Introduction
Exposure to ionizing radiation during reactor accidents, nuclear weapon detonations, or dirty bombs leads to a symptom complex that is summarized as acute radiation syndrome (ARS). Among the organ systems that determine the survival of the individual, the response of the small intestines has been well studied [1, 2]. The small intestines are organized hierarchically with leucine rich repeat containing G protein-coupled receptor 5+ (Lgr5+) crypt base columnar (CBC) cells wedged between Paneth cells at the base of the crypts, forming the intestinal stem cell (ISC) population [3]. Progeny of CBC cells migrate in a conveyor-like fashion to the apex of the intestinal villi, where the cells are eventually shed off and die. Following ionizing radiation exposure, epithelial cell death in the small intestines occurs rapidly, leading to the loss of villi and failure of the intestine’s resorption and barrier functions [2]. This results in fluid loss, bleeding, and bacterial translocation, which can cause sepsis and death unless the surviving ISCs can reconstitute tissue integrity in time.
The ability of surviving CBC cells to repair intestinal tissue integrity is influenced not simply by the radiation dose received by the epithelial cells but also by the response of various other tissue components of the intestinal wall. For instance, the death of endothelial cells in the villi capillaries affects the radiation response of the small intestine [4], and crypts in proximity to Peyer’s patches are more radioresistant than those further from these immune hubs [5, 6]. However, the overall interplay of these multiple tissue components in response to radiation is incompletely understood.
In the event of mass casualties resulting from nuclear incidents, there is an urgent need for drugs capable of mitigating the effects of lethal radiation doses [7]. The logistical challenges, such as mass panic or infrastructure collapse, necessitate the development of drugs that can be administered up to 24 hours after exposure to a lethal dose of radiation (e.g., 70% lethal dose within 10 days [LD70/10]). Our previous efforts to identify such drugs have involved high-throughput screening of drug libraries, using lymphocyte death as the primary endpoint. Among the compounds identified, 1-[(4-nitrophenyl)sulfonyl]-4-phenylpiperazine (NSPP) has shown the most efficacy in mitigating hematopoietic and gastrointestinal (GI)-ARS, which is usually accompanied by dehydration, diarrhea, or electrolyte disturbance, and supported by laboratory or endoscopic evidence of mucosal injury [8, 9]. However, this approach did not fully capture the complex responses of intestinal tissue to radiation exposure [10].
In this study, we performed a comprehensive gene expression analysis of the murine duodenum in C3Hf/Sed/Kam mice to uncover the early transcriptomic changes triggered by a lethal dose of total body irradiation (TBI) and the subsequent recovery processes. We confirmed that death following TBI was primarily due to GI-ARS based on clinical signs (severe diarrhea, weight loss, and dehydration) and histopathological evidence of small intestinal injury, rather than late hematopoietic failure. Our analysis revealed dynamic shifts in gene expression over time, with early activation of pathways associated with cell death, cell cycle arrest, compromised hematopoiesis, and immune deficiencies. These early disruptions were then gradually subsided/restored, followed by the upregulation of genes involved in cell-cell adhesion, protein digestion and absorption, critical signaling pathways, and ISC-associated gene signatures.
Importantly, treatment with NSPP further enhanced the activation of ISC signatures, expediting tissue repair and recovery. Notably, the expression of proline and serine rich coiled-coil 1 (Psrc1) was significantly elevated with NSPP treatment, suggesting its pivotal role in promoting the regenerative effects observed. These findings highlight the complex and coordinated response of intestinal tissues to radiation-induced damage and underscore the therapeutic potential of NSPP in mitigating GI-ARS. By advancing our understanding of the molecular mechanisms involved in intestinal recovery, this study lays the groundwork for developing more effective and innovative radiation countermeasures.
Materials and Methods
1. Animals
C3Hf/Sed/Kam mice were bred and maintained in a pathogen-free environment in the American Association of Laboratory Animal Care-accredited Animal Facilities of Department of Radiation Oncology, University of California, Los Angeles, in accordance with all local and national guidelines for the care of animals, and were approved by the University of California Los Angeles’ Institutional Animal Care and Use Committee (Protocol #: ARC-2015-065).
2. Irradiation
C3Hf/Sed/Kam mice were used because this strain has been historically well characterized in radiation response studies, particularly GI-ARS models, and provides consistent, reproducible outcomes. Female mice were selected to reduce aggression and stress-related variability. Twelve-week-old animals were chosen to represent young adults, providing a mature yet resilient baseline for assessing radiation-induced injury. Mice were irradiated using an experimental X-ray irradiator (Gulmay Medical Inc.) at a dose rate of 2.8159 Gy/min for the time required to apply a prescribed dose. The X-ray beam was operated at 300 kV and hardened using a 4 mm Be, a 3 mm Al, and a 1.5 mm Cu filter. National Institute of Standards and Technology (NIST)-traceable dosimetry was performed using a micro-ionization chamber, and dosimetry was confirmed using films (Ashland Specialty Ingredients G.P.) placed at five positions inside the abdomen of mouse cadavers.
Mice were anesthetized prior to irradiation with an intra-peritoneal injection of 30 μL of a ketamine (100 mg/mL, Phoenix; Medivant Healthcare) and xylazine (20 mg/mL, AnaSed; Akorn) mixture at a 4:1 ratio. Following anesthesia, mice were placed on a tray with their tails and limbs secured using tape to minimize movement during irradiation. Sham-irradiated mice underwent the same anesthesia and restraint procedures but were not exposed to irradiation.
3. In Vivo Drug Administration
Twenty-four hours after irradiation, the mice received three daily subcutaneous injections of 5 mg/kg NSPP (Vitascreen) to the scruff of the neck. This compound was dissolved in 15 μL dimethyl sulfoxide (DMSO) and then suspended in 1 mL of 1% cremophor EL, with each injection spaced 24 hours apart. As a vehicle, mock-treated control, animals received injections of the DMSO/cremophor EL solution without the compound.
4. RNA Isolation
Twenty-eight, 72, and 96 hours after irradiation or sham irradiation, the animals were euthanized using isoflurane and cervical dislocation. The abdominal cavity was immediately opened, and the small intestines were mobilized, harvested, and flushed with phosphate-buffered saline (PBS). Even under homeostasis, the activity of signaling pathways changes dramatically along the murine small intestines [11]. In order to minimize this effect, we restricted the analysis to the duodenum. The proximal half of the duodenum of each mouse was cut and minced, and RNA was extracted using Trizol (Thermo Fisher Scientific).
5. Bulk RNA Sequencing
Bulk RNA sequencing (RNASeq) was performed by Novogene. Quality and integrity of total RNA were controlled on Agilent Technologies 2100 Bioanalyzer (Agilent Technologies). The RNASeq library was generated using NEBNext Ultra RNA Library Prep Kit (New England Biolabs) according to the manufacturer’s protocols. The library concentration was quantified using a Qubit 3.0 fluorometer (Life Technologies) and then diluted to 1 ng/μL before checking insert size on an Agilent Technologies 2100 Bioanalyzer and quantifying to greater accuracy by quantitative polymerase chain reaction (qPCR) (library molarity >2 nM). The library was sequenced on Illumina NovaSeq6000 with an average of 20 M reads per RNA sample. Reads were then mapped to the mouse genome (mg39) following their standard pipeline [12].
Read counts were analyzed using the integrated Differential Expression and Pathway (iDEP) package version 2.0 [13]. Differentially expressed genes (DEGs) were calculated using the DESeq2 algorithm with a minimum of a 2-fold change and a false discovery rate (FDR) of 0.1. Enrichment p-values were calculated based on a one-sided hypergeometric test. p-values were then adjusted for multiple testing using the Benjamini-Hochberg procedure and converted to FDR. Fold enrichment was defined as the percentage of genes in the list belonging to a pathway, divided by the corresponding percentage in the background.
6. Hematoxylin and Eosin Staining
Small intestines were harvested from mice at designated time points post-irradiation. Tissues were flushed with cold PBS to remove contents, fixed in 10% formalin for 24 hours at room temperature, and then processed for paraffin embedding using standard histological procedure. Four μm sections were cut and baked for 1 hour at 65 °C, deparaffinized in two successive xylene baths for 5 minutes each, followed by hydration through an alcohol gradient (ethanol 100%, 90%, 70%, 50%, and 25%) for 5 minutes each, followed by rinsing in distilled water. Slides were then stained in Mayer’s hematoxylin (Sigma-Aldrich) for 5 minutes, rinsed in running tap water for 5 minutes, and differentiated in 0.3% acid alcohol (1% HCl in 70% ethanol) for 30 seconds. After a second rinse in tap water, slides were blued in 0.1% sodium bicarbonate solution for 30 seconds and rinsed again. Eosin Y (Sigma-Aldrich) counterstaining was performed for 2 minutes, followed by dehydration through graded ethanol (70%, 95%, and 100%) and clearing in xylene. Coverslips were mounted with Permount mounting medium (Fisher Scientific).
7. TdT-Mediated dUTP Nick-End Labeling Staining
Twenty-four hours after TBI with 16 Gy, the mice were administered NSPP on a daily basis. Twenty-eight hours and 72 hours post-irradiation, the mice were euthanized, and their small intestines were immediately harvested and fixed in formalin. The tissues were then embedded in paraffin, and 4 μm sections were prepared for terminal deoxynucleotidyl transferase (TdT) in Situ Apoptosis Detection (#4812-30-K; R&D Systems). Briefly, the slides were baked for 1 hour in an oven at 65 °C, then dewaxed in two successive xylene baths for 5 minutes each, followed by hydration through an alcohol gradient (ethanol 100%, 90%, 70%, 50%, and 25%) for 5 minutes each. The sections were subsequently processed for labeling and viewing as per the manufacturer’s instructions.
8. Statistics
All data are presented as mean±standard error of the mean from a minimum of three biologically independent experiments. Statistical significance was determined using two-way analysis of variance for multiple comparisons, with a p≤0.05 considered significant. Graphs and statistical analyses were performed using GraphPad Prism software version 10.2.0 (GraphPad Software Inc.). For bulk RNASeq, DEGs were identified with a minimum fold-change threshold of 2 and a FDR cut-off of 0.1.
Results
1. Radiation-Induced Apoptosis in Murine Small Intestine and Its Mitigation by NSPP
C3Hf/Sed/Kam mice were exposed to 16 Gy TBI, a dose corresponding to the LD70/10 for this strain (based on our institutional pilot data and reference [14] providing LD50/10 values for different experimental conditions, such as mouse strain and irradiator setup). This dose results in the manifestation of a full GI-ARS, with 70% of the mice expected to die within 10 days post-irradiation. Histological examination revealed severe intestinal injury: by 28 hours post-TBI the villus-crypt architecture had largely collapsed and the epithelial lining was disrupted. Limited mucosal regeneration was evident at 72 hours, characterized by the appearance of shortened villus-crypt units (Fig. 1A). In parallel, TdT-mediated dUTP nick-end labeling (TUNEL) staining demonstrated the widespread apoptotic cells detected in the epithelial layer at 28 hours, and although apoptotic activity declined by 72 hours, it remained significantly elevated relative to sham-irradiated controls (Fig. 1B and 1C).
Our previous studies provided proof-of-concept that GI injury can be mitigated when treatment is initiated 24 hours after irradiation [5, 8] and demonstrated that NSPP has the potential to expand the ISC pool, increase the number of regenerating crypts, and reduce GI-ARS mortality [12]. Here, NSPP was again administered 24 hours post-TBI, and intestinal histology was assessed at 28 hours and 72 hours post-TBI. No distinct difference was observed in the disruption of epithelial lining at 28 hours post-TBI after NSPP treatment. However, at 72 hours post-TBI, NSPP-treated mice displayed a greater number of newly formed villus-crypt units compared with irradiated controls (Fig. 1A). Consistently, TUNEL staining revealed significantly fewer apoptotic cells in the NSPP group at both time points, confirming its mitigating effects (Fig. 1B and 1C).
2. Total Body Irradiation Induces Distinct Gene Expression Profiles in Murine Small Intestines
To better understand the effects of radiation mitigators on the recovery process, we first examined early transcriptomic changes in the small intestines following a lethal dose of TBI, as well as changes during subsequent recovery without interventions. We performed bulk RNASeq at three time points (28, 72, and 96 hours post-TBI), as shown in Fig. 2A. Principal component analysis demonstrated that biologically independent replicates clustered closely, indicating small variation between individual animals (Fig. 2B). Hierarchical clustering of DEGs revealed distinct gene expression profiles as early as 28 hours post-TBI (Fig. 2C), with 178 genes upregulated and 568 genes downregulated compared to the sham-irradiated controls (Fig. 2D). Next, we conducted a Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of the DEGs. The analysis revealed that the up-regulated genes significantly overlapped with gene sets involved in the p53 signaling pathway and cell cycle regulation (Fig. 2E). This finding aligns with the established understanding of the biological response to DNA damage and stress caused by radiation, which includes the activation of p53 signaling to mediate cell cycle arrest, DNA repair, or apoptosis [15]. However, the down-regulated genes were significantly associated with gene sets involved in hematopoietic cell lineage, primary immunodeficiency, and T cell receptor signaling pathway (Fig. 2F). This observation was in line with the well-known impact of radiation on the immune system, as it often results in compromised hematopoiesis and immune deficiencies [16] and the role of the intestines as a major part of the immune system.
To explore the subsequent transcriptomic changes during the recovery process, we analyzed samples at 72 hours and 96 hours post-irradiation. Again, principal component analysis showed that biologically independent replicates clustered closely, with distinct separation between different groups (Fig. 2G). Hierarchical clustering of DEGs revealed unique profiles among the groups (Fig. 2H). At 72 hours post-irradiation, 797 genes were upregulated and 506 genes were downregulated compared to the sham-irradiated control animals (Fig. 2I). At 96 hours post-irradiation, 2,565 genes were upregulated and 1,464 genes were downregulated compared to the sham-irradiated controls (Fig. 2J). This indicated dynamic transcriptomic changes during the recovery phase.
To gain a better understanding of the dynamic transcriptomic changes, we conducted KEGG enrichment analysis of DEGs from all three time points post-irradiation compared to the corresponding sham-irradiated groups. Our findings showed that the initially upregulated p53 signaling pathway and cell cycle modulation gradually subsided with the completion of DNA repair. Concurrently, the downregulated pathways related to hematopoietic cell lineage and immunodeficiencies showed a gradual restoration over time, indicating recovery of immune function and the residing hematopoietic cell compartment (Fig. 2K). Additionally, we observed an upregulation in pathways related to cytokine-cytokine receptor interaction, cell adhesion molecules, focal adhesion, extracellular matrix (ECM)-receptor interaction, and protein digestion and absorption during the recovery process, with a more pronounced increase at 96 hours post-irradiation (Fig. 2K). This aligns with the gradual recovery of the intestinal epithelial lining, which is crucial for nutrient absorption. The enhanced expression of these pathways suggests a robust effort by the tissues to restore integrity and function, facilitating overall recovery from radiation-induced damage. Conversely, we noted downregulation of metabolic pathways during the intestinal recovery process post-irradiation, indicating a prioritization of repairing damaged tissues and restoring cellular function over energy-consuming metabolic activities. Furthermore, we also observed activation of the phosphoinositide 3-kinase-protein kinase B (PI3K-Akt), transforming growth factor-beta, Janus kinase-signal transducer and activator of transcription, mitogen-activated protein kinase, cyclic adenosine monophosphate, and Hippo signaling pathways during the recovery process, with a more pronounced signature at 96 hours post-irradiation (Fig. 2K), indicating a coordinated activation of multiple signaling pathways known to support tissue repair and regeneration [17, 18].
3. Upregulation of the Lgr5+ Intestinal Stem Cell Gene Signature during Recovery
The gene signature of Lgr5+ ISCs has been extensively studied and characterized [19–21]. Integrating transcriptomics with proteomics analyses defined a molecular signature for Lgr5+ CBC cells, consisting of 510 stem cell-enriched genes [19]. To extend our analysis, we compared our lists of DEG from three post-irradiation time points with these ISC signature genes. We found that several of these signature genes were upregulated during the recovery process, with the expression of an increasing number of genes becoming more pronounced over time (Fig. 3). This indicated active participation of Lgr5+ ISCs in tissue regeneration and repair. Notably, the gene Psrc1 consistently showed strong activation throughout the recovery process, highlighting its potential role in this regenerative response.
Recently, two landmark studies published in Cell provided significant insights into the biology of ISCs [22, 23]. These studies identified and characterized the presence of multipotent ISCs located in the upper crypt zone. These undifferentiated cells play a pivotal role in maintaining intestinal homeostasis [22] and facilitating regeneration after irradiation-induced injury [23]. The cells, marked by fibroblast growth factor binding protein 1 (Fgfbp1), have been shown to repopulate the intestinal epithelium, including regenerating the well-known Lgr5+ CBC cells. Additionally, the sequencing data from the isthmus progenitor cells uncovered a novel signature of genes associated with intestinal stemness. To explore potential overlaps, we compared our upregulated DEG lists (Supplementary Table 1) to their defined stemness signature. Interestingly, we found that two genes, amphiregulin (Areg) and clusterin (Clu), were prominently present in our DEG lists at both 72 hours and 96 hours post-TBI time points, suggesting their potential involvement in the regenerative response following TBI.
4. Early Transcriptomic Changes in the Small Intestines Following NSPP Mitigation
Previously, we have reported that NSPP has the potential to expand the ISC pool, increase the number of regenerating crypts, and prevent the GI-ARS [12]. To identify novel intervention targets influenced by NSPP that benefit the subsequent recovery process, we first explored the early transcriptomic changes at 28 hours post-TBI (4 hours after NSPP application). Again, principal component analysis showed close clustering of biologically independent replicates, with distinct separation between groups (Fig. 4A). Hierarchical clustering of DEGs revealed unique gene expression profiles among the groups (Fig. 4B). However, the drug effect at this early time point was minimal, with only seven differentially downregulated genes in comb 28 hours post-TBI (the NSPP plus radiation group) compared to radiation alone (Fig. 4C), and 14 upregulated and two downregulated genes in the NSPP-only group compared to the sham-irradiated group (Fig. 4D). We also compared the NSPP plus radiation treated animals to the sham-irradiated group, identifying 222 differentially upregulated and 373 downregulated genes, as shown in the volcano plot (Fig. 4E). KEGG enrichment analysis of these DEGs revealed a significant overlap with gene sets involved in hematopoietic cell lineage and the intestinal immune network for immunoglobulin A production (Fig. 4F). This suggests that NSPP may potentially boost intestinal recovery not only by increasing the number of ISCs but also by recovering resident immune cells, a function of NSPP described previously [8].
5. Transcriptomic Changes during Recovery Following NSPP Mitigation
Next, we examined transcriptomic changes at 72 hours (48 hours after NSPP application) and 96 hours (72 hours after NSPP application) post-TBI to identify potentially novel targets that facilitate recovery. Again, principal component analysis revealed partial clustering of samples, suggesting modest transcriptional differences between groups (Fig. 5A), and hierarchical clustering of DEGs revealed characteristic gene expression profiles for the NSPP-treated animals (Fig. 5B). At 72 hours post-TBI, differences were subtle, with only two upregulated genes and three downregulated genes in the NSPP plus radiation group compared to radiation alone (Fig. 5C), and 11 downregulated genes in the NSPP-only group compared to sham-irradiated control (Fig. 5D). Comparing the NSPP plus radiation group to the sham-irradiated control, we found 922 upregulated and 517 downregulated genes (Fig. 5E). KEGG enrichment analysis revealed significant overlap of upregulated DEGs with gene sets involved in focal adhesion, cytokine-cytokine receptor interaction, ECM-receptor interaction, and phosphoinositide 3-kinase-protein kinase B (PI3K-Akt) signaling pathway (Fig. 5F). In contrast, downregulated genes overlapped with metabolism-related pathways (Fig. 5G), consistent with observations from the radiation-only group compared to the sham-irradiated group in Fig. 2K.
Ninety-six hours post-TBI is a crucial time point for observing GI-ARS recovery, characterized by the appearance of regenerative crypts and notable changes in gene expression and cellular pathways during this period [23]. In our study, the application of NSPP resulted in 101 significantly upregulated and 66 downregulated genes compared to sham-irradiated controls (Fig. 5H). Additionally, applying NSPP 24 hours post-TBI revealed a distinct gene signature, with 2,616 upregulated and 1,422 downregulated genes relative to the sham-irradiated group (Fig. 5I). KEGG enrichment analysis showed that the upregulated genes were involved in ECM-receptor interaction, focal adhesion, cytokine-cytokine receptor interaction, complement and coagulation cascade, and protein digestion and absorption (Fig. 5J). In contrast, the downregulated DEGs were associated with metabolism (Fig. 5K). Furthermore, NSPP application after TBI led to 24 significantly upregulated and eight downregulated genes compared to the radiation-only group (Fig. 5L). The upregulated genes were linked to neuroactive ligand-receptor interaction and protein digestion and absorption (Fig. 5M), while the downregulated DEGs were associated with metabolism and interleukin-17 signaling pathway (Fig. 5N).
6. Lgr5+ Intestinal Stem Cell Gene Signature Changes during Recovery Following NSPP Mitigation
Lastly, in order to investigate the impact of NSPP mitigation on ISC recovery, we compared the DEGs at 72 hours and 96 hours after TBI compared to their corresponding sham-irradiated controls with the list of Lgr5+ ISC signature genes. Our analysis revealed a progressive and robust activation of these ISC signature genes, with the number of activated genes increasing as the recovery process advanced following NSPP treatment (Fig. 6). Importantly, this activation became more pronounced over time when compared to the effects of radiation alone (Fig. 3). These findings suggest that NSPP treatment significantly enhances the recovery of the Lgr5+ ISC population, which is critical for effective tissue regeneration and repair. Among the key findings, Psrc1 expression was notably higher following NSPP treatment, emphasizing its potential role in mediating the observed regenerative effects.
Further analysis using gene signatures associated with isthmus progenitor cells revealed that the intestinal stemness-associated gene lymphocyte antigen 6 family member A (Ly6a) was already upregulated at 28 hours after TBI (4 hours after NSPP application), marking an early regenerative response. At 72 hours after TBI (48 hours after NSPP application), we observed a significant upregulation of Areg and annexin A2 (Anxa2). At 96 hours after TBI (72 hours after NSPP application), we found further increases in the expression of nucleolin (Ncl), S100 calcium binding protein A6 (S100a6), and Clu (Corresponding upregulated DEGs can be found in Supplementary Table 1). These findings underscore the ability of NSPP to increase the expression of key ISC-associated genes, highlighting its potential to enhance tissue regeneration and repair in the irradiated small intestines. The progressive upregulation of these critical genes provides a molecular basis for the observed improvements in recovery and reinforces the therapeutic potential of NSPP in mitigating radiation-induced damage.
Discussion
In this study, we provide a comprehensive characterization of the transcriptomic changes in the murine duodenum following exposure to a lethal dose of TBI. Our findings reveal dynamic shifts in gene expression over time, highlight the complex and coordinated response of intestinal tissues to radiation-induced damage, and underscore the therapeutic potential of NSPP in mitigating GI-ARS.
A key observation in the study was the activation of ISC-associated genes, such as Lgr5+ population signatures, which were well-known to play a crucial role in epithelial repair and regeneration. However, Lgr5+ ISCs are known to be highly sensitive to radiation and other external stimuli [24, 25], often leading to their depletion following exposure. This raises important questions about the involvement of alternative cell populations in maintaining and restoring intestinal integrity under severe damage conditions. One recent study has highlighted Tuft cells as a potential alternative regenerative population after radiation exposure. Tuft cells, which were traditionally considered as long-lived, postmitotic cells, exhibited an unexpected ability to revert to a stem-like state following radiation exposure [26]. These cells are highly resistant to radiation-induced damage and capable of generating organoids containing all intestinal epithelial cell types. Similarly, a recent study described that the Fgfbp1+ ISC population in the upper crypt region generates progeny, propagating bi-directionally along the crypt-villus axis and replenishing Lgr5+ cells in the crypt base. The high-resolution single cell profiling and in vivo lineage tracing in this report demonstrated that isthmus cells possess functional stem cell properties and participate in cellular turnover during both homeostasis and regeneration. Additional evidence of plasticity comes from delta like canonical Notch ligand 1+ (Dll1+) crypt cells and quiescent +4 label-retaining cells (LRCs) [27–29]. Under homeostatic conditions, Dll1+ cells generate short-lived secretory clones, while quiescent +4 LRCs co-express Lgr5 and markers of secretory lineages, such as Paneth and enteroendocrine cells. Both populations have been shown to regain stemness and contribute to regeneration following injury. These findings illustrate the hierarchical and flexible interplay of various cell types during tissue repair, adding new dimensions to our understanding of intestinal regeneration.
Our transcriptomic analysis revealed the activation of gene signatures associated with traditional Lgr5+ ISC populations, suggesting that Lgr5+ cells contribute to recovery and regeneration following radiation exposure. Additionally, the upregulation of the Areg and Clu genes—markers of isthmus progenitor cells [23]—supports the presence of a reserve stem cell population likely involved in recovery and regeneration. More importantly, NSPP treatment further amplified the elevation of Lgr5+ gene signatures and those associated with isthmus progenitor cells as early as 28 hours after TBI (4 hours after NSPP application). Furthermore, the expression of stemness-related genes [22, 23, 26] such as Ly6a, Anxa2, Ncl, S100a6, and Clu was increased after NSPP treatment, indicating that NSPP enhances stem cell activation and the regenerative capacity of the intestinal epithelium following radiation-induced injury. Notably, the transcriptomic differences observed in comb (ionizing radiation [IR]+NSPP) versus control comparisons were less apparent when compared to the IR group alone, suggesting that NSPP primarily modulates radiation-induced responses rather than activating broad independent transcriptional changes.
Among the genes upregulated during recovery in our study, Psrc1 emerged as a particularly noteworthy candidate for its potential role in intestinal regeneration. Psrc1 encodes a proline-rich protein regulated by the tumor suppressor protein p53 [30, 31]. It plays a crucial role in cell cycle progression and mitotic spindle function, both essential processes for ISC proliferation and differentiation. Psrc1 marks a subset of ISCs enriched with major histocompatibility complex (MHC) class II molecules involved in antigen presentation and interactions with immune cells [32]. These ISC-III cells, which are progressively more differentiated, also possess the capacity to de-differentiate and regain stem-like properties. In our study, NSPP treatment significantly amplified Psrc1 expression, suggesting that this gene may serve as a key mediator of stem cell activation and epithelial repair. The increased expression of Psrc1 likely contributes to the enhanced regenerative capacity observed with NSPP treatment, emphasizing its critical role in stem cell-mediated recovery. Future studies should explore the mechanistic role of Psrc1 in ISC biology, particularly its influence on cellular plasticity and its interactions with other regenerative pathways and compartments within the GI system. Understanding how Psrc1 contributes to ISC functionality and its cross-talk with immune cells may reveal novel strategies for enhancing intestinal repair and mitigating damage following radiation or other injuries.
We have previously demonstrated that NSPP functions as an activator of the Hedgehog signaling pathway by directly binding to the transmembrane domain of Smoothened, a key upstream effector in this pathway [8]. Hedgehog signaling is well-established as a critical regulator of ISC proliferation, crypt regeneration, and epithelial maintenance, all of which are essential for recovery from radiation-induced damage. In addition to its role in promoting epithelial regeneration, NSPP also appears to influence the gut’s immune microenvironment. In our earlier studies, we observed increased cellularity in Peyer’s patches, the lymphoid structures that serve as an immunological defense hub in the small intestine [5]. This suggests that NSPP not only supports epithelial repair but may also enhance mucosal immune responses, potentially contributing to improved barrier function and host defense during recovery from GI-ARS. In the present study, transcriptomic profiling of intestinal tissues further identified Psrc1 as a gene potentially regulated by NSPP, highlighting new avenues for exploring the molecular targets of NSPP and refining its therapeutic potential for radiation-induced intestinal injury.
While our study provides valuable insights into the transcriptomic changes in the murine duodenum following TBI and highlights the potential of NSPP as a radiation mitigator, several limitations should be considered. These findings need to be validated in other regions of the intestine to determine whether similar regenerative mechanisms are consistent throughout the GI tract. Additionally, assessing the long-term effects of NSPP treatment, including its impact on survival, tissue functionality, and potential side effects (fibrosis, chronic inflammation, or altered epithelial homeostasis), is crucial for evaluating its therapeutic viability. Finally, the use of bulk RNASeq in this study does not provide information about the interactions between different cellular compartments within the epithelium, such as immune cells and endothelial cells. Single cell RNASeq or spatial transcriptomics could be beneficial, as they have the potential to unravel complex cellular interactions by providing high-resolution data on gene expression at the individual cell level and preserving spatial context within the tissue. This could lead to a more comprehensive understanding of the cellular dynamics and molecular mechanisms involved in intestinal regeneration and how NSPP influences these processes following radiation exposure.
Conclusion
Our study highlights the complex and dynamic molecular mechanisms underlying intestinal recovery following radiation exposure. The contributions of Lgr5+ ISCs, and possibly other plasticity-driven cell populations, are essential for tissue repair. NSPP’s ability to amplify these regenerative processes underscores its therapeutic potential. These findings enhance our understanding of radiation-induced damage and recovery, providing a foundation for developing targeted therapies to mitigate radiation injuries and improve outcomes in radiological emergencies.
Supplementary Materials
Supplementary materials can be found via https://doi.org/10.14407/jrpr.2025.00108.
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