Application of nobiletin in prevention and treatment of ionizing radiation damage

Abstract

The invention relates to the technical field of biological medicine, in particular to application of nobiletin in prevention and treatment of ionizing radiation damage. The nobiletin provided by the invention is extracted from plants, is of a natural flavonoid structure, has high compatibility with endogenous metabolites of a human body, and has good biocompatibility. Animal experiments prove that when the medicine is administrated at the optimal effective dose (200mg / kg) and 12 hours before irradiation, no obvious toxic reaction is shown, the immunogenicity is low, the medicine is suitable for long-term or conventional radiation protection application, the common problems of adverse reaction and accumulated toxicity in synthesis of protective medicines are solved, and meanwhile, the medicine can be in the optimal action state when being exposed to radiation, so that the medicine can be applied to radiation protection for a long time. The survival time of irradiated mice is remarkably prolonged, acute radiation injury pathological symptoms such as intestinal edema and villus shortening are effectively improved, intestinal crypt regeneration and stem cell proliferation are promoted, and reliable radiation protection efficiency is shown.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, specifically to the application of noriheptacorlin in the prevention and treatment of ionizing radiation damage. Background Technology

[0002] With the widespread application of nuclear technology in medicine, energy, and defense, the risk of ionizing radiation exposure has increased significantly. Ionizing radiation can damage multiple systems in a living organism, with the gastrointestinal system and bone marrow hematopoietic system being the most sensitive. The main pathological features of acute gastrointestinal radiation sickness (GI-ARS) include intestinal edema, villus shortening, crypt necrosis, and impaired regeneration. In severe cases, it can lead to intestinal barrier dysfunction, causing infection and death. Damage to the bone marrow hematopoietic system manifests as a decrease in hematopoietic stem cells, a decline in peripheral blood leukocytes and erythrocytes, leading to weakened immunity and anemia. Currently, research on radiation protectants mainly focuses on synthetic compounds (such as amifostine) and some natural products. However, existing technologies have significant limitations: while synthetic compounds have some protective effects, they have significant side effects and a strict dosing window, requiring administration shortly before radiation exposure, limiting their application scenarios; many reported natural product-based protectants suffer from unclear protective effects, ambiguous mechanisms of action, and unoptimized dosing regimens, failing to effectively protect key intestinal cells (such as intestinal stem cells) and thus failing to meet practical radiation protection needs.

[0003] While existing synthetic radiation protection drugs (such as amifostine) offer some radiation protection, their complex chemical structures and poor biocompatibility lead to significant side effects (such as gastrointestinal reactions and blood pressure fluctuations), limiting their application in specific populations (such as the elderly and those with weakened constitutions). This is because the structure of synthetic drugs differs significantly from the body's own metabolites, easily triggering immune responses and metabolic disorders. For existing natural product-based radiation protection agents, an optimal dosing regimen is not clearly defined; excessively high or low doses can affect protective efficacy, and the dosing time window is often poorly designed, failing to establish an effective protective barrier before radiation damage occurs. This is due to a lack of systematic dose-effect and time-effect studies, making it impossible to precisely match the peak drug effect with the radiation exposure time. Current technologies lack in-depth research on the protective mechanisms against radiation damage, often focusing on simple antioxidant effects and neglecting key aspects such as intestinal stem cell protection, mitophagy regulation, and hematopoietic system repair. This results in limited protective effects and an inability to comprehensively address multi-system damage caused by radiation. This is because current research has not fully revealed the connection between the core pathological mechanisms of radiation damage and the targets of natural products. Existing protective agents are insufficient in protecting against intestinal epithelial crypt regeneration and cannot effectively promote the repair of intestinal barrier function and the recovery of peripheral blood counts after radiation. This is because their targets are not focused on key processes such as intestinal stem cell survival and hematopoietic stem cell proliferation, making it difficult to fundamentally repair radiation-induced tissue and organ damage.

[0004] Therefore, developing a radiation protection technology based on natural active ingredients, with a clear mechanism of action, significant protective effect, and high safety is of great practical significance. Summary of the Invention

[0005] In view of this, the technical problem to be solved by the present invention is to provide the application of noriheptacortine in the prevention and treatment of ionizing radiation damage. The noriheptacortine provided by the present invention has high safety, few side effects, high biocompatibility and high stability, and has excellent effect in the prevention and treatment of ionizing radiation damage.

[0006] This invention provides the application of noriheptacorlin in the preparation of drugs for the prevention and / or treatment of ionizing radiation damage and related diseases.

[0007] In some embodiments, the ionizing radiation damage includes gastrointestinal damage and / or bone marrow hematopoietic dysfunction.

[0008] In some embodiments, the associated disease includes acute radiation sickness of the gastrointestinal tract.

[0009] In some embodiments, the ionizing radiation includes at least one of alpha radiation, gamma radiation, X-ray radiation, and neutron radiation.

[0010] In some embodiments, the dose of ionizing radiation includes 5 to 10 Gy.

[0011] In some embodiments, the effective dose of the nobiletin is 100-200 mg / kg based on mouse body weight.

[0012] In some embodiments, the drug is administered once or twice 12 to 24 hours before the ionizing radiation is applied.

[0013] In some specific embodiments, the effective dose of the nodosumetin is 200 mg / kg, and the drug is administered once 12 hours before ionizing radiation.

[0014] In some embodiments, the medicament further includes pharmaceutically acceptable excipients;

[0015] The excipients include one or more of the following: diluent, stabilizer, osmotic pressure regulator, pH regulator, preservative, and antioxidant.

[0016] In some embodiments, the dosage form of the drug includes an injection, an oral dose, or a topical preparation;

[0017] The topical preparations include gels, creams, patches, or sprays.

[0018] In some embodiments, the drug regulates mitophagy, reduces ROS levels, and decreases cellular DNA damage and apoptosis by activating the NRF2 signaling pathway.

[0019] In some embodiments, the drug inhibits apoptosis of intestinal epithelial cells and small intestinal crypt cells induced by ionizing radiation.

[0020] In some embodiments, the drug promotes the proliferation of small intestinal crypt cells induced by ionizing radiation.

[0021] Compared with the prior art, the beneficial effects of the present invention include:

[0022] 1. The hesperidin provided by this invention is derived from plant extracts and has a natural flavonoid structure. It exhibits high compatibility with endogenous human metabolites and good biocompatibility. Animal experiments have confirmed that at the optimal effective dose (200 mg / kg) and administered 12 hours before irradiation, no significant toxic reactions were observed. It has low immunogenicity and is suitable for long-term or routine radiation protection applications. It overcomes the common adverse reactions and cumulative toxicity problems of synthetic protective drugs. At the same time, it allows the drug to be in its optimal state of action during radiation exposure, significantly prolonging the survival time of irradiated mice, effectively improving acute radiation injury pathological symptoms such as intestinal edema and villus shortening, and promoting intestinal crypt regeneration and stem cell proliferation, demonstrating reliable radiation protection efficacy.

[0023] 2. This invention confirms that noriheptacorlina exerts its radiation protection effect by activating the NRF2 signaling pathway, regulating mitophagy, and scavenging reactive oxygen species (ROS). This mechanism has been verified by inhibitor experiments, providing a solid theoretical basis for further optimization and development of the technical solution and avoiding the problem of unstable protective effects caused by unclear mechanisms of action.

[0024] 3. Nobiletin not only protects against acute radiation injury to the gastrointestinal tract but also protects the bone marrow hematopoietic system, making it widely applicable in various radiation exposure scenarios such as nuclear medicine diagnosis and treatment, nuclear industry operations, national defense, and emergency protection. Furthermore, its oral administration is convenient, resulting in good patient compliance and suitability for different groups and environments.

[0025] 4. The hesperidin used in this invention is of natural origin, with abundant raw materials, mature extraction and purification processes, and low production costs. The optimal dosing regimen is simple and easy to implement, requiring no complex equipment or operating procedures, which is conducive to large-scale production and clinical translation, and has significant market application potential. Attached Figure Description

[0026] Figure 1 The study showed the inhibitory effects of five compounds at different concentrations on tumor cells.

[0027] Figure 2This study shows the effects of five compounds on organoid budding at a dose of 25 μM.

[0028] Figure 3 This study shows the effects of five compounds on organoid budding at a dose of 100 μM.

[0029] Figure 4 This shows the effect of five compounds on organoid budding at a dose of 200 μM;

[0030] Figure 5 The results of MTT activity assays for five compounds at different concentrations are shown.

[0031] Figure 6 The results of using an irradiated mouse model to investigate the dosage and timing of drug administration are shown. Figure A shows the technical route, Figure B shows the survival time of mice in different groups, and Figure C shows the HE staining results of intestinal pathology in mice in different groups.

[0032] Figure 7 A diagram showing the KEGG pathway enrichment analysis of differentially expressed genes in stem cells after Nob treatment of organoids;

[0033] Figure 8 Volcano plot showing differentially expressed genes between the NOB-treated and control groups after NOB treatment of organoids;

[0034] Figure 9 Hierarchical clustering heatmap of differentially expressed genes between the NOB-treated group and the control group after NOB treatment of organoids;

[0035] Figure 10 Volcano diagram showing differentially expressed genes between the NOB-treated and control groups in crypt cells;

[0036] Figure 11 A graph showing the enrichment of differentially expressed genes in the KEGG pathway in an in vivo stem cell model.

[0037] Figure 12 Hierarchical clustering heatmap showing differentially expressed genes in stem cells between the NOB-treated group and the control group;

[0038] Figure 13 The figure shows that NOB pretreatment inhibits radiation-induced death of normal intestinal epithelial cells. Figure A shows the effect of NOB on intestinal epithelial cell viability under non-radiation conditions, Figure B shows the protective effect of NOB pretreatment on intestinal epithelial cell viability under radiation conditions, and Figure C shows the protective effect of NOB pretreatment on intestinal epithelial cell clonogenic ability.

[0039] Figure 14 The study shows the inhibitory effect of NOB pretreatment on radiation-induced apoptosis in IEC6 cells. The left figure shows the cell apoptosis status of different treatment groups detected by flow cytometry, and the right figure is a bar chart of quantitative apoptosis rate.

[0040] Figure 15 The study shows the alleviating effect of NOB pretreatment on DNA double-strand breaks (DSB) in intestinal crypt cells of mice after radiation. Figure A shows the results of immunofluorescence staining, and Figure B shows the quantitative statistical results.

[0041] Figure 16 The study showed the inhibitory effect of NOB pretreatment on early apoptosis of intestinal crypt cells in mice after radiation. Figure C shows the TUNEL immunofluorescence staining results, Figure D shows the quantitative statistics of TUNEL positive cells, Figure E shows the immunohistochemical results, and Figure G shows the Cleaved-caspase-3 protein blot.

[0042] Figure 17 The results show that NOB pretreatment enhances intracellular mitochondrial autophagy. The left image shows the Western blot results, and the right image shows the immunofluorescence colocalization results.

[0043] Figure 18 The study shows the inhibitory effect of NOB pretreatment on intracellular reactive oxygen species (ROS) levels after radiation. Figure A shows the results of immunofluorescence staining, and Figure B shows the quantitative box plot.

[0044] Figure 19 This study validates the molecular mechanism by which NOB promotes mitophagy through activation of the NRF2 pathway. Figure A shows the Western blot results, Figure B shows the immunofluorescence staining results, and Figure C shows the quantitative box plot.

[0045] Figure 20 The NRF2 inhibitor ML385 reversed the NOB-induced mitophagy effect;

[0046] Figure 21 This study demonstrates the in vivo protective effect of NRF2 activation against NOB-induced intestinal radiation damage in mice. Detailed Implementation

[0047] This invention provides the application of noriheptacorlin in the prevention and treatment of ionizing radiation damage. Those skilled in the art can refer to the content of this document and appropriately modify the process parameters to achieve the desired result. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments. Those skilled in the art can obviously make modifications or appropriate alterations and combinations to the methods and applications described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention.

[0048] Terminology Explanation:

[0049] Noriheptacorlin (NOB): It belongs to the flavonoid compound class and has a basic C6-C3-C6 skeleton. It is the core active ingredient of this invention.

[0050] Ionizing radiation: refers to radiation that can ionize the atoms or molecules of matter, such as X-rays, which can damage the cells and tissues of organisms, causing problems such as intestinal epithelial damage and bone marrow hematopoietic dysfunction.

[0051] Gastrointestinal acute radiation sickness (GI-ARS): Acute damage to the gastrointestinal system caused by ionizing radiation, mainly manifested as intestinal edema, villus damage, and impaired crypt regeneration.

[0052] Mitochondrophagy: The process by which cells maintain cellular homeostasis by degrading damaged mitochondria, and is an important defense mechanism for cells to cope with damage.

[0053] NRF2: Nuclear factor E2-related factor 2, an important transcription factor, is involved in regulating the expression of genes related to antioxidation, anti-inflammation and cell protection, and plays a key role in radiation damage protection.

[0054] Intestinal crypt cells: A group of cells located in the crypts of the intestinal mucosa, including intestinal stem cells, which are the basis for the regeneration and repair of intestinal epithelial cells.

[0055] 16SRNA sequencing: a molecular biology technique used to analyze the composition and diversity of the gut microbiota.

[0056] This invention provides a radiation protection technology based on the natural compound nobiletin (NOB). This technology has high safety and few side effects, and solves the problem of poor biocompatibility of existing synthetic drugs.

[0057] This invention clarifies the optimal dosing regimen for NOB (including dosage and dosing time) to maximize radiation protection and solves the problems of unclear dosing regimens and unstable protective effects of existing natural product-based protective agents.

[0058] This invention reveals that NOB achieves precise protection of intestinal epithelial cells and intestinal stem cells by activating NRF2 to regulate mitophagy, thus solving the problems of vague protection mechanisms and limited protective effects in existing technologies.

[0059] This invention develops a radiation protection scheme that can effectively improve the symptoms of acute radiation sickness in the gastrointestinal tract and prolong the survival time of radiation-damaged mice, meeting the radiation protection needs in clinical and practical scenarios.

[0060] This invention selects noriheptacorlina (NOB) as the core active ingredient for radiation protection. Its natural source, high biocompatibility, and clearly defined NRF2-mitochondrial autophagy regulatory pathway target are the core foundation for achieving highly efficient and low-toxicity radiation protection. The optimal dosing regimen of "single administration 12 hours before irradiation + 200 mg / kg dose," which has undergone systematic dose and time screening, maximizes the protective effect of NOB and is a key process to ensure radiation protection efficacy. The multi-dimensional protective mechanism, centered on NRF2 signaling pathway activation and linked to mitophagy regulation, ROS scavenging, and DNA damage repair, is the core structure for achieving intestinal epithelial protection. Any competitor seeking equivalent radiation protection must employ the core combination of "natural flavonoid NOB as the active ingredient + specific dosing time and dose + NRF2-mitochondrial autophagy regulatory mechanism," otherwise, the radiation protection effect of this invention cannot be achieved.

[0061] To further verify the technical effects of the present invention, the following technical means were adopted:

[0062] Adding intestinal stem cell stemness testing (OLFM4 staining): The reason is that intestinal stem cells are key to tissue repair and functional recovery after radiation damage. Adding this test can accurately assess the protective effect of NOB on key stem cell populations and further optimize the protection plan.

[0063] Adding NRF2 inhibitor reverse verification experiments: The reason is that by blocking the NRF2 pathway with inhibitors, it can be clearly seen that the protective effect of NOB depends on the activation of NRF2, thereby elucidating the core mechanism of action and providing theoretical support for the stability and reproducibility of the protocol.

[0064] The linkage between in vitro cell experiments and in vivo animal experiments is enhanced because in vitro experiments can clarify the direct protective effect of NOB at the cellular level (such as inhibiting apoptosis and reducing DNA damage), while in vivo experiments can verify its protective effect in the whole organism (such as improving intestinal pathology and promoting blood cell recovery). The combination of the two ensures the comprehensiveness and practicality of the technical solution.

[0065] The validation of multiple radiation dose models (7Gy, 7.5Gy, 10Gy) was added because radiation doses vary in different scenarios. Validation of multiple dose models can broaden the applicability of the technical solution and ensure that it can play an effective protective role under different radiation intensities.

[0066] All the test materials used in this invention are common commercial products and are readily available in the market. Naringenin (Nar) and hesperitin (Hes, used in this cell experiment) were purchased from Mce, while tangeritin (Tan), hesperitin (Hes, used in this cell experiment), nobiletin (NOB), and rutin (Rut) were purchased from Selleck. All flavonoids had a purity of over 95%. The invention is further illustrated below with reference to examples.

[0067] Example 1

[0068] Nar (naringenin), Hes (hesperidin), Rut (rutin), Tan (tangeretin), and NOB (nosperidin) are all flavonoids, all possessing a basic C6-C3-C6 skeleton. These five flavonoids were preliminarily screened out. HCT116 cells were passaged and seeded into 96-well plates when they reached 80% confluence in 10cm culture dishes. 3000-5000 cells were seeded per well. Cell density was observed under a microscope (cells were evenly distributed in the wells, with sufficient space for growth without being too sparse). Cell adhesion was observed 12-24 hours after seeding. Once complete adhesion was achieved, the cells were treated with Nar, Tan, NOB, Hes, and Rut at concentrations of 0μM, 0.98μM, 1.95μM, 3.91μM, 7.81μM, 15.63μM, 31.25μM, 62.5μM, 125μM, 250μM, and 50μM, respectively. Drugs were administered at a concentration of 0 μM, with 3-4 accessory wells for each drug concentration. The dose-response relationship of several flavonoid compounds on HCT116 tumor cells was observed. It was found that the responsiveness of HCT116 cells to different drugs varied with increasing drug concentration. All drugs showed no significant inhibitory effect on HCT116 cells at concentrations below 10 μM. With further increases in drug concentration, drugs such as Tan, NOB, Hes, and Nar showed some inhibitory effect on tumor cells at around 40 μM. Rut showed no significant inhibitory effect on tumor cells, but all showed inhibitory effects on cell proliferation after further increases in drug concentration.

[0069] Following the above experiments, we further explored the effects of these compounds on radiotherapy for colorectal tumors and radiation protection of the intestinal epithelium. Small intestinal organoids are derived from stem cells isolated from the small intestinal crypts and cultured in vitro. They are crucial for analyzing the effects of drugs on intestinal stem cells. We used flavonoid compounds Tan, NOB, Hes, Nar, and Rut in our experiments, selecting drug concentrations of 0 μM, 25 μM, 100 μM, and 200 μM for validation. Drugs were administered 24 hours after plating, and the effect of the drugs on the budding rate of organoids was observed 24 hours after administration. Simultaneously, organoid activity was assessed using MTT staining at 72 hours after administration to evaluate organoid proliferation. We found that the budding rate changed in all treatment groups with increasing dosage. NOB and Tan at 25 μM had a certain inhibitory effect on organoids, manifested as a decrease in budding rate and a reduction in the number of buds per organoid (e.g., ...). Figure 2 As shown in the figure). At 100 μM, the budding of organoids in each group showed significant differences. Tan, NOB, and Nar all showed significant inhibitory effects on organoids, but Hes and Rut had little effect on organoid budding (e.g., Figure 3 As shown in the figure). At 200 μM, Tan had already precipitated into crystals, and organoid growth was significantly inhibited; organoids in the NOB group basically stopped growing and became completely round with the central region turning black; organoids in the Nar group also partially became round, and the buds became shorter; the Hes group showed slight inhibition, but organoids in the Rut group could still sprout normally (as shown in the figure). Figure 4 (As shown). Comprehensive analysis revealed that NOB exhibited the strongest inhibitory effect on the stemness of intestinal epithelial organoids.

[0070] After 72 hours of culture, intestinal organoids were subjected to MTT activity assays. Results are as follows: Figure 5 As shown, the effects of different flavonoid compounds on the activity of small intestinal crypts varied depending on the drug, with NOB exhibiting a significant inhibitory effect on organoids. Among these results, NOB, at concentrations with significant in vitro antitumor effects, induced growth inhibition of intestinal organoids, providing a new reference for in vitro studies of colorectal tumor cells, especially in the study of tumor stem cells. To explore whether its in vivo effects and mechanisms are consistent with those in vitro, and whether an appropriate drug concentration is required for its tumor-inhibiting effect, while also providing a reference for the study of stem cell inhibition mechanisms, we ultimately chose NOB for our study, investigating its mechanism of action on intestinal stem cells.

[0071] Example 2

[0072] The following group experiment is designed, and the experimental procedure is as follows: Figure 6 As shown in Figure A:

[0073] Experiment 1: Screening of Dosing Time: Three dosing time regimens were designed: single gavage administration 24 hours before irradiation, two gavage administrations 24 hours and 12 hours before irradiation, and single gavage administration 12 hours before irradiation. The dosage was 200 mg / kg. A mouse model of whole-body irradiation of 7.5 Gy was constructed. The effect of dosing time was investigated by observing the survival of the mice.

[0074] Experiment 2: Three NOB dosages of 100 mg / kg, 150 mg / kg, and 200 mg / kg were designed. At the same time, a group that only received the excipient (0.5% sodium carboxymethyl cellulose) (Veh group) was set up to construct a mouse model of 10 Gy whole body irradiation. The effect of drug dosage was investigated by observing indicators such as intestinal pathological sections (HE staining) and crypt regeneration.

[0075] The results are as follows Figure 6 As shown in Figures B and C, although the intervention group mice did not survive to 30 days, administering NOB once 24 hours before ionizing radiation (IR) or twice, 24 hours and 12 hours before IR, prolonged the time to death of mice after 7.5 Gy ionizing radiation. Furthermore, administering NOB once 12 hours before irradiation was slightly more effective than administering it twice, 24 hours and 2 hours before irradiation. Preliminary screening suggested that the irradiation dose might be too high; subsequent mouse survival studies will reduce the irradiation dose and select models that are administered NOB 12 hours before irradiation. Simultaneously, HE staining of intestinal pathology 84 hours after IR showed that crypt regeneration was better at 200 mg / kg than at 150 and 100 mg / kg. In conclusion, based on the screening results, we selected animal models administered NOB 12 hours before irradiation at a concentration of 200 mg / kg for systematic research.

[0076] Example 3

[0077] In the in vivo transcriptomics sample delivery experiment, a control group (treated with sodium carboxymethyl cellulose) and a NOB (200 mg / kg) treatment group were set up, with 4 mice in each group. All mice were treated by gavage and samples were collected 12-14 hours later. The small intestinal crypts were collected, and the specific steps are as follows:

[0078] 1) Mice were euthanized by cervical dislocation after being anesthetized with isoflurane; all subsequent procedures were performed on ice.

[0079] 2) Soak the mice in 75% ethanol for 30 seconds, then quickly cut open the abdominal cavity of the mice with intestinal scissors, and open the skin-peritoneal layer in sequence, without damaging the integrity of the intestinal lumen. Remove the small intestine 2 cm below the stomach, bluntly separate the mesentery, and take it 1 cm above the cecum. Place it in a 10 cm culture dish containing pre-cooled sterile PBS.

[0080] 3) Use a sterile 20mL syringe to quickly flush out feces from the intestines. Longitudinally cut open the small intestine to expose the intestinal lumen. Then, wash the intestines sequentially with 2-4 10cm culture dishes containing ice-cold DPBS until no intestinal contents remain in the DPBS.

[0081] 4) Fold both ends of the small intestine in half, and cut the folded section into a 0.5 cm long segment. Slide the segment into a centrifuge tube containing 40 mL of chelation solution, and incubate on ice with a shaker for 40 min. Wash three times with 1× dissociation buffer and let stand for 15 min.

[0082] 5) Wrap the centrifuge tube with three layers of absorbent paper, shake it up and down 100-120 times, filter it with a 70μm filter to remove villous debris, and evaluate the crypt detachment under a microscope. Collect the filtered suspension (containing crypts).

[0083] 6) After inverting the centrifuge, centrifuge at 60g, 4℃, for 5 minutes using a pre-cooled centrifuge;

[0084] 7) Discard the supernatant, leaving a small amount of liquid to prevent the precipitate from becoming loose. Use a 1mL pipette tip to aspirate 0.5mL of DPBS to resuspend the crypt precipitate in a 1.5mL EP tube. Centrifuge at 110g, 4℃, for 5min.

[0085] 8) Discard the supernatant, collect 60 μL of crypt cells from each mouse, add 1 mL of TRIZOL, thoroughly pipette and freeze at -80°C for subsequent transcriptome sequencing.

[0086] In vivo transcriptomics experiments: RNA samples from the small intestinal crypts of different groups were purified, reverse transcribed, library constructed, and sequenced by Shanghai Meiji Biopharmaceutical Biotechnology Co., Ltd. These procedures followed the instructions provided by Illumina, the manufacturer located in San Diego, California. The quality of the sequencing data output and sequencing reads from the RNA samples was evaluated. The reference gene source for this analysis was mice, with the reference genome version Grcm39. The reference genome source can be found at http: / / asia.ensembl.org / Mus_musculus / Info / Index. After screening and quality control of the raw data, the quality of the secondary reverse transcriptome sequencing results was evaluated using HISAT2. Gene and transcript expression levels were quantified separately using RESS software. This software used Deseq2 with the following filtering criteria to analyze gene expression differences between samples: FDR < 0.05 and |log2Fc| ≥ 1 (where Fc represents fold change). In addition, functional condensation analyses, including GO and KEGG analyses, were performed to identify significantly abundant DEGs (differentially expressed genes) in GO terms and signaling pathways. Comparisons were made with the whole transcriptome background using Bonferroni-corrected p≤0.05. GO functional enrichment analysis was performed using Gotools, and KEGG path analysis was performed using KOBAS. All data were analyzed on the MajorBio Cloud Platform (https: / / cloud.majorbio.com / ).

[0087] KEEG signal path analysis results are as follows: Figure 7 As shown, differentially expressed genes in stem cells are significantly enriched in pathways regulating stem cell pluripotency, signal transduction, and cancer-related processes, suggesting that these genes play important roles in maintaining stem cell stemness, regulating cell fate, and participating in disease development (especially tumors). The Hippo pathway showed the most significant differences, and the Wnt pathway also exhibited differences.

[0088] Volcanic map as follows Figure 8 As shown, the study revealed the changes in gene expression profiles induced by NOB treatment, identifying a total of 858 significantly differentially expressed genes, including 410 upregulated genes and 448 downregulated genes. Significant differential expression was observed in core genes such as Egr1 and Aldh1a1. The results indicated that, compared to the control group, several characteristic genes of ISCs, particularly Ifimt3, were significantly reduced in the NOB group.

[0089] Results of stem cell-related gene clustering analysis: Figure 9As shown, the expression levels of stem cell-related genes are also downregulated. The figure indicates that NOB treatment significantly and specifically remodeled the gene expression profile of the samples, forming an expression pattern that is drastically different from that of the control group. The 25 core DEGs can be divided into upregulated and downregulated co-expression modules, among which stem cell and Wnt pathway-related genes such as Lgr5, Myc, and Ascl2 are significantly upregulated.

[0090] To further analyze the role of NOB in vivo, we collected crypt cells from mice in the NOB-treated and control groups and performed RNA-seq.

[0091] Volcano image Figure 10 As shown, NOB treatment induced changes in gene expression profiles, identifying a total of 327 significantly differentially expressed genes, including 196 upregulated genes and 131 downregulated genes. Among them, the significantly differentially expressed core genes such as Aldh1a1, Zbtb16, Reg3b and Reg3g, as well as genes related to retinol metabolism pathways, were observed.

[0092] KEGG analysis results are as follows: Figure 11 As shown, the main differences lie in antigen presentation and Th17 cell progression, which are not directly related to stem cells. KEGG pathway enrichment analysis revealed that differentially expressed genes in the in vivo stem cell model were significantly enriched in immune regulation, longevity / aging, hormone signaling, and core cell signaling pathways, with antigen processing and presentation and longevity regulation being the most prominent enriched pathways. These results reveal the multi-pathway synergistic characteristics of stem cell function regulation in vivo and clarify that the immune-metabolic-stem network is a key target for intervention factors (NOB) in regulating in vivo stem cell function.

[0093] like Figure 12 As shown in the hierarchical clustering heatmap, NOB treatment significantly and specifically remodeled the gene expression profile of stem cells in vivo, resulting in an expression pattern drastically different from the control group. The 30 core DEGs can be divided into upregulated and downregulated co-expression modules, with significant upregulation of stem cell and Wnt pathway-related genes such as Lgr5, Ascl2, and Axin2.

[0094] Example 4

[0095] 1. To investigate the radiation protection potential of nobiletin (NOB) on normal intestinal epithelial cells, we assessed its biosafety and then examined its protective effect on cell survival and proliferation under radiation-induced damage conditions. The specific experimental procedures were as follows: NOB was administered 24 hours after cell plating, followed by ionizing radiation (IR) treatment 12 hours after administration. Fresh culture medium was immediately replaced after irradiation.

[0096] To determine the experimentally safe concentration of NOB, we first treated normal intestinal epithelial cells with different concentrations (1, 5, 10, 20, 50 μM) of NOB under radiation-free (NO-IR) conditions, using the solvent-treated group (Vehicle) as a control. Figure 13 As shown in Figure A, compared with the control group, the cell viability of each NOB treatment group did not change significantly and remained near the baseline level. This result indicates that within the concentration range selected in this experiment, NOB itself has no significant cytotoxic effect on normal intestinal epithelial cells and possesses good biocompatibility. Given the safety of NOB, we further verified whether it could alleviate radiation-induced cell damage. Cells were pretreated with the above-mentioned different concentrations of NOB before receiving ionizing radiation. The results showed ( Figure 13 (Figure B) Compared with the unirradiated group, the cell viability of the Vehicle+IR group was significantly decreased, indicating that the radiation damage model was successfully established. NOB pretreatment significantly improved cell survival after radiation in a concentration-dependent manner. Specifically, the cell viability of the 10, 20, and 50 μM NOB pretreatment groups was significantly higher than that of the radiation control group (***P < 0.001), and at concentrations of 20 μM and 50 μM, cell viability almost recovered to near-normal unirradiated levels. This result suggests that NOB pretreatment can effectively alleviate radiation-induced loss of intestinal epithelial cell viability. However, the instantaneous increase in cell viability does not completely equate to the recovery of long-term proliferative capacity; therefore, we used a colony formation assay to further evaluate the protective effect of NOB on cell regeneration potential. Figure 13 As shown in Figure C, the left image represents representative colony formation, and the right image shows the corresponding quantitative statistics of colony formation rate. Under 0 Gy (no radiation) conditions, there was no significant difference in colony formation ability between the Vehicle group and the NOB pretreatment group, further verifying the safety of NOB. However, after 5 Gy radiation, the colony formation rate of the Vehicle group decreased sharply, while the colony formation rate of the NOB pretreatment group significantly increased. This indicates that NOB pretreatment not only alleviated immediate cell death but, more importantly, protected the proliferation and regeneration potential of intestinal epithelial cells after radiation damage. These results demonstrate that norimethamine pretreatment can significantly inhibit radiation-induced death of normal intestinal epithelial cells, exerting a cytoprotective effect in radiation damage by maintaining cell viability and protecting colony formation ability, and this effect is concentration-dependent.

[0097] 2. To further explore the potential mechanism by which nobiletin (NOB) alleviates radiation-induced cell damage, we used flow cytometry to detect the effect of NOB pretreatment on ionizing radiation (IR)-induced apoptosis in IEC6 cells. Cells were pretreated with NOB for 12 hours before irradiation. The medium was changed immediately after irradiation, and apoptosis was then detected by Annexin V-FITC / PI double staining.

[0098] like Figure 14 As shown in the left-middle figure, the experiment was conducted with four treatment groups: solvent control without radiation (Veh-NO-IR), NOB pretreatment without radiation (NOB-NO-IR), solvent control with radiation (Veh-IR), and NOB pretreatment with radiation (NOB-IR). The results showed that under radiation-free conditions, the proportion of apoptotic cells in both the Veh-NO-IR and NOB-NO-IR groups was extremely low, with no significant difference between them, indicating that NOB itself does not induce apoptosis in IEC6 cells and has good biocompatibility. In contrast, the Veh-IR group showed a significant apoptotic cell population, with a significantly increased proportion of both early and late apoptotic cells, confirming that ionizing radiation can effectively induce apoptosis in IEC6 cells. In the NOB-IR group, the proportion of both early and late apoptotic cells was significantly reduced compared to the Veh-IR group, and the apoptotic cell population was significantly smaller, suggesting that NOB pretreatment can effectively inhibit radiation-induced apoptosis. To further verify these observations, we quantitatively calculated the total apoptosis rate (the sum of the proportions of early apoptotic cells and late apoptotic / necrotic cells) in each group, and the results are shown below. Figure 14 As shown in the right figure, under radiation-free conditions, the apoptosis rate in the Veh-NO-IR group remained at approximately 5% of the normal baseline level, with no significant difference compared to the NOB-NO-IR group, further confirming that NOB itself has no pro-apoptotic effect. After radiation exposure, the apoptosis rate in the Veh-IR group increased sharply to approximately 30%, consistent with the scatter plot results, indicating that radiation caused severe apoptotic damage. In contrast, the apoptosis rate in the NOB-IR group significantly decreased to approximately 18%, a decrease of approximately 40% compared to the Veh-IR group, a statistically significant difference. In conclusion, ionizing radiation can induce a significant apoptotic response in IEC6 cells, while pretreatment with noriheptacortine can effectively inhibit this process and significantly reduce the radiation-induced apoptosis rate.

[0099] 3. DNA double-strand breaks (DSBs) are a key initiation event leading to cell damage and even death caused by ionizing radiation. To investigate the molecular mechanism of the radioprotective effect of nobiletin (NOB), we evaluated the effect of NOB pretreatment on the level of DNA damage in intestinal crypt cells of mice after radiation by detecting γ-H2AX focal spot formation. Mice were pretreated with NOB and then subjected to whole-body ionizing radiation. Intestinal tissue was collected 4 hours after radiation (IR-4h). Immunofluorescence staining was used to label the DNA damage marker γ-H2AX (green fluorescence), and the cell nuclei were counterstained with DAPI (blue fluorescence). The intestinal crypt region was observed in particular.

[0100] Immunofluorescence staining results showed ( Figure 15(Figure A) Four hours after irradiation, dense γ-H2AX green fluorescence signals were observed in the intestinal crypts of mice in the solvent control group, indicating that radiation induced numerous DNA double-strand breaks; while the number of γ-H2AX focal points in the crypts of the NOB pretreated group was significantly reduced. Quantitative statistical analysis further confirmed ( Figure 15 (Figure B) The number of γ-H2AX foci in each crypt cell of the NOB pretreatment group was significantly reduced by approximately 40% compared to the radiation control group, a statistically significant difference. These results indicate that noriheptacorline pretreatment can effectively alleviate radiation-induced DNA double-strand breaks in mouse intestinal crypt cells, suggesting that it may exert a radiation-protective effect on normal intestinal epithelial cells by protecting genomic integrity and blocking the radiation damage cascade at its source. This demonstrates that NOB pretreatment reduces DNA damage in mouse intestinal crypt cells after radiation exposure.

[0101] 4. To evaluate the inhibitory effect of hesperidin pretreatment on radiation-induced early apoptosis of mouse intestinal crypt cells, this study employed multiple techniques for systematic validation at the histological, cellular, and molecular levels. C57BL / 6 mice were pretreated with NOB or a solvent control before receiving whole-body ionizing radiation. Small intestinal tissue was harvested 4 hours post-irradiation. Apoptotic cells were labeled with TUNEL immunofluorescence (red fluorescence), and cell nuclei were counterstained with DAPI (blue fluorescence). The apoptosis status in the intestinal crypt region was observed, and the proportion of TUNEL-positive cells was quantitatively analyzed. Simultaneously, immunohistochemistry was used to detect the expression of activated caspase-3 to assess the activation level of the apoptosis execution pathway. Furthermore, Western blotting was used to detect the protein expression level of cleaved-caspase-3 in different treatment groups, with TUBULIN as an internal control, to validate the regulatory effect of NOB on radiation-induced apoptosis signaling pathways at the molecular level.

[0102] The results showed that pretreatment with noriheptacorline significantly inhibited radiation-induced early apoptosis of mouse intestinal crypt cells. Figure 16 As shown in Figure C, TUNEL immunofluorescence staining revealed that 4 hours after irradiation, the number of TUNEL-positive cells (red fluorescence) in the intestinal crypts and epithelial region of the solvent control group mice was significantly increased, while the number of TUNEL-positive cells in the crypt region of the NOB pretreatment group was significantly reduced. Figure 16 Quantitative statistical analysis of the D-plot further confirmed that the proportion of TUNEL-positive cells in the NOB pretreatment group significantly decreased to approximately 8%, a reduction of about 55% compared to the 18% in the solvent control group, a statistically significant difference. In the validation of the apoptosis execution pathway, Figure 16 Immunohistochemical results from the E-plot showed that extensive Cleaved-caspase-3 positive staining was observed in the intestinal epithelium and crypt regions of the solvent control group, while the positive signal was significantly reduced in the NOB pretreatment group. Figure 16Western blot results from the G-plot further confirmed at the molecular level that NOB pretreatment significantly inhibited radiation-induced upregulation of cleaved-caspase-3 protein expression, and that NOB itself did not induce this protein expression under radiation-free conditions. These results indicate that noriheptacortine pretreatment effectively blocks the radiation-induced apoptosis cascade by inhibiting caspase-3 activation, thereby protecting mouse intestinal crypt cells from radiation damage.

[0103] 5. To explore the molecular mechanism by which hesperidin pretreatment enhances intracellular mitophagy, this study employed Western blot and immunofluorescence colocalization techniques for systematic validation at the molecular and cellular levels. Cells were divided into non-radiated and radiated groups, each further subdivided into solvent control and NOB pretreatment subgroups. Western blot was used to detect the protein expression level of LC3, a core marker of autophagy, with the degree of LC3-I to LC3-II conversion reflecting autophagy activity. Tubulin was used as an internal control protein. Simultaneously, immunofluorescence colocalization was used for visualization analysis of mitophagy: mitochondria were labeled with Mito-Tracker (red fluorescence), autophagosomes with LC3-GFP (green fluorescence), and the cell nucleus with DAPI (blue fluorescence). The occurrence level of mitophagy was assessed by observing the colocalization of red and green fluorescence.

[0104] The results are as follows Figure 17 As shown, pretreatment with noriheptacorlins significantly enhances intracellular mitophagy, especially under radiation stress. Figure 17 In the left-middle panel, under non-radiation conditions, the LC3-II band in the NOB-treated group was significantly stronger than that in the solvent control group, suggesting that NOB can induce autophagy activation at a basal level. Under radiation conditions, the LC3-II expression level in the NOB pretreated group was further increased, significantly higher than that in the solvent control radiation group, indicating that NOB can further enhance the autophagy response under radiation stress. Immunofluorescence colocalization map ( Figure 17 The right-hand side of the figure further validated this finding: in the solvent control group, the co-localization signal between mitochondria and autophagosomes was weak; in the NOB group, the LC3 fluorescence signal was enhanced, and the co-localization with mitochondria was significantly increased; although the solvent control radiation group showed some degree of autophagy activation, co-localization was still limited; while in the NOB pretreated radiation group, the LC3 fluorescence signal was significantly enhanced, forming obvious yellow / orange co-localized punctate aggregates with mitochondria. These results indicate that NOB pretreatment significantly enhances intracellular mitochondrial autophagy levels by promoting the conversion of LC3-I to LC3-II and increasing the co-localization of mitochondria and autophagosomes. This may be one of the important mechanisms by which NOB maintains mitochondrial homeostasis and exerts its cytoprotective role under radiation stress.

[0105] 6. To investigate the effect of noriheptacorline pretreatment on inhibiting radiation-induced intracellular reactive oxygen species (ROS) accumulation, this study systematically validated the effect from both qualitative and quantitative perspectives using immunofluorescence staining combined with quantitative analysis. Cells were pretreated with NOB or a solvent control followed by ionizing radiation. Intracellular ROS were then labeled with ROS-specific fluorescent probes, and the cell nuclei were counterstained with DAPI. The intracellular distribution and fluorescence intensity of ROS were observed using fluorescence microscopy, and the ROS fluorescence intensity of each group was quantitatively and statistically analyzed. The IntDen / Cell no. value reflected the relative content of intracellular ROS.

[0106] The results are as follows Figure 18 As shown, pretreatment with nobiletin significantly inhibited radiation-induced intracellular reactive oxygen species accumulation, demonstrating a strong antioxidant capacity. Figure 18 As shown in the immunofluorescence staining in Figure A, the solvent control irradiation group showed high-intensity and widely distributed green ROS fluorescence signals in the cells, indicating that radiation induced the generation of a large amount of ROS, causing severe oxidative stress; while the NOB pretreated irradiation group showed significantly reduced ROS fluorescence signal intensity and sparse distribution in the cells, indicating that NOB effectively cleared radiation-induced ROS. Figure 18 Quantitative statistical analysis of Figure B further confirmed this observation: the median ROS level in the solvent control radiation group was approximately 300,000 IntDen / Cell, with a wide data distribution, suggesting that radiation induced significant and heterogeneous oxidative stress; while the median ROS level in the NOB pretreated radiation group significantly decreased to approximately 100,000 IntDen / Cell, a reduction of approximately 67% compared to the solvent control group, a statistically significant difference. These results indicate that noriheptacorline pretreatment significantly reduces intracellular oxidative stress levels by effectively scavenging radiation-induced reactive oxygen species, which may be one of the important upstream mechanisms by which it alleviates DNA damage, inhibits apoptosis, and maintains mitochondrial homeostasis.

[0107] 7. To verify the molecular mechanism by which hesperidin promotes mitophagy through activation of the NRF2 pathway, this study systematically validated the mechanism at the molecular expression and subcellular localization levels using nucleocytoplasmic separation Western blot, immunofluorescence staining, and quantitative analysis. Cytoplasmic and nuclear components were separated using a nucleocytoplasmic separation kit. Western blot was used to detect the protein expression distribution of NRF2 in different treatment groups (non-radiation group and 4-hour post-radiation group, each including a solvent control and a NOB pretreatment subgroup), with Lamin B1 used as an internal nuclear reference to correct the loading amount. Simultaneously, immunofluorescence staining was used to analyze the subcellular localization of NRF2: Vehicle and different concentrations of NOB treatment groups were set up. NRF2 protein was labeled with NRF2 antibody (green fluorescence), and the cell nucleus was counterstained with DAPI (blue fluorescence). The nuclear translocation of NRF2 was observed using Merge images. Furthermore, the NRF2 nuclear / cytoplasmic fluorescence intensity ratio in each group was quantitatively analyzed, and the degree of NRF2 nuclear translocation was presented in box plot form.

[0108] The results are as follows Figure 19 As shown, noriheptacorline significantly promotes NRF2 nuclear translocation and activates its transcriptional activity in a concentration-dependent manner. Figure 19 As shown in Figure A, the nuclear-cytoplasmic separation Western blot revealed that under non-radiation conditions, NRF2 in the solvent control group was mainly retained in the cytoplasm, with extremely low levels in the nucleus; while the expression of NRF2 in the nucleus was significantly increased in the NOB pretreatment group. Four hours after radiation, the expression of NRF2 in the nucleus of the NOB pretreatment group was further significantly enhanced, significantly higher than that in the solvent control radiation group, indicating that NOB can further enhance NRF2 nuclear translocation under radiation stress. Figure 19 Immunofluorescence staining in Figure B further validated this finding: in the solvent control group, NRF2 (green) was mainly distributed in the cytoplasm, with weak signal in the nucleus; as the NOB concentration increased from 5 μM to 20 μM and 50 μM, the NRF2 signal in the nucleus increased in a concentration-dependent manner, forming obvious nuclear aggregation. Figure 19 Quantitative statistical analysis of the C-plot confirmed this trend: the NRF2 nucleus / cytoplasm ratio remained at a low level of approximately 0.5 in the solvent control group; it significantly increased to approximately 2 in the NOB 5 μM group; and further increased to approximately 4 and 8 in the NOB 20 μM and 50 μM groups, respectively, all with statistical significance. These results suggest that noriheptacortine activates its transcriptional function by promoting NRF2 nuclear translocation, which may be the upstream molecular mechanism by which it upregulates the expression of mitophagy-related genes and enhances mitophagy activity.

[0109] 8. To verify the molecular mechanism by which noriheptacorlin promotes mitophagy through activation of the NRF2 pathway, this study used the NRF2-specific inhibitor ML385 for reverse verification. Immunofluorescence co-localization technology was used to assess the regulatory role of NRF2 in NOB-induced mitophagy at the cellular level. The experiment was divided into four groups: solvent control radiation group, NOB pretreatment radiation group, ML385 inhibitor radiation group, and NOB and ML385 co-treatment radiation group. Mitochondria were labeled with Mito-Tracker, autophagosomes were labeled with LC3-GFP, and cell nuclei were counterstained with DAPI. The co-localization of mitochondria and autophagosomes was observed using fluorescence microscopy to assess the level of mitophagy.

[0110] The results are as follows Figure 20 As shown, the NRF2-specific inhibitor ML385 significantly reversed the NOB-induced enhanced mitophagy effect under radiation conditions. As indicated by the immunofluorescence colocalization map, in the solvent control radiation group, mitochondria and LC3 showed a certain degree of colocalization, suggesting that radiation induced basal levels of mitophagy as a cellular stress defense response. In the NOB pretreated radiation group, LC3 fluorescence signal was significantly enhanced, and colocalization with mitochondria increased significantly, forming significant yellow / orange dotted clusters, indicating that NOB significantly enhanced mitophagy under radiation conditions. In the ML385 inhibitor radiation group, LC3 fluorescence signal was weakened, and colocalization between mitochondria and autophagosomes was significantly reduced, suggesting that inhibiting NRF2 can reduce radiation-induced mitophagy levels. In the NOB and ML385 co-treated radiation group, both LC3 fluorescence signal and mitochondrial-autophagosome colocalization were significantly reduced, significantly weaker than the NOB pretreated radiation group, recovering to levels close to those of the solvent control radiation group. The above results indicate that ML385 almost completely reversed the enhancing effect of NOB on mitophagy, confirming the molecular mechanism by which NOB promotes mitophagy by activating the NRF2 pathway.

[0111] 9. To verify the key role of NRF2 activation in nobiletin-mediated intestinal radiation protection, this study used the NRF2-specific inhibitor ML385 for in vivo reverse verification. Histological morphological analysis was used to assess the regulatory function of NRF2 in the NOB radiation protection effect at the tissue level. Mice were divided into three groups: solvent control radiation group, NOB pretreatment radiation group, and NOB and ML385 co-treatment radiation group. After receiving the corresponding pretreatment, mice in each group underwent whole-body ionizing radiation. Small intestinal tissue was collected at specific time points after radiation, and H&E staining was performed. Intestinal villus structure, crypt morphology, and epithelial integrity were observed using an optical microscope to assess the degree of radiation-induced intestinal damage and the protective effect of NOB.

[0112] The results are as follows Figure 21As shown, the NRF2-specific inhibitor ML385 significantly reversed the protective effect of NOB against radiation-induced intestinal damage in mice. As indicated by H&E staining, the solvent control radiation group exhibited typical radiation-induced intestinal mucosal damage characteristics: significantly shortened and broken intestinal villi, severely disrupted crypt structures, extensive epithelial cell shedding, and significant inflammatory cell infiltration in the lamina propria, indicating severe structural and functional damage caused by radiation. The NOB pretreatment radiation group, however, showed a significant protective effect: the length and morphology of intestinal villi were essentially restored to normal, the crypt structures remained intact, epithelial cells were neatly arranged, and no significant shedding or inflammatory infiltration was observed, effectively maintaining the integrity of the intestinal mucosal barrier. In the NOB and ML385 co-treatment radiation group, the protective effect of NOB was significantly reversed: intestinal villi again shortened and merged, crypt structures became disordered, epithelial cell shedding and inflammatory cell infiltration reappeared, and the degree of intestinal damage returned to levels close to those of the solvent control radiation group. The above results indicate that inhibiting NRF2 activity almost completely eliminates the intestinal radioprotective effect of NOB, and histologically confirms that NRF2 activation is the core mechanism by which NOB exerts its intestinal radioprotective effect.

[0113] The above are merely preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. Application of noriheptacorlin in the preparation of drugs for the prevention and / or treatment of ionizing radiation damage and related diseases.

2. The application according to claim 1, characterized in that, The ionizing radiation damage includes gastrointestinal damage and / or bone marrow hematopoietic dysfunction.

3. The application according to claim 1, characterized in that, The related diseases include acute radiation sickness of the gastrointestinal tract.

4. The application according to claim 1, characterized in that, The ionizing radiation includes at least one of alpha radiation, gamma radiation, X-ray radiation, and neutron radiation.

5. The application according to claim 4, characterized in that, The dose of ionizing radiation is 5 to 10 Gy.

6. The application according to claims 1 to 5, characterized in that, The effective dose of the nodosumetin, based on mouse body weight, is 100-200 mg / kg.

7. The application according to claims 1-5, characterized in that, The drug is administered once or twice, 12 to 24 hours before ionizing radiation.

8. The application according to claim 6 or 7, characterized in that, The effective dose of the tangeretin is 200 mg / kg, and the drug is administered once 12 hours before ionizing radiation.

9. The application according to claims 1-5, characterized in that, The drug also includes pharmaceutically acceptable excipients; The excipients include one or more of the following: diluent, stabilizer, osmotic pressure regulator, pH regulator, preservative, and antioxidant.

10. The application according to any one of claims 1 to 5, characterized in that, The dosage forms of the drug include injections, oral preparations, or topical formulations; The topical preparations include gels, creams, patches, or sprays.

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