背景：放射治疗作为癌症治疗的重要手段，在腹腔肿瘤的治疗中起着至关重要的作用，但放疗或突发辐射暴露后，常会引起放射性肠损伤(radiation-induced intestinal injury, RIII)。近年来，单细胞 RNA 测序技术（single-cell RNA sequencing, scRNA-seq）的迅速发展为在单细胞水平上解析辐射损伤后细胞的动态变化及修复过程的关键分子机制提供了技术支持。Wnt3/β-catenin 信号通路和 MIF-CD74/CD44 轴作为调控细胞增殖和炎症反应的重要机制，在放射性肠损伤的发生和修复中具有重要作用，但其具体的时空动态变化与交互调控机制仍未被完全阐明。因此，系统性地探讨辐射损伤后细胞的动态变化、Wnt3/β-catenin信号通路在不同剂量照射下的响应及其与MIF-CD74/CD44轴在放射性肠损伤中的交互关系，对加深放射性肠损伤修复机制的认识及制定精准的辐射防护策略具有重要的临床意义。

目的：本研究旨在利用scRNA-seq技术，从单细胞水平全面解析低剂量（13 Gy） 与高剂量（15 Gy）腹部照射后不同时间点小鼠肠隐窝各细胞群的动态变化及其异质性。重点解析Wnt3/β-catenin信号通路在不同剂量照射下的双重作用及MIF-CD74/CD44轴在高剂量照射后介导炎症反应导致小鼠死亡中的作用，并探讨药物干预的最佳时间窗口和关键调控节点，以期为意外辐射暴露或临床上辐射损伤的精准治疗提供新思路和新的干预策略。

方法：本研究选用 SPF 级的 C57BL/6 小鼠为实验动物模型。小鼠随机分为13 Gy 组与 15 Gy 组，各组小鼠分别在照射后第 1、3、5 天采集肠隐窝进行单细胞测序，或在照射后第 1、2、4 天给予 LGK974、LF3、CD44 单克隆抗体或氨磷汀进行药物干预。利用scRNA-seq技术对采集的样本进行测序，用 Seurat 软件包对测序数据进行细胞聚类及差异表达基因（differentially expressed genes, DEGs）的分析；对 DEGs 进行基因本体（Gene Ontology, GO）富集分析，以识别与损伤和修复相关的关键基因及涉及的重要生物过程；应用 scVelo 进行 RNA 速度分析，以解析瞬时扩增（Transit-Amplifying, TA）细胞群在损伤修复过程中的分化趋势。采用CellChat 工具对测序后的数据构建细胞间通讯网络，重点解析 Wnt3/β-catenin 信号通路与 MIF-CD74/CD44 轴在各细胞群之间的配体-受体相互作用，以揭示肠隐窝各细胞群之间及它们与巨噬细胞、中性粒细胞等免疫细胞群之间的信号传递。最后利用药物干预实验进行功能验证，小鼠在照射后第 1、2、4 天给予 LGK974、LF3、CD44 单克隆抗体以及氨磷汀进行药物干预。利用免疫荧光染色、小鼠生存率实验及类器官培养等方法，对干预后肠组织DNA 损伤、肠隐窝细胞的增殖、生存率水平进行评估，以探讨Wnt3/β-catenin 信号通路与 MIF-CD74/CD44 轴在放射性肠损伤修复中的作用。

结果：本研究利用scRNA-seq技术对肠隐窝进行测序，用 Seurat 软件包对测序数据进行分析，并利用生物学实验进行验证，得出以下结果，隐窝基底柱状细胞（crypt-based columnar cell, CBC）与 TA 细胞的比例在13 Gy 照射后第 1 天显著下降，之后缓慢恢复，在第 5 天恢复至对照水平；第三天主要激活DNA复制及细胞增值相关生物过程以修复损伤，第五天进行抗菌防御。15Gy中隐窝结构严重破坏，CBC 细胞修复受阻；巨噬细胞中 CD44 异常上调，抗菌肽基因（Defa/Reg 家族）及抗菌防御反应相关生物过程持续激活。RNA速度分析发现辐射暴露显著增加了 TA 细胞的异质性。一方面，TA 细胞可以分化为成熟的肠上皮细胞，维持屏障完整性；另一方面，在 CBC 细胞受损时，TA 细胞可通过去分化成为干细胞，补充干细胞库，加快肠损伤的修复。低剂量（13 Gy）照射后，Wnt3/β-catenin通路促进肠道干细胞的增值修复和抗菌防御，提高小鼠生存率；高剂量（15 Gy）照射后，Wnt3/β-catenin通路异常激活促进炎症反应，加重DNA 损伤，使肠道稳态失衡，导致小鼠死亡，表明Wnt3/β-catenin 通路在高低剂量照射后表现出双重效应。15 Gy 照射后巨噬细胞上CD44的表达显著升高，同时巨噬细胞迁移抑制因子MIF（macrophage migration inhibitory factor, MIF       ）与 CD44 的结合增强，巨噬细胞与中性粒细胞的相互作用也显著增强。CD44 单抗干预显示，照射后第 2 天阻断 CD44 可显著提高 15 Gy 照射后小鼠的生存率，死亡率从 66.7% 降至 33.3%。表明MIF-CD74/CD44 轴在介导炎症级联反应引起小鼠死亡中发挥重要作用。抗氧化剂氨磷汀可在一定程度上抑制巨噬细胞中 CD44 的上调及其与 MIF 的结合，抑制MIF介导的促炎信号；氨磷汀可通过清除自由基缓解氧化应激引起的DNA损伤，减轻放射性肠损伤；此外，氨磷汀还可以促进 MIF 与肠上皮 CD44 结合，通过Wnt3/β-catenin通路保护干细胞，改善类器官生长，促进肠损伤的修复，发挥多重保护作用。

结论：本研究利用单细胞 RNA 测序技术和生物信息学分析系统揭示了不同照射剂量下肠隐窝各细胞群的修复动态和放射性肠损伤的关键调控机制。明确了TA细胞具有显著的异质性，不仅可以分化为成熟的肠上皮细胞而且可以逆分化为肠干细胞。结合生物学实验，阐明了 Wnt3/β-catenin 通路的双重作用，即在低剂量照射下的促修复作用及高剂量照射下的加重损伤作用。证明了Wnt3/β-catenin信号通路的过度激活和 MIF-CD74/CD44 轴介导的炎症级联反应是高剂量照射导致肠损伤修复失败和小鼠死亡的重要原因，并发现照射后第 2 天为最佳药物干预窗口，此时干预可显著改善放射性肠损伤，提升小鼠的存活率。此外，氨磷汀作为辐射防护剂可有效保护 DNA免受氧化损伤并抑制了巨噬细胞中CD44的上调，同时缓解MIF介导的炎症级联反应。本研究深化了我们对放射性肠损伤分子机制的认识，具有重要的科学意义和潜在的临床转化价值。

Background: Radiotherapy represents a cornerstone in the management of abdominal malignancies; however, its clinical efficacy is frequently constrained by dose-limiting RIII. Recent breakthroughs in scRNA-seq have revolutionized our understanding of dynamic cellular changes at the single-cell level, particularly illuminating crypt epithelial regeneration processes following radiation insult. Emerging evidence highlights the pivotal roles of the Wnt3/β-catenin pathway and the MIF-CD74/CD44 axis in orchestrating cellular proliferation, lineage specification, and inflammatory homeostasis during intestinal repair. While their spatiotemporal dynamics and interactive regulatory mechanisms remain incompletely characterized. Therefore, a comprehensive investigation of cellular dynamics post-radiation, the dual role of the Wnt3/β-catenin pathway under different radiation doses, and the functional crosstalk between the Wnt3/β-catenin pathway and the MIF-CD74/CD44 axis in RIII is of great clinical significance for understanding the intestinal radiobiology and developing precise radiation protection strategies targeting key molecular nodes in RIII pathogenesis.

Objective: This study aims to leverage scRNA-seq technology to comprehensively profile the dynamic changes and heterogeneity of different cell populations in the intestinal crypts of mice following low-dose (13 Gy) and high-dose (15 Gy) abdominal irradiation at multiple time points post-irradiation. Our research efforts are centered on clarifying the dual-function of the Wnt3/β-catenin signaling pathway under different radiation doses and the role of the MIF-CD74/CD44 axis in mediating inflammation-induced mortality after high-dose radiation. Additionally, we explore the optimal timing for drug intervention and key regulatory nodes to provide new insights and therapeutic strategies for the precise treatment of radiation damage.

Methods: SPF-grade C57BL/6 mice were used as the experimental animal model and randomly divided into 13 Gy and 15 Gy groups. Intestinal crypt samples were harvested for scRNA - seq on days 1, 3, and 5 post-irradiation. Drug interventions were carried out using LGK974, LF3, CD44 monoclonal antibody, or amifostine on days 1, 2, and 4 after irradiation. The obtained sequencing data were analyzed with the Seurat software package. This analysis involved cell clustering and the identification of differentially expressed genes. GO enrichment analysis was performed to pinpoint key genes and biological processes associated with radiation - induced damage and subsequent repair mechanisms. RNA velocity analysis was conducted using scVelo to clarify the differentiation trajectories of TA cells. To analyze ligand - receptor interactions between the Wnt3/β-catenin pathway and the MIF - CD74/CD44 axis across different cell populations, cell - cell communication networks were constructed with CellChat. This approach enabled the revelation of signaling communication between intestinal crypt cell populations and immune cell populations, such as macrophages and neutrophils. Subsequently, functional validation was carried out through drug intervention experiments. Mice were administered LGK974, LF3, CD44 monoclonal antibody, or amifostine on days 1, 2, and 4 post-irradiation. Immunofluorescence staining, mouse survival experiments, and organoid culture were utilized to assess the impacts of these interventions on DNA damage in intestinal tissues, crypt cell proliferation, and survival rates. These comprehensive methods were employed to explore the roles of the Wnt3/β- catenin signaling pathway and the MIF - CD74/CD44 axis in the repair of radiation - induced intestinal injury.

Results: In this study, single-cell RNA sequencing was applied to sequence the intestinal crypts, and the resulting data were analyzed using the Seurat software package. The findings were further corroborated by biological experiments. The results demonstrated that following 13 Gy radiation, the ratio of CBCs and TA cells decreased significantly on day 1 and gradually returned to control levels by day 5. On day 3, processes related to DNA replication and cell proliferation were predominantly activated for repair, while antimicrobial defense mechanisms were enhanced on day 5. In contrast, 15 Gy radiation severely damaged the crypt structure, hindering the repair of CBCs. Macrophage CD44 expression was significantly upregulated, and antimicrobial peptide genes (Defa/Reg family) along with associated processes were persistently activated. RNA velocity analysis indicated that radiation exposure increased the heterogeneity of TA cells. These cells could either differentiate into mature enterocytes to maintain the integrity of the intestinal barrier or dedifferentiate into stem cells to replenish the stem cell pool and accelerate the repair process. Low-dose (13 Gy) radiation activated the Wnt3/β-catenin pathway, which promoted stem cell proliferation and antimicrobial defense, thereby improving the survival rate of mice. However, high-dose (15 Gy) radiation aberrantly activated the Wnt3/β-catenin pathway, exacerbating inflammation and DNA damage. This led to an imbalance in intestinal homeostasis and ultimately resulted in mortality, highlighting the dual effects of the Wnt3/β - catenin pathway under low- and high-dose radiation. After 15 Gy irradiation, CD44 expression in macrophages increased significantly, enhancing MIF-CD44 binding and the interaction between macrophages and neutrophils. Intervention with a CD44 monoclonal antibody on day 2 after 15 Gy radiation significantly increased the mouse survival rate from 33.3% to 66.7%, suggesting that the MIF-CD74/CD44 axis plays a pivotal role in mediating inflammation-induced mortality in mice. Amifostine, an antioxidant, inhibited the upregulation of macrophage CD44 and MIF binding. It also reduced oxidative stress-induced DNA damage and promoted MIF-CD44 binding on enterocytes, protecting stem cells via the Wnt3/β-catenin pathway. This promoted organoid growth and facilitated intestinal repair, demonstrating the multifaceted radioprotective effects of amifostine.

Conclusion: This study systematically elucidated the repair dynamics of different cell populations in the intestinal crypts and key regulatory mechanisms under different radiation doses using scRNA-seq and bioinformatics analyses. Notably, TA cells were found to display substantial heterogeneity, possessing the ability to differentiate into mature enterocytes as well as dedifferentiate into intestinal stem cells.​In conjunction with biological experiments, the dual-function of the Wnt3/β-catenin pathway was clarified. Specifically, it was shown to promote repair processes under low-dose radiation conditions, yet exacerbate injury under high-dose radiation. The study demonstrated that the over-activation of the Wnt3/β-catenin signaling pathway, along with the inflammatory cascade mediated by the MIF - CD74/CD44 axis, represents a critical factor contributing to the failure of intestinal injury repair and subsequent mortality in mice following high-dose radiation. Furthermore, the second day post - radiation was identified as the optimal intervention window. Intervention during this period was shown to significantly ameliorate RIII and increase the survival rate of mice. Additionally, amifostine, a well-known radioprotective agent, was found to effectively safeguard DNA from oxidative damage, inhibit the upregulation of CD44 in macrophages, and concomitantly alleviate the MIF-mediated inflammatory cascade.​ Collectively, this study provides novel insights into the molecular mechanisms underlying radiation-induced intestinal injury. As such, it holds significant scientific importance and substantial potential for clinical translation.