The evolving geopolitical landscape has heightened the probability of nuclear incidents, including accidental release or deliberate detonation, which can cause acute, life-threatening radiation injury to large populations. High-dose ionizing radiation (IR) is highly likely to cause radiation injury to the intestines and lead to intestinal radiation sickness. This study systematically explored the protective effect of cobalt chloride (CoCl2) on intestinal radiation injury and its preliminary mechanism from multiple levels, including cells, intestinal tissues, intestinal organoids, and live mice. The results showed that CoCl2 pretreatment could significantly enhance the radiation tolerance of mice, not only greatly improving the survival rate and multiple indicators such as intestinal injury score, but also maintaining the integrity of the small intestinal epithelial villi structure. At the same time, it promotes the proliferation and differentiation of intestinal organoids, inhibits the apoptosis of intestinal epithelial cells, and enhances the expression of barrier protection genes, thereby enhancing the intestinal resistance to radiation injury. Mechanism studies have shown that CoCl2 can up-regulate the expression of hypoxia-inducible factor-2 α (HIF-2α) via hypoxia-mimetic action and activate downstream signaling pathways related to cell proliferation, anti-apoptosis, and angiogenesis. This study initially clarified the mechanism by which CoCl2 protects the intestinal tract from radiation injury, providing a scientific basis and strategic support for the development of new radiation protection targets. Its in-depth research and application transformation are expected to play an important role in the future field of nuclear radiation protection.

BACKGROUND:The hematopoietic system is extremely sensitive to ionizing radiation(IR) and is easily damaged after exposure. Recent studies [1, 2] have shown that hematopoietic lineage differentiation may play a key role in Ionizing radiation -induced acute injury. METHODS:C57BL/6 mice matched for age, sex and weight were randomly grouped and intraperitoneal injected with PBS, Evolocumab (80.0 mg/kg) or MMP9-IN (20.0 mg/kg). Survival time, body weight, pathology, hematopoietic cell differentiation changes and organoids of the mice after IR (10.0 Gy) were compared, and the mechanism of action in tissues was verified by transcriptome sequencing. RESULTS:Ionizing radiation can inhibit hematopoietic cell lineage differentiation and affect cholesterol metabolism. The expression of the Pcsk9 gene was significantly upregulated after IR. Treatment with a Pcsk9 monoclonal antibody (Evolocumab) exerted significant radioprotective effects, including improving the survival rate of irradiated mice, reducing body weight loss, preserving the integrity of the bone marrow cavity, and promoting cellular proliferation. Flow cytometric analysis showed that Evolocumab significantly increased hematopoietic precursor cells and hematopoietic stem cells and promoted their differentiation into ST-LSKs and MPPs. In addition, the proportions of CMPs and GMPs were increased, whereas the proportion of MEPs was decreased. RNA sequencing results suggested that the IR-induced downregulation of Cd3e within the hematopoietic cell lineage pathway could be rescued by Evolocumab, which was further validated at the cellular level. CD3e is a T-cell-associated marker, and Evolocumab treatment was associated with an increased proportion of CD3e+ T cells. Analysis of previously reported hematopoiesis-related factors [3-5] indicated that Evolocumab markedly upregulated Mmp9 expression. Correlation analysis revealed that Pcsk9 was negatively correlated with Cd3e, whereas Cd3e was positively correlated with Mmp9. Following ionizing radiation, Mmp9 knockout (Mmp9 KO) mice exhibited reduced survival compared with wild-type mice. Moreover, in wild-type mice, administration of an MMP9 inhibitor (MMP9-IN) attenuated the radioprotective effects of Evolocumab, indicating that Mmp9 contributes to Evolocumab-mediated radioprotection. CONCLUSION:Evolocumab exerts a positive effect on promoting the proliferation and differentiation of hematopoietic stem cells against IR-induced hematopoietic system injury, with its protective mechanism likely involving the activation of hematopoietic cell lineages to upregulate CD3e+ T cells and Mmp9 expression.

Pelvic radiotherapy, a key treatment for pelvic malignancies, frequently causes iatrogenic premature ovarian insufficiency (POI) in young female patients, highlighting the urgent need for targeted, effective, and low-toxic radioprotectants. Zymosan-A, a TLR2/6 agonist, has potential radioprotective effects. We established a 60Co γ-radiation-induced murine POI model and evaluated Zymosan-A’s efficacy. Results showed Zymosan-A mitigated granulosa cell apoptosis, improved ovarian hormone levels and estrous cycle regularity, increased embryo number, and enhanced offspring weight. Its radioprotective effect against ovarian injury, mediated via the TLRs-NF-κB pathway, was abolished in TLR2-knockout mice. Thus, Zymosan-A effectively protects against radiotherapy-induced POI primarily through the TLR2-NF-κB pathway.

Rationale Parkinson’s disease (PD), a globally prevalent neurodegenerative disorder, is characterized by substantia nigra dopaminergic neuron degeneration and striatal dopamine depletion. While microglial pyroptosis is implicated in neuroinflammation and neural injury via inflammatory cytokine release, the role of the CASPASE-1/GSDMD pathway in PD pathogenesis remains incompletely defined. Methods 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) was used to construct PD model i n vivo , GSDMD-knockout mice was employed to assess pyroptotic mechanisms. MPP⁺-stimulated BV2 microglia were treated with a CASPASE-1 inhibitor in vitro . Microglia-specific GSDMD conditional knockout mice were generated to evaluate cell-type contributions to neuroinflammation and motor deficits. Results GSDMD deficiency attenuated MPTP-induced neuroinflammation, dopaminergic neuron loss, and motor dysfunction in vivo. MPP⁺ exposure triggered NLRP3 inflammasome activation and pyroptosis in BV2 microglia, which was suppressed by CASPASE-1 inhibition. Critically, microglia-specific GSDMD ablation mitigated nigrostriatal degeneration and dyskinesia in PD mice, confirming the centrality of microglial pyroptosis. Conclusion Our findings demonstrate that microglia drive neuroinflammation in PD via CASPASE-1/GSDMD-mediated pyroptosis, directly linking this pathway to dopaminergic neurodegeneration and motor impairment. Targeting GSDMD-dependent pyroptosis represents a promising therapeutic strategy. * PD : Parkinson’s disease MPTP : 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine MPP+iodide : 1-methyl-4-phenylpyridinium iodide VX-765 : Belnacasan SN : substantia nigra GSDMD : Gasdermin-D NLRP3 : NOD-like receptor thermal protein domain associated protein 3 PAMP : Pathogen-Associated Molecular Patterns DApMP : Damage-Associated Molecular Patterns TH : Tyrosine hydroxylase GFAP : Glial Fibrillary Acidic Protein α-Syn : α-Synuclein AD : Alzheimer’s disease

Severe ionizing radiation (IR) causes the acute lethal damage of hematopoietic system and gastrointestinal tract. By establishing a radiation injury model, we found that Diprovocim, a TLR2 agonist, protected mice against the lethal damage of hematopoietic system and gastrointestinal tract. Diprovocim inhibited the IR-induced damage, promoted erythrocyte differentiation and elevated the proportion of hematopoietic stem cells (HSCs) in irradiated mice, and promoted the proliferation and differentiation of intestinal stem cells (ISCs). In addition, the RNA seq results suggested that Diprovocim significantly upregulated the TLR2 signaling pathway, and Diprovocim had no radioprotective effect on TLR2 KO mice, suggesting that Diprovocim activated TLR2 signaling pathway to exert its radioprotective function. The RNA sequencing results also suggested that Diprovocim significantly up-regulated the expression of SOX9. Diprovocim had no radioprotective effect after SOX9 knockdown. In conclusion, we demonstrated that Diprovocim protected the radiation-induced damage and upregulated targeting TLR2-SOX9 axis and that Diprovocim might be a potential high-efficiency selective agent.

Ionizing radiation (IR) induced damages are common complications of radiotherapy for tumors, severely limiting the intensity and therapeutic efficacy of the radiotherapy program. Emerging data indicated that the cGAS-STING pathway has paradoxical effects on IR-induced damage. SR-717, as a non-nucleotide, small-molecule stimulator of interferon genes (STING) agonist, has been proven that it could activate the STING signaling pathway. In this work, we try to explore the radioprotection of the STING signaling pathway and figure out whether SR-717 could be a potential intestinal radioprotective agent. C57BL/6 mice were intraperitoneally treated with SR-717 or normal saline (NS). By analyzing the survival rate, body weight, and the number of peripheral blood cells after IR exposure, we found that SR-717 improved the survival rate and body weight of mice, protected the intestine from IR-induced damage as well as hematopoietic damage, and promoted the regeneration of intestinal stem cells (ISCs). Cell viability and apoptosis after irradiation were detected after stimulation of MODE-K cells with SR-717 or PBS. We found that SR-717 increased cell viability and inhibited apoptosis in vitro. The mechanism of SR-717 in intestinal radiation protection was investigated by RNA-seq. The results of RNA-seq and qRT-PCR suggested that SR-717 significantly activated the immune system via the STING-IL-6 signaling pathway. In addition, we discussed the role of TLR2 in SR-717-mediated anti-radiation activity, and TLR2 deletion significantly reversed the radioprotective effects of SR717. In conclusion, we proved STING signaling activation displayed anti-radiation activity and found SR-717 displayed anti-radiation activity via the STING-IL-6 signaling pathway, suggesting SR-717 could be a potential intestinal radioprotective agent.

Ionising radiation exposure can lead to acute haematopoietic radiation syndrome. Despite significant advancements in the field of radioprotection, no drugs with high efficacy and low toxicity have yet been approved by the Food and Drug Administration. FG-4592, as a proline hydroxylase inhibitor, may play an important role in radioprotection of the haematopoietic system. Mice were peritoneal injected with FG-4592 or normal saline. After irradiation, the survival time, body weight, peripheral blood cell and bone marrow cell (BMC) count, cell apoptosis, pathology were analysed and RNA-sequence technique (RNA-Seq) was conducted to explore the mechanism of FG-4592 in the haematopoietic system. Our results indicated that FG-4592 improved the survival rate and weight of irradiated mice and protected the spleen, thymus and bone marrow from IR-induced injury. The number of BMCs was increased and protected against IR-induced apoptosis. FG-4592 also promoted the recovery of the blood system and erythroid differentiation. The results of RNA-Seq and Western blot showed that the NF-κB signalling pathway and hypoxia-inducible factor-1 (HIF-1) signalling pathway were upregulated by FG-4592. Meanwhile, RT-PCR results showed that FG-4592 could promote inflammatory response significantly. FG-4592 exhibited radioprotective effects in the haematopoietic system by promoting inflammatory response and targeting the NF-κB, HIF signalling pathway.

Intestinal stem cells (ISCs) are responsible for intestinal homeostasis and are important for the regeneration of damaged intestine. We established an ionizing radiation (IR)-induced intestinal injury model and observed that Gelsolin KO mice had increased radiosensitivity. The deletion of Gelsolin aggravated intestinal damage and reduced the number of ISCs after lethal IR. The intestinal organoid experiments showed that Gelsolin deletion inhibited ISCs function after IR. Notably, RNA sequencing and RT-PCR results showed IL-17 signaling pathway was down-regulated and Th17 cells differentiation was inhibited in Gelsolin KO mice. Moreover, recombinant IL-17 A ameliorated IR-induced intestinal injury and promoted ISCs regeneration. To figure out the role of Gelsolin in Th17 cells differentiation, flow cytometry was used and we found that Gelsolin targets Th17 cells functionality via the p-STAT3/RORγt axis. By establishing the co-culture system, we proved that Th17 cells promoted self-renewal and budding abilities in Gelsolin-deficient organoids. Finally, we found that Gelsolin was protective against DSS-induced colitis and that this protective effect was not specific or limited to the IR induced intestinal injury model. Based on these results, we proved Gelsolin maintained the regeneration of ISCs by sustaining Th17 cells functions via the p-STAT3/RORγt axis.

Background: Intestinal tissue is extremely sensitive to ionizing radiation (IR), which is easy to cause intestinal radiation sickness, and the mortality rate is very high after exposure. Recent studies have found that intestinal immune cells and intestinal stem cells (ISCs) may play a key role in IR-induced intestinal injury. Methods: C57BL6 mice matched for age, sex and weight were randomly grouped and intraperitoneal injected with PBS, Scleroglucan (125.0 mg/kg) or Anti-mouse IL-17A -InVivo (10 mg/kg), the number of mice in each group was n >= 3.Survival time, body weight, pathology, organoids and immune cell markers of the mice after IR (10.0 Gy) were compared, and the mechanism of action in intestinal tissues was verified by transcriptome sequencing. Results: Scleroglucan has significant radiation protective effects on the intestine, including improving the survival rate of irradiated mice, inhibiting the radiation damage of intestinal tissue, and promoting the proliferation and differentiation of intestinal stem cells (ISCs). The results of RNA sequencing suggested that Scleroglucan could significantly activate the immune system and up-regulate the IL-17 and NF-kappa B signaling pathways. Flow cytometry showed that Scleroglucan could significantly up-regulate the number of Th17 cells and the level of IL17A in the gut. IL-17A provides radiation protection. After intraperitoneal injection of Scleroglucan and Antimouse IL-17A -InVivo, mice can significantly reverse the radiation protection effect of Scleroglucan, downregulate the molecular markers of intestinal stem cells and the associated markers of DC, Th1 and Th17 cells, and up-regulate the associated markers of Treg and Macrophage cells. Conclusion: Scleroglucan may promote the proliferation and regeneration of ISCs by regulating the activation of intestinal immune function mediated by IL-17 signaling pathway and play a protective role in IR-induced injury.

Intestinal stem cells (ISCs) play a crucial role in maintaining the equilibrium and regenerative potential of intestinal tissue, thereby ensuring tissue homeostasis and promoting effective tissue regeneration following injury. It has been proven that targeting Toll-like receptors (TLRs) can help prevent radiation-induced damage to the intestine. In this study, we established an intestinal injury model using IR and evaluated the effects of CL429 on ISC regeneration both in vivo and in vitro. Following radiation exposure, mice treated with CL429 showed a significant increase in survival rates (100% survival in the treated group compared to 54.54% in the control group). CL429 also showed remarkable efficacy in inhibiting radiation-induced intestinal damage and promoting ISC proliferation and regeneration. In addition, CL429 protected intestinal organoids against IR-induced injury. Mechanistically, RNA sequencing and Western blot analysis revealed the activation of the Wnt and Hippo signaling pathways by CL429. Specifically, we observed a significant upregulation of YAP1, a key transcription factor in the Hippo pathway, upon CL429 stimulation. Furthermore, knockdown of YAP1 significantly attenuated the radioprotective effect of CL429 on intestinal organoids, indicating that CL429-mediated intestinal radioprotection is dependent on YAP1. In addition, we investigated the relationship between TLR2 and YAP1 using TLR2 knockout mice, and our results showed that TLR2 knockout abolished the activation of CL429 on YAP1. Taken together, our study provides evidence supporting the role of CL429 in promoting ISC regeneration through activation of TLR2-YAP1. And further investigation of the interaction between TLRs and other signaling pathways may enhance our understanding of ISC regeneration after injury.

BackgroundIonizing radiation (IR), including radiotherapy, can exert lasting harm on living organisms. While liposaccharide (LPS) offers resistance to radiation damage, it also induces toxic responses. Thankfully, an LPS analogue called N-formylmethionine-leucyl-phenylalanine (fMLP) holds the potential to mitigate this toxicity, offering hope for radiation protection.MethodsSurvival of C57BL/6 mice exposed to IR after administration with fMLP/LPS/WR-2721 or saline was recorded. Cell viability and apoptosis assay of bone marrow (BMC), spleen and small intestinal epithelial (HIECs) cells were tested by Cell Counting Kit-8 (CCK-8) and flow cytometry assay. Tissue damage was evaluated by Hematoxilin and Eosin (H&E), Ki-67, and TUNEL staining. RNA sequencing was performed to reveal potential mechanisms of fMLP-mediated radiation protection. Flow cytometry and western blot were performed to verify the radiation protection mechanism of fMLP on the cell cycle.ResultsThe survival rates of C57BL/6 mice exposed to ionizing radiation after administering fMLP increased. fMLP demonstrated low toxicity in vitro and in vivo, maintaining cell viability and mitigating radiation-induced apoptosis. Moreover, it protected against tissue damage in the hematopoietic and intestinal system. RNA sequencing shed light on fMLP's potential mechanism, suggesting its role in modulating innate immunity and cell cycling. This was evidenced by its ability to reverse radiation-induced G2/M phase arrests in HIECs.ConclusionfMLP serves as a promising radioprotective agent, preserving cells and radiosensitive tissues from IR. Through its influence on the cell cycle, particularly reversing radiation-induced arrest in G2/M phases, fMLP offers protection against IR's detrimental effects.

Accidental radiation exposure causes the acute lethal damage of hematopoietic system and gastrointestinal tract1,2. By establishing an ionizing radiation (IR) induced injury model, we found macrophage-activating lipopeptide-2 (MALP-2) exhibited significant radioprotective effects in mice. MALP-2 improved the survival of irradiated mice, inhibited the radiation-induced gastrointestinal tract damage. Through intestinal organoid experiments, we found that MALP-2 protected the intestinal organoid against IR-induced injury. Next, we identified the differentially expressed genes (DEGs) between PBS and MALP-2 groups based on the RNA sequencing result3. And the RNA-seq results showed that MALP-2 increased the levels of interleukin 6 (IL-6), IL-12, G-CSF, GM-CSF, TNF-α, CCL-3, PGE-2 and SOD2. KEGG enrichment analysis showed that DEGs were significantly enriched in Toll-like receptor signaling pathway and NF-κB signaling pathway. In line with these observations, the expression level of IL-6 and GM-CSF were increased by using flow cytometry. Moreover, MALP-2 protected WT mice from IR induced death but had no radioprotective effects on the TLR2 KO and IL-6 KO mice, suggesting that the radioprotection of MALP-2 was mediated by activating TLR2/IL-6 axis. In conclusion, our data suggested that the MALP-2 could induce significant radioprotective effects and MALP-2 might be a potential radioprotective agent.

本教研室创新性地建立了现场技能训练-虚拟仿真实践教学-部队防护训练基地"三位一体"的混合式综合能力培养模式,发现新的培训模式虽然提高了学员核应急医学救援的综合实践能力,但由于将学员实践能力的教学划分为三个部分,使得原来单一的教学评价模式已经不能够适用现有教学模式.基于此,本教研室构建了以教学评价指标化为核心的核应急医学救援任职教育实践教学评价体系.本教研室将评价维度分为三个:教学支持、教学效果、学员满意度,同时结合评价维度最终确定了九个核心教学评价指标,并将所构建的教学评价体系运用于教学评价实践,对所存在的问题给出相应的建议.本教研室通过该体系的构建与实施,对现有的教学进一步评价与优化,以更好地加强教学效果,提升教学质量.

BACKGROUND:The incidence of inflammatory bowel disease (IBD) is growing in the population. At present, the etiology of inflammatory bowel disease remains unclear, and there is no effective and low-toxic therapeutic drug. The role of the PHD-HIF pathway in relieving DSS-induced colitis is gradually being explored.METHODS:Wild-type C57BL/6 mice were used as a model of DSS-induced colitis to explore the important role of Roxadustat in alleviating DSS-induced colitis. High-throughput RNA-Seq and qRT-PCR methods were used to screen and verify the key differential genes in the colon of mice between normal saline (NS) and Roxadustat groups.RESULTS:Roxadustat could alleviate DSS-induced colitis. Compared with the mice in the NS group, TLR4 were significantly up-regulated in the Roxadustat group. TLR4 KO mice were used to verify the role of TLR4 in the alleviation of DSS-induced colitis by Roxadustat.CONCLUSION:Roxadustat has a repairing effect on DSS-induced colitis, and may alleviate DSS-induced colitis by targeting the TLR4 pathway and promote intestinal stem cell proliferation.

Intestinal stem cells (ISCs) are responsible for intestinal tissue homeostasis and are important for the regeneration of damaged intestinal epithelia. Through the establishment of ionizing radiation (IR) induced intestinal injury model, we found that the radiosensitivity of the intestine of GSN KO mice increases significantly. It means that GSN played a key role in the process of radiation-induced intestinal injury. The deletion of GSN aggravated the radiation damage of the intestine and decreased the survival rate of intestinal crypts and the number of ISCs after lethal IR in vivo. Through intestinal organoid experiments, we found that the deletion of GSN inhibited the proliferation and differentiation of ISCs after IR. Remarkably, the results of RNA sequencing and RT-PCR showed that IL-17 signaling pathway was down-regulated and Th17 cells differentiation was inhibited in GSN KO mice. Moreover, recombinant IL-17A ameliorated IR-induced intestinal injury and promoted the ISCs regeneration in GSN KO mice and GSN-deficient organoids. To figure out the role of GSN in Th17 cells differentiation, flow cytometry was used and we found that Gelsolin targets Th17 cells functionality via p-STAT3/RORγt signaling pathway. Finally, by establishing the co-culture system of intestinal organoids and Th17 cells, we found that decreased self-renewal and budding efficiency in GSN-deficient organoids were rescued by Th17 cells. In conclusion, we demonstrated that GSN maintained the regeneration of ISCs by sustaining Th17 cells functions via p-STAT3/RORγt axis.Funding: This study was supported in part by the grants from Youth Training Program (No. 21QNPY034), Shanghai Sailing Program (No. 19YF1459100), and National Natural Science Foundation of China (No. 81903260, No. 81872559, No. 82173459, No.81372932). These support the experimental research and data collection of the subject.Declaration of Interest: The authors confirmed that there are no conflicts of interest.Ethical Approval: All animal experiments conformed to the National Institute of Health Guide for the Care and Use of Laboratory Animals' (NIH Publication No. 85-23, National Academy Press, Washington, DC, revised 1996), with the approval of the Laboratory Animal Center of the Naval Medical University, Shanghai.

Abstract Background Hematopoiesis have been proved that it could be changed in the patients with advanced cancer. In this study, we investigated the changes of HSCs differentiation in advanced tumor-bearing mice. Methods The tumor-bearing mice model was established by subcutaneously inoculating with xenografts of B16-F10 mouse melanoma cells into the right back of male wild-type C57BL/6 mice. Hematopoietic stem cells and multi-lineage differentiation were evaluated using blood routine, HE-staining, flow cytometry assay and HSCs culture technology. Results The mice model exhibited hematopoietic suppression, marked by a severe anemia, and the multi-lineage differentiation of hematopoietic stem cells was disturbed. Especially, the differentiation of megakaryocyte and erythrocyte were blocked, while myeloid cell and lymphoid cell differentiation was encouraged in advanced tumor-bearing mice. Conclusion In this study we showed that the differentiation of hematopoietic stem cells was disturbed in advanced tumor-bearing mice, which provided new knowledge about cancer cachexia related hematopoietic abnormality.

Ionizing radiation (IR)-induced intestinal injury is usually accompanied by high lethality. Intestinal stem cells (ISCs) are critical and responsible for the regeneration of the damaged intestine. Astragalus polysaccharide (APS), one of the main active ingredients of Astragalus membranaceus (AM), has a variety of biological functions. This study was aimed to investigate the potential effects of APS on IR-induced intestine injury via promoting the regeneration of ISCs. We have established models of IR-induced intestinal injury and our results showed that APS played great radioprotective effects on the intestine. APS improved the survival rate of irradiated mice, reversed the radiation damage of intestinal tissue, increased the survival rate of intestinal crypts, the number of ISCs and the expression of intestinal tight junction-related proteins after IR. Moreover, APS promoted the cell viability while inhibited the apoptosis of MODE-K. Through organoid experiments, we found that APS promoted the regeneration of ISCs. Remarkably, the results of network pharmacology, RNA sequencing and RT-PCR assays showed that APS significantly upregulated the HIF-1 signalling pathway, and HIF-1 inhibitor destroyed the radioprotection of APS. Our findings suggested that APS promotes the regeneration of ISCs through HIF-1 signalling pathway, and it may be an effective radioprotective agent for IR-induced intestinal injury.

Background Severe ionizing radiation (IR)-induced intestinal injury associates with high mortality, which is a worldwide problem requiring urgent attention. In recent years, studies have found that the PHD-HIF signaling pathway may play key roles in IR-induced intestinal injury, and we found that FG-4592, the PHD inhibitor, has significant radioprotective effects on IR-induced intestinal injury. Methods In the presence or absence of FG-4592 treatment, the survival time, pathology, cell viability, cell apoptosis, and organoids of mice after irradiation were compared, and the mechanism was verified after transcriptome sequencing. The data were analyzed using SPSS ver. 19 software. Results Our results show that FG-4592 had significant radioprotective effects on the intestine. FG-4592 improved the survival of irradiated mice, inhibited the radiation damage of intestinal tissue, promoted the regeneration of intestinal crypts after IR and reduced the apoptosis of intestinal crypt cells. Through organoid experiments, it is found that FG-4592 promoted the proliferation and differentiation of intestinal stem cells (ISCs). Moreover, the results of RNA sequencing and Western blot showed that FG-4592 significantly upregulated the TLR4 signaling pathway, and FG-4592 had no radioprotection on TLR4 KO mice, suggesting that FG-4592 may play protective role against IR by targeting TLR4. Conclusion Our work proves that FG-4592 may promote the proliferation and regeneration of ISCs through the targeted regulation of the TLR4 signaling pathway and ultimately play radioprotective roles in IR-induced injury. These results enrich the molecular mechanism of FG-4592 in protecting cells from IR-induced injury and provide new methods for the radioprotection of intestine.

Abstract Background radiation-induced intestinal injury (RIII) is an important cause of death in nuclear accidents and common complication after radiotherapy in patients with pelvic, abdominal, or retroperitoneal tumors. Up to now there is no effective means to prevent or treat RIII due to its complex mechanism, in which the death of intestinal cells is the main reason. Recently, GSDMD-mediated pyroptosis was identified as an important type of cell death and play a role in many diseases. However, the effect of pyroptosis on RIII is still unclear. Method using GSDMD knockout mice, the role of pyroptosis in the RIII was investigated. By detecting the release of LDH, expression of GSDMD, Caspase-11, Caspase-1 and absorption rate of SYTOX Green, the pyroptosis of radiated Mode-k cells was determined, simultaneously the common pyroptosis induced by LPS was conducted as positive control. Further, the upstream of GSDMD were screened by predictive analysis of transcription factors combing RNA-seq. Results we showed that GSDMD-mediated pyroptosis is involved in the process of RIII, and unexpectedly found that radiation induced a delayed pyroptosis that is substantially different from common pyroptosis induced by such as LPS. Further investigation revealed that radiation-induced DNA damage up-regulated the expression of P53, which subsequently transcribes GSDMD. In addition, the up-regulated GSDMD led to pyroptosis simultaneously promoting Ca2+ influx that afterwards enhanced apoptosis induced by radiation. Finally, targeting GSDMD, disulfiram displayed a potential protection for RIII. Conclusion radiation could induce delayed pyroptosis in the intestinal epithelial cell that is greatly different from common pyroptosis. During that process, GSDMD was cleaved and had inducible high expression which was mainly mediated by P53 transcription.

Intestinal stem cells (ISCs) are responsible for intestinal tissue homeostasis and are important for the regeneration of the damaged intestinal epithelia. Through the establishment of ionizing radiation (IR) induced intestinal injury model, we found that a TLR2 agonist, Zymosan-A, promoted the regeneration of ISCs in vivo and in vitro. Zymosan-A improved the survival of abdominal irradiated mice (81.82% of mice in the treated group vs. 30% of mice in the PBS group), inhibited the radiation damage of intestinal tissue, increased the survival rate of intestinal crypts and the number of ISCs after lethal IR in vivo. Through organoid experiments, we found that Zymosan-A promoted the proliferation and differentiation of ISCs after IR. Remarkably, the results of RNA sequencing and Western Blot (WB) showed that Zymosan-A reduced IR-induced intestinal injury via TLR2 signaling pathway and Wnt signaling pathway and Zymosan-A had no radioprotection on TLR2 KO mice, suggesting that Zymosan-A may play a radioprotective role by targeting TLR2. Moreover, our results revealed that Zymosan-A increased ASCL2, a transcription factor of ISCs, playing a core role in the process of Zymosan-A against IR-induced intestinal injury and likely contributing to the survival of intestinal organoids post-radiation. In conclusion, we demonstrated that Zymosan-A promotes the regeneration of ISCs by upregulating ASCL2.