\u003eIn vitro\u003c/em\u003e experiments confirmed reduced proliferation and increased mortality rates of post-irradiated basal cells (Supplementary Fig.\u0026nbsp;2d-f). The effects of ferroptosis inhibitor ferrostatin-1, apoptosis inhibitor Z-Val-Ala-Asp fluoromethylketone (Z-VAD-fmk), necroptosis inhibitor necrostatin-1, and pyroptosis inhibitor Belnacasan (VX-765) on the survival of post-irradiated basal cells were further evaluated. Treatment with ferrostatin-1 or Z-VAD-fmk partially restored the clonogenic survival rate of basal cells that was reduced by radiation exposure. The ferroptosis inhibitors demonstrated a more pronounced restorative effect than other cell death inhibitors (Supplementary Fig.\u0026nbsp;2g). There was a significant increase in labile iron and ROS levels within the basal cells; these features were substantially reduced by ferrostatin-1 treatment (Supplementary Fig.\u0026nbsp;2h, i). Collectively, these data support ferroptosis in basal cells as a primary component of radiation-induced epithelial cell death.\u003c/p\u003e\n\u003ch3\u003ePotential early-stage protective phenomenon in RIOM\u003c/h3\u003e\n\u003cp\u003eMurine tongue mucosal specimens were analyzed at sequential time-points to elucidate post-radiation basal cell ferroptosis. Expression of PTGS2, a ferroptosis biomarker\u003csup\u003e13\u003c/sup\u003e, did not rise markedly in basal cells during early radiation phase, but increased in the late phase (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eg). Importantly, enhanced mucosal thickness and robust basal cell proliferation without DNA damage suggested early phase hyperactivity in RIOM (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eh and Supplementary Fig.\u0026nbsp;3a, b). Single-cell sequencing of oral mucosal tissues was conducted at different time-points after radiation (Supplementary Fig.\u0026nbsp;3c, d). There was an increase in basal cell numbers in the early-phase, followed by a decline in the late-phase (Supplementary Fig.\u0026nbsp;3e). In agreement with these results, GSVA of basal cells indicated active enzymatic and protein folding functions in the early-phase of post-radiation. There was also regulation of the cell cycle to enhance proliferative and suppress apoptosis (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ei and Supplementary Fig.\u0026nbsp;3f). In the late stage, the decline in protein folding, wound healing processes, and basement membrane functions was observed (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ei and Supplementary Fig.\u0026nbsp;3g). These results suggest that a protective mechanism may be activated during the early phase of RIOM. This protective mechanism promotes compensatory proliferation of basal cells to counteract adverse stimuli.\u003c/p\u003e\n\u003ch3\u003eEffect of microenvironment factors on basal cell ferroptosis\u003c/h3\u003e\n\u003cp\u003eBasal cells are crucial for epithelium renewal and injury response. They are controlled by localized cues within their microenvironment\u003csup\u003e14\u003c/sup\u003e. Understanding the cellular and molecular environment that signals post-injury repair is essential for appreciating basal cell homeostasis and mucosal regeneration after injury\u003csup\u003e15\u003c/sup\u003e. Accordingly, the cellular components of the microenvironment were analyzed to investigate the protective mechanisms for basal cells in the early phase of RIOM. Notably, in irradiated mouse and human tongue mucosal specimens, fibroblasts were closely located around basal cells, with numbers peaking in the early phase and fibrosis enhanced in the late phase (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea, b and Supplementary Fig.\u0026nbsp;4a). There was sustained high-level fibroblast-basal cell interaction during the early phase. This activity declined markedly in the late phase (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ee). When basal cells were co-cultured with fibroblasts under irradiation exposure, the proliferative activity of the basal cells was significantly higher compared to that of basal cells cultured alone (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ef, g). Indeed, when basal cells were co-cultured with fibroblasts and subsequently irradiated, there was a significant reduction in iron content and lipid peroxidation levels in the basal cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eh, i). These data demonstrate that post-radiation crosstalk between fibroblasts and basal cells plays a critical role in controlling compensatory protection against oral mucosal injury.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFunctional analysis of fibroblasts was performed to elucidate their protective mechanism. The analysis revealed distinct patterns of functional clustering at different post-radiation time-points. Apart from their role in secreting collagen and extracellular matrix components for connective tissue integrity, fibroblasts displayed active metabolic functions at the early phase after exposure to ionizing radiation. They transitioned to an immunomodulatory role, particularly T-cell regulation, in the late phase (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec, d and Supplementary Fig.\u0026nbsp;4b). A trajectory of fibroblast state transitions at different post-radiation times was used for gene enrichment analysis (Supplementary Fig.\u0026nbsp;4c). Differential gene expression was noted along the fibroblast differentiation trajectory. Metabolism and epithelial protection genes (e.g., ODC1, CRCT1, SLPI) were upregulated in the early phase, while inflammation-related genes (e.g., CCL4, CXCL13, LCN2) were enriched in the late phase (Supplementary Fig.\u0026nbsp;4d). This indicates that fibroblasts exert protective effects on basal cells in the early phase of RIOM. These protective effects may be associated with metabolic adjustments.\u003c/p\u003e\n\u003ch3\u003eMechanism by which fibroblasts inhibit ferroptosis\u003c/h3\u003e\n\u003cp\u003eThe mechanism by which fibroblasts inhibit ferroptosis was subsequently investigated. On the ninth day after irradiation, single-cell sequencing identified ferroptosis-related differentially expressed genes in the basal cells. This finding indicated a reduced protective effect of fibroblasts on the basal cells and increased ferroptosis. Among these genes, the spermidine/spermine N1-acetyltranferase 1 (SAT1), ACSL4, and solute carrier family 39 member 14 (SLC39A14) demonstrated the most significant upregulation (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea). Further validation using basal cells co-cultured with fibroblasts revealed that fibroblasts had the most pronounced inhibitory effect on SAT1 expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb). Flow cytometry cell sorting of epithelial cells also identified a significant increase in SAT1 expression (Supplementary Fig.\u0026nbsp;5a, b). Likewise, there was pronounced elevation of SAT1 protein levels in the basal cell lines after exposure to ionizing radiation (Supplementary Fig.\u0026nbsp;5c). Co-culturing basal epithelial cells with fibroblasts resulted in a rapid, transient decline in SAT1 expression, followed by gradual recovery (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec). Interference with SAT1 reduced lipid peroxidation levels in the basal cells (Supplementary Fig.\u0026nbsp;5d). This finding suggests that fibroblasts suppress SAT1 expression to inhibit ferroptosis. Mice deficient in SAT1 were used to study the role of SAT1 in radiation-induced ferroptosis \u003cem\u003ein vivo\u003c/em\u003e. The mucosal epithelium of SAT1-deficient mice maintained its thickness and resilience against radiation. The basal cells in these mice preserved a high proliferative capacity during the late phase (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed, e). This finding validates that SAT1 contributes to the development of radiation injury.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe downstream targets of SAT1 were subsequently examined to identify the mediator responsible for fibroblast-induced suppression of ferroptosis. Studies have shown that arachidonate 15-lipoxygenase (ALOX15) is a downstream molecule of SAT1 that promotes lipid peroxidation of cell membranes and ferroptosis\u003csup\u003e16, 17\u003c/sup\u003e. However, single-cell sequencing and immunofluorescence failed to detect ALOX15 protein expression in the basal cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ef and Supplementary Fig.\u0026nbsp;5e). This prompted re-evaluation of the SAT1 downstream pathway in regulating ferroptosis. Protein interaction analysis was performed on all ferroptosis-relevant differentially expressed genes within the basal cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eg). Among these genes, ACSL4 was identified as the most strongly interacting partner of SAT1. Concurrently, levels of ACSL4 were markedly elevated after radiation exposure (Supplementary Fig.\u0026nbsp;5f). This finding highlights the important role of ACSL4 in the ferroptosis cascade in basal cells. The relationship between SAT1 and ACSL4 was further elucidated using small interfering RNA (siRNA) to silence their endogenous expressions in the basal cells. Silencing SAT1 significantly suppressed ACSL4 expression, whereas silencing ACSL4 had negligible effects on SAT1 levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eh). In SAT1-deficient mice exposed to radiation, a marginal increase in ACSL4 expression was identified during the late post-radiation phase (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ei). These observations substantiate that SAT1 exerts regulatory control over its downstream target, ACSL4.\u003c/p\u003e \u003cp\u003eFinally, the protein expression levels of ACSL4 in basal cells co-cultured with fibroblasts were monitored to examine if fibroblasts exert their protective influence by targeting ACSL4. Basal cells that were co-cultured with fibroblasts displayed a marked decrease in ACSL4 expression. However, this inhibitory effect could not be sustained over time, with ACSL4 expression recovering after an initial decline after radiation exposure (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ej). These \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e experiments consistently demonstrate that fibroblasts alleviate ferroptosis in basal cells through suppression of the SAT1-ACSL4 axis in the early phase of RIOM. However, this protective mechanism appears to weaken in the late phase, exacerbating ferroptosis in the basal cells.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eThe role of fibroblast-derived polyamines and JunD in regulating basal cell ferroptosis\u003c/h2\u003e \u003cp\u003eFibroblasts exhibit increased metabolic activity in the early phase. They also regulate SAT1, a key enzyme in polyamine metabolism (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea). Hence, it is hypothesized that this regulation may occur through the secretion of polyamines by fibroblasts. Accordingly, polyamine metabolism in fibroblasts was quantified using the scMetabolism analytical tool. There was a relatively high level during the early phase after radiation exposure. This activity decreased over time (Supplementary Fig.\u0026nbsp;6a).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe polyamine content between fibroblasts and basal cells was compared after radiation exposure. When cultivated independently, radiation led to an increase in polyamines within fibroblasts, and a decrease in basal cells and cellular supernatant. However, basal cells that were co-cultured with fibroblasts displayed an initial elevation in spermidine/spermine content, followed by a reduction at later stages (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb and Supplementary Fig.\u0026nbsp;6b). Similarly, spermidine/spermine was detected in the supernatant of co-cultured cells, with elevated levels at the early stage and significantly-reduce levels at the later stage (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb and Supplementary Fig.\u0026nbsp;6c). Putrescine levels within the basal cells remained high during the early phase after radiation exposure; these levels decreased in the later stage. Compared to culturing alone, the levels of polyamines within the basal cells were higher when they were co-cultured with fibroblasts (Supplementary Fig.\u0026nbsp;6d).\u003c/p\u003e \u003cp\u003eOrnithine decarboxylase 1 (ODC1), a major gene in the synthesis pathway\u003csup\u003e18, 19\u003c/sup\u003e, was substantially up-regulated after radiation (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ee). This was confirmed at the protein level by immunofluorescence imaging. A pronounced reduction in ODC1 expression was observed in fibroblasts derived from late-stage RIOM mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ee). ODC1 gene knockdown was performed in fibroblasts, followed by co-culture with basal cells before radiation (Supplementary Fig.\u0026nbsp;6f). The supernatant showed higher levels of spermidine/spermine when ODC1 was expressed in fibroblasts. In contrast, spermidine levels significantly decreased when ODC1 expression was reduced (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ef). Immunofluorescence staining for spermidine/spermine in basal cells supported these findings, showing high polyamine concentrations during the early post-radiation phase. The polyamine concentrations were notably reduced in human mucositis specimens and in mice in the later phase, confirming the observed decline in spermidine/spermine levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec and Supplementary Fig.\u0026nbsp;6e). These results validate that fibroblasts regulate the polyamine level in basal cells in a paracrine manner. In agreement with these results, pre-treatment of basal cells with different concentrations of polyamines before radiation exposure showed that spermidine and spermine effectively suppressed the expression of SAT1 and ACSL4 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eg). In addition, when ODC1 expression in fibroblasts was inhibited, the expression of SAT1 and ACSL4 in the basal cells increased following irradiation (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eh). This treatment also reduced lipid peroxidation (Supplementary Fig.\u0026nbsp;6g), and enhanced cell proliferation. Spermidine and spermine demonstrated better efficacy than putrescine (Supplementary Fig.\u0026nbsp;6h). These findings confirm the function of fibroblast-derived polyamines in regulating the SAT1-ACSL4 axis to protect basal cells against ferroptosis.\u003c/p\u003e \u003cp\u003eImmunofluorescence observations further indicated that spermidine and spermine accumulated in both the cytoplasm and the nucleus of basal cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec, d). As highly-charged aliphatic polycations, polyamines are capable of binding to negatively-charged nucleic acids. This interaction potentially modulates gene expression by altering transcription factor specificity\u003csup\u003e20,21\u003c/sup\u003e. Because of the observed nuclear localization, it was further hypothesized that polyamines regulate the upstream transcription factors of SAT1.\u003c/p\u003e \u003cp\u003eAnalysis of single cell RNA-sequencing data through the Single-Cell Regulatory Network Inference and Clustering (Scenic) platform identified JunD and JunB as potential transcription factors that control SAT1. In particular, JunD had a higher regulon activity score (RAS) compared to JunB (Supplementary Fig.\u0026nbsp;7a). Expression of JunD was upregulated in murine basal cells during the late stage of radiation exposure (Supplementary Fig.\u0026nbsp;7b). Small interfering RNA-mediated knockdown was subsequently used to investigate the role of JunD in basal cell ferroptosis. Knockdown of JunD resulted in the reduction of lipid peroxidation levels (Supplementary Fig.\u0026nbsp;7c). Transcription factor binding analysis of the SAT1 promoter using Joint Accessible Sites of Promoters and Regulators (JASPER) identified JunD as binding sequences of SAT1 promoter (Fig.\u0026nbsp;6a, b). The ChIP analysis identified increased enrichment of JUND at the indicated binding sites in the SAT1 gene promoter, which was further increased upon radiation treatment (Fig.\u0026nbsp;6c). The protein levels of both SAT1 and ACSL4 were substantially diminished after JunD disruption (Fig.\u0026nbsp;6d, e). These findings clarify that JunD modulates SAT1 expression. Activation of JUND dictates the onset of ferroptosis in basal cells.\u003c/p\u003e \u003cp\u003eTaken together, the results identified sophisticated crosstalk among fibroblast-secreted polyamines, JunD-directed transcriptional regulation, and the regulation of the SAT1-ACSL4 pathway.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003ePolyamines in mucosal protection against ionizing radiation-induced damage\u003c/h3\u003e\n\u003cp\u003eTo further validate the role of polyamines in mucosal injury, difluoromethylornithine (DFMO), an ODC1 inhibitor, was used to see if polyamines inhibit mucosal injury by regulating basal cell ferroptosis. Administration of DFMO reduced mucosal thickness and weakened basal cell proliferation in the early phase after ionizing radiation exposure (Fig.\u0026nbsp;6f and Supplementary Fig.\u0026nbsp;7d). In addition, JunD was prematurely activated in basal cells treated with DFMO after radiation exposure. This was accompanied by a significant up-regulation of ACSL4 (Fig.\u0026nbsp;6g, h).\u003c/p\u003e \u003cp\u003eThe potential of spermidine and spermine as a preventive strategy against mucositis was investigated due to their ability to block ferroptosis in basal cells during the early phase of RIOM. Local injections of polyamines were performed prior to the onset of ionizing radiation exposure and continued until day 9 after radiation. Tongue tissues were examined 3 days and 9 days post-radiation (Fig.\u0026nbsp;6i). Polyamine supplementation suppressed JunD activation in the basal cells until the late phase of radiation (Fig.\u0026nbsp;6j). This inhibition reduced ACSL4 expression and markedly lowered ferroptosis levels (Fig.\u0026nbsp;6k and Supplementary Fig.\u0026nbsp;7e). In order to validate the therapeutic efficacy of polyamines compared to commonly used clinical drugs, two additional groups received KGF-1 (6.25mg/kg) intraperitoneal injection and 0.15% benzydamine (BDM) mouthwash treatment daily from 7 days before radiation to 9 days after the last dose of radiation (Fig.\u0026nbsp;6I). Spermidine treatment exhibited the best therapeutic effect, surpassing that of the ferroptosis inhibitor LIP-1, spermine, KGF-1 and BDM mouthwash. Immunofluorescence staining revealed that spermidine-treated mucosa exhibited significantly greater proliferation 9 days after radiation (Fig.\u0026nbsp;6l and Supplementary Fig.\u0026nbsp;7f). What\u0026rsquo;s more, spermidine treatment resulted in thicker mucosa, increased basal layer cell structure, and improved continuity in the late post-radiation phase (Fig.\u0026nbsp;6m). These findings demonstrate that spermidine supplementation effectively inhibits ferroptosis and promotes mucosal proliferation. This therapeutic strategy is potentially promising for mitigating RIOM.\u003c/p\u003e \u003cp\u003eFurther, we explored the therapeutic potential of polyamines in mitigating damage to other tissues during abdominal radiation. Mice were intraperitoneally injected with saline, spermidine (10mg/kg), and amifostine (AM) (10mg/kg) and subsequently subjected to a single 10Gy abdominal radiation. On the eighth day post-radiation, we observed changes in the colon. We found that the colon length of mice supplemented with spermidine was significantly longer (Supplementary Fig.\u0026nbsp;8a), indicating reduced inflammatory damage. H\u0026amp;E staining confirmed the protective effect of spermidine against radiation-induced colon injury. Spermidine treatment notably safeguarded the colonic mucosa, reducing the shedding of intestinal villi (Supplementary Fig.\u0026nbsp;8b). Concurrently, crypt base columnar stem cells maintained a higher proliferative capacity (Supplementary Fig.\u0026nbsp;8c). To confirm the impact of spermidine on inhibiting ferroptosis, we assessed the protein expression levels of key biomarkers involved in the ferroptosis pathway in radiated colon tissues. We observed a significant decrease in ACSL4 expression levels in radiated colon tissues following spermidine treatment (Supplementary Fig.\u0026nbsp;8d, e), suggesting that polyamines can suppress ferroptosis to inhibit radiation-induced colon injury. Additionally, the protective effects of spermidine on liver and lung tissues post-irradiation were observed (Supplementary Fig.\u0026nbsp;8f). The results indicated that spermidine inhibited vacuolar degeneration in the liver following radiation and reduced the degree of alveolar collapse, demonstrating a promising protective effect. In summary, polyamines, through the inhibition of ferroptosis, also provide good protection against radiation-induced injuries to the colon as well as the liver and lungs. Overall, treatment targeting ferroptosis with polyamines holds promise in minimizing normal tissue damage caused by radiation therapy.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003ePrevention and treatment of RIOM must address the problem of epithelial ablation caused by massive basal cell death\u003csup\u003e22\u003c/sup\u003e. However, the mechanisms and processes behind basal cell death caused by ionizing radiation remain largely unknown. In this study, scRNA-seq, functional analysis, and \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e experiments were used to demonstrate that ferroptosis is the major cell death pathway of oral epithelial basal cells. More importantly, the study focused on the crosstalk between fibroblasts and basal cells. The study identified that polyamines produced by ODC1 activation in fibroblasts are absorbed by basal cells during the early radiation stage. These polyamines bind to JUND in the basal cells and inhibit expression of the SAT1-ACSL4 signaling pathway. This mechanism protects basal cells from ferroptosis. The focus on prevention in RIOM treatment led to the use of polyamines to prevent mucosal injury. This strategy produced promising results, and highlights the potential of polyamines for wide-ranging clinical applications.\u003c/p\u003e \u003cp\u003ePrevious studies have suggested that strong ionizing radiation induces DNA double-strand breaks. This causes cell cycle arrest, senescence, and different modes of cell death, including apoptosis, necrosis, autophagy, and mitotic catastrophe. Apart from direct DNA damage, ionizing radiation can produce ROS such as hydroxyl radicals and hydrogen peroxide through radiolysis of cellular water and stimulation of oxidases. These ROS species may damage nucleic acids, proteins, and lipids. Indirect damage is the primary mode of cell death\u003csup\u003e4,23\u003c/sup\u003e. Through single-cell sequencing and \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e experiments, ferroptosis was identified as the primary form of cell death in the basal layer of oral epithelial cells after their exposure to ionizing radiation. Ferroptosis is a unique form of iron-dependent cell death driven by the accumulation of lipid peroxides, due to the imbalance of the cellular antioxidant system\u003csup\u003e24,25\u003c/sup\u003e. Ferroptosis is distinct from apoptosis, necroptosis, and pyroptosis, which have specific functions during development and immune processes and respond to particular physiological stimuli\u003csup\u003e26,27\u003c/sup\u003e. Results of the present study provide a possible explanation for the occurrence of mucosal damage following massive cell death events induced by ionizing radiation. Radiation may cause a widespread elevation of ROS, and ferroptosis can propagate through ROS waves at a constant rate over long distances among human cells. This makes cell populations a medium for ROS propagation\u003csup\u003e28,29\u003c/sup\u003e. The rupture of cell membranes following ferroptosis further disrupts the barrier formed by epithelial cells, allowing invasion of microorganisms and infiltration of harmful substance. This subsequently triggers an inflammatory response of monocytes and the release of pro-inflammatory cytokines that exacerbates tissue damage\u003csup\u003e30,31\u003c/sup\u003e. However, the specific mechanisms by which ionizing radiation induces ferroptosis in oral mucosal epithelial cells remain largely unknown. Single-cell RNA-sequencing analysis performed in the present work revealed the important roles of ferroptosis-related molecular targets (such as SAT1, ACSL4, JunD, and PTGS2) in promoting basal cell death. These findings establish a foundation for understanding the pathogenesis of RIOM, and offer novel molecular biomarkers for prognostic assessment in individuals who are exposed to ionizing radiation.\u003c/p\u003e \u003cp\u003eRadiation-induced cell death is a complex biological process involving a dynamic sequence of interrelated events throughout the mucosa. These events ultimately target highly proliferative epithelial basal cells\u003csup\u003e2\u003c/sup\u003e. Time-series analysis using sc-RNA seq was used to track the dynamic process of ferroptosis in basal cells following radiation stress over time. Unexpectedly, ferroptosis in basal cells was not prominently associated with mucosal thickening during the early stages of radiation exposure. Concurrently, a substantial increase in the number of fibroblasts within the microenvironment was observed. This finding indicates that the organism is in a compensatory phase. This compensatory mechanism appears to be crucial for inhibiting ferroptosis in basal cells. Understanding this mechanism may aid in developing early intervention strategies against RIOM. Fibroblasts in the epithelial microenvironment play a significant role during the compensatory phase by secreting metabolites to inhibit ferroptosis in the basal cells. Recent single-cell analyses have shown that fibroblasts undergo transcriptional changes that resemble the processes seen in cell differentiation. These changes help promote tissue repair during injury, suggesting that fibroblasts adapt their gene expression to support healing\u003csup\u003e32,33\u003c/sup\u003e. Because the functional state of fibroblasts is dynamic during tissue repair, these cells can interact with the epithelium to promote successful resistance to injury\u003csup\u003e34\u003c/sup\u003e. For example, viral transduction with keratinocyte lineage-related transcription factors such as Dnp63a, Grhl2, Tfap2a, and Myc can reprogram fibroblasts in situ within skin wounds. This process generates fibroblast-derived epidermis and enhances re-epithelialization of the wound\u003csup\u003e35\u003c/sup\u003e. This complex cell-to-cell communication includes not only traditional protein-ligand-receptor pairs, but also small metabolites that diffuse within the microenvironment. Metabolic interactions are important regulators in tissue repair\u003csup\u003e36\u003c/sup\u003e. Specifically, ferroptosis is regulated by multiple metabolic pathways, including redox balance, iron handling, mitochondrial activity, and metabolism of amino acids, lipids, and sugars\u003csup\u003e10\u003c/sup\u003e. Metabolic regulation is a key mechanism for modulating cell susceptibility to ferroptosis\u003csup\u003e37\u0026ndash;39\u003c/sup\u003e. Fibroblasts act as signaling contributors to the epithelial microenvironment by secreting metabolites that regulate epithelial cell function\u003csup\u003e33\u003c/sup\u003e. In this work, fibroblasts were found to secrete polyamines that regulate basal cells after radiation damage. This discovery uncovers a new mechanism by which fibroblasts maintain the compensatory capacity of basal cells through metabolic support.\u003c/p\u003e \u003cp\u003eIn this work, ionizing radiation was found to activate ODC1 in fibroblasts, causing them to produce polyamines. These polyamines are then absorbed and accumulated in basal cells. Ornithine decarboxylase 1 is a key enzyme in polyamine synthesis. For example, the aryl hydrocarbon receptor binds to the xenobiotic response element sequence in the ODC1 promoter region. This binding promotes the transcription of ODC1. As a result, downstream metabolites such as putrescine, spermidine, and spermine are produced. These metabolites can inhibit pyroptosis in macrophages\u003csup\u003e40\u003c/sup\u003e. Polyamines regulate arginine metabolism and enhance the antioxidant capacity of glutathione\u003csup\u003e41\u0026ndash;44\u003c/sup\u003e. This study identified a direct link between polyamine metabolism and ferroptosis and suggested that using polyamines to inhibit ferroptosis may be a new way to protect against radiation damage. This discovery has important implications for treating not only RIOM, but also radiation-induced damage to the skin, intestines, and other tissues. Polyamines have strong affinity for nucleic acids, enabling them to regulate fundamental cellular processes such as DNA replication, gene transcription regulation, and control of translation elongation and termination\u003csup\u003e45\u003c/sup\u003e. Consistent with the findings of those studies, JUND was identified in the present work as a transcription factor upstream of the SAT1-ACSL4 signaling pathway. Polyamines can regulate JUND expression at the transcriptional level and mediate interactions between fibroblasts and basal cells. This helps to inhibit ferroptosis in basal cells. These findings reveal a new compensatory function of polyamines produced by fibroblasts and emphasize the importance of the inhibited JUND-SAT1-ACSL4 signaling axis in promoting radiation-induced oral mucosal injury.\u003c/p\u003e \u003cp\u003eThe present study identified that under radiation stress, fibroblasts in the microenvironment inhibit ferroptosis in basal cells by producing polyamines. This discovery introduces a safer and more effective therapeutic approach for RIOM. In the context of RIOM, polyamine supplementation may support the natural healing process. Overall, these results increase the potential for leveraging the body\u0026rsquo;s own protective mechanisms for stress protection in clinical settings. These insights may also contribute to the design and development of treatment for radiation-related diseases.\u003c/p\u003e \u003cp\u003eDespite the promising findings, there are limitations to this study. One such limitation is the unclear mechanism by which polyamines are transported from fibroblasts to basal cells, whether through passive diffusion or the release of extracellular vesicles\u003csup\u003e46\u003c/sup\u003e. Understanding this process may result in improved strategies for delivering polyamines to enhance mucosal protection. Another area for future research is the interaction between polyamine metabolism and the oral microbiome. This has not been investigated in the present work. Future experiments should examine how specific microorganisms influence polyamine production and their impact on radiation-induced mucosal injury. This may result in novel treatment regimens that combine microbiome modulation with polyamine supplementation to improve therapeutic outcomes. Addressing these limitations in future research will be essential for optimizing polyamine-based therapy and fully understanding fibroblast-basal cell interactions during radiation-induced injury.\u003c/p\u003e "},{"header":"Methods","content":"\u003cp\u003eThe methods in the present study are provided in Supplementary Information.\u003c/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData availability\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe main data supporting the results of this study are available within the paper and its Supplementary Information. The raw and analyzed datasets generated during the study are available for research purposes from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgment\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the National Natural Science Foundation of China (no. 82325012 to Lina Niu).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eW.Y., K.J., K.W., X.L., Y.M., and L.N. designed and organized experiments. W.Y., K.W., X.L., and X. H. performed experiments and analyzed data. W.Y., K.J., K.W., Q.W., M.W., J.W., F.T., and L.N. prepared figures and edited manuscript. W.Y., Q.W., J.W., Q.L., M.S., and L.N. wrote the paper. L.N. conceived, supervised, and directed the study.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclare of interest \u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors do not declare competing financial interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003e Elad, S., Yarom, N., Zadik, Y., Kuten-Shorrer, M., \u0026amp; Sonis, S.T. The broadening scope of oral mucositis and oral ulcerative mucosal toxicities of anticancer therapies. \u003cem\u003eCA Cancer J Clin.\u003c/em\u003e \u003cb\u003e72\u003c/b\u003e,57\u0026ndash;77 (2022).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Sonis, S.T. The pathobiology of mucositis. \u003cem\u003eNat. Rev. Cancer.\u003c/em\u003e \u003cb\u003e4\u003c/b\u003e, 277\u0026ndash;284 (2004).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Spielberger, R. et al. 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Polyamine-mediated ferroptosis amplification acts as a targetable vulnerability in cancer. \u003cem\u003eNat Commun.\u003c/em\u003e \u003cb\u003e15\u003c/b\u003e, 2461 (2024).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Compensation, ferroptosis, fibroblasts-epithelium interaction, metabolic milieu, polyamine metabolism, microenvironment, radiation-induced oral mucositis","lastPublishedDoi":"10.21203/rs.3.rs-5617929/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5617929/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eIonizing radiation is effective in combating cancer but inflicts severe damage on the oral mucosa. The mechanisms behind this damage remain unclear, and current treatment modalities are primarily palliative. This study revealed that ferroptosis is the predominant reason for oral-radiation depletion of oral mucosal epithelial cells. More importantly, compensatory mechanisms are activated in the organism during the early stage after radiation exposure. These compensatory mechanisms arise from the metabolic support provided by fibroblasts. In the early post-radiation stage, fibroblasts supply polyamines, which are readily absorbed by basal epithelial cells, protecting them from ferroptosis. Local supplementation of polyamines effectively mitigates mucosal damage. This study emphasizes the important role of fibroblast-mediated metabolic support in protecting the oral mucosa from radiation-induced damage. Results of the study provide new insights into combating radiation-related diseases by enhancing the self-protective responses of living organisms.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e","manuscriptTitle":"Metabolic support protects oral mucosa from ferroptosis in radiation-induced mucositis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-01-31 07:13:45","doi":"10.21203/rs.3.rs-5617929/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-communications","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"NCOMMS","sideBox":"Learn more about [Nature Communications](http://www.nature.com/ncomms/)","snPcode":"","submissionUrl":"https://mts-ncomms.nature.com/","title":"Nature Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Communications","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"fa76e99b-eb1f-4f87-8a96-d8c757f41ee1","owner":[],"postedDate":"January 31st, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":41462639,"name":"Health sciences/Medical research/Drug development"},{"id":41462640,"name":"Biological sciences/Cell biology/Cell death"}],"tags":[],"updatedAt":"2026-01-15T08:05:59+00:00","versionOfRecord":{"articleIdentity":"rs-5617929","link":"https://doi.org/10.1038/s41467-025-67214-5","journal":{"identity":"nature-communications","isVorOnly":false,"title":"Nature Communications"},"publishedOn":"2025-12-08 05:00:00","publishedOnDateReadable":"December 8th, 2025"},"versionCreatedAt":"2025-01-31 07:13:45","video":"","vorDoi":"10.1038/s41467-025-67214-5","vorDoiUrl":"https://doi.org/10.1038/s41467-025-67214-5","workflowStages":[]},"version":"v1","identity":"rs-5617929","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5617929","identity":"rs-5617929","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}