Radiotherapy is a primary treatment modality for malignant tumors and remains the most widely used therapeutic approach (Schaue and McBride, 2015). Approximately 40% of long-term survivors benefit from radiotherapy, and around 60–70% of cancer patients receive radiotherapy at various stages of their disease (Baumann et al., 2016; Overgaard and Bartelink, 1995). The radiation dose is a critical factor influencing the effectiveness of tumor treatment. As the dose increases, both the local control rate of the tumor and the patient’s survival rate may improve; however, the risk of side effects to normal tissues within the irradiated field also rises (Melia and Parsons, 2023). Despite its efficacy, one of the significant limitations of radiation therapy is the potential damage it can cause to surrounding healthy tissues, particularly the intestines in abdominal and pelvic treatments (Levy et al., 2020). Acute radiation intestinal injury is a common complication characterized by inflammation, epithelial cell loss, and compromised barrier function, often leading to symptoms such as diarrhea, abdominal pain, and malabsorption (Lu et al., 2023). These effects not only impact patient quality of life but can also necessitate treatment interruptions or dose reductions, potentially compromising therapeutic outcomes (Bao et al., 2019).

FLASH radiotherapy (FLASH-RT) is a revolutionary new technology in tumor radiotherapy that can deliver ultra-high dose rates (≥40 Gy/s) within an extremely short duration (milliseconds to microseconds) (Favaudon et al., 2014). Compared to conventional dose-rate radiotherapy (CONV-RT), FLASH-RT offers two major advantages: an extremely brief treatment time and enhanced protection for normal tissues, while maintaining the same tumor cell-killing efficacy as CONV-RT (Hughes and Parsons, 2020). In summary, FLASH-RT can improve the efficiency of radiotherapy, ensure treatment efficacy, and reduce toxicity to normal tissues (Chow and Ruda, 2024; Srinivasan et al., 2024).

Despite these promising results, the mechanisms underlying the FLASH effect remain incompletely understood (Shiraishi et al., 2024). Research indicates that FLASH-RT may modify cellular and molecular responses to radiation, including oxidative stress, DNA damage repair, and immune responses (Hageman et al., 2022; Ma et al., 2024). However, these effects are intricate and necessitate further investigation. A deeper understanding of these mechanisms could enhance clinical applications and facilitate the broader adoption of FLASH-RT in oncology (Yan et al., 2024).

Electrons, kilovolt low-energy X-rays, and protons have been used in preclinical studies of FLASH-RT, but these radiation types are not widely applied in clinical practice (Bourhis et al., 2019; Favaudon et al., 2014; Montay-Gruel et al., 2019; Montay-Gruel et al., 2018; Ryoo et al., 2016; Simmons et al., 2019; Vozenin et al., 2019). Electrons and low-energy X-rays are typically used to treat superficial tumors, but their limited penetration makes them unsuitable for tumors located deeper within the body (Eling et al., 2019; Rahman et al., 2022). Flash protons can be used to treat deep-seated tumors, but their high construction and operational costs hinder widespread adoption (Montay-Gruel et al., 2022). High-energy X-rays are the most commonly used type of radiation in clinical radiotherapy, as they offer deep penetration, small divergence, low radiation intensity, and are affordable for patients (Nath et al., 1984). However, generating ultra-high dose-rate high-energy X-rays is challenging, limiting further research in this area. This study utilized a compact, clinical-grade single high-energy X-ray source (CHEXs) FLASH-RT device. The device is capable of generating ultra-high dose-rate high-energy X-rays and has been validated to induce FLASH effect (Shan et al., 2023). While murine models remain a cornerstone for preclinical FLASH research, there is a growing body of work utilizing in vitro 3D models such as spheroids and organoids, which better mimic tissue structure and tumor microenvironments. Durak-Kozica et al. (2023) applied FLASH proton irradiation to 3D cancer spheroids, demonstrating preserved structural integrity and suggesting promising biological effects. Moreover, PET-based studies by Cesar et al. (2024) and image-guided FLASH discussions by Lang (2024) further exemplify the relevance of in vitro FLASH systems in translational settings. This study preliminarily examines the potential correlation between the gut microbiome and the protective effects of FLASH-RT on intestinal tissue using metagenomic analysis.

This study tested three different doses to evaluate the protective effect of FLASH-RT on normal tissues. At a dose of 10 Gy, no mortality was observed in any group, while at 14 Gy, the entire conventional treatment group died, and only one mouse in the FLASH group survived. This suggests that the 14 Gy dose was too high, leading to near-total mortality, which was not conducive to observing the protective effects. Therefore, in subsequent experiments, we used a dose of 12 Gy, which resulted in approximately 50% mortality in the CONV group. Although no statistically significant difference in survival was found between the FLASH and CONV groups at the 12 Gy dose—likely due to the small sample size (n = 10)—the survival trends clearly indicate that FLASH-RT provides substantial protective effects. Further examination of the intestinal tissue from the 12 Gy group via HE staining revealed less tissue damage and fewer inflammatory cell infiltrations in the FLASH group. These findings further support the notion that high-energy X-ray FLASH-RT can confer protective effects on normal intestinal tissue.

LEfSe analysis revealed that several beneficial bacterial species in the FLASH group, including Weissella, Ligilactobacillus ruminis and Lactobacillus taiwanensis, were relatively enriched compared to the CONV group, based on their proportional abundance within the microbial community. Numerous studies have shown that Weissella produces antimicrobial exopolysaccharides, bacteriocins, hydrogen peroxide, and organic acids (Fusco et al., 2015), exhibiting antibacterial, antifungal, antioxidant, and anti-inflammatory properties, as well as enhancing the intestinal epithelial barrier function (Prado et al., 2020). The bacteriocins synthesized by Weissella demonstrate significant antimicrobial or bactericidal activity (Klaenhammer, 1993). The released organic acids also exert bactericidal effects against Vibrio parahaemolyticus T.11 (Ahmed et al., 2022), while byproducts such as lactic acid and ethanol may enhance the anti-mycobacterial effect of lactic acid alone (Stedman et al., 2020). Ligilactobacillus ruminis, on the other hand, reduces the levels of pro-inflammatory cytokines such as IL-1β, TNF-α, and IL-17, thereby mitigating intestinal tissue damage. Additionally, L. ruminis increases the levels of short-chain fatty acids (SCFAs) in mouse feces, further modulating the gut microbiota balance and alleviating intestinal inflammation (Yang et al., 2021). Overall, L. ruminis exerts its beneficial effects by regulating inflammatory responses and restoring gut microbiota balance, thereby reducing inflammation and inhibiting intestinal tissue damage (Thorakkattu et al., 2022). These findings suggest that the intestinal protective effect of FLASH-RT may be associated with changes in the microbiome and its metabolites.

Interestingly, we observed that the genus Alistipes, which is known to exert protective effects against colitis and hepatic inflammation, was significantly depleted in the conventional irradiation group but relatively preserved following FLASH exposure. This finding aligns with prior studies indicating that Alistipes contributes to gut immune homeostasis, partly through production of short-chain fatty acids (SCFAs) and modulation of tryptophan metabolism. The preservation of Alistipes in FLASH-treated mice may thus underlie part of the observed reduction in intestinal injury, and warrants further mechanistic exploration in future studies.

LEfSe pathway analysis indicates that the protective effect of FLASH-RT on normal intestinal tissues might be related to several signaling pathways. Glycosaminoglycans (GAGs) are essential extracellular matrix components that play pivotal roles in intestinal barrier integrity and tissue repair (Tang et al., 2018). Studies indicate that GAG degradation exacerbates murine colitis symptoms, whereas inhibiting this process attenuates inflammatory responses (Belmiro et al., 2005). Specifically, probiotic lactobacilli that suppress GAG degradation ameliorate colitis by inhibiting both pro-inflammatory cytokine expression and bacterial GAG catabolism (Lee et al., 2009). This finding appears to contradict the protective effects of FLASH radiotherapy. However, another study suggests that reactive oxygen species (ROS) play a significant role in GAG degradation. ROS, with their high reactivity, can engage in multiple chemical reactions with GAGs, particularly hydroxyl radicals (•OH), which can break the glycosidic bonds in GAGs, thereby reducing their molecular weight and leading to moderate GAG degradation (Fuchs and Schiller, 2014). The consumption of ROS may mitigate oxidative stress damage to normal cells, creating favorable conditions for tissue repair and regeneration. While existing evidence shows a link between ROS and GAG degradation, further research is needed to better understand the specific role and extent of this pathway in the protective mechanism of FLASH radiotherapy.

The PI3K/Akt signaling pathway orchestrates critical cellular processes including proliferation, survival, and tissue repair. In inflammatory bowel disease, upregulation of the NRG1/ERBB₃ axis activates PI3K/Akt signaling, sustaining intestinal epithelial proliferation and damage repair (Qiu et al., 2024). Studies have shown that activation of the PI3K/Akt/mTOR pathway is a key mechanism underlying radiation resistance in tumor cells across multiple radiotherapy models (Su et al., 2022). Activated Akt exerts its effects by stimulating the downstream mTOR pathway or by inhibiting proteins such as Bad and Caspase-9, which regulate cell proliferation, differentiation, apoptosis, and migration, contributing to radiation resistance in tumor cells (Chang et al., 2014; Chang et al., 2015; Horn et al., 2015). Based on these findings, we hypothesize that FLASH radiotherapy may enhance intestinal radioprotection by activating PI3K/Akt signaling in normal enterocytes, thereby promoting epithelial regeneration and repair.

Arabinogalactan (AG), a soluble dietary fiber with an excellent safety profile, has demonstrated immunomodulatory (Hamed et al., 2022), anticancer (Gong et al., 2020), anti-inflammatory, and antioxidant properties (Zheng et al., 2023). It also alleviates cisplatin-induced intestinal damage. Mechanistically, AG mitigates lipopolysaccharide induced damage to the small intestinal epithelial barrier by reducing inflammation and oxidative stress through modulation of the AMPK/SIRT1/NF-κB signaling pathway. In dry eye disease models, AG significantly reduced uric acid levels by 27% and reactive oxygen species (ROS) by 38%, thereby attenuating oxidative stress (Silvani et al., 2020). In acute liver injury models, AG treatment not only elevated levels of antioxidant enzymes, non-enzymatic antioxidants, and total hepatic antioxidant capacity but also significantly decreased lipid peroxidation in liver tissue (Sun et al., 2018). The enrichment of the arabinogalactan biosynthesis Mycobacterium signaling pathway in the flash group suggests that the protective effect of FLASH-RT on intestinal tissue may be related to the anti-inflammatory and antioxidant properties of arabinogalactan. These findings suggest that FLASH-RT may influence immune and stress-response pathways, including glycosaminoglycan degradation, PI3K/Akt and arabinogalactan biosynthesis Mycobacterium signaling, although the activation of these pathways could also reflect microbial stress or host-pathogen interaction rather than beneficial effects.

The high number of differentially detected species between irradiated and non-irradiated groups, as shown in Figure 3C, may initially appear surprising given the controlled housing conditions. However, this is likely attributable to deep sequencing sensitivity and the emergence of rare taxa after dominant species were depleted. Most of these species were detected at extremely low abundance, and their biological role remains uncertain. It is possible that microbial niche space became available following radiation-induced community collapse, allowing transient or low-abundance microbes to expand. Although FLASH-irradiated mice exhibited a microbiome profile that differed from that of conventionally irradiated animals, notable deviations from the control group remained. Alpha diversity in the FLASH group was partially restored compared to the CONV group, but remained lower than that of the unirradiated controls. NMDS and heatmap analyses also revealed that FLASH samples clustered closer to the CONV group than to the control group, indicating incomplete recovery. These findings suggest that FLASH-RT mitigates but does not fully reverse radiation-induced alterations in the gut microbiota.

Although metagenomic data preliminarily suggest that the protective effect of FLASH-RT on normal intestinal tissue may be linked to the modulation of gut microbiota composition, several limitations exist in this study. The diversity analysis results show substantial within-group variation in the FLASH group, and this high heterogeneity may impact the statistical significance of specific microbial changes, thereby interfering with the stability of the conclusions. However, due to the limited number of experimental animal samples, excluding samples that deviate from the norm may introduce substantial selection bias. Furthermore, inherent differences in the microbiota state across different irradiation batches of mice complicate sample addition, potentially increasing systemic bias and affecting the reliability and comparability of the results. In this experiment, 80% of the mice survived after receiving 12 Gy FLASH-RT, while 20% died, suggesting individual variability in the protective effect of FLASH. Combined with the observed heterogeneity in the microbiota species of the FLASH group, we hypothesize that the heterogeneity within the FLASH group may be related to differences in individual microbiota recovery abilities, which could be a key factor in the protective effect of FLASH. Therefore, excluding individuals with large differences might obscure the biological significance of individual microbiota recovery processes. To more accurately reflect the individual variability after FLASH-RT, we retained all raw data and approached data analysis with caution, avoiding overinterpretation of potential causal relationships. Through this approach, we believe that this study provides preliminary insights into the changes in gut microbiota following FLASH-RT intervention and offers new directions for exploring the mechanisms behind individual responses to FLASH-RT.

In summary, the protective effects of FLASH-RT on the intestine may be associated with multiple microbiota and signaling pathways. Gut microbiota may exert their effects through specific metabolites, such as short-chain fatty acids and polysaccharides. However Liberles et al. (2013) proposed that confirming causal mechanisms requires further experimental validation. This study is based on metagenomic analyses and does not include targeted metabolomics; therefore, functional inferences derived solely from metagenomic pathway annotations should be interpreted with caution. Causal relationships between microbial community changes and host effects require experimental validation. Future research should further explore the specific roles and mechanisms of different microbiota in this process to better understand the potential of FLASH-RT in intestinal protection.

Compared to CONV-RT, high-energy X-ray FLASH irradiation exerts radioprotective effects on normal intestinal tissue. Alterations in the gut microbiota and associated signaling pathways may be linked to the protective effects of FLASH.