目的：作为电离辐射高敏感器官，肠道在盆腹部肿瘤患者放射治疗中高度易损，会诱发电离辐射急性暴露后延迟效应（delayed effects of acute radiation exposure，DEARE）形成电离辐射肠道远期损伤，严重危害患者生存和生活质量。目前尚无有效治疗手段，迫切需要针对性治疗药物和治疗方案。电离辐射诱导的衰老是DEARE的主要机制之一，以此靶点开发治疗策略极具潜力。作为一种高效的烟酰胺腺嘌呤二核苷酸(nicotinamide adenine dinucleotide，NAD)补充剂，烟酰胺核糖（Nicotinamide riboside, NR）在调控细胞能量代谢和延缓衰老进程方面表现出显著的生物学效应。本研究旨在明确NR对电离辐射远期肠损伤的治疗作用，并进一步阐明其潜在的作用机制，以期为肠道DEARE的临床治疗提供新的理论依据和实验基础。

方法：体外实验以正常人肠上皮细胞（HIEC-6）为研究对象，通过cck-8法检测NR剂量梯度下的HIEC-6细胞毒性；通过流式细胞术检测NR对辐射暴露后HIEC-6细胞凋亡和DNA损伤水平；通过SA-β-Gal染色检测辐射暴露后远期的HIEC-6细胞衰老改变。体内实验以C57BL/6J雄性小鼠为研究对象，通过6 Gy一次性全身辐射暴露诱导小鼠辐射远期损伤模型。单次照射8周后，NR灌胃给药3周，并于取材前进行前肢抓力检测及形态观察。通过H&E染色、免疫组织化学染色检测肠道组织学改变。通过SA-β-Gal染色小肠组织冰冻切片、蛋白免疫印迹和RT-qPCR衰老标志物p21,p16表达水平分析、以及组织抗体芯片检测肠道组织(senescence-associated secretory phenotype, SASP)因子水平变化评估肠道衰老改变。通过免疫组织化学染色干细胞功能标志物、BrdU标记实验、以及隐窝细胞体外小肠类器官形成实验检测小肠干细胞功能及表型变化。通过16S rRNA测序和代谢物分析手段探究相关机制并进行分子生物学验证。

结果：体外实验中，多浓度NR对HIEC-6细胞无明显的毒性作用，采用250 μM NR能够显著降低急性电离辐射暴露后的细胞凋亡和DNA损伤水平并抑制远期细胞衰老表型。体内实验中，NR给药可以显著抑制辐射暴露小鼠整体衰老表型、改善被毛状态、提高前肢抓力。对于DEARE导致的肠道损伤，NR显著抑制结肠缩短、抑制绒毛增长并显著改善小肠组织衰老表型，表现为小肠隐窝SA-β-Gal染色阳性、衰老标记物p21,p16表达水平以及SASP水平抑制。在肠道干细胞方面，NR能够显著抑制辐射远期损伤导致的小肠干细胞数量减少和增殖功能障碍，并增强小肠隐窝细胞类器官形成能力。机制上，NR有效抑制辐射远期损伤诱导的氧化应激，减轻肠道长期氧化损伤和肠道干细胞DNA损伤。此外，NR还可通过改善肠道菌群失调、调控肠道代谢微环境发挥其治疗作用，这一过程与激活肠道SIRT6、SIRT7信号通路以及抑制mTORC1通路的活性密切相关。

结论：电离辐射远期损伤诱导肠道发生衰老，NR可通过降低肠道氧化应激损伤、缓解肠道共生态紊乱、调节肠道代谢抑制肠道衰老进而救治辐射远期肠损伤。本研究为NR治疗肠道DEARE提供理论支持和实验基础，同时有利于更好地理解电离辐射急性暴露后延迟效应以进行治疗药物和策略开发。

Objectives: As a highly radiosensitive organ, the intestine is particularly vulnerable during radiotherapy for abdominopelvic tumors, which can induce delayed effects of acute radiation exposure (DEARE), leading to long-term radiation-induced intestinal injury that severely compromises patient survival and quality of life. Currently, there are no effective therapeutic approaches available, creating an urgent need for targeted pharmacological interventions and treatment strategies. Radiation-induced senescence represents one of the primary mechanisms underlying DEARE, making it a promising therapeutic target. Nicotinamide riboside (NR), as a potent nicotinamide adenine dinucleotide (NAD) booster, has demonstrated significant biological effects in regulating cellular energy metabolism and decelerating aging processes. This study aims to investigate the therapeutic potential of NR against radiation-induced long-term intestinal injury and elucidate its underlying mechanisms, with the goal of providing novel theoretical foundations and experimental evidence for the clinical management of intestinal DEARE.

Methods: We utilized human intestinal epithelial cells (HIEC-6) as the model system in vitro. The cytotoxicity of NR at various doses was assessed using the CCK-8 assay. Apoptosis and DNA damage levels in HIEC-6 cells post-radiation were evaluated via flow cytometry, while long-term cellular senescence was detected using SA-β-Gal staining. For in vivo experiments, a long-term radiation injury model was established in male C57BL/6J mice through a single 6 Gy whole-body irradiation. After 8 weeks, NR was administered via oral gavage for 3 weeks, and forelimb grip strength was measured prior to sacrifice. Intestinal histological changes were examined using H&E staining and immunohistochemistry. Intestinal senescence was assessed through SA-β-Gal staining of frozen tissue sections, Western blot and RT-qPCR analysis of senescence-associated markers, and tissue antibody arrays to measure senescence-associated secretory phenotype (SASP) factors. Stem cell functionality was evaluated via immunohistochemical staining of stem cell markers, BrdU labeling assays, and crypt cell-derived organoid formation experiments. Mechanisms were explored using 16S rRNA sequencing and metabolomic analysis, followed by molecular biology validation.

Results: In vitro experiments showed that NR had no obvious toxic effect on HIEC-6 cells. Meanwhile, it could significantly reduce the levels of cell apoptosis and DNA damage after acute ionizing radiation exposure and inhibit the long-term senescence phenotype of cells. In vivo experiments demonstrated that NR administration could significantly suppress the overall aging phenotype of radiation-exposed mice, improve their fur condition, and enhance the grasping force of their forelimbs. Regarding the intestinal damage caused by DEARE, NR could significantly inhibit colon shortening, suppress villus growth, and significantly improve the senescence phenotype of small intestinal tissue, as indicated by the reduction of SA-β-Gal staining in small intestinal crypts, the expression levels of senescence markers p21 and p16, and the SASP levels. In terms of intestinal stem cells, NR could significantly inhibit the reduction in the number of small intestinal stem cells and the impairment of their proliferation function caused by long-term radiation damage, and enhance the organoid formation ability of small intestinal crypt cells.

Conclusions: Delayed effect acute radiation injury induces intestinal senescence, and NR mitigates radiation-induced intestinal damage by reducing oxidative stress, alleviating gut dysbiosis, and regulating intestinal metabolism, thereby inhibiting intestinal senescence. This study provides theoretical and experimental support for the use of NR in treating intestinal DEARE, while also advancing the understanding of delayed effects of acute radiation exposure, facilitating the development of therapeutic drugs and strategies.