Preliminary mechanistic exploration of mitochondrial function in intestinal protection mediated by high-energy X-ray FLASH radiotherapy

Preliminary mechanistic exploration of mitochondrial function in intestinal protection mediated by high-energy X-ray FLASH radiotherapy | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Preliminary mechanistic exploration of mitochondrial function in intestinal protection mediated by high-energy X-ray FLASH radiotherapy Xiaofei Hao, Huan Du, Binwei Lin, Decai Wang, Wei Wu, Mingming Tang, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7650752/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 28 Feb, 2026 Read the published version in Radiation Oncology → Version 1 posted 11 You are reading this latest preprint version Abstract Purpose Ultra-high dose rate (UHDR) radiation retains tumor-killing efficacy while reducing toxicity to normal tissues, holding a promising transformative radiotherapy paradigm. This study aimed to explore the potential role of mitochondria in intestinal protection conferred by high-energy X-ray FLASH radiotherapy (FLASH-RT) and the associated signaling pathways. Method The entire abdomen of healthy female C57BL/6 mice was irradiated using three modes: ultra-high dose rate irradiation (FLASH-RT), conventional dose rate irradiation (CONV-RT), and sham irradiation (Control). Mouse survival status and body weight changes were monitored within 15 days post-irradiation. At 72 hours post-irradiation, whole blood was collected for hematological analysis, and intestinal tissues were harvested for pathological detection, transmission electron microscopy (TEM) observation of mitochondrial changes, and two types of mitochondria- targeted metabolomic assays. Results A Compact single High-energy X-ray Source FLASH-RT device(CHEx-FLASH) was used, with a dose rate of 200 Gy/s. At 15 days post-irradiation, the survival rates of the Control group (100%, 10/10) and FLASH-RT group (80%, 8/10) were significantly higher than that of the CONV-RT group (30%, 2/10). Body weight decreased in the early post-irradiation period across groups, but the decline was milder in FLASH-RT with greater late-stage recovery. Hematological results at 72 hours showed that CONV-RT induced more severe bone marrow suppression compared to FLASH-RT. Intestinal histopathological analysis revealed that FLASH-RT alleviated intestinal inflammation and promoted enterocyte proliferation, while DNA double-strand breaks and apoptosis levels did not differ significantly between the two irradiated groups. FLASH-RT mitigated mitochondrial damage, reduced reactive oxygen species (ROS) levels and mildly activated mitophagy. Mitochondria-related energy metabolomics sequencing of intestinal tissues showed that the mitochondrial damage marker malonic acid was significantly lower in FLASH-RT than in CONV-RT, and differentially expressed metabolites were primarily enriched in mitochondrial antioxidant pathways. Additionally, the increased expression of the antioxidant protein NRF2 and superoxide dismutase ༈SOD) were verified. Conclusion CHEx-FLASH achieves UHDR irradiation and alleviates radiation-induced intestinal injury. The protective effect of FLASH-RT on intestinal tissues may be mediated by mitigating mitochondrial damage and enhancing antioxidant pathways through improved mitochondrial energy metabolism. ultra-high dose rate X-ray FLASH effect intestine mitochondria metabolism Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction The global incidence of cancer continues to rise, and it has become one of the main causes of human death, posing a serious threat to human health [ 1 ]. Concurrently, radiation-induced toxicity has emerged as a worldwide concern. Radiation enteritis—an inflammatory disorder that arises when ionizing radiation injures the intestinal mucosa and adjacent tissues during treatment of pelvic, abdominal, or retroperitoneal malignancies (e.g., cervical, rectal, or prostate cancer), and it is one of the most frequent gastrointestinal complications of radiotherapy [ 2 – 4 ]. Achieving optimal tumour control while minimising intestinal toxicity has therefore become a central objective of contemporary radiotherapy. Ultra-high dose rate radiotherapy (UHDR, ≥ 40 Gy/s), also known as FLASH radiotherapy (FLASH-RT), has attracted extensive attention in recent years due to its ability to retain tumor therapeutic effects while having a unique normal tissue protection effect [ 5 ], and is expected to become a disruptive radiotherapy paradigm. Although some mechanistic hypotheses have been proposed, such as oxygen consumption, DNA damage repair, free radical-free radical interaction, and immune mediation [ 6 – 8 ], there are still conflicting positive and negative research results, and the underlying biology remains incompletely understood. Historically, the biological effects of ionizing radiation were attributed primarily to nuclear DNA damage and its subsequent repair. However, current molecular biology studies have shown that the results of ionizing radiation exposure are highly dependent on the activation and regulation of other molecular components of organelles, which determine the cell's survival and proliferation ability. For example, mitochondria, as the "energy factories" of eukaryotic cells, play a particularly critical role in regulating cellular metabolism, energy supply, and radiation-induced signal transduction [ 9 ]. Surprisingly, mitochondria have recently been implicated in the normal-tissue protection conferred by FLASH-RT. Guo Z et al. [ 10 ] exposed normal human lung fibroblasts (IMR90) to 100 Gy/s proton irradiation and observed significant attenuation of mitochondrial injury—manifest as preserved mitochondrial membrane potential (MMP), restored mitochondrial DNA (mtDNA) copy number and reduced reactive oxygen species (ROS)—whereas malignant cells showed no such sparing. These findings open a new avenue for elucidating the mechanistic basis of FLASH-RT selectivity. In recent years, pre-clinical studies have consistently demonstrated FLASH-RT-mediated sparing of normal brain [ 11 ], lung [ 12 ], intestine [ 13 , 14 ], skin [ 15 ] and bone [ 16 ]. Validation has extended to large mammals [ 17 ] and, more recently, to early-phase human trials [ 18 ]. Among the available beam modalities—electrons, protons and high-energy photons—electrons are restricted to superficial targets by their limited penetration; protons incur high facility costs; whereas high-energy X-rays combine deep penetration with minimal beam divergence and favourable economics, positioning them as the most clinically scalable option for FLASH-RT. However, high-energy X-ray sources capable of achieving UHDR remain scarce worldwide. To better adapt to clinical practice, we have developed the Compact single High-energy X-ray Source FLASH-RT device(CHEx-FLASH), which is economical and small in size and can generate ultra-high dose rates (≥ 40 Gy/s). Based on this equipment, we verified the protective effect of FLASH-RT on normal intestinal tissues in mice, and conducted the first in vivo study to explore the underlying mitochondrial mechanisms, providing mechanistic insights for more adequate FLASH-RT research. Methods and materials Irradiation device and dosimetry monitoring The irradiation device used in this study was a 10 MeV CHEx-FLASH (Zhongjiu Flash Medical Technology Co., Ltd.) with a source surface distance (SSD) of 50 cm. Two dose-rate modalities were compared on the same platform: UHDR (FLASH-RT, ≥ 40 Gy s⁻¹) and conventional dose-rate (CONV-RT, 0.185 Gy/s). We used a current transformer (BCT) to monitor the beam current, and a diamond detector installed downstream of the main collimator to monitor the X-ray beam. Gafchromic™ EBT-XD radiographic films (Ashland Inc., Covington, Kentucky, USA) were placed in solid water at isocentre, as previously described [ 12 ]. Figure 1 a depicts the in vivo irradiation geometry; the experimental set-up is shown in Fig. 1 b. After being anesthetized with isoflurane gas (3–4%, Isoflurane, R510-22-10), the mice were placed prone on a 0.5 cm custom polymethyl methacrylate (PMMA) platform; a 1 cm water-equivalent bolus was applied dorsally for build-up. The RGB values of the EBT-XD films were scanned 5 minutes post-irradiation using an Epson Expression 12000XL scanner (Seiko Epson Corporation, Japan), and analyzed using SNC patient software (Sun Nuclear Corporation, USA). Additionally, before the FLASH experiment, the EBT-XD films were also calibrated using a clinically applied 6 MV Elekta Precise linac (Elekta AB, Stockholm, Sweden). Animal model and euthanasia method Female C57BL/6 mice aged 6–8 weeks were purchased from SPF (Beijing) Biotechnology Co., Ltd. (Beijing, China) and housed at Mianyang Central Hospital (Mianyang, China). All animal experiments were conducted in strict accordance with ethical standards and approved by the Animal Ethics Committee of Mianyang Central Hospital (approval number: S20240203-02). Prior to euthanasia, the experimental animals were anesthetized with isoflurane (R510-22-10) gas (Shenzhen RWD Life Science Co., Ltd.). This approach effectively prevents animals from experiencing pain, suffocation, or stress responses during euthanasia. Furthermore, isoflurane undergoes rapid metabolism, which minimizes interference of the anesthetic with subsequent experimental test results. During the anesthetic induction phase, the initial concentration of isoflurane was set at 4%–5%. Adequate anesthetic depth was confirmed when the animals exhibited loss of corneal reflex and cessation of limb movements. Following this, procedures including irradiation and blood collection were performed, and euthanasia was ultimately achieved via cervical dislocation. The criteria for confirming death were loss of consciousness, respiratory arrest, and cardiac arrest. whole-abdominal irradiation In this study, mice were subjected to three treatments: FLASH-RT (12 Gy/1F), CONV-RT (12 Gy/1F), or Sham (0 Gy, control). Irradiations were delivered to a 4 cm × 4 cm abdominal field extending from the xiphoid process to the upper anal margin. Hair depigmentation, evident several months later, delineated the irradiated region (Fig. 1 c). In addition, the irradiation dose distribution and percent depth dose (PDD) curves are shown in Figs. 1 d-g. Figure 1 h presents the study design: at 72 hours after irradiation, whole blood and small intestinal tissues were collected (n = 5), while the remaining mice (n = 10) were observed twice weekly for survival and body weight changes during the acute phase (15 days). The experiment endpoints were defined as tumor body weight loss > 40%, severe deterioration in overall health, or death. Whole blood count detection At 72 hours after irradiation, mice were anesthetized with isoflurane gas, and the peripheral blood from the eyeballs was collected into EDTA-coated capillary tubes. A URIT-5160Vet auto hematology analyzer (URIT, Guilin, China) was used to determine the white blood cell count (WBC), hemoglobin (HGB), platelet count (PLT), lymphocyte count (LYM), neutrophil percentage (NEU%), and eosinophil percentage (EOS%). Hematoxylin-eosin staining (H&E staining) Following euthanasia, the abdominal cavity was opened and the intestine photographed in situ. The small bowel was then excised, rinsed in ice-cold phosphate-buffered saline(PBS), and fixed overnight in 4% universal fixative (Biosharp BL539A). Paraffin-embedded tissues were sectioned at 4–5 µm and stained with H&E. Subsequently, digital images were acquired with a pathological section scanner (NanoZoomer S360, C13220-01, Hamamatsu Photonics K.K., Japan). Acute radiation-induced intestinal injury was quantified based on the number of intestinal crypts per unit area [ 14 ], villus height, and intestinal injury pathological score [ 14 ]. The semi-quantitative pathological score was determined according to the proportion of the damaged intestinal segment to the total length:0 = normal; 1 = 2/3 affected). Immunohistochemical staining (IHC) and image analysis Following deparaffinization and rehydration, antigen retrieval was performed and sections were incubated with primary antibodies. After washing, species-appropriate secondary antibodies were applied, chromogenic or fluorescent detection was carried out, and nuclei were counterstained. Sections were dehydrated, cleared and coverslipped before digital imaging with either a NanoZoomer S360 bright-field scanner (Hamamatsu Photonics, Japan) or a Pannoramic SCAN II fluorescence scanner (3DHISTECH, Hungary). Among them, Ki-67 (CST, CST9129) was used for proliferation; γH2AX (Sigma-Aldrich, SAB5600038) for double-strand breaks; cleaved caspase-3 (Affinity, AF7022) and TUNEL (BrightGreen Apoptosis Detection kit, A112) for apoptosis; Parkin (Abmart, T56641S), PINK1 (Abmart, TD7742S) and LC3 (ABclonal, A15591) for mitophagy; NRF2 (Proteintech, 80593-1-RR) for antioxidant capacity. Positive-staining areas were quantified in ImageJ (1.54f). Transmission Electron Microscope Fresh small-intestinal samples (~ 1–3 mm³) were excised and immersed within 1 min in ice-cold 2.5% glutaraldehyde fixative (Yuanye Bio-Technology, Shanghai, R20510). After overnight fixation at 4°C, tissues were post-fixed in 0.1 M phosphate buffer, dehydrated in graded ethanol, infiltrated with resin and polymerised at 60°C for 48 h. Ultra-thin sections (60–80 nm) were cut, stained with uranyl acetate and lead citrate, and examined using a TEM(Hitachi, HT7800). Measurement of MMP, ROS and superoxide dismutase (SOD) The MMP, ROS and SOD of fresh small-intestinal samples were detected using the Mitochondrial Membrane Potential Assay Kit with JC-1 (Beyotime, C2006), the Tissue ROS Test Kit (DHE) (Bjbalb, HR8821), and the SOD Assay Kit (Solarbio, BC0170).A multimode microplate reader (BioTek, Synergy LX, 23101208) was used. When the mitochondrial membrane potential is high, JC-1 accumulates in the mitochondrial matrix to form polymers (J-aggregates) that emit red fluorescence; when the mitochondrial membrane potential is low, JC-1 cannot accumulate in the mitochondrial matrix and exists as monomers, which emit green fluorescence. The level of ROS in tissues can be determined by detecting the fluorescence of DHE products. SOD is a metalloenzyme widely present in organisms, serving as an important scavenger of oxygen free radicals. It can catalyze the dismutation of superoxide anions to generate H₂O₂ and O₂. SOD is not only a superoxide anion-scavenging enzyme but also a major H₂O₂-generating enzyme, playing a crucial role in the biological antioxidant system. Mitochondria-targeted metabolomics(P650) Small-intestinal samples were rinsed in PBS, snap-frozen in liquid nitrogen, and analysed by targeted mitochondrial metabolomics (P650 panel, Panomix Biomedical Technology, Suzhou). The panel quantifies > 600 metabolites, including carbohydrates, organic acids, amino acids, bile acids, indoles, purine nucleotides and lipids. Frozen tissues were thawed at 4°C, mixed with cold methanol/acetonitrile/water (2:2:1, v/v/v), vortexed, sonicated on ice for 30 min, held at − 20°C for 10 min, and centrifuged (14 000 g, 4°C, 20 min). Supernatants were vacuum-dried and reconstituted in 100 µL acetonitrile/water (1:1, v/v), vortexed and re-centrifuged (14 000 g, 4°C, 15 min). Metabolites were separated on an ultra-high-performance liquid chromatograph coupled to a triple-quadrupole mass spectrometer (UHPLC-QTRAP MS). Peak areas were extracted with MultiQuant/Analyst; concentrations were calculated against internal-standard calibration curves. Mitochondria-targeted energy metabolomics(Kit 100) The Kit-100 panel quantifies 100 mitochondrial energy metabolites, covering glycolysis (EMP), the pentose phosphate pathway (PPP), the tricarboxylic acid (TCA) cycle, and selected intermediates from fatty-acid oxidation and amino-acid transamination. Intestinal tissue was homogenised in extraction buffer, freeze-ground and centrifuged; the supernatant was diluted four-fold, spiked with internal standards, vortex-mixed and dried. Samples were reconstituted in either 50% acetonitrile/water (alkaline loading) or 0.1% formic acid / 40% methanol/water (acidic loading), vortexed and centrifuged (12000 rpm, 4°C, 10 min). The resulting supernatants were transferred to LC-MS vials. Data were acquired on a SCIEX OS platform and processed for qualitative and quantitative analysis. Dimensionality reduction (PCA, PLS-DA and OPLS-DA) was performed with the R ropls package. Between-group comparisons used two-sample t-tests or Mann–Whitney–Wilcoxon tests; three or more groups were analysed by one-way ANOVA or Kruskal–Wallis tests. Pathway annotation employed the KEGG Mapper tool. Statistical analysis Statistical analyses were performed using Prism version 8.4.0 (GraphPad Software). All data are expressed as the mean ± standard error of the mean (SEM). Comparisons among FLASH-RT, CONV-RT and Control groups were performed using one-way analysis of variance (ANOVA), while two groups using unpaired t-test .Survival analysis was depicted using Kaplan–Meier curves and assessed with log-rank test analysis. A p value of < 0.05 was considered statistically significant. Results Dosimetry Irradiation parameters are listed in Supplementary Tables 1 and 2. The CHEx-FLASH system delivered 200 Gy/s for FLASH-RT and 0.185 Gy/s for CONV-RT. Because both modalities used the same linac, the prescribed dose was identical (FLASH-RT: 12.01 ± 0.12 Gy; CONV-RT: 12.03 ± 0.12 Gy). Lateral and longitudinal dose profiles measured with EBT-XD films were uniform and symmetric (Fig. 1 f). The PDD curve is presented in Fig. 1 g, where the blue box represents the dose distribution within the range of the abdominal depth of the mice. FLASH-RT enhanced survival rates and mitigates myelosuppression. To compare the toxicity and mortality under a whole abdominal irradiation dose of 12 Gy, mice were subjected to irradiation in the FLASH-RT group, CONV-RT group, and Control group. Peripheral blood cell tests were performed at 3 days post-irradiation (n = 5), and survival rate and body weight changes within 15 days were evaluated (n = 10). The survival rates of the FLASH-RT group, CONV-RT group, and Control group were 100% (10/10), 80% (8/10), and 30% (3/10) respectively, with significant differences among the three groups (Fig. 2 a, P < 0.001). Normalized body weight rose steadily in Controls, declined transiently in both irradiated groups. Specifically, the FLASH-RT group decreased within 5 days after irradiation and then increased, whereas the body weight of the CONV-RT group decreased within 8 days after irradiation and then increased, with the increasing trend being less pronounced than that of the FLASH-RT group (Fig. 2 b, P < 0.0001). Blood analysis (Fig. 2 c) revealed marked WBC and LYM in both irradiated groups, and WBC counts were lower in CONV-RT (0.39 ± 0.09 × 10⁹/L) than in FLASH-RT (0.88 ± 0.21 × 10⁹/L). NEU % and EOS % were also higher in CONV-RT than in FLASH-RT mice ( P 0.05). FLASH-RT alleviated radiation-induced intestinal injury. At 72 hours post-irradiation, we dissected the abdominal cavity of mice in each group (Fig. 2 d). Macroscopically, the intestines in the CONV-RT group showed obvious edema and dilation, while the FLASH-RT group was milder. Further H&E staining of intestinal (Fig. 3 a) revealed that the CONV-RT group had more extensive intestinal structural disorder, villous atrophy, and epithelial damage compared with the FLASH-RT group. Quantitative histology (Fig. 3 b) confirmed that villus height in FLASH-RT (335.99 ± 45.81 µm) matched that of Controls (343.62 ± 36.20 µm) and exceeded the CONV-RT value ( P < 0.01). Crypt density (per 50 µm) was highest in Controls, intermediate in FLASH-RT and lowest in CONV-RT ( P < 0.01), while the intestinal injury score was correspondingly lowest in FLASH-RT ( P < 0.01). IHC demonstrated that proliferative activity (Ki-67) in FLASH-RT was comparable to Controls and significantly higher than in CONV-RT ( P 0.05; Figs. 3 e, f). Cleaved caspase-3 and TUNEL signals were low and did not differ among the three groups ( P > 0.05; Figs. 3 g–j), indicating minimal apoptosis. FLASH-RT mitigated mitochondrial damage and reduces reactive oxygen species (ROS) levels in intestinal tissues. We observed the microstructure of intestinal tissues in the three groups at 72 hours post-irradiation using TEM (Fig. 4 a). The results showed that in the CONV-RT group, mitochondria were extensively abnormally shaped, with mild swelling, dissolved and pale matrix, dilated cristae or vacuolar changes, and local damage to the membrane structure. In contrast, mitochondria in the FLASH-RT group were similar to those in the Control group, with no obvious abnormal changes. The mitochondrial membrane potential in the CONV-RT group was significantly lower than that in the FLASH-RT group and the Control group (Fig. 4 b). Additionally, CONV-RT induced more ROS, while there was no significant difference between the FLASH-RT group and the Control group (Fig. 4 c). The levels of Parkin, PINK1, and LC3 in the FLASH-RT group were lower than those in the CONV-RT group ( P < 0.05). The levels of Parkin and PINK1 in the FLASH-RT group were higher than those in the Control group, while there was no significant difference in LC3 levels (Figs. 4 d and 4 e). These findings indicate that both FLASH-RT and CONV-RT can activate mitophagy, but the latter is more pronounced. FLASH-RT may confered intestinal protection by enhancing antioxidant defenses against oxidative damage. Given the differences in mitochondrial damage between FLASH-RT and CONV-RT, we performed mitochondria-targeted metabolomics. A total of 538 metabolites were identified in the P650 project, and their classification based on chemical taxonomy is detailed in Supplementary Fig. 1. PCA was conducted on all samples and quality control (QC) samples using the quantitative results of the identified metabolites. The experimental results (Fig. 5 a) showed that the samples were closely clustered, indicating good reproducibility of the experiment. We compared the differential metabolites among the groups (Fig. 5 b, P < 0.05) and found that there were 106 differential metabolites between the CONV-RT group and the Control group, and 106 differential metabolites between the FLASH-RT group and the Control group. In the comparison between the FLASH-RT group and the CONV-RT group, a total of 8 differentially expressed metabolites were identified, among which 6 were upregulated and 2 were downregulated. We intuitively displayed the fold changes of the significant differential metabolites identified in the two groups using a bar chart (Fig. 5 c). Further hierarchical cluster analysis was performed on each group of samples to form a cluster tree showing the similarity between samples, and the results are shown in Fig. 5 d, where samples clustered in the same group have higher similarity. The 6 upregulated differential metabolites in the FLASH-RT group were adenosine diphosphate ribose (ADP-ribose), aspartylphenylalanine, betaine, gamma-glutamyl-phenylalanine, glycyl-phenylalanine, and hypotaurine; the 2 downregulated differential metabolites were apocholic acid and tauro-α-muricholic acid (α-TMCA), with their quantitative analysis results shown in Fig. 5 e. KEGG pathway enrichment analysis of the list of differential metabolites using MetaboAnalyst showed (Fig. 5 f) that the FLASH group was enriched in 2 pathways, namely glycine, serine and threonine metabolism, and taurine and hypotaurine metabolism, which are mainly related to the regulation of mitochondrial antioxidant function. In addition, we detected 100 substances targeting mitochondrial energy metabolism. The PCA score plot of all samples showed obvious clustering among the groups, indicating good reproducibility of the analysis (Fig. 6 a). Further OPLS-DA showed significant separation between the Control group, FLASH-RT group, and CONV-RT group (Fig. 6 a). Figure 6 b shows the Z-score plot of all metabolites; the closer to the right, the higher the relative content of the current metabolite in the sample, and the closer to the left, the lower the content of the current metabolite. Quantitative analysis revealed that malonic acid in the CONV-RT group was significantly higher than that in the FLASH-RT group (Fig. 6 c). KEGG pathway enrichment analysis of the metabolites was performed, and the network diagram results (Fig. 6 d) showed that the FLASH group was most enriched in 5 pathways, namely central carbon metabolism in cancer, protein digestion and absorption, aminoacyl-tRNA biosynthesis, glucagon signaling pathway, and alanine, aspartate and glutamate metabolism, which are also related to mitochondrial oxidative stress function. Furthermore, the expression level of the antioxidant protein NRF2 in intestinal tissues at 72 hours post-irradiation was determined by IHC. We found that the Control group had the highest level, and the FLASH-RT group was also significantly higher than the CONV-RT group ( P < 0.01). In addition, the SOD content in intestinal tissues of the FLASH-RT group (4.17 ± 1.05 U/mg prot) was significantly lower than that of the CONV-RT group (8.17 ± 1.83 U/mg prot). These results suggest that FLASH-RT can activate mitochondrial antioxidant function to cope with radiation damage. Discussion Due to its biological function of protecting normal tissues, FLASH-RT is expected to become a disruptive radiotherapy modality, but its underlying biological mechanisms remain to be explored. In this study, we utilized the CHEx-FLASH device, focusing on confirming the protective effect of FLASH-RT on normal intestinal tissues in mice. For the first time, we observed the phenomenon of alleviated intestinal mitochondrial damage, and further analyzed and verified the differential mitochondrial metabolites between the FLASH-RT and CONV-RT groups, revealing that FLASH-RT may achieve intestinal protection by enhancing mitochondrial antioxidant function. We investigated mortality, body weight change curves, acute hematological toxicity, and intestinal damage following 12Gy whole abdominal irradiation. The results of this study showed that compared with CONV-RT, FLASH-RT significantly improved survival and body weight gain, which is consistent with previous studies on intestinal FLASH irradiation with protons or electrons [ 13 , 19 ]. We also observed that FLASH-RT could reduce myelosuppression, which has been reported in a study by Zhu H. et al. [ 20 ], who further found that CONV-RT significantly reduced WBC and LYM counts, with a more pronounced decrease at 48h post-irradiation compared to 3h. We found that NEU% increased significantly after CONV-RT, which may be due to the relative increase in the proportion of neutrophils caused by lymphopenia, or inflammatory responses induced by intestinal tissue damage after radiotherapy. This is supported by our abdominal dissection of mice at 72h post-irradiation, where we observed obvious intestinal edema and dilation in CONV-RT mice, while no significant swelling was noted in the intestines of FLASH-RT and Control group mice. Histopathological examination of intestinal tissues revealed that FLASH-RT alleviated post-radiation intestinal damage, as evidenced by lower intestinal injury scores, and significantly better intestinal villus height, number of intestinal crypts, and intestinal cell proliferation capacity compared to CONV-RT. This confirms that the CHEx-FLASH device exhibits a significant intestinal protective effect, consistent with the abdominal irradiation effects of other electrons, protons, or X-rays [ 13 , 14 , 21 – 23 ]. In addition, we found no statistically significant difference in γH2AX expression levels between FLASH-RT and CONV-RT (P > 0.05), indicating that the degree of DNA double-strand damage was comparable between the two groups. However, since our detection time point was 72h post-irradiation, it is possible that DNA damage may have been repaired. Zhu H. et al. [ 20 ] found that γH2AX levels were relatively high at 3h post-irradiation and decreased at 48h, but γH2AX levels in CONV-RT remained higher than those in FLASH-RT. However, comparative studies on DNA damage are inconsistent: Shi X. et al. [ 23 ] irradiated intestinal organoids and human intestinal epithelial cells HIEC-6 with X-ray FLASH-RT and CONV-RT, and found that the expression levels of γH2AX and 53BP1 were almost identical in both groups. In contrast, other researchers observed that acute irradiation with electrons in human lung cells [ 24 ] and intestinal crypt stem cells [ 13 ] resulted in lower levels of 53BP1 or γH2AX in FLASH-RT compared to CONV-RT. In general, discrepancies in studies on DNA damage may be related to factors such as time points, dose rate levels, and differences in radiation sources. Additionally, there are still inconsistencies regarding whether cell apoptosis is induced after irradiation with the two dose rates, so the mechanism underlying the protective effect of FLASH-RT on normal organs requires further exploration. Currently, researchers have proposed several hypotheses [ 6 , 25 ], including oxygen depletion, free radical-free radical interaction, and immune factors, but there are conflicting positive and negative research results reported. Surprisingly, in recent years, a hypothesis has emerged that mitochondria are involved in the protective mechanism of FLASH-RT. A research team irradiated normal human lung fibroblasts (IMR90) with protons and found that FLASH-RT (100 Gy/s) could alleviate mitochondrial damage, including morphological and functional changes such as mitochondrial membrane potential, mtDNA copy number, and ROS [ 10 ], while this phenomenon was not observed in cancer cells. A pre-published article also reported that compared with low-dose-rate electron irradiation (0.36 Gy/s), FLASH electron irradiation at 61 or 610 Gy/s significantly enhanced cytochrome c release from mitochondria in human breast cancer cells MCF-10A, thereby triggering massive caspase activation and inhibiting cytoplasmic mtDNA accumulation and interferon-β(IFN-β) secretion [ 26 ]. Another in vivo study on the protective effect of electron FLASH-RT on the esophagus showed via TEM that CONV-RT induced mitochondrial swelling, blurred structure, disappearance or break of cristae, reduced matrix electron density, and vacuolation, while mitochondrial swelling and structural blurring were less obvious in the FLASH-RT group. Proteomic analysis revealed that CONV-RT downregulated the expression of key proteins involved in the TCA cycle and oxidative phosphorylation in mice [ 27 ]. In our study, we observed changes in mitochondrial structure following high-energy X-ray irradiation for the first time. TEM also revealed similar obvious characteristics of intestinal mitochondrial damage in the CONV-RT group, while mitochondrial damage in the FLASH-RT group was mild. Meanwhile, compared with FLASH-RT, CONV-RT disrupted mitochondrial membrane potential and increased ROS, confirming functional impairment. In addition, CONV-RT exhibited higher expression of mitophagy-related proteins Parkin and PINK1 than FLASH-RT, indicating that mitophagy is activated to remove dysfunctional mitochondria after mitochondrial damage [ 28 ]. Mitochondria are small double-membraned organelles in the cytoplasm of eukaryotic cells, coupled with the electron transport chain (ETC) and oxidative phosphorylation (OXPHOS) through the tricarboxylic acid (TCA) cycle, and are known as the "energy factories" of the cell. Using targeted metabolomics, we performed qualitative and quantitative analysis of mitochondria-related metabolites and found that FLASH-RT expressed lower levels of malonic acid (an endogenous competitive inhibitor of succinate dehydrogenase), which is a marker of mitochondrial damage [ 29 ]. Malonate causes rapid collapse of MMP and ROS production, thereby overwhelming mitochondrial antioxidant capacity and leading to mitochondrial swelling [ 30 ]. We also conducted pathway enrichment analysis of other differential mitochondrial metabolites, which mainly enriched the "Glycine, serine and threonine metabolism" and "Taurine and hypotaurine metabolism" pathways. From previous literature, we know that "Glycine/serine/threonine metabolism" can affect carbohydrate patterns and energy utilization efficiency, and ultimately delay intervertebral disc degeneration through antioxidant effects [ 31 ], while the "serine/glycine pathway" can maintain cell function, oxidative stress tolerance, and survival [ 32 ]. "Taurine and hypotaurine metabolism" can also protect mitochondria from oxidative and sub-oxidative stress [ 33 , 34 ]. Metabolite analysis indicated that FLASH-RT enhances antioxidant capacity, so we further verified that FLASH-RT expressed higher levels of NRF2 and SOD than CONV-RT, which are antioxidant genes produced in response to ionizing radiation-induced oxidative stress to protect cells from damage [ 35 , 36 ]. In summary, we propose the following hypothesis (Fig. 7 ): high-energy X-ray FLASH-RT can reduce ROS production, mitigate mitochondrial damage, and promote the metabolism of mitochondrial antioxidant substances, thereby enhancing antioxidant capacity, reducing intestinal cell damage, and achieving intestinal protective effects. Of course, this study has some limitations. For example, the detection of ROS, myelosuppression, and γH2AX breakage indices lacks dynamic detection at multiple time points, and multi-factor exploration of FLASH-RT dose parameters such as dose rate and fractionation methods is not conducted. However, we innovatively proposed that FLASH-RT achieves normal tissue protection by enhancing mitochondrial antioxidant function, which provides a favorable research basis for further mechanistic studies by researchers. In addition, for research institutions lacking FLASH-RT equipment, combining CONV-RT with antioxidants may be a potential approach to reduce radiotherapy-induced tissue damage in the future. Conclusion CHEx-FLASH achieves UHDR irradiation and alleviates radiation-induced intestinal injury. The protective effect of FLASH-RT on intestinal tissues may be mediated by mitigating mitochondrial damage and enhancing antioxidant pathways through improved mitochondrial energy metabolism. Abbreviations CHEx-FLASH Prototype of Compact High-energy X-ray Flash Radiotherapy Equipment UHDR ultra-high dose rate FLASH-RT FLASH Radiotherapy CONV-RT Conventional Dose Rate Radiotherapy SSD Source Surface Distance PDD Percent Depth Dose TEM Transmission Electron Microscope MMP Mitochondrial Membrane Potential ROS Reactive Oxygen Species SOD Superoxide Dismutase. Declarations Ethical approval All mouse experiments adhered to the approved guidelines of the Ethics Committee of Mianyang Central Hospital (approval number: S20240203-02). Consent for publication Not applicable. Competing interests The authors declare that they have no competing interests. Funding: This work was financially supported by the Projects of National Natural Science Foundation of China (Grant NO. U2330122) and the General Program of the Sichuan Natural Science Foundation (Grant No. 2023NSFSC0710). Author Contribution X. Hao, H. Du, D. Wang, M. Tang, H. Zhang and Y. Zhu conducted the animal experiments and biological analyses. X. Hao, B. Lin, W. Wu, Y. Yang and X. D were responsible for irradiation beam control and performed the physics-related analyses. B. Lin, D. Wang,Y. Zhang and X. Du contributed to the experimental design and provided critical revisions to the manuscript. X. Hao drafted the main manuscript text and prepared all figures. All authors reviewed and approved the final version of the manuscript. Data Availability All data generated or analysed during this study are included in this published article and its supplementary information files References Bray F, Laversanne M, Sung H, Ferlay J, Siegel RL, Soerjomataram I, et al. Global cancer statistics 2022: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin. 2024;74(3):229–63. 10.3322/caac.21834 . Lu Q, Liang Y, Tian S, Jin J, Zhao Y, Fan H. Radiation-Induced Intestinal Injury: Injury Mechanism and Potential Treatment Strategies. Toxics. 2023;11(12):1011. 10.3390/toxics11121011 . Yang XF, Zheng MY, An LY, Sun JM, Hei QW, Ji YH, et al. Quality evaluation of guidelines for the diagnosis and treatment of radiation enteritis. Radiat Oncol. 2023;18(1):14. 10.1186/s13014-023-02204-9 . Araujo IK, Muñoz-Guglielmetti D, Mollà M. Radiation-induced damage in the lower gastrointestinal tract: Clinical presentation, diagnostic tests and treatment options. Best Pract Res Clin Gastroenterol. 2020;48–49:101707. 10.1016/j.bpg.2020.101707 . Favaudon V, Caplier L, Monceau V, Pouzoulet F, Sayarath M, Fouillade C, et al. Ultrahigh dose-rate FLASH irradiation increases the differential response between normal and tumor tissue in mice. Sci Transl Med. 2014;6(245):245ra93. 10.1126/scitranslmed.3008973 . Lin B, Gao F, Yang Y, Wu D, Zhang Y, Feng G, et al. FLASH Radiotherapy: History and Future. Front Oncol. 2021;11:644400. 10.3389/fonc.2021.644400 . Spitz DR, Buettner GR, Petronek MS, St-Aubin JJ, Flynn RT, Waldron TJ, et al. An integrated physico-chemical approach for explaining the differential impact of FLASH versus conventional dose rate irradiation on cancer and normal tissue responses. Radiother Oncol. 2019;139:23–7. 10.1016/j.radonc.2019.03.028 . Ohsawa D, Hiroyama Y, Kobayashi A, Kusumoto T, Kitamura H, Hojo S, et al. DNA strand break induction of aqueous plasmid DNA exposed to 30 MeV protons at ultra-high dose rate. J Radiat Res. 2022;63(2):255–60. 10.1093/jrr/rrab114 . Averbeck D, Rodriguez-Lafrasse C. Role of Mitochondria in Radiation Responses: Epigenetic, Metabolic, and Signaling Impacts. Int J Mol Sci. 2021;22(20):11047. 10.3390/ijms222011047 . Guo Z, Buonanno M, Harken A, Zhou G, Hei TK. Mitochondrial Damage Response and Fate of Normal Cells Exposed to FLASH Irradiation with Protons. Radiat Res. 2022;197(6):569–82. 10.1667/RADE-21-00181.1 . Montay-Gruel P, Acharya MM, Gonçalves Jorge P, Petit B, Petridis IG, Fuchs P, et al. Hypofractionated FLASH-RT as an Effective Treatment against Glioblastoma that Reduces Neurocognitive Side Effects in Mice. Clin Cancer Res. 2021;27(3):775–84. 10.1158/1078-0432.CCR-20-0894 . Gao F, Yang Y, Zhu H, Wang J, Xiao D, Zhou Z, et al. First demonstration of the FLASH effect with ultrahigh dose rate high-energy X-rays. Radiother Oncol. 2022;166:44–50. 10.1016/j.radonc.2021.11.004 . Levy K, Natarajan S, Wang J, Chow S, Eggold JT, Loo PE, et al. Abdominal FLASH irradiation reduces radiation-induced gastrointestinal toxicity for the treatment of ovarian cancer in mice. Sci Rep. 2020;10(1):21600. 10.1038/s41598-020-78017-7 . Zhu H, Xie D, Yang Y, Huang S, Gao X, Peng Y, et al. Radioprotective effect of X-ray abdominal FLASH irradiation: Adaptation to oxidative damage and inflammatory response may be benefiting factors. Med Phys. 2022;49(7):4812–22. 10.1002/mp.15680 . Soto LA, Casey KM, Wang J, Blaney A, Manjappa R, Breitkreutz D, Skinner L, et al. FLASH Irradiation Results in Reduced Severe Skin Toxicity Compared to Conventional-Dose-Rate Irradiation. Radiat Res. 2020;194(6):618–24. 10.1667/RADE-20-00090 . Verginadis II, Velalopoulou A, Kim MM, Kim K, Paraskevaidis I, Bell B et al. FLASH proton reirradiation, with or without hypofractionation, mitigates chronic toxicity in the normal murine intestine, skin, and bone. bioRxiv [Preprint]. 2024.07.08.602528. 10.1101/2024.07.08.602528 Vozenin MC, De Fornel P, Petersson K, Favaudon V, Jaccard M, Germond JF, et al. The Advantage of FLASH Radiotherapy Confirmed in Mini-pig and Cat-cancer Patients. Clin Cancer Res. 2019;25(1):35–42. 10.1158/1078-0432.CCR-17-3375 . Bourhis J, Sozzi WJ, Jorge PG, Gaide O, Bailat C, Duclos F, et al. Treatment of a first patient with FLASH-radiotherapy. Radiother Oncol. 2019;139:18–22. 10.1016/j.radonc.2019.06.019 . Lim TL, Morral C, Verginadis II, Kim K, Luo L, Foley CJ et al. Early Inflammation and Interferon Signaling Direct Enhanced Intestinal Crypt Regeneration after Proton FLASH Radiotherapy. bioRxiv [Preprint]. 2024.08.16.608284. 10.1101/2024.08.16.608284 Zhu H, Liu S, Qiu J, Hu A, Zhou W, Wang J, et al. Instantaneous dose rate as a crucial factor in reducing mortality and normal tissue toxicities in murine total-body irradiation: a comparative study of dose rate combinations. Mol Med. 2025;31(1):79. 10.1186/s10020-025-01135-3 . Eggold JT, Chow S, Melemenidis S, Wang J, Natarajan S, Loo PE, et al. Abdominopelvic FLASH Irradiation Improves PD-1 Immune Checkpoint Inhibition in Preclinical Models of Ovarian Cancer. Mol Cancer Ther. 2022;21(2):371–81. 10.1158/1535-7163.MCT-21-0358 . Valdés Zayas A, Kumari N, Liu K, Neill D, Delahoussaye A, Gonçalves Jorge P, et al. Independent Reproduction of the FLASH Effect on the Gastrointestinal Tract: A Multi-Institutional Comparative Study. Cancers (Basel). 2023;15(7):2121. 10.3390/cancers15072121 . Shi X, Yang Y, Zhang W, Wang J, Xiao D, Ren H, et al. FLASH X-ray spares intestinal crypts from pyroptosis initiated by cGAS-STING activation upon radioimmunotherapy. Proc Natl Acad Sci U S A. 2022;119(43):e2208506119. 10.1073/pnas.2208506119 . Fouillade C, Curras-Alonso S, Giuranno L, Quelennec E, Heinrich S, Bonnet-Boissinot S, et al. FLASH Irradiation Spares Lung Progenitor Cells and Limits the Incidence of Radio-induced Senescence. Clin Cancer Res. 2020;26(6):1497–506. 10.1158/1078-0432.CCR-19-1440 . Bogaerts E, Macaeva E, Isebaert S, Haustermans K. Potential Molecular Mechanisms behind the Ultra-High Dose Rate FLASH Effect. Int J Mol Sci. 2022;23(20):12109. 10.3390/ijms232012109 . Lv J, Sun J, Luo Y, Liu J, Wu D, Fang Y et al. FLASH Irradiation Regulates IFN-β induction by mtDNA via Cytochrome c Leakage.bioRxiv 2024.04.10.588811; doi: https://doi.org/10.1101/2024.04.10.588811 Ren W, Hou L, Zhang K, Chen H, Feng X, Jiang Z, et al. The sparing effect of ultra-high dose rate irradiation on the esophagus. Front Oncol. 2024;14:1442627. 10.3389/fonc.2024.1442627 . Scheibye-Knudsen M, Fang EF, Croteau DL, Wilson DM 3rd, Bohr VA. Protecting the mitochondrial powerhouse. Trends Cell Biol. 2015;25(3):158–70. 10.1016/j.tcb.2014.11.002 . Yuan C, Zheng L, Zhao Y. Protective effect of 3-n-butylphthalide against intrastriatal injection of malonic acid-induced neurotoxicity and biochemical alteration in rats. Biomed Pharmacother. 2022;155:113664. 10.1016/j.biopha.2022.113664 . Fernandez-Gomez FJ, Galindo MF, Gómez-Lázaro M, Yuste VJ, Comella JX, Aguirre N, et al. Malonate induces cell death via mitochondrial potential collapse and delayed swelling through an ROS-dependent pathway. Br J Pharmacol. 2005;144(4):528–37. 10.1038/sj.bjp.0706069 . Wu X, Liu C, Yang S, Shen N, Wang Y, Zhu Y, et al. Glycine-Serine-Threonine Metabolic Axis Delays Intervertebral Disc Degeneration through Antioxidant Effects: An Imaging and Metabonomics Study. Oxid Med Cell Longev. 2021;2021:5579736. 10.1155/2021/5579736 . Ost M, Keipert S, van Schothorst EM, Donner V, van der Stelt I, Kipp AP, Petzke KJ, et al. Muscle mitohormesis promotes cellular survival via serine/glycine pathway flux. FASEB J. 2015;29(4):1314–28. 10.1096/fj.14-261503 . Seneff S, Kyriakopoulos AM. Taurine prevents mitochondrial dysfunction and protects mitochondria from reactive oxygen species and deuterium toxicity. Amino Acids. 2025;57(1):6. 10.1007/s00726-024-03440-3 . Baliou S, Adamaki M, Ioannou P, Pappa A, Panayiotidis MI, Spandidos DA, Christodoulou I, Kyriakopoulos AM, Zoumpourlis V. Protective role of taurine against oxidative stress (Review). Mol Med Rep. 2021;24(2):605. 10.3892/mmr.2021.12242 . Kaya H, Delibas N, Serteser M, Ulukaya E, Ozkaya O. The effect of melatonin on lipid peroxidation during radiotherapy in female rats. Strahlenther Onkol. 1999;175(6):285–8. 10.1007/BF02743581 . Liu S, Zhang HL, Li J, Ye ZP, Du T, Li LC, et al. Tubastatin A potently inhibits GPX4 activity to potentiate cancer radiotherapy through boosting ferroptosis. Redox Biol. 2023;62:102677. 10.1016/j.redox.2023.102677 . Additional Declarations No competing interests reported. Supplementary Files Supplementarymaterials.docx Cite Share Download PDF Status: Published Journal Publication published 28 Feb, 2026 Read the published version in Radiation Oncology → Version 1 posted Editorial decision: Revision requested 24 Nov, 2025 Reviews received at journal 24 Nov, 2025 Reviews received at journal 18 Nov, 2025 Reviewers agreed at journal 10 Nov, 2025 Reviews received at journal 06 Nov, 2025 Reviewers agreed at journal 27 Oct, 2025 Reviewers agreed at journal 27 Oct, 2025 Reviewers invited by journal 23 Oct, 2025 Editor assigned by journal 01 Oct, 2025 Submission checks completed at journal 29 Sep, 2025 First submitted to journal 27 Sep, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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16:24:33","extension":"html","order_by":19,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":136195,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7650752/v1/0b915777bf49a969e6fd01d4.html"},{"id":95225417,"identity":"03c210ee-0d48-4e34-a285-d6804cfeaddb","added_by":"auto","created_at":"2025-11-05 16:25:01","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":578821,"visible":true,"origin":"","legend":"\u003cp\u003eThe experimental setup diagrams, parameters, and design for FLASH-RT and CONV-RT. (a) Schematic diagram of the experimental setup. (b) Actual in vivo irradiation scenario: the mouse was placed on a PMMA plate, covered with a 1-cm-thick compensator for dose accumulation, and an EBTXD film was positioned between the compensator and the mouse's dorsal surface. (c) The irradiation field was a full abdominal field of 4 cm × 4 cm, with the upper boundary set below the xiphoid process of the mouse and the lower boundary at the upper end of the anus. (d) EBTXD film was used to assess the dose distribution. (e) EBTXD film was applied to evaluate the Percentage Depth Dose (PDD). (f) Horizontal and vertical dose curves. (g) PDD curve, where the area within the blue dashed box reflects the dose at a depth of 1 cm in the mouse abdomen. (h) Schematic diagram of the research design.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-7650752/v1/c49b21c9d0db678e671e7eb1.png"},{"id":95224647,"identity":"fefd93b2-4e0d-4a69-bbba-90a59bf0d3a8","added_by":"auto","created_at":"2025-11-05 16:24:06","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":691098,"visible":true,"origin":"","legend":"\u003cp\u003eSurvival status, body weight, and hematological results post irradiation. (a) Survival curves within 15 days post irradiation. (b) Standardized body weight change graphs within 15 days after irradiation. (c) Results of blood routine tests at 72 hours post irradiation. (d) In vivo photographs of the full abdomen of mice at 72 hours post irradiation. *\u003cem\u003eP\u003c/em\u003e≤0.05, **\u003cem\u003eP\u003c/em\u003e≤0.01, ***\u003cem\u003eP\u003c/em\u003e≤0.001, ****\u003cem\u003eP\u003c/em\u003e≤0.0001, ns: no significance.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-7650752/v1/65605360622f205da14c4244.png"},{"id":95123393,"identity":"7e7a34ae-d97f-4d89-bb81-9d20f4b7aded","added_by":"auto","created_at":"2025-11-04 14:25:16","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1060951,"visible":true,"origin":"","legend":"\u003cp\u003ePathological staining results of intestinal tissues at 72 h post irradiation. (a) Representative H\u0026amp;E staining images (scale bars, 100×, 250 μm; 300×, 50 μm). (b) Comparative analysis of intestinal villus length, number of intestinal crypts, and intestinal tissue damage scores. (c) Representative immunohistochemical (IHC) stained Ki-67 image (scale bars, 200×, 100 μm). (d) Positive Ki-67 area analysis. (e) Representative IHC stained γH2AX image (scale bars, 200×, 100 μm). (f) Positive γH2AX area analysis. (g) Representative IHC stained cleaved-caspase3 (c-caspase3) image (scale bars, 200×, 100 μm). (h) Positive c-caspase3 area analysis. (i) Representative IHC stained Tunel image (scale bars, 200×, 50 μm). (j) Positive Tunel area analysis. *\u003cem\u003eP\u003c/em\u003e≤0.05, **\u003cem\u003eP\u003c/em\u003e≤0.01, ***\u003cem\u003eP\u003c/em\u003e≤0.001, ****\u003cem\u003eP\u003c/em\u003e≤0.0001, ns: no significance.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-7650752/v1/1119a37b14d992c63e743d6b.png"},{"id":95225111,"identity":"1265450a-d7ca-49f5-9a62-3d8cfeba3dcf","added_by":"auto","created_at":"2025-11-05 16:24:36","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1019893,"visible":true,"origin":"","legend":"\u003cp\u003eDetection of mitochondrial-related indices in intestinal tissues post irradiation. (a) Representative transmission electron microscopy (TEM) images of intestinal tissue mitochondria (scale bars, 100000×, 1 μm). (b) Quantitative analysis of mitochondrial membrane potential levels in intestinal tissues. (c) Quantitative analysis of reactive oxygen species (ROS) levels in intestinal tissues. (d) Representative IHC stained images of Parkin, PINK1, and LC3 (scale bars, 200×, 100 μm). (e) Positive area analysis of Parkin, PINK1, and LC3. *\u003cem\u003eP\u003c/em\u003e≤0.05, **\u003cem\u003eP\u003c/em\u003e≤0.01, ***\u003cem\u003eP\u003c/em\u003e≤0.001, ****\u003cem\u003eP\u003c/em\u003e≤0.0001, ns: no significance.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-7650752/v1/57667109e30c278245645ed9.png"},{"id":95123371,"identity":"6d2a0ce4-6361-45d8-80f1-341c9da0ad0b","added_by":"auto","created_at":"2025-11-04 14:25:15","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":570842,"visible":true,"origin":"","legend":"\u003cp\u003eMitochondria-targeted metabolism analysis of intestinal tissues post irradiation (P650). (a) Principal Component Analysis (PCA) of the overall samples. (b) Volcano plot of differentially expressed energy metabolites between different groups. (c) Linear Discriminant Analysis Effect Size (LEfSe) showing substances with significant differences between the FLASH-RT and CONV-RT groups. (d) Hierarchical clustering heatmap of significantly different metabolites between the FLASH-RT and CONV-RT groups. (e) Quantitative analysis of substances with significant differences between the FLASH-RT and CONV-RT groups. (f) Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment pathway map (bubble chart) for the FLASH-RT vs. CONV-RT groups. *\u003cem\u003eP\u003c/em\u003e≤0.05, **\u003cem\u003eP\u003c/em\u003e≤0.01, ***\u003cem\u003eP\u003c/em\u003e≤0.001, ****\u003cem\u003eP\u003c/em\u003e≤0.0001, ns: no significance.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-7650752/v1/cbe6b7c187d419ffe71597b6.png"},{"id":95123372,"identity":"23ed7858-421d-4d6d-96bf-e7c5378ac913","added_by":"auto","created_at":"2025-11-04 14:25:15","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":617032,"visible":true,"origin":"","legend":"\u003cp\u003eMitochondria-targeted energy metabolism analysis of intestinal tissues post irradiation (Kit 100). (a) PCA and Orthogonal Partial Least Squares Discriminant Analysis (OPLS-DA) score plots for each group. (b) Z-score plot comparing the CONV-RT and Control groups. The X-axis represents the Z-score of the relative abundance of metabolites, and the Y-axis shows the metabolite names. The further to the right, the higher the relative abundance of the metabolite in the sample. (c) Box plot of malonic acid quantification in the CONV-RT and Control groups. (d) Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment pathway map (network diagram) for the FLASH-RT vs. CONV-RT groups. Green dots represent pathways, and other dots represent metabolites. The size of the pathway dots indicates the number of connected metabolites (larger dots signify more connections), and the color of metabolite dots represents the magnitude of the log2FC value. (e) Representative IHC stained NRF2 image (scale bars, 200×, 100 μm). (f) Positive NRF2 area analysis. (g) Quantitative analysis of SOD content. *\u003cem\u003eP \u003c/em\u003e≤ 0.05, **\u003cem\u003eP \u003c/em\u003e≤ 0.01, ***\u003cem\u003eP \u003c/em\u003e≤ 0.001\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-7650752/v1/442173f50326c86d4e8e4e52.png"},{"id":95123373,"identity":"85153098-7541-437d-8d70-454ce65e3a1f","added_by":"auto","created_at":"2025-11-04 14:25:15","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":415459,"visible":true,"origin":"","legend":"\u003cp\u003eResearch hypothesis diagram.\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-7650752/v1/a3ab67ce602e2a3177658a59.png"},{"id":103765677,"identity":"e138838c-492c-4dbd-843e-8dcdcb6b0a99","added_by":"auto","created_at":"2026-03-02 16:07:20","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5855515,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7650752/v1/1c9148f1-4a30-41a5-8c63-19fe4fed0115.pdf"},{"id":95225990,"identity":"9953ccd7-64ec-440c-932c-56787f2b58ae","added_by":"auto","created_at":"2025-11-05 16:25:56","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":193351,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarymaterials.docx","url":"https://assets-eu.researchsquare.com/files/rs-7650752/v1/f4c6d2d08a3f7321d499f10a.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Preliminary mechanistic exploration of mitochondrial function in intestinal protection mediated by high-energy X-ray FLASH radiotherapy","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe global incidence of cancer continues to rise, and it has become one of the main causes of human death, posing a serious threat to human health [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Concurrently, radiation-induced toxicity has emerged as a worldwide concern. Radiation enteritis\u0026mdash;an inflammatory disorder that arises when ionizing radiation injures the intestinal mucosa and adjacent tissues during treatment of pelvic, abdominal, or retroperitoneal malignancies (e.g., cervical, rectal, or prostate cancer), and it is one of the most frequent gastrointestinal complications of radiotherapy [\u003cspan additionalcitationids=\"CR3\" citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Achieving optimal tumour control while minimising intestinal toxicity has therefore become a central objective of contemporary radiotherapy.\u003c/p\u003e\u003cp\u003eUltra-high dose rate radiotherapy (UHDR, \u0026ge;\u0026thinsp;40 Gy/s), also known as FLASH radiotherapy (FLASH-RT), has attracted extensive attention in recent years due to its ability to retain tumor therapeutic effects while having a unique normal tissue protection effect [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e], and is expected to become a disruptive radiotherapy paradigm. Although some mechanistic hypotheses have been proposed, such as oxygen consumption, DNA damage repair, free radical-free radical interaction, and immune mediation [\u003cspan additionalcitationids=\"CR7\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e], there are still conflicting positive and negative research results, and the underlying biology remains incompletely understood.\u003c/p\u003e\u003cp\u003eHistorically, the biological effects of ionizing radiation were attributed primarily to nuclear DNA damage and its subsequent repair. However, current molecular biology studies have shown that the results of ionizing radiation exposure are highly dependent on the activation and regulation of other molecular components of organelles, which determine the cell's survival and proliferation ability. For example, mitochondria, as the \"energy factories\" of eukaryotic cells, play a particularly critical role in regulating cellular metabolism, energy supply, and radiation-induced signal transduction [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eSurprisingly, mitochondria have recently been implicated in the normal-tissue protection conferred by FLASH-RT. Guo Z et al. [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e] exposed normal human lung fibroblasts (IMR90) to 100 Gy/s proton irradiation and observed significant attenuation of mitochondrial injury\u0026mdash;manifest as preserved mitochondrial membrane potential (MMP), restored mitochondrial DNA (mtDNA) copy number and reduced reactive oxygen species (ROS)\u0026mdash;whereas malignant cells showed no such sparing. These findings open a new avenue for elucidating the mechanistic basis of FLASH-RT selectivity.\u003c/p\u003e\u003cp\u003eIn recent years, pre-clinical studies have consistently demonstrated FLASH-RT-mediated sparing of normal brain [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e], lung [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e], intestine [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e], skin [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e] and bone [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Validation has extended to large mammals [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e] and, more recently, to early-phase human trials [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Among the available beam modalities\u0026mdash;electrons, protons and high-energy photons\u0026mdash;electrons are restricted to superficial targets by their limited penetration; protons incur high facility costs; whereas high-energy X-rays combine deep penetration with minimal beam divergence and favourable economics, positioning them as the most clinically scalable option for FLASH-RT.\u003c/p\u003e\u003cp\u003eHowever, high-energy X-ray sources capable of achieving UHDR remain scarce worldwide. To better adapt to clinical practice, we have developed the Compact single High-energy X-ray Source FLASH-RT device(CHEx-FLASH), which is economical and small in size and can generate ultra-high dose rates (\u0026ge;\u0026thinsp;40 Gy/s). Based on this equipment, we verified the protective effect of FLASH-RT on normal intestinal tissues in mice, and conducted the first in vivo study to explore the underlying mitochondrial mechanisms, providing mechanistic insights for more adequate FLASH-RT research.\u003c/p\u003e"},{"header":"Methods and materials","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eIrradiation device and dosimetry monitoring\u003c/h2\u003e\u003cp\u003eThe irradiation device used in this study was a 10 MeV CHEx-FLASH (Zhongjiu Flash Medical Technology Co., Ltd.) with a source surface distance (SSD) of 50 cm. Two dose-rate modalities were compared on the same platform: UHDR (FLASH-RT, \u0026ge; 40 Gy s⁻\u0026sup1;) and conventional dose-rate (CONV-RT, 0.185 Gy/s). We used a current transformer (BCT) to monitor the beam current, and a diamond detector installed downstream of the main collimator to monitor the X-ray beam. Gafchromic\u0026trade; EBT-XD radiographic films (Ashland Inc., Covington, Kentucky, USA) were placed in solid water at isocentre, as previously described [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Figure\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea depicts the in vivo irradiation geometry; the experimental set-up is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eAfter being anesthetized with isoflurane gas (3\u0026ndash;4%, Isoflurane, R510-22-10), the mice were placed prone on a 0.5 cm custom polymethyl methacrylate (PMMA) platform; a 1 cm water-equivalent bolus was applied dorsally for build-up. The RGB values of the EBT-XD films were scanned 5 minutes post-irradiation using an Epson Expression 12000XL scanner (Seiko Epson Corporation, Japan), and analyzed using SNC patient software (Sun Nuclear Corporation, USA). Additionally, before the FLASH experiment, the EBT-XD films were also calibrated using a clinically applied 6 MV Elekta Precise linac (Elekta AB, Stockholm, Sweden).\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eAnimal model and euthanasia method\u003c/h3\u003e\n\u003cp\u003eFemale C57BL/6 mice aged 6\u0026ndash;8 weeks were purchased from SPF (Beijing) Biotechnology Co., Ltd. (Beijing, China) and housed at Mianyang Central Hospital (Mianyang, China). All animal experiments were conducted in strict accordance with ethical standards and approved by the Animal Ethics Committee of Mianyang Central Hospital (approval number: S20240203-02).\u003c/p\u003e\u003cp\u003ePrior to euthanasia, the experimental animals were anesthetized with isoflurane (R510-22-10) gas (Shenzhen RWD Life Science Co., Ltd.). This approach effectively prevents animals from experiencing pain, suffocation, or stress responses during euthanasia. Furthermore, isoflurane undergoes rapid metabolism, which minimizes interference of the anesthetic with subsequent experimental test results. During the anesthetic induction phase, the initial concentration of isoflurane was set at 4%\u0026ndash;5%. Adequate anesthetic depth was confirmed when the animals exhibited loss of corneal reflex and cessation of limb movements. Following this, procedures including irradiation and blood collection were performed, and euthanasia was ultimately achieved via cervical dislocation. The criteria for confirming death were loss of consciousness, respiratory arrest, and cardiac arrest.\u003c/p\u003e\n\u003ch3\u003ewhole-abdominal irradiation\u003c/h3\u003e\n\u003cp\u003eIn this study, mice were subjected to three treatments: FLASH-RT (12 Gy/1F), CONV-RT (12 Gy/1F), or Sham (0 Gy, control). Irradiations were delivered to a 4 cm \u0026times; 4 cm abdominal field extending from the xiphoid process to the upper anal margin. Hair depigmentation, evident several months later, delineated the irradiated region (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec). In addition, the irradiation dose distribution and percent depth dose (PDD) curves are shown in Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed-g. Figure\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eh presents the study design: at 72 hours after irradiation, whole blood and small intestinal tissues were collected (n\u0026thinsp;=\u0026thinsp;5), while the remaining mice (n\u0026thinsp;=\u0026thinsp;10) were observed twice weekly for survival and body weight changes during the acute phase (15 days). The experiment endpoints were defined as tumor body weight loss\u0026thinsp;\u0026gt;\u0026thinsp;40%, severe deterioration in overall health, or death.\u003c/p\u003e\n\u003ch3\u003eWhole blood count detection\u003c/h3\u003e\n\u003cp\u003eAt 72 hours after irradiation, mice were anesthetized with isoflurane gas, and the peripheral blood from the eyeballs was collected into EDTA-coated capillary tubes. A URIT-5160Vet auto hematology analyzer (URIT, Guilin, China) was used to determine the white blood cell count (WBC), hemoglobin (HGB), platelet count (PLT), lymphocyte count (LYM), neutrophil percentage (NEU%), and eosinophil percentage (EOS%).\u003c/p\u003e\n\u003ch3\u003eHematoxylin-eosin staining (H\u0026E staining)\u003c/h3\u003e\n\u003cp\u003eFollowing euthanasia, the abdominal cavity was opened and the intestine photographed in situ. The small bowel was then excised, rinsed in ice-cold phosphate-buffered saline(PBS), and fixed overnight in 4% universal fixative (Biosharp BL539A). Paraffin-embedded tissues were sectioned at 4\u0026ndash;5 \u0026micro;m and stained with H\u0026amp;E. Subsequently, digital images were acquired with a pathological section scanner (NanoZoomer S360, C13220-01, Hamamatsu Photonics K.K., Japan). Acute radiation-induced intestinal injury was quantified based on the number of intestinal crypts per unit area [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e], villus height, and intestinal injury pathological score [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. The semi-quantitative pathological score was determined according to the proportion of the damaged intestinal segment to the total length:0\u0026thinsp;=\u0026thinsp;normal; 1\u0026thinsp;=\u0026thinsp;\u0026lt;\u0026thinsp;1/3 segment affected; 2\u0026thinsp;=\u0026thinsp;1/3\u0026ndash;2/3 affected; 3\u0026thinsp;=\u0026thinsp;\u0026gt;\u0026thinsp;2/3 affected).\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eImmunohistochemical staining (IHC) and image analysis\u003c/h2\u003e\u003cp\u003eFollowing deparaffinization and rehydration, antigen retrieval was performed and sections were incubated with primary antibodies. After washing, species-appropriate secondary antibodies were applied, chromogenic or fluorescent detection was carried out, and nuclei were counterstained. Sections were dehydrated, cleared and coverslipped before digital imaging with either a NanoZoomer S360 bright-field scanner (Hamamatsu Photonics, Japan) or a Pannoramic SCAN II fluorescence scanner (3DHISTECH, Hungary). Among them, Ki-67 (CST, CST9129) was used for proliferation; γH2AX (Sigma-Aldrich, SAB5600038) for double-strand breaks; cleaved caspase-3 (Affinity, AF7022) and TUNEL (BrightGreen Apoptosis Detection kit, A112) for apoptosis; Parkin (Abmart, T56641S), PINK1 (Abmart, TD7742S) and LC3 (ABclonal, A15591) for mitophagy; NRF2 (Proteintech, 80593-1-RR) for antioxidant capacity. Positive-staining areas were quantified in ImageJ (1.54f).\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eTransmission Electron Microscope\u003c/h3\u003e\n\u003cp\u003eFresh small-intestinal samples (~\u0026thinsp;1\u0026ndash;3 mm\u0026sup3;) were excised and immersed within 1 min in ice-cold 2.5% glutaraldehyde fixative (Yuanye Bio-Technology, Shanghai, R20510). After overnight fixation at 4\u0026deg;C, tissues were post-fixed in 0.1 M phosphate buffer, dehydrated in graded ethanol, infiltrated with resin and polymerised at 60\u0026deg;C for 48 h. Ultra-thin sections (60\u0026ndash;80 nm) were cut, stained with uranyl acetate and lead citrate, and examined using a TEM(Hitachi, HT7800).\u003c/p\u003e\n\u003ch3\u003eMeasurement of MMP, ROS and superoxide dismutase (SOD)\u003c/h3\u003e\n\u003cp\u003eThe MMP, ROS and SOD of fresh small-intestinal samples were detected using the Mitochondrial Membrane Potential Assay Kit with JC-1 (Beyotime, C2006), the Tissue ROS Test Kit (DHE) (Bjbalb, HR8821), and the SOD Assay Kit (Solarbio, BC0170).A multimode microplate reader (BioTek, Synergy LX, 23101208) was used.\u003c/p\u003e\u003cp\u003eWhen the mitochondrial membrane potential is high, JC-1 accumulates in the mitochondrial matrix to form polymers (J-aggregates) that emit red fluorescence; when the mitochondrial membrane potential is low, JC-1 cannot accumulate in the mitochondrial matrix and exists as monomers, which emit green fluorescence. The level of ROS in tissues can be determined by detecting the fluorescence of DHE products. SOD is a metalloenzyme widely present in organisms, serving as an important scavenger of oxygen free radicals. It can catalyze the dismutation of superoxide anions to generate H₂O₂ and O₂. SOD is not only a superoxide anion-scavenging enzyme but also a major H₂O₂-generating enzyme, playing a crucial role in the biological antioxidant system.\u003c/p\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eMitochondria-targeted metabolomics(P650)\u003c/h2\u003e\u003cp\u003eSmall-intestinal samples were rinsed in PBS, snap-frozen in liquid nitrogen, and analysed by targeted mitochondrial metabolomics (P650 panel, Panomix Biomedical Technology, Suzhou). The panel quantifies\u0026thinsp;\u0026gt;\u0026thinsp;600 metabolites, including carbohydrates, organic acids, amino acids, bile acids, indoles, purine nucleotides and lipids. Frozen tissues were thawed at 4\u0026deg;C, mixed with cold methanol/acetonitrile/water (2:2:1, v/v/v), vortexed, sonicated on ice for 30 min, held at \u0026minus;\u0026thinsp;20\u0026deg;C for 10 min, and centrifuged (14 000 g, 4\u0026deg;C, 20 min). Supernatants were vacuum-dried and reconstituted in 100 \u0026micro;L acetonitrile/water (1:1, v/v), vortexed and re-centrifuged (14 000 g, 4\u0026deg;C, 15 min). Metabolites were separated on an ultra-high-performance liquid chromatograph coupled to a triple-quadrupole mass spectrometer (UHPLC-QTRAP MS). Peak areas were extracted with MultiQuant/Analyst; concentrations were calculated against internal-standard calibration curves.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003eMitochondria-targeted energy metabolomics(Kit 100)\u003c/h2\u003e\u003cp\u003eThe Kit-100 panel quantifies 100 mitochondrial energy metabolites, covering glycolysis (EMP), the pentose phosphate pathway (PPP), the tricarboxylic acid (TCA) cycle, and selected intermediates from fatty-acid oxidation and amino-acid transamination. Intestinal tissue was homogenised in extraction buffer, freeze-ground and centrifuged; the supernatant was diluted four-fold, spiked with internal standards, vortex-mixed and dried. Samples were reconstituted in either 50% acetonitrile/water (alkaline loading) or 0.1% formic acid / 40% methanol/water (acidic loading), vortexed and centrifuged (12000 rpm, 4\u0026deg;C, 10 min). The resulting supernatants were transferred to LC-MS vials. Data were acquired on a SCIEX OS platform and processed for qualitative and quantitative analysis. Dimensionality reduction (PCA, PLS-DA and OPLS-DA) was performed with the R ropls package. Between-group comparisons used two-sample t-tests or Mann\u0026ndash;Whitney\u0026ndash;Wilcoxon tests; three or more groups were analysed by one-way ANOVA or Kruskal\u0026ndash;Wallis tests. Pathway annotation employed the KEGG Mapper tool.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003eStatistical analysis\u003c/h2\u003e\u003cp\u003eStatistical analyses were performed using Prism version 8.4.0 (GraphPad Software). All data are expressed as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error of the mean (SEM). Comparisons among FLASH-RT, CONV-RT and Control groups were performed using one-way analysis of variance (ANOVA), while two groups using unpaired t-test .Survival analysis was depicted using Kaplan\u0026ndash;Meier curves and assessed with log-rank test analysis. A \u003cem\u003ep\u003c/em\u003e value of \u0026lt;\u0026thinsp;0.05 was considered statistically significant.\u003c/p\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003eDosimetry\u003c/h2\u003e\u003cp\u003eIrradiation parameters are listed in Supplementary Tables\u0026nbsp;1 and 2. The CHEx-FLASH system delivered 200 Gy/s for FLASH-RT and 0.185 Gy/s for CONV-RT. Because both modalities used the same linac, the prescribed dose was identical (FLASH-RT: 12.01\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12 Gy; CONV-RT: 12.03\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12 Gy). Lateral and longitudinal dose profiles measured with EBT-XD films were uniform and symmetric (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ef). The PDD curve is presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eg, where the blue box represents the dose distribution within the range of the abdominal depth of the mice.\u003c/p\u003e\u003cp\u003e\u003cb\u003eFLASH-RT enhanced survival rates and mitigates myelosuppression.\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo compare the toxicity and mortality under a whole abdominal irradiation dose of 12 Gy, mice were subjected to irradiation in the FLASH-RT group, CONV-RT group, and Control group. Peripheral blood cell tests were performed at 3 days post-irradiation (n\u0026thinsp;=\u0026thinsp;5), and survival rate and body weight changes within 15 days were evaluated (n\u0026thinsp;=\u0026thinsp;10). The survival rates of the FLASH-RT group, CONV-RT group, and Control group were 100% (10/10), 80% (8/10), and 30% (3/10) respectively, with significant differences among the three groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea, P\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Normalized body weight rose steadily in Controls, declined transiently in both irradiated groups. Specifically, the FLASH-RT group decreased within 5 days after irradiation and then increased, whereas the body weight of the CONV-RT group decreased within 8 days after irradiation and then increased, with the increasing trend being less pronounced than that of the FLASH-RT group (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb, P\u0026thinsp;\u0026lt;\u0026thinsp;0.0001).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eBlood analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec) revealed marked WBC and LYM in both irradiated groups, and WBC counts were lower in CONV-RT (0.39\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09 \u0026times; 10⁹/L) than in FLASH-RT (0.88\u0026thinsp;\u0026plusmn;\u0026thinsp;0.21 \u0026times; 10⁹/L). NEU % and EOS % were also higher in CONV-RT than in FLASH-RT mice (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Haemoglobin and platelet levels did not differ among groups (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05).\u003c/p\u003e\u003cp\u003e\u003cb\u003eFLASH-RT alleviated radiation-induced intestinal injury.\u003c/b\u003e\u003c/p\u003e\u003cp\u003eAt 72 hours post-irradiation, we dissected the abdominal cavity of mice in each group (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed). Macroscopically, the intestines in the CONV-RT group showed obvious edema and dilation, while the FLASH-RT group was milder. Further H\u0026amp;E staining of intestinal (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea) revealed that the CONV-RT group had more extensive intestinal structural disorder, villous atrophy, and epithelial damage compared with the FLASH-RT group. Quantitative histology (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb) confirmed that villus height in FLASH-RT (335.99\u0026thinsp;\u0026plusmn;\u0026thinsp;45.81 \u0026micro;m) matched that of Controls (343.62\u0026thinsp;\u0026plusmn;\u0026thinsp;36.20 \u0026micro;m) and exceeded the CONV-RT value (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01). Crypt density (per 50 \u0026micro;m) was highest in Controls, intermediate in FLASH-RT and lowest in CONV-RT (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01), while the intestinal injury score was correspondingly lowest in FLASH-RT (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01). IHC demonstrated that proliferative activity (Ki-67) in FLASH-RT was comparable to Controls and significantly higher than in CONV-RT (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01; Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec, d). γH2AX staining indicated irradiation-induced DNA damage in both irradiated groups, with no difference between them (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05; Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ee, f). Cleaved caspase-3 and TUNEL signals were low and did not differ among the three groups (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05; Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eg\u0026ndash;j), indicating minimal apoptosis.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eFLASH-RT mitigated mitochondrial damage and reduces reactive oxygen species (ROS) levels in intestinal tissues.\u003c/b\u003e\u003c/p\u003e\u003cp\u003eWe observed the microstructure of intestinal tissues in the three groups at 72 hours post-irradiation using TEM (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea). The results showed that in the CONV-RT group, mitochondria were extensively abnormally shaped, with mild swelling, dissolved and pale matrix, dilated cristae or vacuolar changes, and local damage to the membrane structure. In contrast, mitochondria in the FLASH-RT group were similar to those in the Control group, with no obvious abnormal changes. The mitochondrial membrane potential in the CONV-RT group was significantly lower than that in the FLASH-RT group and the Control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb). Additionally, CONV-RT induced more ROS, while there was no significant difference between the FLASH-RT group and the Control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec). The levels of Parkin, PINK1, and LC3 in the FLASH-RT group were lower than those in the CONV-RT group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). The levels of Parkin and PINK1 in the FLASH-RT group were higher than those in the Control group, while there was no significant difference in LC3 levels (Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ee). These findings indicate that both FLASH-RT and CONV-RT can activate mitophagy, but the latter is more pronounced.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eFLASH-RT may confered intestinal protection by enhancing antioxidant defenses against oxidative damage.\u003c/b\u003e\u003c/p\u003e\u003cp\u003eGiven the differences in mitochondrial damage between FLASH-RT and CONV-RT, we performed mitochondria-targeted metabolomics. A total of 538 metabolites were identified in the P650 project, and their classification based on chemical taxonomy is detailed in Supplementary Fig.\u0026nbsp;1. PCA was conducted on all samples and quality control (QC) samples using the quantitative results of the identified metabolites. The experimental results (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea) showed that the samples were closely clustered, indicating good reproducibility of the experiment. We compared the differential metabolites among the groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb, P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) and found that there were 106 differential metabolites between the CONV-RT group and the Control group, and 106 differential metabolites between the FLASH-RT group and the Control group. In the comparison between the FLASH-RT group and the CONV-RT group, a total of 8 differentially expressed metabolites were identified, among which 6 were upregulated and 2 were downregulated. We intuitively displayed the fold changes of the significant differential metabolites identified in the two groups using a bar chart (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec). Further hierarchical cluster analysis was performed on each group of samples to form a cluster tree showing the similarity between samples, and the results are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ed, where samples clustered in the same group have higher similarity. The 6 upregulated differential metabolites in the FLASH-RT group were adenosine diphosphate ribose (ADP-ribose), aspartylphenylalanine, betaine, gamma-glutamyl-phenylalanine, glycyl-phenylalanine, and hypotaurine; the 2 downregulated differential metabolites were apocholic acid and tauro-α-muricholic acid (α-TMCA), with their quantitative analysis results shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ee. KEGG pathway enrichment analysis of the list of differential metabolites using MetaboAnalyst showed (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ef) that the FLASH group was enriched in 2 pathways, namely glycine, serine and threonine metabolism, and taurine and hypotaurine metabolism, which are mainly related to the regulation of mitochondrial antioxidant function.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eIn addition, we detected 100 substances targeting mitochondrial energy metabolism. The PCA score plot of all samples showed obvious clustering among the groups, indicating good reproducibility of the analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea). Further OPLS-DA showed significant separation between the Control group, FLASH-RT group, and CONV-RT group (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea). Figure\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb shows the Z-score plot of all metabolites; the closer to the right, the higher the relative content of the current metabolite in the sample, and the closer to the left, the lower the content of the current metabolite. Quantitative analysis revealed that malonic acid in the CONV-RT group was significantly higher than that in the FLASH-RT group (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ec). KEGG pathway enrichment analysis of the metabolites was performed, and the network diagram results (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ed) showed that the FLASH group was most enriched in 5 pathways, namely central carbon metabolism in cancer, protein digestion and absorption, aminoacyl-tRNA biosynthesis, glucagon signaling pathway, and alanine, aspartate and glutamate metabolism, which are also related to mitochondrial oxidative stress function. Furthermore, the expression level of the antioxidant protein NRF2 in intestinal tissues at 72 hours post-irradiation was determined by IHC. We found that the Control group had the highest level, and the FLASH-RT group was also significantly higher than the CONV-RT group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01). In addition, the SOD content in intestinal tissues of the FLASH-RT group (4.17\u0026thinsp;\u0026plusmn;\u0026thinsp;1.05 U/mg prot) was significantly lower than that of the CONV-RT group (8.17\u0026thinsp;\u0026plusmn;\u0026thinsp;1.83 U/mg prot). These results suggest that FLASH-RT can activate mitochondrial antioxidant function to cope with radiation damage.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eDue to its biological function of protecting normal tissues, FLASH-RT is expected to become a disruptive radiotherapy modality, but its underlying biological mechanisms remain to be explored. In this study, we utilized the CHEx-FLASH device, focusing on confirming the protective effect of FLASH-RT on normal intestinal tissues in mice. For the first time, we observed the phenomenon of alleviated intestinal mitochondrial damage, and further analyzed and verified the differential mitochondrial metabolites between the FLASH-RT and CONV-RT groups, revealing that FLASH-RT may achieve intestinal protection by enhancing mitochondrial antioxidant function.\u003c/p\u003e\u003cp\u003eWe investigated mortality, body weight change curves, acute hematological toxicity, and intestinal damage following 12Gy whole abdominal irradiation. The results of this study showed that compared with CONV-RT, FLASH-RT significantly improved survival and body weight gain, which is consistent with previous studies on intestinal FLASH irradiation with protons or electrons [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. We also observed that FLASH-RT could reduce myelosuppression, which has been reported in a study by Zhu H. et al. [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e], who further found that CONV-RT significantly reduced WBC and LYM counts, with a more pronounced decrease at 48h post-irradiation compared to 3h. We found that NEU% increased significantly after CONV-RT, which may be due to the relative increase in the proportion of neutrophils caused by lymphopenia, or inflammatory responses induced by intestinal tissue damage after radiotherapy. This is supported by our abdominal dissection of mice at 72h post-irradiation, where we observed obvious intestinal edema and dilation in CONV-RT mice, while no significant swelling was noted in the intestines of FLASH-RT and Control group mice. Histopathological examination of intestinal tissues revealed that FLASH-RT alleviated post-radiation intestinal damage, as evidenced by lower intestinal injury scores, and significantly better intestinal villus height, number of intestinal crypts, and intestinal cell proliferation capacity compared to CONV-RT. This confirms that the CHEx-FLASH device exhibits a significant intestinal protective effect, consistent with the abdominal irradiation effects of other electrons, protons, or X-rays [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan additionalcitationids=\"CR22\" citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eIn addition, we found no statistically significant difference in γH2AX expression levels between FLASH-RT and CONV-RT (P\u0026thinsp;\u0026gt;\u0026thinsp;0.05), indicating that the degree of DNA double-strand damage was comparable between the two groups. However, since our detection time point was 72h post-irradiation, it is possible that DNA damage may have been repaired. Zhu H. et al. [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e] found that γH2AX levels were relatively high at 3h post-irradiation and decreased at 48h, but γH2AX levels in CONV-RT remained higher than those in FLASH-RT. However, comparative studies on DNA damage are inconsistent: Shi X. et al. [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e] irradiated intestinal organoids and human intestinal epithelial cells HIEC-6 with X-ray FLASH-RT and CONV-RT, and found that the expression levels of γH2AX and 53BP1 were almost identical in both groups. In contrast, other researchers observed that acute irradiation with electrons in human lung cells [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e] and intestinal crypt stem cells [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e] resulted in lower levels of 53BP1 or γH2AX in FLASH-RT compared to CONV-RT. In general, discrepancies in studies on DNA damage may be related to factors such as time points, dose rate levels, and differences in radiation sources. Additionally, there are still inconsistencies regarding whether cell apoptosis is induced after irradiation with the two dose rates, so the mechanism underlying the protective effect of FLASH-RT on normal organs requires further exploration.\u003c/p\u003e\u003cp\u003eCurrently, researchers have proposed several hypotheses [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e], including oxygen depletion, free radical-free radical interaction, and immune factors, but there are conflicting positive and negative research results reported. Surprisingly, in recent years, a hypothesis has emerged that mitochondria are involved in the protective mechanism of FLASH-RT. A research team irradiated normal human lung fibroblasts (IMR90) with protons and found that FLASH-RT (100 Gy/s) could alleviate mitochondrial damage, including morphological and functional changes such as mitochondrial membrane potential, mtDNA copy number, and ROS [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e], while this phenomenon was not observed in cancer cells. A pre-published article also reported that compared with low-dose-rate electron irradiation (0.36 Gy/s), FLASH electron irradiation at 61 or 610 Gy/s significantly enhanced cytochrome c release from mitochondria in human breast cancer cells MCF-10A, thereby triggering massive caspase activation and inhibiting cytoplasmic mtDNA accumulation and interferon-β(IFN-β) secretion [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Another in vivo study on the protective effect of electron FLASH-RT on the esophagus showed via TEM that CONV-RT induced mitochondrial swelling, blurred structure, disappearance or break of cristae, reduced matrix electron density, and vacuolation, while mitochondrial swelling and structural blurring were less obvious in the FLASH-RT group. Proteomic analysis revealed that CONV-RT downregulated the expression of key proteins involved in the TCA cycle and oxidative phosphorylation in mice [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eIn our study, we observed changes in mitochondrial structure following high-energy X-ray irradiation for the first time. TEM also revealed similar obvious characteristics of intestinal mitochondrial damage in the CONV-RT group, while mitochondrial damage in the FLASH-RT group was mild. Meanwhile, compared with FLASH-RT, CONV-RT disrupted mitochondrial membrane potential and increased ROS, confirming functional impairment. In addition, CONV-RT exhibited higher expression of mitophagy-related proteins Parkin and PINK1 than FLASH-RT, indicating that mitophagy is activated to remove dysfunctional mitochondria after mitochondrial damage [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Mitochondria are small double-membraned organelles in the cytoplasm of eukaryotic cells, coupled with the electron transport chain (ETC) and oxidative phosphorylation (OXPHOS) through the tricarboxylic acid (TCA) cycle, and are known as the \"energy factories\" of the cell.\u003c/p\u003e\u003cp\u003eUsing targeted metabolomics, we performed qualitative and quantitative analysis of mitochondria-related metabolites and found that FLASH-RT expressed lower levels of malonic acid (an endogenous competitive inhibitor of succinate dehydrogenase), which is a marker of mitochondrial damage [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Malonate causes rapid collapse of MMP and ROS production, thereby overwhelming mitochondrial antioxidant capacity and leading to mitochondrial swelling [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. We also conducted pathway enrichment analysis of other differential mitochondrial metabolites, which mainly enriched the \"Glycine, serine and threonine metabolism\" and \"Taurine and hypotaurine metabolism\" pathways.\u003c/p\u003e\u003cp\u003eFrom previous literature, we know that \"Glycine/serine/threonine metabolism\" can affect carbohydrate patterns and energy utilization efficiency, and ultimately delay intervertebral disc degeneration through antioxidant effects [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e], while the \"serine/glycine pathway\" can maintain cell function, oxidative stress tolerance, and survival [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. \"Taurine and hypotaurine metabolism\" can also protect mitochondria from oxidative and sub-oxidative stress [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Metabolite analysis indicated that FLASH-RT enhances antioxidant capacity, so we further verified that FLASH-RT expressed higher levels of NRF2 and SOD than CONV-RT, which are antioxidant genes produced in response to ionizing radiation-induced oxidative stress to protect cells from damage [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eIn summary, we propose the following hypothesis (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e): high-energy X-ray FLASH-RT can reduce ROS production, mitigate mitochondrial damage, and promote the metabolism of mitochondrial antioxidant substances, thereby enhancing antioxidant capacity, reducing intestinal cell damage, and achieving intestinal protective effects.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eOf course, this study has some limitations. For example, the detection of ROS, myelosuppression, and γH2AX breakage indices lacks dynamic detection at multiple time points, and multi-factor exploration of FLASH-RT dose parameters such as dose rate and fractionation methods is not conducted. However, we innovatively proposed that FLASH-RT achieves normal tissue protection by enhancing mitochondrial antioxidant function, which provides a favorable research basis for further mechanistic studies by researchers. In addition, for research institutions lacking FLASH-RT equipment, combining CONV-RT with antioxidants may be a potential approach to reduce radiotherapy-induced tissue damage in the future.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eCHEx-FLASH achieves UHDR irradiation and alleviates radiation-induced intestinal injury. The protective effect of FLASH-RT on intestinal tissues may be mediated by mitigating mitochondrial damage and enhancing antioxidant pathways through improved mitochondrial energy metabolism.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eCHEx-FLASH\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003ePrototype of Compact High-energy X-ray Flash Radiotherapy Equipment\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eUHDR\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eultra-high dose rate\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eFLASH-RT\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eFLASH Radiotherapy\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eCONV-RT\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eConventional Dose Rate Radiotherapy\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eSSD\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eSource Surface Distance\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003ePDD\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003ePercent Depth Dose\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eTEM\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eTransmission Electron Microscope\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eMMP\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eMitochondrial Membrane Potential\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eROS\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eReactive Oxygen Species\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eSOD\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eSuperoxide Dismutase.\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthical approval\u003c/strong\u003e\u003cp\u003e All mouse experiments adhered to the approved guidelines of the Ethics Committee of Mianyang Central Hospital (approval number: S20240203-02).\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003cp\u003eNot applicable.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eFunding:\u003c/h2\u003e\u003cp\u003eThis work was financially supported by the Projects of National Natural Science Foundation of China (Grant NO. U2330122) and the General Program of the Sichuan Natural Science Foundation (Grant No. 2023NSFSC0710).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eX. Hao, H. Du, D. Wang, M. Tang, H. Zhang and Y. Zhu conducted the animal experiments and biological analyses. X. Hao, B. Lin, W. Wu, Y. Yang and X. D were responsible for irradiation beam control and performed the physics-related analyses. B. Lin, D. Wang,Y. Zhang and X. Du contributed to the experimental design and provided critical revisions to the manuscript. X. Hao drafted the main manuscript text and prepared all figures. All authors reviewed and approved the final version of the manuscript.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eAll data generated or analysed during this study are included in this published article and its supplementary information files\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eBray F, Laversanne M, Sung H, Ferlay J, Siegel RL, Soerjomataram I, et al. Global cancer statistics 2022: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin. 2024;74(3):229\u0026ndash;63. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3322/caac.21834\u003c/span\u003e\u003cspan address=\"10.3322/caac.21834\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLu Q, Liang Y, Tian S, Jin J, Zhao Y, Fan H. Radiation-Induced Intestinal Injury: Injury Mechanism and Potential Treatment Strategies. Toxics. 2023;11(12):1011. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3390/toxics11121011\u003c/span\u003e\u003cspan address=\"10.3390/toxics11121011\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eYang XF, Zheng MY, An LY, Sun JM, Hei QW, Ji YH, et al. Quality evaluation of guidelines for the diagnosis and treatment of radiation enteritis. Radiat Oncol. 2023;18(1):14. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1186/s13014-023-02204-9\u003c/span\u003e\u003cspan address=\"10.1186/s13014-023-02204-9\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAraujo IK, Mu\u0026ntilde;oz-Guglielmetti D, Moll\u0026agrave; M. Radiation-induced damage in the lower gastrointestinal tract: Clinical presentation, diagnostic tests and treatment options. Best Pract Res Clin Gastroenterol. 2020;48\u0026ndash;49:101707. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.bpg.2020.101707\u003c/span\u003e\u003cspan address=\"10.1016/j.bpg.2020.101707\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eFavaudon V, Caplier L, Monceau V, Pouzoulet F, Sayarath M, Fouillade C, et al. Ultrahigh dose-rate FLASH irradiation increases the differential response between normal and tumor tissue in mice. Sci Transl Med. 2014;6(245):245ra93. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1126/scitranslmed.3008973\u003c/span\u003e\u003cspan address=\"10.1126/scitranslmed.3008973\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLin B, Gao F, Yang Y, Wu D, Zhang Y, Feng G, et al. FLASH Radiotherapy: History and Future. Front Oncol. 2021;11:644400. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3389/fonc.2021.644400\u003c/span\u003e\u003cspan address=\"10.3389/fonc.2021.644400\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSpitz DR, Buettner GR, Petronek MS, St-Aubin JJ, Flynn RT, Waldron TJ, et al. An integrated physico-chemical approach for explaining the differential impact of FLASH versus conventional dose rate irradiation on cancer and normal tissue responses. Radiother Oncol. 2019;139:23\u0026ndash;7. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.radonc.2019.03.028\u003c/span\u003e\u003cspan address=\"10.1016/j.radonc.2019.03.028\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eOhsawa D, Hiroyama Y, Kobayashi A, Kusumoto T, Kitamura H, Hojo S, et al. DNA strand break induction of aqueous plasmid DNA exposed to 30 MeV protons at ultra-high dose rate. J Radiat Res. 2022;63(2):255\u0026ndash;60. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1093/jrr/rrab114\u003c/span\u003e\u003cspan address=\"10.1093/jrr/rrab114\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAverbeck D, Rodriguez-Lafrasse C. Role of Mitochondria in Radiation Responses: Epigenetic, Metabolic, and Signaling Impacts. Int J Mol Sci. 2021;22(20):11047. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3390/ijms222011047\u003c/span\u003e\u003cspan address=\"10.3390/ijms222011047\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGuo Z, Buonanno M, Harken A, Zhou G, Hei TK. Mitochondrial Damage Response and Fate of Normal Cells Exposed to FLASH Irradiation with Protons. Radiat Res. 2022;197(6):569\u0026ndash;82. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1667/RADE-21-00181.1\u003c/span\u003e\u003cspan address=\"10.1667/RADE-21-00181.1\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMontay-Gruel P, Acharya MM, Gon\u0026ccedil;alves Jorge P, Petit B, Petridis IG, Fuchs P, et al. Hypofractionated FLASH-RT as an Effective Treatment against Glioblastoma that Reduces Neurocognitive Side Effects in Mice. Clin Cancer Res. 2021;27(3):775\u0026ndash;84. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1158/1078-0432.CCR-20-0894\u003c/span\u003e\u003cspan address=\"10.1158/1078-0432.CCR-20-0894\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGao F, Yang Y, Zhu H, Wang J, Xiao D, Zhou Z, et al. First demonstration of the FLASH effect with ultrahigh dose rate high-energy X-rays. Radiother Oncol. 2022;166:44\u0026ndash;50. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.radonc.2021.11.004\u003c/span\u003e\u003cspan address=\"10.1016/j.radonc.2021.11.004\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLevy K, Natarajan S, Wang J, Chow S, Eggold JT, Loo PE, et al. Abdominal FLASH irradiation reduces radiation-induced gastrointestinal toxicity for the treatment of ovarian cancer in mice. Sci Rep. 2020;10(1):21600. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1038/s41598-020-78017-7\u003c/span\u003e\u003cspan address=\"10.1038/s41598-020-78017-7\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eZhu H, Xie D, Yang Y, Huang S, Gao X, Peng Y, et al. Radioprotective effect of X-ray abdominal FLASH irradiation: Adaptation to oxidative damage and inflammatory response may be benefiting factors. Med Phys. 2022;49(7):4812\u0026ndash;22. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1002/mp.15680\u003c/span\u003e\u003cspan address=\"10.1002/mp.15680\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSoto LA, Casey KM, Wang J, Blaney A, Manjappa R, Breitkreutz D, Skinner L, et al. FLASH Irradiation Results in Reduced Severe Skin Toxicity Compared to Conventional-Dose-Rate Irradiation. Radiat Res. 2020;194(6):618\u0026ndash;24. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1667/RADE-20-00090\u003c/span\u003e\u003cspan address=\"10.1667/RADE-20-00090\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eVerginadis II, Velalopoulou A, Kim MM, Kim K, Paraskevaidis I, Bell B et al. FLASH proton reirradiation, with or without hypofractionation, mitigates chronic toxicity in the normal murine intestine, skin, and bone. bioRxiv [Preprint]. 2024.07.08.602528. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1101/2024.07.08.602528\u003c/span\u003e\u003cspan address=\"10.1101/2024.07.08.602528\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eVozenin MC, De Fornel P, Petersson K, Favaudon V, Jaccard M, Germond JF, et al. The Advantage of FLASH Radiotherapy Confirmed in Mini-pig and Cat-cancer Patients. Clin Cancer Res. 2019;25(1):35\u0026ndash;42. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1158/1078-0432.CCR-17-3375\u003c/span\u003e\u003cspan address=\"10.1158/1078-0432.CCR-17-3375\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBourhis J, Sozzi WJ, Jorge PG, Gaide O, Bailat C, Duclos F, et al. Treatment of a first patient with FLASH-radiotherapy. Radiother Oncol. 2019;139:18\u0026ndash;22. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.radonc.2019.06.019\u003c/span\u003e\u003cspan address=\"10.1016/j.radonc.2019.06.019\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLim TL, Morral C, Verginadis II, Kim K, Luo L, Foley CJ et al. Early Inflammation and Interferon Signaling Direct Enhanced Intestinal Crypt Regeneration after Proton FLASH Radiotherapy. bioRxiv [Preprint]. 2024.08.16.608284. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1101/2024.08.16.608284\u003c/span\u003e\u003cspan address=\"10.1101/2024.08.16.608284\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eZhu H, Liu S, Qiu J, Hu A, Zhou W, Wang J, et al. Instantaneous dose rate as a crucial factor in reducing mortality and normal tissue toxicities in murine total-body irradiation: a comparative study of dose rate combinations. Mol Med. 2025;31(1):79. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1186/s10020-025-01135-3\u003c/span\u003e\u003cspan address=\"10.1186/s10020-025-01135-3\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eEggold JT, Chow S, Melemenidis S, Wang J, Natarajan S, Loo PE, et al. Abdominopelvic FLASH Irradiation Improves PD-1 Immune Checkpoint Inhibition in Preclinical Models of Ovarian Cancer. Mol Cancer Ther. 2022;21(2):371\u0026ndash;81. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1158/1535-7163.MCT-21-0358\u003c/span\u003e\u003cspan address=\"10.1158/1535-7163.MCT-21-0358\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eVald\u0026eacute;s Zayas A, Kumari N, Liu K, Neill D, Delahoussaye A, Gon\u0026ccedil;alves Jorge P, et al. Independent Reproduction of the FLASH Effect on the Gastrointestinal Tract: A Multi-Institutional Comparative Study. Cancers (Basel). 2023;15(7):2121. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3390/cancers15072121\u003c/span\u003e\u003cspan address=\"10.3390/cancers15072121\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eShi X, Yang Y, Zhang W, Wang J, Xiao D, Ren H, et al. FLASH X-ray spares intestinal crypts from pyroptosis initiated by cGAS-STING activation upon radioimmunotherapy. Proc Natl Acad Sci U S A. 2022;119(43):e2208506119. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1073/pnas.2208506119\u003c/span\u003e\u003cspan address=\"10.1073/pnas.2208506119\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eFouillade C, Curras-Alonso S, Giuranno L, Quelennec E, Heinrich S, Bonnet-Boissinot S, et al. FLASH Irradiation Spares Lung Progenitor Cells and Limits the Incidence of Radio-induced Senescence. Clin Cancer Res. 2020;26(6):1497\u0026ndash;506. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1158/1078-0432.CCR-19-1440\u003c/span\u003e\u003cspan address=\"10.1158/1078-0432.CCR-19-1440\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBogaerts E, Macaeva E, Isebaert S, Haustermans K. Potential Molecular Mechanisms behind the Ultra-High Dose Rate FLASH Effect. Int J Mol Sci. 2022;23(20):12109. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3390/ijms232012109\u003c/span\u003e\u003cspan address=\"10.3390/ijms232012109\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLv J, Sun J, Luo Y, Liu J, Wu D, Fang Y et al. FLASH Irradiation Regulates IFN-β induction by mtDNA via Cytochrome c Leakage.bioRxiv 2024.04.10.588811; doi: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1101/2024.04.10.588811\u003c/span\u003e\u003cspan address=\"10.1101/2024.04.10.588811\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eRen W, Hou L, Zhang K, Chen H, Feng X, Jiang Z, et al. The sparing effect of ultra-high dose rate irradiation on the esophagus. Front Oncol. 2024;14:1442627. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3389/fonc.2024.1442627\u003c/span\u003e\u003cspan address=\"10.3389/fonc.2024.1442627\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eScheibye-Knudsen M, Fang EF, Croteau DL, Wilson DM 3rd, Bohr VA. Protecting the mitochondrial powerhouse. Trends Cell Biol. 2015;25(3):158\u0026ndash;70. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.tcb.2014.11.002\u003c/span\u003e\u003cspan address=\"10.1016/j.tcb.2014.11.002\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eYuan C, Zheng L, Zhao Y. Protective effect of 3-n-butylphthalide against intrastriatal injection of malonic acid-induced neurotoxicity and biochemical alteration in rats. Biomed Pharmacother. 2022;155:113664. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.biopha.2022.113664\u003c/span\u003e\u003cspan address=\"10.1016/j.biopha.2022.113664\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eFernandez-Gomez FJ, Galindo MF, G\u0026oacute;mez-L\u0026aacute;zaro M, Yuste VJ, Comella JX, Aguirre N, et al. Malonate induces cell death via mitochondrial potential collapse and delayed swelling through an ROS-dependent pathway. Br J Pharmacol. 2005;144(4):528\u0026ndash;37. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1038/sj.bjp.0706069\u003c/span\u003e\u003cspan address=\"10.1038/sj.bjp.0706069\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWu X, Liu C, Yang S, Shen N, Wang Y, Zhu Y, et al. Glycine-Serine-Threonine Metabolic Axis Delays Intervertebral Disc Degeneration through Antioxidant Effects: An Imaging and Metabonomics Study. Oxid Med Cell Longev. 2021;2021:5579736. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1155/2021/5579736\u003c/span\u003e\u003cspan address=\"10.1155/2021/5579736\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eOst M, Keipert S, van Schothorst EM, Donner V, van der Stelt I, Kipp AP, Petzke KJ, et al. Muscle mitohormesis promotes cellular survival via serine/glycine pathway flux. FASEB J. 2015;29(4):1314\u0026ndash;28. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1096/fj.14-261503\u003c/span\u003e\u003cspan address=\"10.1096/fj.14-261503\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSeneff S, Kyriakopoulos AM. Taurine prevents mitochondrial dysfunction and protects mitochondria from reactive oxygen species and deuterium toxicity. Amino Acids. 2025;57(1):6. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1007/s00726-024-03440-3\u003c/span\u003e\u003cspan address=\"10.1007/s00726-024-03440-3\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBaliou S, Adamaki M, Ioannou P, Pappa A, Panayiotidis MI, Spandidos DA, Christodoulou I, Kyriakopoulos AM, Zoumpourlis V. Protective role of taurine against oxidative stress (Review). Mol Med Rep. 2021;24(2):605. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3892/mmr.2021.12242\u003c/span\u003e\u003cspan address=\"10.3892/mmr.2021.12242\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKaya H, Delibas N, Serteser M, Ulukaya E, Ozkaya O. The effect of melatonin on lipid peroxidation during radiotherapy in female rats. Strahlenther Onkol. 1999;175(6):285\u0026ndash;8. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1007/BF02743581\u003c/span\u003e\u003cspan address=\"10.1007/BF02743581\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLiu S, Zhang HL, Li J, Ye ZP, Du T, Li LC, et al. Tubastatin A potently inhibits GPX4 activity to potentiate cancer radiotherapy through boosting ferroptosis. Redox Biol. 2023;62:102677. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.redox.2023.102677\u003c/span\u003e\u003cspan address=\"10.1016/j.redox.2023.102677\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"radiation-oncology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"raon","sideBox":"Learn more about [Radiation Oncology](http://ro-journal.biomedcentral.com/)","snPcode":"13014","submissionUrl":"https://submission.nature.com/new-submission/13014/3","title":"Radiation Oncology","twitterHandle":"@OncoBioMed","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"ultra-high dose rate, X-ray, FLASH effect, intestine, mitochondria, metabolism","lastPublishedDoi":"10.21203/rs.3.rs-7650752/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7650752/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003ePurpose\u003c/h2\u003e\u003cp\u003eUltra-high dose rate (UHDR) radiation retains tumor-killing efficacy while reducing toxicity to normal tissues, holding a promising transformative radiotherapy paradigm. This study aimed to explore the potential role of mitochondria in intestinal protection conferred by high-energy X-ray FLASH radiotherapy (FLASH-RT) and the associated signaling pathways.\u003c/p\u003e\u003ch2\u003eMethod\u003c/h2\u003e\u003cp\u003eThe entire abdomen of healthy female C57BL/6 mice was irradiated using three modes: ultra-high dose rate irradiation (FLASH-RT), conventional dose rate irradiation (CONV-RT), and sham irradiation (Control). Mouse survival status and body weight changes were monitored within 15 days post-irradiation. At 72 hours post-irradiation, whole blood was collected for hematological analysis, and intestinal tissues were harvested for pathological detection, transmission electron microscopy (TEM) observation of mitochondrial changes, and two types of mitochondria- targeted metabolomic assays.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e\u003cp\u003eA Compact single High-energy X-ray Source FLASH-RT device(CHEx-FLASH) was used, with a dose rate of 200 Gy/s. At 15 days post-irradiation, the survival rates of the Control group (100%, 10/10) and FLASH-RT group (80%, 8/10) were significantly higher than that of the CONV-RT group (30%, 2/10). Body weight decreased in the early post-irradiation period across groups, but the decline was milder in FLASH-RT with greater late-stage recovery. Hematological results at 72 hours showed that CONV-RT induced more severe bone marrow suppression compared to FLASH-RT. Intestinal histopathological analysis revealed that FLASH-RT alleviated intestinal inflammation and promoted enterocyte proliferation, while DNA double-strand breaks and apoptosis levels did not differ significantly between the two irradiated groups. FLASH-RT mitigated mitochondrial damage, reduced reactive oxygen species (ROS) levels and mildly activated mitophagy. Mitochondria-related energy metabolomics sequencing of intestinal tissues showed that the mitochondrial damage marker malonic acid was significantly lower in FLASH-RT than in CONV-RT, and differentially expressed metabolites were primarily enriched in mitochondrial antioxidant pathways. Additionally, the increased expression of the antioxidant protein NRF2 and superoxide dismutase ༈SOD) were verified.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e\u003cp\u003eCHEx-FLASH achieves UHDR irradiation and alleviates radiation-induced intestinal injury. The protective effect of FLASH-RT on intestinal tissues may be mediated by mitigating mitochondrial damage and enhancing antioxidant pathways through improved mitochondrial energy metabolism.\u003c/p\u003e","manuscriptTitle":"Preliminary mechanistic exploration of mitochondrial function in intestinal protection mediated by high-energy X-ray FLASH radiotherapy","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-11-04 14:25:09","doi":"10.21203/rs.3.rs-7650752/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-11-24T16:06:07+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-11-24T15:20:27+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-11-18T19:37:26+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"268520046143191966051703199326443123396","date":"2025-11-10T16:34:40+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-11-06T12:31:52+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"270839835614498498082462690919829633966","date":"2025-10-27T19:35:48+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"340180569298683401481492919792195432096","date":"2025-10-27T08:12:47+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-10-23T12:25:21+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-10-01T07:15:00+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-09-29T04:45:58+00:00","index":"","fulltext":""},{"type":"submitted","content":"Radiation Oncology","date":"2025-09-27T05:27:00+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"radiation-oncology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"raon","sideBox":"Learn more about [Radiation Oncology](http://ro-journal.biomedcentral.com/)","snPcode":"13014","submissionUrl":"https://submission.nature.com/new-submission/13014/3","title":"Radiation Oncology","twitterHandle":"@OncoBioMed","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"59b5d1d6-b22c-43c9-943a-5db9267cb267","owner":[],"postedDate":"November 4th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-03-02T16:04:06+00:00","versionOfRecord":{"articleIdentity":"rs-7650752","link":"https://doi.org/10.1186/s13014-026-02809-w","journal":{"identity":"radiation-oncology","isVorOnly":false,"title":"Radiation Oncology"},"publishedOn":"2026-02-28 15:57:24","publishedOnDateReadable":"February 28th, 2026"},"versionCreatedAt":"2025-11-04 14:25:09","video":"","vorDoi":"10.1186/s13014-026-02809-w","vorDoiUrl":"https://doi.org/10.1186/s13014-026-02809-w","workflowStages":[]},"version":"v1","identity":"rs-7650752","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7650752","identity":"rs-7650752","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}