Intestinal Radiation Research Articles

An Active Fraction of Trillium tschonoskii Promotes the Regeneration of Intestinal Epithelial Cells After Irradiation.

Despite significant scientific advances toward the development of safe and effective radiation countermeasures, no drug has been approved for use in the clinic for prevention or treatment of radiation-induced acute gastrointestinal syndrome (AGS). Thus, there is an urgent need to develop potential drugs to accelerate the repair of injured intestinal tissue. In this study, we investigated that whether some fractions of Traditional Chinese Medicine (TCM) have the ability to regulate intestinal crypt cell proliferation and promotes crypt regeneration after radiation. By screening the different supplements from a TCM library, we found that an active fraction of the rhizomes of Trillium tschonoskii Maxim (TT), TT-2, strongly increased the colony-forming ability of irradiated rat intestinal epithelial cell line 6 (IEC-6) cells. TT-2 significantly promoted the proliferation and inhibited the apoptosis of irradiated IEC-6 cells. Furthermore, in a small intestinal organoid radiation model, TT-2 promoted irradiated intestinal organoid growth and increased Lgr5+ intestinal stem cell (ICS) numbers. More importantly, the oral administration of TT-2 remarkably enhanced intestinal crypt cell proliferation and promoted the repair of the intestinal epithelium of mice after abdominal irradiation (ABI). Mechanistically, TT-2 remarkably activated the expression of ICS-associated and proliferation-promoting genes and inhibited apoptosis-related gene expression. Our data indicate that active fraction of TT can be developed into a potential oral drug for improving the regeneration and repair of intestinal epithelia that have intestinal radiation damage.

Low-Intensity Exercise Modulates Gut Microbiota to Fight Against Radiation-Induced Gut Toxicity in Mouse Models.

Radiation-induced gastrointestinal (GI) tract toxicity halts radiotherapy and degrades the prognosis of cancer patients. Physical activity defined as “any bodily movement produced by skeletal muscle that requires energy expenditure” is a beneficial lifestyle modification for health. Here, we investigate whether walking, a low-intensity form of exercise, could alleviate intestinal radiation injury. Short-term (15 days) walking protected against radiation-induced GI tract toxicity in both male and female mice, as judged by longer colons, denser intestinal villi, more goblet cells, and lower expression of inflammation-related genes in the small intestines. High-throughput sequencing and untargeted metabolomics analysis showed that walking restructured the gut microbiota configuration, such as elevated Akkermansia muciniphila, and reprogramed the gut metabolome of irradiated mice. Deletion of gut flora erased the radioprotection of walking, and the abdomen local irradiated recipients who received fecal microbiome from donors with walking treatment exhibited milder intestinal toxicity. Oral gavage of A. muciniphila mitigated the radiation-induced GI tract injury. Importantly, walking did not change the tumor growth after radiotherapy. Together, our findings provide novel insights into walking and underpin that walking is a safe and effective form to protect against GI syndrome of patients with radiotherapy without financial burden in a preclinical setting.

Abstract PO-081: LR-IL-22 protects the intestine to facilitate whole abdomen irradiation in ovarian cancer

Abstract Ovarian cancer is the most lethal gynecological cancer worldwide with an estimated 152,000 deaths per year. Despite optimal management with radical cytoreductive surgery and subsequent platinum/taxane-based chemotherapy, most patients, will suffer recurrence within 18 months. Our laboratory has recently discovered a new therapeutic agent for intestinal radiation protection, namely the novel second-generation probiotic Lactobacillus reuteri (LR) genetically engineered to produce the radioprotective cytokine Interleukin-22 (IL-22) (Zhang et al. In Vivo, 34(1):39-50, 2020 Jan-Feb). To demonstrate that LR-IL-22 could protect the intestines from irradiation, we used three mouse models (total body irradiation (TBI), whole abdomen irradiation (WAI) and partial body irradiation (PBI)). For TBI, C57BL/6 mice were irradiated to 9.25 Gy to the entire body. For WAI, C57BL/6 mice were irradiated using a linear accelerator so that only the abdomen was irradiated to 19.75 Gy with the remainder of the body shielded from the irradiation. PBI was performed with the right rear leg shielded with the rest of the body irradiated to 15 Gy. In all three models the mice were gavaged 24 hours after irradiation with 1 × 109 LR-IL-22 cells. The mice were followed for development of either the hematopoietic syndrome (TBI) or gastrointestinal syndrome (WAI or PBI). In separate experiments we determined if mice irradiated as above had decreased irradiation induced inflammation using a Luminex assay on the intestine and blood plasma from mice sacrificed on days 0, 1, 2, 3, 5 and 7 following irradiation. We also determined whether intraoral gavage of LR-IL-22 24 hours prior to irradiation might protect the tumor in a mouse ovarian tumor model. Murine ovarian tumor cells 2F8-cis were injected intraperitoneally into Muc1 transgenic mice. Seventy-two hours later the mice were irradiated to 16 Gy WAI and followed for tumor growth. In the TBI model, mice treated with LR-IL-22 24 hours prior to irradiation had an increased survival of 80% compared to 0% in control irradiated mice (p = 0.0001). In the WAI model, mice treated with LR-IL-22 had a 40% survival following 19.75 Gy compared to 0% in the control irradiated group (p = 0. 0100). Following the PBI dose of 15 Gy, mice treated with LR-IL-22 had a 70% survival compared to 0% for the control irradiation only mice (p = 0.0006). Decreased expression of several inflammatory proteins such as TNF-α, IL-6 and IFN-γ (p = 0.0423, 0.0473 and 0.0024, respectively) was also detected in mice treated with LR-IL-22 24 hours prior to WAI compared to control irradiated mice. Furthermore, two weeks after injecting Muc1 transgenic mice with tumors, the nonirradiated mice had more than 200 small tumor nodules disseminated throughout the peritoneum while control WAI mice or mice treated with intraoral LR-IL-22 prior to WAI had no more than 10 tumor nodules. Hence, intraoral LR-IL-22 prior to chemoradiation may protect the intestines and result in increased survival of ovarian cancer patients. Citation Format: Diala Fatima Hamade, Renee Fisher, Wen Hou, Donna Shields, Michael W. Epperly, Joel S. Greenberger. LR-IL-22 protects the intestine to facilitate whole abdomen irradiation in ovarian cancer [abstract]. In: Proceedings of the AACR Virtual Special Conference on Radiation Science and Medicine; 2021 Mar 2-3. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(8_Suppl):Abstract nr PO-081.

Biological Effects of Abdominal Irradiation on Intestinal Barrier Breakdown Identified By Second-Generation Probiotic, LR-IL-22

Toxic effects of total body irradiation and whole abdomen irradiation include breakdown of the intestinal barrier function and bacterial translocation into the abdominal lymphatic and systemic vascular circulations. Therapeutic effects of countermeasures against intestinal radiation damage have relied upon serum assays for bacterial endotoxin and citrulline, biomarkers of relatively late effects in barrier dysfunction. Intestinal barrier breakdown effects on mesenteric lymph nodes and spleen have been incompletely documented. In the present experiments, we gavaged C57BL/6NTac female mice one hour before irradiation (19.75 Gy whole abdomen) with 100 microliters containing 109 second-generation probiotic, Lactobacillus reuteri-Interleukin-22 (LR-IL-22) containing transgenes for both mammalian IL-22 and Rapamycin resistance (Alexander LM, et. al., Applied and Environmental Microbiology 85(10): e02335-18, 2019), LR alone, or sham-gavage. Twenty-four hours later, Peyer's patches, mesenteric lymph nodes, spleen, and peripheral blood were collected at serial times after gavage and assayed for growth of Rapamycin-resistant bacteria, and for relative compositions of immunocytes in mesenteric lymph nodes spleen and blood including: CD3, CD19, CD8, CD45, NK1.1, CD25, Foxp3, CD103, and PD-1+ lymphocytes. Compared to non-irradiated, non-gavaged mice, animals receiving LR or LR-IL-22 showed no significant changes in weight or length of the spleen or lymph nodes 24 hr after gavage. There was an increase in PD-1 expression in the spleen of the mice gavaged with LR-IL-22. There were no other significant changes in the composition of immunocytes in abdominal lymph nodes, or spleen and detectable second-generation probiotic growth in these organs. At 24 hr after irradiation, abdominal irradiated mice demonstrated a significant loss of weight and length of the spleen (p < 0.05) as well as decreased lymphocytes in the blood (p < 0.05). Mice gavaged with LR-IL-22 and irradiated to 19.75 Gy had a significant increase in lymphocytes and red blood cells compared to irradiation only (p < 0.05). There was no significant change in the size or weight of the lymph nodes. In the lymph nodes there was an increase in CD8+ cells after irradiation while the LR-IL-22 gavaged mice had an increase in PD-1 expression. There was no detectable growth of Rapamycin resistant bacteria in non-gavaged animals. Mice gavaged with LR-IL-22, but not LR showed decreased biomarkers of intestinal barrier breakdown. Thus, gavage of genetically labeled second-generation probiotics represents a sensitive assay system for irradiation changes in the intestinal barrier function, translocation of bacteria into the abdominal lymphatic system, and therapeutic effect of enteric delivered radiation countermeasures. Supported by U19-AI1068021 Grant. Disclosures No relevant conflicts of interest to declare.

The Protection Effect of Resveratrol Against Radiation-Induced Inflammatory Bowel Disease via NLRP-3 Inflammasome Repression in Mice.

With the extensive application of radiotherapy in various cancers, its side effects in tissues adjacent to cancers are garnering much attention. Intestines are sensitive to irradiation due to its rapid proliferation, and irradiation-induced enteric inflammation is common in patients with pelvic peritoneal tumors. Sirt1, class III protein deacetylase, could lead to transcriptional repression of various inflammation-associated genes, and our previous study has proved its relationship with interleukin (IL)-1β. Here we show that resveratrol, the activator of Sirt1, could alleviate the bowel inflammation induced by irradiation and the expression of Sirt1 is consistent with the inflammation level. We further identified in vivo that Sirt1 repress the expression of IL-1β by the repression of NLR Family, Pyrin Domain Containing protein 3 (NLRP3) expression. In conclusion, this study confirms resveratrol acts against radiation-induced inflammatory bowel disease via NLRP-3 inflammasome repression in mice and supports Sirt1 as a potential biomarker and therapy target in intestinal radiation protection.

THE COMBINATION OF EXTERNAL RADIATION THERAPY WITH HIGH DOSE RATE BRAHYTERAPY BOOST FOR PROSTATE CANCER: A COMPARATIVE CHARACTERISTIC OF TWO FRACTION MODES

The purpose of the study was to analyze treatment outcomes in prostate cancer patients, who received external radiotherapy combined with high dose rate (HDR) 198 Ir brachytherapy boost of 10 Gy in 2 fractions or 15 Gy in 1 fraction. Material and methods. Between July 2012 and June 2017, 98 patients with prostate cancer received radiation therapy at N.N. Petrov National Medical Research Center of Oncology (St-Petersburg, Russia). Intensitymodulated radiation therapy (IMRT) at a total dose of 46–50 Gy was delivered to the prostate, seminal vesicles and regional lymph outflow areas (RTOG, 2009). The patients were then divided into 2 groups. Group I patients (81 patients, 82.7 %) received HDR brachytherpy boost of 10 Gy in 2 fractions, Group II patients (17 patients, 17.3 %) received HDR brachytherpy boost of 15 Gy in 1 fraction. Inclusion criteria were: high or extremely high risk of relapse according to NCCN (2010), no evidence of distant metastases and no evidence of pronounced urinary tract disorders (residual urine volume less than 50 ml, IPSS less than 17 points, maximum urination rate ≥10 ml / sec). Assessment of radiation-induced complications (toxicity) was carried out in accordance with generally accepted RTOG / EORTC (1995) criteria and taking into account the terminological recommendations of CTCAE v 4.0 (2009). Results. The median follow-up time was 39.2 [30.7; 48.7] (20.3–69.8) months in Group I and 37.0 [30.9; 47.9] (23.7–50.7) months in Group II. The 3-year disease-free survival rate in the study groups regardless of the boost was 77.1 % (75.9 % in Group I and 77.8 % in group II, p=0.7). The 3-year disease-free survival rates in patients with high and extremely high risk regardless of the radiotherapy regimen were 88.6 % and 70.8 %, respectively (р=0.04). Genitourinary early radiation toxicity and rectal intestinal early radiation toxicity of grade 3 were observed in 13.5% and 12.3 % of Group I patients and in 17.6 and 5.9 % of Group II patients. The most significant late complications were the formation of urethral stricture (1 patient of group I) and postradiation rectal stenosis (1 patient of group II). Evaluation of the level of erectile function in both groups showed a significant decrease in satisfaction with the quality of sexual life, which, apparently, can be explained not only by radiation damage to the structures responsible for hemodynamics of the penis, but also by castration syndrome due to prolonged hormonal deprivation in the vast majority of patients. Conclusion . The combination of external beam radiation therapy with HDR brachytherapy boost is an effective method of radical treatment of prostate cancer patients with high and extremely high risk of recurrence.

Me6TREN targets β-catenin signaling to stimulate intestinal stem cell regeneration after radiation.

Background: Acute gastrointestinal syndrome (AGS) is one of the most severe clinical manifestations after exposure to high doses of radiation, and is life-threatening in radiological emergency scenarios. However, an unmet challenge is lacking of an FDA-approved drug that can ameliorate the damage of radiation-exposed intestinal tissues and accelerate the regeneration of injured epithelia. In this study, we investigated whether the small molecule Me6TREN (Me6) can regulate intestinal stem cell (ISC) proliferation and promote crypt regeneration after irradiation.Methods: Lethally irradiated mice were administered with Me6 or PBS to study the survival rate, and sections of their small intestine were subjected to immunostaining to evaluate epithelial regeneration. An intestinal organoid culture system was employed to detect the role of Me6 in organoid growth and ISC proliferation. We further investigated the key signaling pathways associated with Me6 using microarray, western blotting, and RNA interference techniques.Results: We identified the small molecule Me6 as a potent intestinal radiation countermeasure. Systemic administration of Me6 significantly improved ISC and crypt cell regeneration and enhanced the survival of mice after high doses of radiation. Using an in vitro intestinal organoid culture system, we found that Me6 not only induced ISC proliferation but also increased the budding rate of intestinal organoids under unirradiated and irradiated conditions. Me6 remarkably activated the expression of ISC-associated and proliferation-promoting genes, such as Ascl2, Lgr5, Myc, and CyclinD1. Mechanistically, Me6 strongly stimulated the phosphorylation of β-catenin at the S552 site and increased the transcriptional activity of β-catenin, a key signaling pathway for ISC self-renewal and proliferation. This is further evidenced by the fact that knockdown of β-catenin abolished the effect of Me6 on intestinal organoid growth in vitro and crypt regeneration in irradiated mice.Conclusion: The small molecule Me6TREN induced ISC proliferation, enhanced intestinal organoid growth in vitro, and promoted intestinal tissue regeneration after radiation injury by activating β-catenin signaling.

Radiation mitigation of the intestinal acute radiation injury in mice by 1-[(4-nitrophenyl)sulfonyl]-4-phenylpiperazine.

The objective of the study was to identify the mechanism of action for a radiation mitigator of the gastrointestinal (GI) acute radiation syndrome (ARS), identified in an unbiased high‐throughput screen. We used mice irradiated with a lethal dose of radiation and treated with daily injections of the radiation mitigator 1‐[(4‐nitrophenyl)sulfonyl]‐4‐phenylpiperazine to study its effects on key pathways involved in intestinal stem cell (ISC) maintenance. RNASeq, quantitative reverse transcriptase‐polymerase chain reaction, and immunohistochemistry were performed to identify pathways engaged after drug treatment. Target validation was performed with competition assays, reporter cells, and in silico docking. 1‐[(4‐Nitrophenyl)sulfonyl]‐4‐phenylpiperazine activates Hedgehog signaling by binding to the transmembrane domain of Smoothened, thereby expanding the ISC pool, increasing the number of regenerating crypts and preventing the GI‐ARS. We conclude that Smoothened is a target for radiation mitigation in the small intestine that could be explored for use in radiation accidents as well as to mitigate normal tissue toxicity during and after radiotherapy of the abdomen.

Propolis extract protects against radiation-induced intestinal mucositis through anti-apoptotic mechanisms.

Intestinal mucositis is a common side effect during radiotherapy that could be largely prevented by compounds possessing anti-inflammatory or anti-oxidant properties, including extracts of propolis containing a high proportion of flavonoids. A specially formulated aqueous extract of propolis (PWE) has been prepared in such a way to preclude the inclusion of flavonoids but contain mostly organic aromatic acids to study whether it would still protect against radiation-induced intestinal mucositis and to study the possible involvement of apoptotic pathways. Rats were exposed to a gamma radiation dose of 8Gy from a Cesium-137 source in order to inflict intestinal mucositis. Three days before exposure, rats were given PWE orally and treatment continued for 2 more days. Twenty-four hours later, rats were sacrificed, the small intestine was excised, and sections were examined histologically. Different parameters for apoptosis, inflammation, and oxidative stress were determined in the serum and in intestinal homogenates. Radiation exposure led to histological and biochemical signs of intestinal damage. This was associated with an increase in apoptotic indicators and derangement in oxidative stress parameters. All deranged parameters were largely prevented by PWE. The findings provide evidence that the protective effect of PWE against intestinal radiation damage involves not only its anti-inflammatory and anti-oxidant effects but also its anti-apoptotic properties as well.

TLR4 Agonist Monophosphoryl Lipid A Alleviated Radiation-Induced Intestinal Injury.

The small intestine is one of the most sensitive organs to irradiation injury, and the development of high effective radioprotectants especially with low toxicity for intestinal radiation sickness is urgently needed. Monophosphoryl lipid A (MPLA) was found to be radioprotective in our previous study, while its effect against the intestinal radiation injury remained unknown. In the present study, we firstly determined the intestinal apoptosis after irradiation injury according to the TUNEL assay. Subsequently, we adopted the immunofluorescence technique to assess the expression levels of different biomarkers including Ki67, γ-H2AX, and defensin 1 in vivo. Additionally, the inflammatory cytokines were detected by RT-PCR. Our data indicated that MPLA could protect the intestine from ionizing radiation (IR) damage through activating TLR4 signal pathway and regulating the inflammatory cytokines. This research shed new light on the protective effect of the novel TLR4 agonist MPLA against intestine detriment induced by IR.

Gamma-Tocotrienol Protects the Intestine from Radiation Potentially by Accelerating Mesenchymal Immune Cell Recovery.

Natural antioxidant gamma-tocotrienol (GT3), a vitamin E family member, provides intestinal radiation protection. We seek to understand whether this protection is mediated via mucosal epithelial stem cells or sub-mucosal mesenchymal immune cells. Vehicle- or GT3-treated male CD2F1 mice were exposed to total body irradiation (TBI). Cell death was determined by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. Villus height and crypt depth were measured with computer-assisted software in tissue sections. Functional activity was determined with an intestinal permeability assay. Immune cell recovery was measured with immunohistochemistry and Western blot, and the regeneration of intestinal crypts was assessed with ex vivo organoid culture. A single dose of GT3 (200 mg/kg body weight (bwt)) administered 24 h before TBI suppressed cell death, prevented a decrease in villus height, increased crypt depth, attenuated intestinal permeability, and upregulated occludin level in the intestine compared to the vehicle treated group. GT3 accelerated mesenchymal immune cell recovery after irradiation, but it did not promote ex vivo organoid formation and failed to enhance the expression of stem cell markers. Finally, GT3 significantly upregulated protein kinase B or AKT phosphorylation after TBI. Pretreatment with GT3 attenuates TBI-induced structural and functional damage to the intestine, potentially by facilitating intestinal immune cell recovery. Thus, GT3 could be used as an intestinal radioprotector.

Amelioration of whole abdominal irradiation-induced intestinal injury in mice with 3,3′-Diindolylmethane (DIM)

Ionizing radiation-induced intestinal injury is a catastrophic disease with limited effective therapies. 3,3′-Diindolylmethane (DIM), a potent antioxidant agent, has previously been shown to ameliorate hematopoietic injury in a murine model of total body radiation injury, but its effects on ionizing radiation-induced intestinal damage are not clear. Here, we demonstrate that administration of DIM not only protects mice against whole abdominal irradiation (WAI)-induced lethality and weight loss but also ameliorates crypt-villus structural and functional injury of the small intestine. In addition, treatment with DIM significant enhances WAI-induced reductions in Lgr5+ ISCs and their progeny cells, including lysozyme+ Paneth cells, Villin+ enterocytes and Ki67+ instantaneous amplifying cells, thus promoting small intestine repair following WAI exposure. Notably, the expression of Nrf2 increased, while the number of apoptotic cells and the expression of γH2AX decreased in the small intestines of DIM-treated mice compared to mice treated with vehicle following WAI. In vitro, we demonstrated that DIM protected human intestinal epithelial cell-6 (HIEC-6) against ionizing radiation, leading to increased cell vitality. Mechanistically, the radioprotective effect of DIM was likely attributable to its anti-DNA damage effects in irradiated HIEC-6 cells. Moreover, these changes were related to reduction in reactive oxygen species (ROS) levels and increased the activities of antioxidant enzymatic in irradiated HIEC-6 cells. Additionally, the DIM radioprotective effects on the intestine resulted in the restoration of the WAI-shifted gut bacteria composition in mice. Collectively, our findings demonstrate that the beneficial properties of DIM mitigate intestinal radiation injury, which provides a novel strategy for improving the therapeutic effects of irradiation-induced intestinal injury.

The Preliminary Application of Faeces Microbiome as Biodosimeters of Intestinal Acute Radiation Injury

Intestine is one of the most sensitive tissues in acute radiation injury(ARI）characterized by infection, diarrhea and lethality. Development of robust biodosimeters based on individual biological response is crucial for accurate assessment of exposure level and important decisions of medical therapy in Intestinal ARI. In our study, with the increase of radiation dosage, faeces flora constitute revealed corresponding variation tendency in accord with Intestinal radiotoxicity ,including diarrhea and weight decline, intestinal proliferation inhibition and DNA damage, apoptosis and inflamation induction. Because of the feasibility in dosage prediction and evaluation of intestinal ARI, such results supported the preliminary application of faeces microbiome as biodosimeters of intestinal ARI. A total of 120 adult SD rats (SPF) were randomly divided into four groups and received respectively whole-body exposure of 6MeV electron beam, namely 0, 4, 8 and 12Gy. The rats were sacrificed at 24h post-radiation. Faeces flora constitution was analyzed with 16SRNA gene sequencing. We evaluated the rats’ general condition by diarrhea and weight, lethality observation. Intestinal proliferation and DNA damage, morphology were respectively examined with Brdu staining, γ-H2AX staining and HE staining. Protein expression was judged by Western blot. Endotoxemia was tested by serum lipopolysaccharide (LPS) with limulus chromogenic assay. A P-value＜0.05 was considered statistically significant. Faeces flora constitution varied corresponding to the radiation dosage of 0, 4, 8 and 12Gy. The decreased rate of faeces flora proportion was as follows: Prevotella (36%,53%,76%), Ruminococcus (24%,33%,81%), Bifidobacterium (61%,89%,95%), Lactobacillus (54%,88%,92%), versus control group. The increased proportion of Bacteroides and Enterubacterium was 0.4%, 1.2%, 3.1 %and 0.7%, 2.8%, 3.6%, versus control group. The ratio of Enterubacterium to Bifidobacterium ranged from 0.26 to 1.6,15,41. With the variation tendency of radiation dosage and faeces flora constitute, intestinal radiotoxicity involving diarrhea and weight decrease, lethality exacerbated rapidly. More and more significant inhibition of intestinal proliferation and obvious DNA damage were observed, accompanied by increasing expression of apoptosis protein caspase3 and inflammatory factors like IL-1β,IL-6,TNFα (p < 0.05). Radiation caused the most dramatic morphology destruction and endotoxemia in the last group than others (p < 0.05). Our study demonstrate that faeces microbiome as biodosimeters is relatively stable and convenient for sample collection compared with conditional technologies ,which may bring about fast and accurate dosage prediction and radiotoxicity evaluation for intestinal ARI .This will provide a proof of principal for future precision medicine of ARI.

Interleukin 6 Signaling Blockade Exacerbates Acute and Late Injury From Focal Intestinal Irradiation

To evaluate the acute changes in leukocyte populations after focal irradiation and to assess the role of interleukin 6 (IL-6) in acute and late radiation injury. Mice were surgically implanted with a radiopaque marker on the surface of the small intestine. Mice were then imaged with cone beam computed tomography to locate the marker and irradiated with 18Gy of 5×5mm collimated x-rays onto the marked intestine using the Small Animal Radiation Research Platform. Intestinal sections and blood were harvested 1, 3.5, 7, and 14days and 2months postirradiation (post-IR) for histology and complete blood count, respectively. Immune cell populations were assessed by immunofluorescence in the acute phase. Collagen deposition was assessed 2months post-IR. IL-6-/- intestinal sections were assessed post-IR for morphology, EdU, Ki67, and TUNEL in comparison to IL-6+/+ mice. Furthermore, a set of IL-6+/+ mice were treated with anti-IL-6R to assess the role of IL-6 in late intestinal injury. Intestinal radiation damage peaked 14days post-IR, and fibrosis had developed by 60days post-IR. There was a marked infiltration of immune cells into the irradiated intestine, with increased neutrophils, macrophages, B-cells, and CD4+ T cells maintained from 3.5 to 14days post-IR. CD8+ T cells were decreased from days 7 to 14 post-IR. Systemically, leukocytes were increased in the peripheral blood 14days post-IR with anemia being maintained from 14days to 2months. IL-6 was significantly increased in the serum post-IR. IL-6-/- mice demonstrated worsened intestinal injury acutely post-IR. Moreover, anti-IL-6R-treated mice presented with worsened intestinal fibrosis 2months post-IR. Focal irradiation of the intestine produced a significant increase in immune cells in the irradiated area and systemic inflammation and anemia. Blockade of IL-6 signaling was found to exacerbate acute intestinal injury and late intestinal injury after focal irradiation.

PO-1037: Intestinal radiation plays a pivotal role in CTLA-4 Ab induced an autoimmune enteritis mouse model

PO-1037: Intestinal radiation plays a pivotal role in CTLA-4 Ab induced an autoimmune enteritis mouse model

Protective Effects of Flagellin A N/C Against Radiation-Induced NLR Pyrin Domain Containing 3 Inflammasome-Dependent Pyroptosis in Intestinal Cells

Toinvestigate changes induced in mouse intestines after irradiation and to explore the potential radioprotective effects of flagellin AN/C (FlaAN/C). A mouse model of radiation-induced enteropathy was used in this study. A 10-Gy abdominal irradiation was performed on FlaAN/C- and vehicle-injected mice to explore the role of FlaAN/C in intestinal radiation injury and to study the molecular mechanism in this process. In the intestinal tissue, pathologic changes were investigated by hematoxylin and eosin staining, TUNEL staining, and immunohistochemistry; Western blotting and quantitative reverse transcription-polymerase chain reaction were used to determine the changes in protein and messenger RNA levels, respectively; and an enzyme-linked immunosorbent assay was performed to determine protein concentration in serum. The involvement of the reactive oxygen species pathway was investigated by determining superoxide dismutase, glutathione (GSH) peroxidase, GSH reductase, and GSH disulfide (GSSG) plus GSH activities. Flagellin A N/C inhibited radiation-induced reactive oxygen species production, decreased NLRP3 activity, and reduced the occurrence of caspase-1-dependent pyroptosis. The results revealed that oxidative stress, bioenergetic impairment, and subsequent NLRP3 inflammasome activation were involved in radiation-induced intestinal injury. Flagellin A N/C exerted a protective effect by blunting the activation of NLRP3 inflammasome signaling, thereby reducing the inflammatory response and occurrence of caspase-1-dependent pyroptosis in the intestine. Therefore, treatment of mice with FlaAN/C reduced radiation-induced intestinal injury. Reactive oxygen species-induced NLRP3 inflammasomes mediated radiation-induced pyroptosis of the intestinal cells, and FlaAN/C suppressed pyroptosis to protect the intestinal tissue. These results provide novel insights into the mechanisms underlying radiation-induced cytotoxicity, and FlaAN/C might be a potential preventive therapy for radiation-induced intestinal injury in patients with cancer.

Geranylgeranylacetone Ameliorates Intestinal Radiation Toxicity by Preventing Endothelial Cell Dysfunction.

Radiation-induced intestinal toxicity is common among cancer patients after radiotherapy. Endothelial cell dysfunction is believed to be a critical contributor to radiation tissue injury in the intestine. Geranylgeranylacetone (GGA) has been used to treat peptic ulcers and gastritis. However, the protective capacity of GGA against radiation-induced intestinal injury has not been addressed. Therefore, we investigated whether GGA affects intestinal damage in mice and vascular endothelial cell damage in vitro. GGA treatment significantly ameliorated intestinal injury, as evident by intestinal crypt survival, villi length and the subsequently prolonged survival time of irradiated mice. In addition, intestinal microvessels were also significantly preserved in GGA-treated mice. To clarify the effect of GGA on endothelial cell survival, we examined endothelial function by evaluating cell proliferation, tube formation, wound healing, invasion and migration in the presence or absence of GGA after irradiation. Our findings showed that GGA plays a role in maintaining vascular cell function; however, it does not protect against radiation-induced vascular cell death. GGA promoted endothelial function during radiation injury by preventing the loss of VEGF/VEGFR1/eNOS signaling and by down-regulating TNFα expression in endothelial cells. This finding indicates the potential impact of GGA as a therapeutic agent in mitigating radiation-induced intestinal damage.

Radiation induces proinflammatory dysbiosis: transmission of inflammatory susceptibility by host cytokine induction

ObjectiveRadiation proctitis (RP) is a complication of pelvic radiotherapy which affects both the host and microbiota. Herein we assessed the radiation effect on microbiota and its relationship to tissue damage...

Recombinant Thrombomodulin (Solulin) Ameliorates Early Intestinal Radiation Toxicity in a Preclinical Rat Model.

Intestinal radiation toxicity occurs during and after abdominopelvic radiotherapy. Endothelial cells play a significant role in modulating radiation-induced intestinal damage. We demonstrated that the endothelial cell surface receptor thrombomodulin (TM), a protein with anticoagulant, anti-inflammatory and antioxidant properties, mitigates radiation-induced lethality in mice. The goal of this study was to determine whether recombinant TM (Solulin) can protect the intestine from toxicity in a clinically relevant rat model. A 4 cm loop of rat small bowel was exposed to fractionated 5 Gy X radiation for 9 consecutive days. The animals were randomly assigned to receive daily subcutaneous injections of vehicle or Solulin (3 mg/kg/day or 10 mg/kg/day) for 27 days starting 4 days before irradiation. Early intestinal injury was assessed two weeks after irradiation by quantitative histology, morphometry, immunohistochemistry and luminol bioluminescence imaging. Solulin treatment significantly ameliorated intestinal radiation injury, made evident by a decrease in myeloperoxidase (MPO) activity, transforming growth factor beta (TGF-β) immunoreactivity, collagen-I deposition, radiation injury score (RIS) and intestinal serosal thickening. These findings indicate the need for further development of Solulin as a prophylactic and/or therapeutic agent to mitigate radiation-induced intestinal damage.

Radiation-induced changes in intestinal and tissue-nonspecific alkaline phosphatase: implications for recovery after radiation therapy

BackgroundExogenous replacement of depleted enterocyte intestinal alkaline phosphatase (IAP) decreases intestinal injury in models of colitis. We determined whether radiation-induced intestinal injury could be mitigated by oral IAP supplementation and the impact on tissue-nonspecific AP. MethodsWAG/RjjCmcr rats (n = 5 per group) received lower hemibody irradiation (13 Gy) followed by daily gavage with phosphate-buffered saline or IAP (40 U/kg/d) for 4 days. Real-time polymerase chain reaction, AP activity, and microbiota analysis were performed on intestine. Lipopolysaccharide and cytokine analysis was performed on serum. Data were expressed as a mean ± SEM with P greater than .05 considered significant. ResultsIntestine of irradiated animals demonstrates lower hemibody irradiation and is associated with upregulation of tissue-nonspecific AP, downregulation of IAP, decreased AP activity, and altered composition of the intestinal microbiome. ConclusionsSupplemental IAP after radiation may be beneficial in mitigating intestinal radiation syndrome as evidenced by improved histologic injury, decreased acute intestinal inflammation, and normalization of intestinal microbiome.