Radiation-induced Intestinal Fibrosis Research Articles

Combining proteomics and Phosphoproteomics to investigate radiation-induced rectal fibrosis in rats and the effects of pSTAT3 inhibitor S3I-201 on human intestinal fibroblasts

ObjectiveTo investigate the regulatory mechanisms of radiation-induced rectal fibrosis (RIRF) and assess the therapeutic potential of S3I-201. MethodsSprague-Dawley rats were divided into control and radiation groups, with the latter exposed to 20 Gray pelvic X-rays. After 10 weeks, rectal tissues were analyzed using tandem mass tag (TMT) proteomics and phosphoproteomics. Pathway enrichment was performed via Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses, with secondary annotation using Cluego. Representative proteins and their phosphorylated counterparts were validated through immunoblotting in another cohort. STAT3 levels in rectal tissues from irradiated and non-irradiated colorectal cancer patients were examined, and the effects of S3I-201 on human rectal fibroblasts were evaluated. ResultsThe radiation group showed significant inflammatory responses and collagen deposition in the rat rectal walls. Enrichment analysis revealed that radiation-induced proteins and phosphoproteins were primarily involved in extracellular matrix-receptor interaction and the MAPK signaling pathway. Immunoblotting indicated increased expression of p-CAMKII, p-MRACKS, p-Cfl1, p-Myl9, and p-STAT3 in the radiation group compared to the control, while p-AKT1 expression decreased. Elevated phosphorylation of STAT3 was observed in submucosal fibroblasts of the post-radiation human rectum. S3I-201 specifically inhibited STAT3 phosphorylation and suppressed activation of human rectal fibroblasts, also inhibiting the pro-fibrotic effects of the classical TGF-β/Smad/CTGF pathway. ConclusionBy integrating phosphoproteomics and proteomics, this study elucidated the protein regulatory network of RIRF and identified the potential therapeutic targets, including phosphoproteins such as STAT3 in managing RIRF. SignificanceIn our research, we employed TMT labeling alongside LC-MS/MS techniques to comprehensively explore the proteomic and phosphoproteomic landscapes in rat models of radiation-induced intestinal fibrosis (RIRF). Our analysis revealed the function and pathways of proteins and phosphorylated proteins triggered by radiation, as well as those with protective roles. We mapped a network of interactions among these proteins and validated key protein expression levels using quantitative methods. Furthermore, we investigated STAT3 as a potential therapeutic target, assessing the efficacy of the inhibitor S3I-201 in laboratory settings, and highlighting its potential for RIRF treatment. Overall, our findings provide groundbreaking insights into the mechanisms underlying RIRF, paving the way for the development of future antifibrotic therapies.

Radiation-induced intestinal fibrosis: pathological assessment and pharmacological prevention

Although radiotherapy can improve the local control rate of tumors and prolong the survival period of patients, it can also lead to long-term adverse effects such as radiation-induced intestinal fibrosis. Radiation-induced intestinal fibrosis has a high incidence and poses significant challenges to treatment, severely impacting the quality of life of patients. Combining findings from domestic and international research, along with experiences of our center, this article mainly discusses the pathological changes of radiation-induced intestinal fibrosis, as well as the current status and challenges of pathological assessment and pharmacological prevention of this condition. At present, there is no definitive method to reverse the fibrotic pathological changes. Thus, the prevention of fibrosis is a crucial issue to be resolved. In the meantime, there is a lack of ideal assessment methods and effective preventive medications in clinical practice. It is necessary to enhance both basic and clinical research, thoroughly investigate the pathogenesis of the disease, and identify effective intervention targets to promote the diagnosis and treatment of radiation-induced intestinal fibrosis.

NMN alleviates radiation-induced intestinal fibrosis by modulating gut microbiota

Aim Radiation-induced intestinal fibrosis, a common complication of long-term survivors after receiving abdominal and pelvic radiotherapy, has no effective clinical drugs at present. Nicotinamide mononucleotide (NMN) has been reported to alleviate a variety of age-related diseases and has potential of regulating gut microbiota. The current study focuses on the role of gut microbiota in chronic radiation induced intestinal fibrosis, and investigates whether NMN plays a protective role in radiation-induced intestinal fibrosis as well as the impact of NMN on radiation-induced dysbiosis of gut microbiota. Materials and methods C57BL/6J mice received 15 Gy abdominal irradiation and NMN (300 mg/kg/day) supplement in drinking water. Feces were collected at 4- and 8-months post-irradiation and performed 16S rRNA sequencing to detect the gut microbiota. Colon tissues were isolated at 12 months after irradiation with or without NMN supplementation for histological analysis. Results We found that irradiation caused intestinal fibrosis, and altered the β diversity and composition of gut microbiota, while the gut microbiota was observed to be affected by time post-irradiation and age of mice. Long-term NMN supplementation alleviated intestinal fibrosis, and reshaped the composition and function of gut microbiota dysregulated by ionizing radiation (IR). In addition, Akkermansia muciniphila, a promising probiotic, and metabolism-related pathways, such as Biosynthesis of other secondary metabolites and Amino acid metabolism, were more abundant after NMN treatment in irradiated mice. Conclusion IR has a long-term effect on the gut microbiota and NMN supplementation can alleviate radiation induced intestinal fibrosis by reshaping the composition of gut microbiota and regulating the metabolic function of the microorganism.

Selectively Targeting Activated Myofibroblasts by ABT-263 Reverses Radiation-Induced Intestinal Fibrosis in Mice

<h3>Purpose/Objective(s)</h3> Radiation induced intestinal fibrosis (RIF) is an irreversible and severe late effect of abdominal and pelvic radiation therapy. The goal of this study was to determine whether clearance of activated fibroblasts with ABT-263, a BCL2/xl inhibitor that can selectively kill myofibroblasts, can reverse RIF. <h3>Materials/Methods</h3> C57BL/6J mice were exposed to a single dose of 12 Gy irradiation on the whole abdomen. The intestinal wall thickness of mice was monitored by MRI at 0, 8^th, 12^th and 18^th week. Eighteen weeks after IR, mice were given 2 cycles of vehicle or ABT-263 treatment by gavage (50 mg/kg per day, 5 days per cycle). Two weeks after last treatment, intestinal sections were harvested for evaluation the effect of ABT-263 on irradiation induced activation of fibroblasts and extracellular matrix deposition by immunohistochemistry and Masson staining. The expression of TGF-β1, α-SMA, BCL-2/BCL-w proteins and phosphorylation of STAT3 in intestinal homogenate were analyzed by western blot analysis. Human and murine fibroblasts were cultured to evaluate the effects of ABT-263 on irradiation or classical TGF-β1-induced (5 ng/ml) activation of fibroblasts. <h3>Results</h3> At 18 weeks after 12Gy abdominal irradiation, C57BL/6J mice developed a significant thickness of the irradiated intestinal wall, with increased number of a-SMA⁺ fibroblasts (activated fibroblasts). Treatment of the RIF mice with ABT-263 after developed RIF could reverse the disease by specific clearance of activated fibroblasts. In vitro, inhibition of Bcl-2 and Bcl-xL by the small molecule BH3 mimetic, ABT-263, or Bcl-xL-specific small interfering RNA induced apoptosis in TGFb1 or irradiation-activated fibroblasts, but not in quiescent fibroblasts. <h3>Conclusion</h3> To our knowledge, this is the first study to demonstrate that RIF can be reversed by a specific myofibroblasts depletion drug ABT-263. ABT-263 has the potential to be developed as a new treatment for RIF.

Coenzyme Q10 attenuates inflammation and fibrosis implicated in radiation enteropathy through suppression of NF-kB/TGF-β/MMP-9 pathways

Radiation enteropathy is one the most common clinical issue for patients receiving radiotherapy for abdominal/pelvic tumors which severely affect the quality of life of cancer patients due to dysplastic lesions (ischemia, ulcer, or fibrosis) that aggravate the radiation damage. Herein, this study demonstrated the prophylactic role of coenzyme Q10 (CoQ10), a powerful antioxidant, against radiotherapy-induced gastrointestinal injury. Male Sprague Dawley rats were divided into four groups: group 1 was defined as control, and group 2 was the irradiated group. Group 3 and 4 were CoQ10 control and radiation plus CoQ10 groups, respectively. CoQ10 (10 mg/kg) was orally administered for 10 days before 10 Gy whole-body radiation and was continued for 4 days post-irradiation. CoQ10 administration protected rats delivered a lethal dose of ϒ-radiation from changes in crypt-villus structures and promoted regeneration of the intestinal epithelium. CoQ10 attenuated radiation-induced oxidative stress by decreasing lipid peroxidation and increasing the antioxidant enzyme catalase activity and reduced glutathione level. CoQ10 also counteracts inflammatory response mediated after radiation exposure through downregulating intestinal NF-ĸB expression which subsequently decreased the level of inflammatory cytokine IL-6 and the expression of COX-2. Radiation-induced intestinal fibrosis confirmed via Masson's trichrome staining occurred through upregulating transforming growth factor (TGF)-β1 and matrix metalloproteinase (MMP)-9 expression, while CoQ10 administration significantly diminishes these effects which further confirmed the anti-fibrotic property of CoQ10. Therefore, CoQ10 is a promising radioprotector that could prevent intestinal complications and enhance the therapeutic ratio of radiotherapy in patients with pelvic tumors through suppressing the NF-kB/TGF-β1/MMP-9 signaling pathway.

TMT-labelled quantitative proteomic analysis to identify the proteins underlying radiation-induced colorectal fibrosis in rats

Radiation-induced colorectal fibrosis (RICF) after pelvic radiotherapy can lead to stenosis and affect patients' quality of life. However, to date, few effective prevention and treatment methods for RICF have been implemented in clinical practice. Therefore, there is a strong need to investigate the molecular events underlying radiation-induced intestinal fibrosis. This study aimed to identify the comprehensive protein expression profiles related to RICF in rats. We performed tandem mass tag (TMT)-labelling proteomic analysis to examine RICF in rats after a single dose of 20 Gy conformal irradiation via X-ray and confirmed the altered proteins with parallel reaction monitoring (PRM). In total, we identified 6692 proteins, of which 5756 were quantified. When the p-value was <0.05, a protein fold change >1.5 or < 0.67 was considered to indicate a differentially abundant protein (DAP). Finally, we detected 227 upregulated proteins and 93 downregulated DAPs. The DAPs were involved in several pathways, including those involved in extracellular space, complement and coagulation cascades pathway, and regulation of response to wounding. Nine proteins in three main pathways were validated by parallel reaction monitoring. Our findings could accelerate the understanding of the mechanism underlying RICF and provide some clues for the identification of potential candidate targets for anti-fibrosis treatment. SignificanceRadiation-induced intestinal fibrosis is a significant problem that causes higher morbidity and affects patients' quality of life. However, there are few effective methods implemented in clinics to prevent and reverse this disease to date. The mechanism of radiation-induced colorectal fibrosis remains unclear, and the proteomic study is very limited. Here, we performed a large-scale comparative quantitative proteomics study on radiation-induced colorectal fibrosis in rats. The results provide an improved understanding of radiation-induced intestinal fibrosis and some clues for the identification of potential candidate targets for anti-fibrosis treatment.

LncRNA WWC2-AS1 functions AS a novel competing endogenous RNA in the regulation of FGF2 expression by sponging miR-16 in radiation-induced intestinal fibrosis

BackgroundRecently, long non-coding RNAs (lncRNAs) were considered as important gene expression regulators involving various biological processes. In this study, we explored the role of lncRNAs in the pathogenesis of radiation-induced intestinal fibrosis (RIF).MethodsLncRNAs were screened by microarray (Human LncRNA Array v3.0, Arraystar, Inc.) and the differentially expressed lncRNAs in RIF and non-RIF were analyzed by bioinformatics methods. The expression of WWC2-AS1/miR-16/FGF2 axis was compared on mRNA and protein level between human intestinal CCD-18Co fibroblasts cell lines and subepithelial SEMFs in response to radiation treatment. The significance of WWC2-AS1 in regulating FGF2 associated proliferation, migration, invasion and fibrosis of CCD-18Co and SEMFs by exposure to radiation was analyzed by shRNA (WWC2-AS1 shRNA) knock-down of endogenous WWC2-AS1.ResultsWWC2-AS1 and FGF2 level was significantly higher while miR-16 was down-regulated in radiation-treated intestinal tissues. WWC2-AS1 more potently boosted FGF2 expression via reducing miR-16, and WWC2-AS1 shRNA remarkably inhibited FGF2 associated proliferation, migration, invasion and fibrosis of radiation treatment in vitro, further demonstrating physical interaction between miR-16 and WWC2-AS1 in radiation-induced fibrosis progress.ConclusionsWWC2-AS1 was highly expressed in RIF, may function as a ceRNA in the regulation of FGF2 by binding miR-16. Targeting WWC2-AS1 thus may benefit radiation-induced fibrosis treatment.

Eosinophil depletion suppresses radiation-induced small intestinal fibrosis.

Radiation-induced intestinal fibrosis (RIF) is a serious complication after abdominal radiotherapy for pelvic tumor or peritoneal metastasis. Herein, we show that RIF is mediated by eosinophil interactions with α-smooth muscle actin-positive (α-SMA+) stromal cells. Abdominal irradiation caused RIF especially in the submucosa (SM) of the small intestine, which was associated with the excessive accumulation of eosinophils in both human and mouse. Eosinophil-deficient mice showed markedly ameliorated RIF, suggesting the importance of eosinophils. After abdominal irradiation, chronic crypt cell death caused elevation of extracellular adenosine triphosphate, which in turn activated expression of C-C motif chemokine 11 (CCL11) by pericryptal α-SMA+ cells in the SM to attract eosinophils in mice. Inhibition of C-C chemokine receptor 3 (CCR3) by genetic deficiency or neutralizing antibody (Ab) treatment suppressed eosinophil accumulation in the SM after irradiation in mice, suggesting a critical role of the CCL11/CCR3 axis in the eosinophil recruitment. Activated α-SMA+ cells also expressed granulocyte-macrophage colony-stimulating factor (GM-CSF) to activate eosinophils. Transforming growth factor-β1 from GM-CSF-stimulated eosinophils promoted collagen expression by α-SMA+ cells. In translational studies, treatment with a newly developed interleukin-5 receptor α-targeting Ab, analogous to the human agent benralizumab, depleted intestinal eosinophils and suppressed RIF in mice. Collectively, we identified eosinophils as a crucial factor in the pathogenesis of RIF and showed potential therapeutic strategies for RIF by targeting eosinophils.

Pirfenidone prevents radiation-induced intestinal fibrosis in rats by inhibiting fibroblast proliferation and differentiation and suppressing the TGF-β1/Smad/CTGF signaling pathway

Radiation-induced intestinal fibrosis (RIF) is a chronic toxicity following radiation, and can be very difficult to treat. Pirfenidone is a promising anti-fibrotic agent that inhibits fibrosis progression in various clinical and experimental studies. This study was aimed to explore whether pirfenidone could protect against RIF, and to evaluate the underlying mechanism. An animal model of RIF was induced by exposure of a single dose of 20 Gy to the pelvis. Rats were orally administered with pirfenidone (200, 400 md/kg/d) for 12 weeks. Primary rat intestinal fibroblasts were cultured to determine the effects of pirfenidone on TGF-β1-induced (5 ng/ml) proliferation and transdifferentiation of fibroblasts. The expression of collagen I, α-SMA, and TGF-β1/Smad/CTGF pathway proteins were analyzed by qRT-PCR and/or western blot analysis. The cell proliferation rate was determined by CCK-8 assay. The results indicated that pirfenidone significantly attenuated fibrotic lesion in irradiated intestines and reduced collagen deposition by inhibiting TGF-β1/Smad/CTGF pathway in rat models. Moreover, in primary rat intestinal fibroblasts, pirfenidone decreased the up-regulation of TGF-β1-induced collagen I and α-SMA by suppressing TGF-β1/Smad/CTGF signaling pathway. Altogether, our findings suggested that pirfenidone attenuated RIF by inhibiting the proliferation and differentiation of intestinal fibroblasts and suppressing the TGF-β1/Smad/CTGF signaling pathway.

Silibinin attenuates radiation-induced intestinal fibrosis and reverses epithelial-to-mesenchymal transition.

Radiotherapy is a common treatment for cancer patients, but its use is often restricted by the tolerance of normal tissue. As cancer patients live longer, delayed radiation effects on normal tissue have become a concern. Radiation-induced enteropathy, including inflammatory bowel disease and fibrosis, are major issues for long-term cancer survivors. To investigate whether silibinin attenuates delayed radiation-induced intestinal injury in mice, we focused on intestinal fibrotic changes. Silibinin improved delayed radiation injuries in mice in association with decreased collagen deposition within the intestines and deceased transforming growth factor (TGF)-β1 levels in the intestine and plasma. Treating mice bearing CT26 mouse colon cancer tumors with both silibinin and radiation stimulated tumor regression more than radiation alone. We also investigated the effect of silibinin on the radiation-induced epithelial-to-mesenchymal transition (EMT), the primary mechanism of fibrosis. We assessed changes in E-cadherin, N-cadherin, and α-smooth muscle actin expression, and demonstrated that silibinin attenuates radiation-induced EMT. Irradiating intestinal epithelial cells increased TGF-β1 levels, but silibinin suppressed TGF-β1 expression by inhibiting Smad2/3 phosphorylation. These results suggest silibinin has the potential to serve as a useful therapeutic agent in patients with radiation-induced intestinal fibrosis.

Soluble Dietary Fiber Ameliorates Radiation-Induced Intestinal Epithelial-to-Mesenchymal Transition and Fibrosis.

Intestinal fibrosis is a late complication of pelvic radiotherapy. Epithelial-to-mesenchymal transition (EMT) plays an important role in tissue fibrosis. The aim of this study was to examine the effect of soluble dietary fiber on radiation-induced intestinal EMT and fibrosis in a mouse model. Apple pectin (4% wt/wt in drinking water) was administered to wild-type and pVillin-Cre-EGFP transgenic mice with intestinal fibrosis induced by a single dose of abdominal irradiation of 10 Gy. The effects of pectin on intestinal EMT and fibrosis, gut microbiota, and short-chain fatty acid (SCFA) concentration were evaluated. Intestinal fibrosis in late radiation enteropathy showed increased submucosal thickness and subepithelial collagen deposition. Enhanced green fluorescent protein (EGFP)+/vimentin+ and EGFP+/α-smooth muscle actin (SMA)+ coexpressing cells were most clearly observed at 2 weeks after irradiation and gradually decreased at 4 and 12 weeks. Pectin significantly attenuated the thickness of submucosa and collagen deposition at 12 weeks (24.3 vs 27.6 µm in the pectin + radiation-treated group compared with radiation-alone group, respectively, P < .05; 69.0% vs 57.1%, P < .001) and ameliorated EMT at 2 and 4 weeks. Pectin also modulated the intestinal microbiota composition and increased the luminal SCFA concentration. The soluble dietary fiber pectin protected the terminal ileum against radiation-induced fibrosis. This effect might be mediated by altered SCFA concentration in the intestinal lumen and reduced EMT in the ileal epithelium.

Molecular aspects of chronic radiation enteritis

Chronic radiation enteritis (CRE) is one of the most feared complications of abdominal or pelvic radiation therapy and the treatment of CRE is difficult and often controversial. Recent progress in molecular biology has shed some light on the pathogenesis of CRE, which is characterized by fibrosis. The purpose of this article is to summarize the current state of knowledge of molecular aspects of radiation induced intestinal fibrosis and to discuss potential therapeutic targets. A review of the up-to-date published literature involving the possible molecular cascades in radiation-induced intestinal fibrosis and prospective targets for CRE were performed using the Pub-Med search engine. Fibrosis development is correlated with transforming growth factor β1 (TGF-β1) and its downstream effector Smad3, which stimulates fibrogenic downstream mediators, such as connective tissue growth factor (CTGF). Ras homologue (Rho) and Rho-associated kinase (ROCK) signaling pathway have been shown to play important roles in the development of CRE. The inhibition of these pathways ameliorated radiation-induced intestinal fibrosis in vitro and in animal studies; however, the relationship between the Smad3 and Rho signaling pathways has not been elucidated. Rho/ROCK and TGF-β1/Smad3 signaling pathways have been shown to play a key role in intestinal fibrogenesis, which might provide with effective possibilities for clinical intervention. Understanding the cooperation between Smad3 and Rho, may therefore be critical to our overall understanding of fibrosis development and maintenance of CRE.

All- trans-Retinoic Acid Attenuates Radiation-Induced Intestinal Fibrosis in Mice

Intestinal fibrosis leading to severe bowel dysmobility or obstruction is a troublesome adverse effect of abdominal or pelvic radiation therapy. We have recently reported that all-trans-retinoic acid (ATRA) prevents radiation- or bleomycin-induced lung fibrosis. Here, we examined the impact of ATRA on the mouse model of radiation-induced intestinal fibrosis. We evaluated the histology of late radiation fibrosis in surgical samples. We then performed histological examinations and quantitative measurements of mRNA of interleukin-6 and transforming growth factor-beta(1) in intestinal tissues of irradiated mice with or without intraperitoneal administration of ATRA and investigated the effect of ATRA on the transdifferentiation and the production of collagen of irradiated human intestinal fibroblasts. Human samples of late radiation enteritis showed thickened submucosa and serosa, consistent with mouse model. Administration of ATRA attenuated irradiation-induced intestinal fibrosis and reduced mRNA of interleukin-6 and transforming growth factor-beta(1). In vitro studies disclosed that ATRA suppressed the transdifferentiation of irradiated intestinal fibroblasts and diminished the production of collagen from the cells. Our findings indicate that ATRA ameliorates irradiation-induced intestinal fibrosis. ATRA could be a novel approach in the treatment of fibrosis associated with chronic radiation enteritis.

Pravastatin Inhibits the Rho/CCN2/Extracellular Matrix Cascade in Human Fibrosis Explants and Improves Radiation-Induced Intestinal Fibrosis in Rats

Intestinal complications after radiotherapy are caused by transmural fibrosis and impair the quality of life of cancer survivors. Radiation fibrosis was considered permanent and irreversible, but recently, its dynamic nature was shown, providing new opportunities for the development of antifibrotic therapies. Among these new targets, we identified the Rho/ROCK pathway and thought to investigate whether pravastatin treatment inhibits Rho pathway activation and elicits an antifibrotic action. Rho and ROCK activities were monitored in human explants presenting radiation fibrosis remodeling after incubation with pravastatin. Subsequent modulation of CCN2, type I collagen, and fibronectin expression were assessed ex vivo and in intestinal smooth muscle cells derived from radiation enteropathy. Then, the therapeutic relevance of the antifibrotic action of pravastatin was explored in vivo in a rat model of chronic radiation fibrosis (19 Gy X-rays) treated with 30 mg/kg/d pravastatin in the drinking water. The results obtained with human explants show that pravastatin specifically inhibits Rho activity in submucosal mesenchymal cells. Pravastatin also elicits ROCK inhibition, and subsequent CCN2 production in human explants and smooth muscle cells isolated from radiation enteropathy. Inhibition of type I collagen and fibronectin does occur, showing that pravastatin modulates the secretory phenotype of mesenchymal cells. Lastly, curative pravastatin administration improves radiation enteropathy in rats. This structural improvement is associated with decreased deposition of CCN2 and subsequent decreased extracellular matrix deposition. Targeting established fibrosis with pravastatin is an efficient and safe antifibrotic strategy in radiation-induced enteropathy, and is easily transferable into the clinic.

Maintenance of radiation-induced intestinal fibrosis: cellular and molecular features.

Recent advances in cell and molecular radiobiology clearly showed that tissue response to radiation injury cannot be restricted to a simple cell-killing process, but depends upon continuous and integrated pathogenic processes, involving cell differentiation and crosstalk between the various cellular components of the tissue within the extracellular matrix. Thus, the prior concept of primary cell target in which a single-cell type (whatever it's epithelial or endothelial cells) dictates the whole tissue response to radiation injury has to be replaced by the occurrence of coordinated multicellular response that may either lead to tissue recovery or to sequel development. In this context, the present review will focus on the maintenance of the radiation-induced wound healing and fibrogenic signals triggered by and through the microenvironment toward the mesenchymal cell compartment, and will highlight how sequential and sustained modifications in cell phenotypes will in cascade modify cell-to-cell interactions and tissue composition.

Heparan mimetic regulates collagen expression and TGF-beta1 distribution in gamma-irradiated human intestinal smooth muscle cells.

Radiation-induced intestinal fibrosis is characterized by collagen accumulation, a process in which TGF-beta1 plays a key role. We analyzed the effects of gamma radiation on collagen expression and TGF-beta1 distribution in human intestinal smooth muscle cells (HISM). We investigated the activity of a carboxymethylated and sulfated dextran (RG-1503), exhibiting antifibrotic properties and promoting in vivo intestinal tissue repair, on irradiated HISM. After (60)Co irradiation (10 Gy), HISM were labeled with [(3)H] proline (+/-RG-1503). Radiolabeled collagen I, III, and V were quantified by SDS-PAGE. TGF-beta1 was quantified by ELISA in culture medium, pericellular and intracellular compartments. Irradiation induced a specific 2.85-fold increase in collagen III production by HISM. Collagen V decreased by 80% 72 h after irradiation. Pericellular TGF-beta1 was increased (up to twofold) in irradiated HISM. RG-1503 added before or after irradiation reversed both mRNA and protein levels of collagen III and V to control values. RG-1503 decreased the amount of TGF-beta1 in the cell layer below the control values. Irradiation of HISM induced the development of a fibrotic phenotype in terms of collagen production and TGF-beta1 distribution. The antifibrotic RG-1503 restored HISM physiological characteristics and may represent a promising therapeutic approach for radiation-induced intestinal fibrosis.