Ionizing radiation has been demonstrated to induce direct DNA damage in esophageal epithelial cells, manifesting as double-strand breaks and base damage, thereby activating DNA damage repair mechanisms (16,17). Concurrently, radiation-induced damage has been shown to disrupt the normal progression of the cell cycle and activate cell cycle checkpoints (18,19). Furthermore, the damage induced by ionizing radiation has been shown to trigger changes in intercellular signaling. TGF-β1 is a pivotal factor in the initiation and termination of cell repair that serves a regulatory role in mucosal tissue repair in conjunction with FN. Activation of the p38MAPK signaling pathway promotes FN production, thereby facilitating rapid repair of the esophageal epithelial mucosa (20).

Epithelial cell damage in the esophagus is an early pathological change of RE, which can lead to the destruction of the epithelial barrier function. This frequently manifests as mucosal congestion, edema, epithelial cell degeneration and necrosis, which in turn triggers an aseptic inflammatory reaction (21). Miao et al (22) previously assessed the intervention effect of morin (3,5,7,2',4'-pentahydroxyflavone; a natural flavonol, predominantly isolated from plants, such as Morus alba and guava leaves;) on the peroxisome proliferator-activated receptor (PPAR)-γ/glutaminolysis/DEPTOR signaling pathway under inflammatory conditions. Using a bleomycin-induced mouse lung fibrosis model (in vivo) and a TGF-β1/hypoxia-stimulated NIH-3T3 fibroblast model (in vitro), it was demonstrated that morin reversed the following three key pathological alterations: i) Suppression of PPAR-γ (bleomycin/TGF-β1 reduced PPAR-γ protein and mRNA expression levels by ~65 and ~58%, respectively and decreased nuclear translocation from 72 to 29%); ii) hyperactivation of glutaminolysis (evidenced by a ~2.3X increase in the activity of the rate-limiting enzyme glutaminase-1); and iii) downregulation of DEPTOR with concomitant mTORC1 activation (DEPTOR protein decreased by ~62%, leading to a ~2.4-fold increase in mTOR phosphorylation). These conclusions were supported by detecting the expression of the myofibroblast marker α-smooth muscle actin (SMA) (22). α-SMA expression was found to be upregulated specifically in lung fibroblasts (and their differentiated myofibroblasts) in bleomycin-induced mouse lungs (in vivo) and in TGF-β1/hypoxia-stimulated NIH-3T3 fibroblasts (a mouse embryonic fibroblast cell line, in vitro) under an inflammatory environment, whereas morin could inhibit the transformation of resident lung fibroblasts (which normally reside in the lung interstitium, the connective tissue between alveolar walls) to myofibroblasts (22). This suggests that factors, such as inflammation, may influence the relevant signaling pathways in lung fibrosis, which details organ-nonspecific cell cycle regulatory mechanisms [including ataxia telangiectasia and rad3-related (ATR)/checkpoint kinase 1 (Chk1)/Wee1 pathways and cyclin-CDK complexes] that control fibroblast survival and activation (23). These mechanisms directly apply to esophageal biology. In RE, esophageal fibroblasts use similar inflammation-modulated cell cycle checkpoints (such as G2-phase activation through ATR/Chk1) and activity-dependent cell cycle progression to proliferate and contribute to esophageal fibrosis, mirroring the fibroblast regulation described in the lung models (23). These factors may activate the fibroblast-to-myofibroblast transformation process by regulating relevant signaling pathways. This transformation is a conserved driver of organ fibrosis, including in the esophagus and aligns with established cell cycle regulatory mechanisms. In esophageal pathologies, such as RE (a precursor to fibrosis), local fibroblasts are stimulated by pro-fibrotic cues (including TGF-β1) to activate the ATR/Chk1/Wee1 signaling axis. This pathway induces G2 checkpoint arrest, which promotes fibroblast survival by facilitating DNA damage repair and subsequently enables their differentiation into myofibroblasts. This regulatory pattern, which mirrors cell cycle-dependent activation processes characterized in general fibrotic disease pathogenesis, directly associates the observed esophageal fibroblast behavior with the well-documented mechanisms of fibroblast-to-myofibroblast transformation (23). Furthermore, as the inflammatory response to RE continues, the activation and transformation of fibroblasts into myofibroblasts may be triggered, resulting in the secretion of substantial amounts of ECM (such as collagen and FN), leading to tissue fibrosis and scar formation (12). The resulting fibrosis imposes limitations on the dilatation function of the esophagus and can also give rise to fibrosis-related symptoms, including esophageal stricture, dysphagia and chronic ulceration, which can have a severely deleterious effect on patients' swallowing function (24). Furthermore, overactivation of the p38MAPK signaling pathway can aggravate the fibrosis (22). Table I systematically associates pathological stages of RE with immune signaling across three phases. During the acute inflammatory phase (1-2 weeks post-radiotherapy), esophageal epithelial necrosis and mucosal edema trigger aseptic inflammation, concomitant with M1 macrophage polarization (characterized by TNF-α and IL-1β secretion) and mild TGF-β1/p38MAPK/FN axis activation, to initiate repair. In the proliferative repair phase (2-4 weeks), epithelial regeneration and granulation tissue formation occur alongside M2 macrophage polarization (marked by IL-10 and TGF-β1 secretion) and regulatory T cell (Treg) expansion. These processes are driven by sustained TGF-β1/p38MAPK activation, which promotes FN synthesis. By the fibrosis phase (>4 weeks), fibroblast transdifferentiation into myofibroblasts results in excessive ECM deposition. Concurrently, macrophages secrete TGF-β1 whilst dendritic cell function is suppressed. Both of the aforementioned phenomena are associated with hyperactivation of the TGF-β1/p38MAPK/FN axis, ultimately exacerbating tissue fibrosis (8,9,12,22,25,26).

To counteract excessive inflammation from M1 macrophage polarization in the acute inflammatory phase, administration of low-dose PPARγ agonist rosiglitazone has been reported to promote early transition to the M2 phenotype, accelerating inflammation resolution (27). In the repair and proliferation phase, modulating T-cell subset balance can enhance tissue repair. Low-dose IL-2 can expand the Tregs population by 15-20%, whilst carefully controlled TGF-β1 activity can avoid pro-fibrotic transformation (28,29). This control is achieved by RE-stage-specific strategies: i) p38MAPK inhibition (using the specific inhibitor SB203580) to reduce excessive FN synthesis; ii) timed administration of the TGF-β1 neutralizing antibody fresolimumab following the initial repair phase; and iii) low-dose TGF-β1 supplementation tailored to RE progression. In addition, targeting hyperactivated TGF-β1/p38MAPK signaling using the monoclonal antibody fresolimumab can block TGF-β1-receptor binding to inhibit fibroblast-to-myofibroblast transdifferentiation and reduce collagen deposition (30).

TGF-β1 is a multifunctional cytokine with several biological functions, including the regulation of cell proliferation, differentiation, apoptosis, migration and immune response (31). Excessive ECM deposition is the main feature of fibrosis (12). In previous studies of experimental liver fibrosis, inhibition of FN deposition has been shown to reduce collagen accumulation and attenuate fibrosis (13,32). Similarly, in RE, TGF-β1 has been found to promote the synthesis and deposition of FN and collagen by activating the p38MAPK signaling pathway (12,26). This observation is supported by studies using the Sprague-Dawley rat RE model, which is established through targeted thoracic radiotherapy (40 Gy single dose) to mimic clinical radiation-induced esophageal injury. Notably, this may induce the transformation of fibroblasts into myofibroblasts and in turn fibrosis of the tissue. In addition, TGF-β1 may inhibit the inflammatory response and promote tissue repair by regulating immune cell function. Specifically, it drives the M1-to-M2 phenotypic transition of macrophages. This process is mediated by activating the p38MAPK pathway, which induces secretion of the anti-inflammatory cytokines IL-10 and TGF-β1. It also induces the differentiation of naive CD4+ T cells into regulatory T cells (Tregs). This differentiation relies on Smad/forkhead box (Fox)p3 signaling to suppress T helper 1 cell (Th1 cell)-mediated excessive inflammation. These regulatory effects of TGF-β1 are consistent with observations in Sprague-Dawley rat RE models and clinical studies (11,12,29).

The p38MAPK signaling pathway has been found to be present in a wide range of eukaryotes (33) and has been identified as pivotal in various biological processes, including cellular stress response, inflammation regulation, cell proliferation, differentiation and apoptosis (25,27,34). Zhang et al (12) previously demonstrated that the TGF-β1/p38MAPK/FN signaling pathway may be implicated in esophageal mucosal repair. To confirm this, a Sprague-Dawley rat RE model was established by 40 Gy single-dose upper esophageal irradiation. H&E staining was used to track temporal changes in esophageal mucosal pathology, whereas reverse transcription-quantitative PCR/Western blotting were used to detect expression dynamics of key pathway molecules (TGF-β1, p38MAPK and FN). TGF-β1 and p38MAPK were observed to be upregulated early (week 1 post-irradiation) during acute mucosal damage, whilst FN expression recovered later (weeks 3-4) alongside epithelial regeneration. These findings suggest that the p38MAPK signaling pathway is closely associated with the development of RE and mucosal repair.

FN is a macromolecular glycoprotein that is a constituent of the ECM and has been reported to be involved in various biological processes, including cell adhesion, migration and differentiation (29). Its role in ulcer healing has been extensively reported, with previous studies demonstrating its ability to promote mucosal repair, contribute to ECM remodeling and regulate cell signaling in the specific context of RE ulcer repair (28). Furthermore, FN can regulate cell physiology through interactions with the TGF-β1 and p38MAPK signaling pathways, thereby contributing to RE ulcer repair (26).

FN is a key downstream molecule of the TGF-β1/p38MAPK/FN signaling axis. Its expression and function are regulated upstream by TGF-β1 activation and the p38MAPK pathway (12). Upon activation, TGF-β1 promotes FN synthesis through the p38MAPK pathway. In turn, FN exerts feedback regulation on the signaling axis primarily through ECM remodeling-mediated mechanotransduction and latent TGF-β1 sequestration (12). Separately, the sequential process of TGF-β1 activation (initiation), p38MAPK phosphorylation (transduction) and FN deposition (execution) constitutes the core pathway driving RE progression (12). The synergistic action of this signaling axis constitutes the core molecular basis for mucosal regeneration, inflammation resolution and fibrosis progression during RE ulcer repair (12). It also lays the groundwork for the subsequent exploration of how immune cells regulate RE repair, which is achieved through their interactions with TGF-β1, p38MAPK and FN (35).

FN functions as a pivotal downstream effector in the TGF-β1/p38MAPK/FN signaling axis. Critically, FN establishes a self-reinforcing feedback loop through the following two mechanistically distinct yet interconnected pathways: i) Mechanotransduction-mediated amplification, where FN deposition increases ECM stiffness, activating integrin β1-dependent focal adhesion kinase/Src signaling (this cascade upregulates TGF-β receptor II expression and enhances cellular sensitivity to TGF-β1, thereby amplifying p38MAPK phosphorylation and driving further FN synthesis, forming a positive feedback circuit) (2,13,26); and ii) biochemical reservoir function, where the ED-A domain of FN binds latent TGF-β-binding protein-1, anchoring latent TGF-β1 within the ECM, which establishes a localized high-concentration reservoir to facilitate sustained TGF-β1 activation and perpetuate pathway signaling (3,13,36). Collectively, this dual feedback mechanism, operating through the ‘signal initiation (TGF-β1 release)/pathway transduction (p38MAPK phosphorylation)/effector execution (FN deposition)’ cascade, constitutes the molecular basis for the transition from physiological repair to pathological fibrosis in chronic RE (Fig. 1) (4).

Macrophages are pivotal in tissue repair, exhibiting both proinflammatory and anti-inflammatory functions through polarization state transition (30). During the early phase of tissue injury and inflammation, macrophages secrete proinflammatory factors (such as TNF-α and IL-1β) through M1 polarization to promote inflammation. As the repair process advances, macrophages then convert to the M2 subtype, which predominantly secrete anti-inflammatory cytokines (including IL-10 and TGF-β1) to inhibit inflammation and promote tissue repair (37). This process (macrophage M1-to-M2 polarization) has been widely observed in other inflammatory diseases, including pulmonary fibrosis, diabetic pulmonary injury and burn wound inflammation (22,26). In these conditions, macrophage phenotypic switching similarly mediates inflammation resolution and tissue repair, which is consistent with the aforementioned mechanisms. However, the specific mechanism in RE in this specific context require further investigation.

In RE, M2-type polarization of macrophages may be closely associated with the TGF-β1/p38MAPK/FN signaling axis. TGF-β1 is a key factor in macrophage polarization and can regulate the inflammatory response of macrophages through the activation of the p38MAPK signaling pathway (13). In addition, TGF-β1 promotes macrophage polarization towards the M2 phenotype by upregulating the expression of key surface receptors, including CD206 (mannose receptor), CD163 (haptoglobin-hemoglobin scavenger receptor), TGF-β receptor II and IL-10 receptor. Concurrently, TGF-β1 activates critical signaling pathways, such as Smad2/3, PI3K/Akt and STAT6, which collectively drive the M2 polarization program (36). Upregulation of FN may synergize with TGF-β1 to promote the restoration of the esophageal mucosal tissue architecture (12,26). During the ulcer repair process in RE, TGF-β1 activates the p38MAPK pathway, promoting FN synthesis. As shown in Fig. 1, M2 macrophages (characterized by IL-10 and TGF-β1 secretion) are activated through this axis, whilst simultaneously inhibiting Th1 cells through IL-10 and TGF-β1, thereby creating an anti-inflammatory microenvironment conducive to tissue repair.

The differentiation and functional regulation of T cell subpopulations in the immune response can result in important effects on tissue repair. Radiation damage activates CD4+ helper T cells and CD8+ cytotoxic T cells through antigen-presenting cells, which participate in the immune response and repair processes (25). CD4+ T cells regulate the activity of other immune cells by secreting cytokines during immune responses, whereas CD8+T cells directly remove radiation-damaged cells through cytotoxic effects (38,39).

In RE, IFN-γ secreted by Th1 cells can promote inflammatory responses and cellular immunity, whereas IL-4, IL-5 and IL-13 secreted by T helper 2 cells (Th2 cells) tend to suppress inflammatory responses and promote humoral immunity (1,40). It has been shown that TGF-β1 can induce T cells to differentiate into Tregs, thereby inhibiting Th1 cell activity and attenuating inflammatory responses (41,42). Furthermore, ovarian cancer cells can drive the differentiation of CD8+ T cells into Tregs with suppressive functionality, a process mediated through activation of the p38MAPK signaling pathway within CD8+ T cells (34,43). This mechanism is distinct from TGF-β-induced CD4+ Treg generation (34). This indicates that activation of the p38MAPK signaling pathway can affect T cell differentiation and function, which can influence the course of the immune response (44-46). The integration of immune regulation and tissue repair is a fundamental aspect of RE therapy, where by regulating the function and subpopulation balance of T cells, it can promote tissue repair and attenuate fibrosis (47).

Dendritic cells are antigen-presenting cells that are pivotal in initiating T-cell immune responses (48). In RE, dendritic cells capture and process antigens from damaged tissues and present them to T cells to activate an immune response (49,50). TGF-β1 has been shown to inhibit the maturation and activation of dendritic cells, reduce the expression of co-stimulatory molecules on their surface, such as CD80 (B7-1), CD86 (B7-2), CD40 and ICOS ligand, thereby inhibit T cell activation (51). Therefore, the TGF-β1 signaling axis can regulate dendritic cell function, affecting their antigen-presenting capacity and immune activation properties (45). Furthermore, TGF-β1 has been shown to activate specific T cell subpopulations, including regulatory Tregs and Th2 cells, through dendritic cells, with the p38MAPK signaling pathway potentially contributing to dendritic cell maturation, migration and antigen presentation (52). The interaction of dendritic cells with TGF-β1/p38MAPK may contribute to the repair of esophagitis and the establishment of immune tolerance by regulating immune homeostasis. Nevertheless, controversy persists regarding the specific role of dendritic cells in RE repair and their relationship with the signaling axis (35,53).