目的：

放射治疗（radiotherapy, RT）作为恶性肿瘤综合治疗的重要手段，在临床应用中面临两个关键科学问题。一方面，肿瘤细胞内在或获得性辐射抗性常常导致治疗失败，严重影响治疗效果。另一方面，辐射对正常组织特别是快速增殖的肠道上皮的损伤限制了治疗剂量的提升，增加了治疗风险。核因子E2相关因子2（nuclear factor erythroid 2-related factor 2, NRF2）作为细胞氧化应激防御系统的核心调控因子，在这两个问题中均发挥着关键但截然相反的作用。

本研究首先聚焦于非小细胞肺癌（non-small cell lung cancer, NSCLC）这一临床治疗难题，旨在评估NRF2抑制剂ML385和核因子E2相关因子1（nuclear factor erythroid 2-related factor 1, NRF1）抑制剂WRR139的辐射增敏效果，探索靶向NRF2或NRF1提高肺癌辐射敏感性的潜在策略，为临床用药提供实验依据。其次，在辐射防护方面，本研究通过对NRF2敲除小鼠的RNA测序结果进行加权基因共表达网络分析（weighted gene co-expression network analysis, WGCNA），重点分析了NRF2调控DNA修复和免疫微环境的分子途径。本研究通过细胞实验与动物模型相结合，系统探讨了NRF2及其抑制剂在肺癌辐射敏感性中的作用，以及NRF2在辐射诱导肠道损伤中的保护机制，为优化放疗策略提供理论依据。

方法：

在细胞实验方面，选用非小细胞肺癌细胞系A549和H460作为研究对象，首先通过CCK-8实验评估NRF2抑制剂ML385和NRF1抑制剂WRR139对细胞增殖的影响，随后利用克隆形成实验检测其联合不同剂量γ射线照射后对细胞辐射敏感性的改变，采用碘化丙啶（propidium iodide, PI）染色结合流式细胞术分析药物处理后的细胞周期分布变化。

在分子层面，通过Western Blotting检测NRF2、NRF1及HO-1的蛋白表达水平，同时运用RT-qPCR技术探究NRF2、NRF1对KEAP1、NQO1、HO-1的转录调控作用。此外，通过免疫荧光实验观察γH2AX foci形成情况，定量评估药物对辐射后DNA损伤的影响。

在动物实验方面，对野生型小鼠和NRF2敲除小鼠给予14 Gy全腹照射后3.5天获取空肠组织进行RNA测序。在数据分析阶段，采用WGCNA筛选差异表达基因模块，并构建竞争性内源RNA（competing endogenous RNA, ceRNA）网络及蛋白-蛋白相互作用（protein-protein interaction, PPI）网络。同时通过流式细胞术动态监测肠道固有层中CD4⁺ T细胞、CD8⁺ T细胞、巨噬细胞及树突状细胞的数量变化，结合基因集变异分析（gene set variation analysis, GSVA）系统评估免疫相关基因与修复通路的关联性。  

结果：  

本研究系统揭示了ML385和WRR139在非小细胞肺癌中的多重调控机制。首先，CCK-8实验显示ML385和WRR139显著抑制非小细胞肺癌细胞的增殖能力，且呈剂量依赖性；进一步通过克隆形成实验证实，两者联合照射能显著增强非小细胞肺癌细胞的辐射敏感性。在分子层面，Western Blotting与RT-qPCR结果显示：ML385降低NRF2蛋白水平，并抑制下游HO-1和NQO1的转录表达；而WRR139降低KEAP1转录水平，但对NRF1蛋白水平无显著影响。同时，免疫荧光实验表明两者显著降低8 Gy照射后细胞中γH2AX foci的形成，提示对DNA损伤修复的抑制作用。

基于对野生型和NRF2敲除小鼠肠组织的RNA测序，发现辐射效应基因Akr1b8、Gsta3表达显著下降，这些基因通过抗氧化作用减轻DNA损伤；通过WGCNA筛选出DNA损伤修复相关基因和免疫调控基因模块，为阐明放射性肠损伤的分子机制提供了重要线索；ceRNA网络揭示lncRNA AK033546和AK020274通过竞争miRNA调控组蛋白和核糖体基因表达，协同促进DNA修复；PPI分析显示核糖体蛋白在辐射后形成独立的功能簇，参与DNA修复过程。流式细胞术检测表明，照射后3.5天CD4⁺ T细胞比例下降，M1型巨噬细胞比例升高。NRF2敲除导致抗氧化基因表达下调，提示其通过调节氧化应激影响炎症微环境。

结论：  

NRF2在肺癌和肠道中具有双重作用——其激活增强肺癌辐射抗性，但保护肠道免受辐射损伤。靶向抑制NRF2/KEAP1通路可增强肺癌放疗效果，而激活NRF2或调控其下游抗氧化基因可能减轻肠道辐射损伤。本研究为NRF2在放疗中的精准调控提供了新思路，为放疗联合靶向治疗策略的优化奠定基础。

Objective:​​

Radiotherapy (RT), as a critical component in the multimodal treatment of malignant tumors, faces two pivotal scientific challenges in clinical practice. First, intrinsic or acquired radioresistance in tumor cells frequently leads to therapeutic failure, significantly compromising treatment efficacy. Second, radiation-induced damage to normal tissues, particularly the rapidly proliferating intestinal epithelium, restricts dose escalation and increases treatment risks. Nuclear factor erythroid 2-related factor 2 (NRF2), a master regulator of cellular oxidative stress defense, plays pivotal yet opposing roles in these dual challenges.

This study first addresses non-small cell lung cancer (NSCLC), a clinical therapeutic challenge, by evaluating the radiosensitizing effects of the NRF2 inhibitor ML385 and the nuclear factor erythroid 2-related factor 1 (NRF1) inhibitor WRR139. This exploration aims to identify potential strategies for enhancing NSCLC radiosensitivity through NRF2/NRF1 targeting and to provide experimental evidence for clinical applications. Additionally, in the context of radioprotection, weighted gene co-expression network analysis (WGCNA) of RNA sequencing data from NRF2-knockout mice was employed to dissect molecular pathways underlying NRF2-mediated regulation of DNA repair and the immune microenvironment. By integrating cellular experiments and animal models, this study systematically investigates the role of NRF2 and its inhibitors in NSCLC radiosensitivity and elucidates NRF2-mediated protective mechanisms against radiation-induced intestinal injury, offering theoretical foundations for optimizing RT strategies.

Methods:​​

For in vitro studies, NSCLC cell lines A549 and H460 were utilized. CCK-8 assays assessed the proliferative effects of ML385 and WRR139, while clonogenic survival assays evaluated radiosensitivity under combined treatment with γ-ray irradiation. Cell cycle distribution was analyzed via propidium iodide (PI) staining and flow cytometry. Mechanistically, Western blotting quantified protein levels of NRF2, NRF1, and HO-1, while RT-qPCR assessed transcriptional regulation of KEAP1, NQO1, and HO-1. Immunofluorescence was performed to quantify γH2AX foci formation, reflecting DNA damage repair inhibition.

For in vivo studies, jejunal tissues from wild-type and NRF2-knockout mice were collected 3.5 days post 14 Gy whole-abdomen irradiation for RNA sequencing. WGCNA identified differentially expressed gene modules, followed by construction of competing endogenous RNA (ceRNA) networks and protein-protein interaction (PPI) networks. Flow cytometry dynamically monitored immune cell populations (CD4⁺/CD8⁺ T cells, macrophages, dendritic cells) in the intestinal lamina propria. Gene set variation analysis (GSVA) correlated immune-related genes with repair pathways.

Results:​​

This study systematically elucidates the multifaceted regulatory mechanisms of ML385 and WRR139 in NSCLC. First, CCK-8 assays demonstrated that ML385 and WRR139 significantly inhibited NSCLC cell proliferation in a dose-dependent manner. Furthermore, colony formation assays confirmed that combinatorial treatment with both agents synergistically enhanced the radiosensitivity of NSCLC cells under irradiation; at the molecular level, Western Blotting and RT-qPCR analyses revealed distinct regulatory pathways: ML385 reduced NRF2 protein levels and suppressed transcriptional expression of downstream targets HO-1 and NQO1, whereas WRR139 downregulated KEAP1 transcriptional levels while exerting no significant effect on NRF1 protein levels; Immunofluorescence assays further demonstrated that both compounds markedly reduced the formation of γH2AX foci in cells following 8 Gy irradiation, indicating their inhibitory effects on DNA damage repair processes. These findings collectively provide mechanistic insights into how ML385 and WRR139 potentiate radiation-induced cytotoxicity through coordinated modulation of NRF2/KEAP1 signaling and impairment of DNA repair capacity.

Based on RNA sequencing analyses of intestinal tissues derived from wild-type and NRF2-knockout murine models, we observed significant downregulation of radiation-responsive genes Akr1b8 and Gsta3, which exert DNA damage mitigation through antioxidative mechanisms. WGCNA identified distinct functional modules enriched in DNA damage repair-associated genes and immune regulatory genes, offering critical insights into the molecular pathogenesis of radiation-induced intestinal injury. The constructed ceRNA network revealed that lncRNAs AK033546 and AK020274 modulate histone variant and ribosomal gene expression via competitive miRNA binding, thereby coordinating DNA repair processes. PPI network analysis further demonstrated that ribosomal proteins formed autonomous functional clusters post-irradiation, implicating their involvement in DNA repair cascades. Flow cytometric quantification revealed a marked decline in CD4⁺ T-cell proportions and elevated infiltration of M1-polarized macrophages at 3.5 days post-irradiation. Notably, NRF2 deficiency resulted in suppressed expression of antioxidant genes, suggesting its regulatory role in inflammatory microenvironment remodeling through oxidative stress modulation. These findings collectively delineate the interplay between redox homeostasis, DNA repair machinery, and immune dysregulation in radiation enteropathy.

Conclusion:​​

NRF2 exhibits dual roles in radiotherapy: its activation promotes radioresistance in NSCLC but protects intestinal epithelium from radiation injury. Pharmacological inhibition of the NRF2/KEAP1 axis enhances NSCLC radiosensitivity, whereas NRF2 activation or downstream antioxidant gene modulation may mitigate intestinal damage. This study provides novel insights into precision NRF2 targeting for optimizing RT efficacy while minimizing toxicity, laying a foundation for combined radiotherapy and molecularly targeted strategies.