Radioactive intestinal injury protection method based on plant active matters

Abstract

The invention discloses a radioactive intestinal injury protection method based on a plant active matter, which is characterized in that the plant active matter is curcumin (Curmin, Curr) loaded ginger outer vesicles (GDNs), the GDNs are separated and purified by adopting a tissue crushing and over-speed differential centrifugation method, and on the basis, the Curr is loaded through a co-incubation method to prepare GDN-Curr. Before an organism is exposed to high-dose ionizing radiation, the plant active matter is orally taken preventively, so that the synergistic anti-inflammatory and anti-oxidation effects are achieved while the Cur oral bioavailability is promoted, and the radioactive intestinal injury is relieved. Besides, the GDN-Cur can regulate and irradiate the intestinal flora of animals and improve the abundance of the Akkermania muciniphila in the intestinal tract, and the Akkermania muciniphila regulates an AKT / NF-kappa B inflammation signal channel by secreting a metabolite, namely Tanespimycin, 17-AAG, so that the intestinal tissue inflammation induced by ionizing radiation is relieved.

Description

Technical Field

[0001] This invention relates to a method for protecting against radiation-induced intestinal injury based on plant active ingredients, wherein the plant active ingredients are ginger-derived nanovesicles (GDNs) loaded with curcumin (Curcumin), and belongs to the field of radiation injury and intervention technology. Background Technology

[0002] High-dose ionizing radiation (IR) induces acute radiation syndrome characterized by dysfunction of metabolism, hematopoiesis, gastrointestinal tract, and immune system. This is a key focus of clinical oncology radiotherapy and the prevention and treatment of accidental irradiation. Developing effective intervention techniques for IR damage is an urgent problem to be solved in the field of radiation medicine (Cancer J. Clin., 2021, 71:437-454). The intestine is the fastest regenerating tissue in adults and is extremely sensitive to radiation. IR exposure easily leads to radiation-induced intestinal damage (IRID), primarily characterized by intestinal dysfunction (Nat. Rev.Dis. Primers, 2019, 5:13). IRID is a significant factor in accidental irradiation-related deaths and a major cause of radiotherapy interruption and impact on the effectiveness of oncology radiotherapy (90% of patients undergoing abdominopelvic radiotherapy experience gastrointestinal symptoms), seriously threatening the quality of life of patients (Clin. Gastroenterol. Hepatol., 2020, 18:A39-A40). Studies have shown that IRID is associated with oxidative stress, inflammatory responses, and immune dysregulation. Besides directly damaging cells, high-energy radiation induces the generation of reactive oxygen species, leading to intracellular homeostasis imbalance and the release of inflammatory mediators, triggering multiple cell death pathways, and inducing crypt cell death and mucosal inflammation (Protein Cell, 2024, 15:83-97). Prevention and treatment of IRID remains a long-standing unresolved issue, and identifying key protective targets and precise intervention technologies is crucial.

[0003] The gut microbiota contains a diverse community of 10 to 100 trillion microorganisms. Gut microbiota and their metabolites are key factors in gastrointestinal digestion and absorption, playing a crucial role in regulating metabolism and immune homeostasis. Therefore, gut microbiota may be involved in the IRID response (Environ. Res., 2023, 229:115947). Stress can reduce gut microbiota diversity and induce the proliferation of specific bacteria, leading to dysbiosis (Ann. Rheum. Dis., 2025, 84:9-13). Previous studies have shown that gut microbiota plays a key role in IR-induced intestinal damage; for example, pelvic and abdominal radiotherapy can induce the proliferation of pathogenic bacteria such as Proteobacteria, Bacillus, Koala, Roche, and Helicobacter (Exp. Hematol. Oncol., 2023,12:48). On the one hand, the reduction of probiotics weakens the gut's resistance to pathogens, while pathogens can induce tight junction protein rearrangement, disrupting the integrity of the intestinal epithelium. On the other hand, pathogenic bacterial lipopolysaccharides activate inflammatory signaling pathways by inducing macrophage M1 polarization, exacerbating IR-induced intestinal inflammation (Gut Microbes, 2025, 17:2458189). Therefore, maintaining gut microbiota homeostasis is crucial for mitigating IR-induced intestinal oxidative damage and inflammation, and gut microbiota provides a new opportunity for IR damage intervention. Guo et al. reported the roles of Trichophyceae and Enterococciaceae and their downstream metabolites in promoting hematopoiesis and alleviating gastrointestinal syndromes, revealing the key role of microbiota metabolism in IR damage protection (Science, 2020, 370:eaay9097). Clinical and animal studies have confirmed that probiotics such as Lactobacillus and Bifidobacterium can improve intestinal stem cell function, regulate T cell differentiation, and maintain intestinal crypt structure through metabolites represented by short-chain fatty acids (SCFAs) and indole derivatives, effectively alleviating intestinal damage caused by intestinal irritation (IR) (ACS Appl. Mater. Interfaces, 2025, 17:10316-10327; GutMicrobes, 2024, 16:2347722). Therefore, gut microbiota and its active metabolites may serve as potential intervention targets for IRID.

[0004] Plants are the main source of carbon and energy for gut microbiota, and diet is a recognized safe and effective means of regulating gut microbiota. Previous studies have shown that some plant extracts (such as polyphenols, flavonoids, anthocyanins, polysaccharides, and vitamins) have unique pharmacological activities as important dietary components (Med. Res. Rev., 2024, 44:539-567). The radioprotective effects of a large number of plant compounds and their derivatives have been confirmed in in vivo and in vitro models, and they are expected to become economical, safe, and efficient radioprotective agents (Sci. Total Environ., 2021, 779:146431). Among them, curcumin (Cur) is a flavonoid polyphenol extract of the natural traditional Chinese medicine turmeric, and it has broad-spectrum antioxidant, anti-inflammatory, antiviral, and antitumor activities (J Control. Release, 2019, 316:359-380). Previous studies have reported that Curl can protect normal tissues from radiotherapy damage by modulating multiple signaling pathways, thereby enhancing the radiosensitization effect of tumors (Phytother. Res., 2024, 38:464-469). Furthermore, studies have reported that Curl can increase the alpha diversity of gut microbiota, increasing the abundance of Lactobacillus, Serratia, Shewanella, Pseudomonas, Helicobacter, and other bacteria, as well as the level of its metabolite butyrate, maintaining intestinal barrier integrity and inhibiting the progression of colitis (Am.J. Physiol. Cell Physiol., 2017, 312:c438-c445). Therefore, Curl may exert its radioprotective effect by mediating gut microbiota regulation. However, Curl has poor water solubility and stability, is easily hydrolyzed and oxidized in the gastrointestinal tract, and is difficult to be effectively taken up by intestinal cells or microbiota, resulting in low oral bioavailability and limiting its clinical application (J Control. Release, 2019, 316:359-380). Therefore, improving the intestinal bioavailability of ur, reducing the frequency and dosage of administration, and enhancing its interaction with intestinal flora or effector cells have become technical challenges that need to be overcome in ur-based radiation protection strategies.

[0005] In recent years, novel intestinal-targeted nanomedicine delivery strategies have attracted widespread attention for their advantages in drug modification, enhanced activity, and reduced adverse reactions, providing a new breakthrough for improving the oral delivery efficiency of ur (Cur) (ACS Nano, 2025, 19:600-620). However, the non-specific toxicity and high cost of synthetic carriers remain major obstacles to the clinical application of some carriers (J. Control. Release, 2022, 348:335-345). Recently, plant-derived nanovesicles (PDNs) have gradually gained attention. Plants, as "nanofactiochemical factories," may produce bioactive vesicles that could become a new generation of therapeutic tools (Adv. Mater., 2023, 35:e2207826). This technology is gradually becoming a cutting-edge hot topic in biomedical and traditional Chinese medicine research, aligning with the national goal of promoting the inheritance and innovative development of traditional Chinese medicine. PDNs possess both intrinsic pharmacological activity and drug delivery potential, are widely available, simple and economical, and pose no safety risks. Evidence suggests that specific PDNs can act on specific gut bacteria, regulating bacterial gene and protein expression (Cell Host Microbe, 2018, 24:637-652.e8), laying a theoretical foundation for exploring radiation protection strategies based on PDNs-gut microbiota regulation. Among these, ginger-derived nanovesicles (GDNs) and their active ingredients have been shown to possess broad anti-inflammatory, antioxidant, and anticancer activities, effectively reducing intestinal tissue damage (Nat. Commun., 2022, 13:7164). Therefore, loading Cur onto GDNs can enhance intestinal retention and targeted accumulation while simultaneously exerting gut microbiota regulation and synergistic anti-radiation efficacy.

[0006] In summary, addressing the key scientific issue of IRID protection, developing GDN-Cur radiation protection agents with gut microbiota regulation capabilities and exploring novel, economical, safe, efficient, and precise medical protection technologies are of great significance for preventing and mitigating radiation-induced intestinal damage. Summary of the Invention

[0007] This invention addresses the problems of radiation-induced intestinal damage caused by ionizing radiation, the potential biotoxicity of synthetic radiation protectants, and the limited protective effect of single radiation protectants. Based on the close relationship between radiation damage and gut microbiota, this invention provides a radiation-induced intestinal damage protection method based on plant active ingredients, namely, a radiation protection technology based on the regulation of gut microbiota by ginger exovesicles and curcumin, thereby preventing and mitigating radiation-induced intestinal damage.

[0008] To achieve the above-mentioned technical objectives, the technical solution adopted by the present invention is as follows:

[0009] Using ginger as raw material, GDNs are isolated and purified, and Cur is loaded to form a radiation protection agent based on ginger exovesicle complex GDN-Cur. The radiation protection agent is used for prophylactic oral administration before the body is exposed to high doses of ionizing radiation to reduce radiation-induced intestinal damage.

[0010] As a further improved technical solution of the present invention, the synthesis method of GDN-Cur includes the following steps:

[0011] GDNs were separated and extracted using a tissue disruption-differential ultracentrifugation method.

[0012] GDNs were purified using sucrose density gradient centrifugation.

[0013] GDN-Cur was prepared by loading Cur using a co-incubation method.

[0014] As a further improved technical solution of the present invention, the separation and extraction of GDNs using the tissue disruption-differential ultracentrifugation method specifically includes the following steps:

[0015] Fresh ginger was purchased from a farmers' market, thoroughly washed with pure water, and soaked in PBS for 2 minutes to remove surface residues. After processing, the ginger was peeled, and a homogenate of ginger tissue was extracted using a juicer. The tissue fluid was filtered through a 0.7 μm filter membrane to remove residues. The filtered tissue fluid was transferred to 50 mL centrifuge tubes and centrifuged at 4°C for 1000 g × 10 min, 3000 g × 20 min, and 6000 g × 40 min, respectively. The supernatant was collected to remove residues, cell debris, and large particles from the precipitate. The obtained supernatant was transferred to 40 mL ultracentrifuge tubes and centrifuged at 4°C for 140000 g × 150 min. After centrifugation, the supernatant was carefully discarded, and the precipitate was resuspended in 1.5–2 mL of PBS to obtain GDNs.

[0016] As a further improved technical solution of the present invention, the purification of GDNs using the sucrose density gradient centrifugation method specifically includes the following steps:

[0017] Prepare sucrose solutions with concentrations of 60%, 45%, 30%, 10%, and 5%, respectively. Slowly add 5 mL of each concentration solution to a 40 mL ultracentrifuge tube to create a density gradient. Slowly add the GDNs obtained in the above steps to the top of the density gradient sucrose solutions and centrifuge at 4°C for 100,000 g × 180 min. After centrifugation, clear stratification of the solutions at different concentration gradients will appear. Take the sample from the brightest sucrose band (30-45%), transfer it to a new 40 mL ultracentrifuge tube, add 20 mL of PBS, centrifuge at 4°C for 100,000 g × 70 min, discard the supernatant, and resuspend the precipitate in 1.5-2 mL of PBS. Filter the sample using a 0.22 μm syringe filter to obtain purified GDNs.

[0018] As a further improvement of the present invention, the method of loading Cur using co-incubation specifically includes the following steps:

[0019] Cur was dissolved in DMSO and diluted to the corresponding concentration with PBS. A certain concentration of Cur was added to the GDNs obtained in step 2 (Cur mass to GDN protein mass ratio 8:1~1:1), and the mixture was vortexed briefly to mix. The mixture was placed in a water bath at 4~37℃ and incubated for 2~24h. The sample was transferred to a 40mL ultracentrifuge tube and centrifuged at 100000g×70min at 4℃. The supernatant was discarded, and the precipitate was resuspended in 1.5-2mL of PBS to obtain the Cur-loaded exovesicular complex GDN-Cur.

[0020] As a further improved technical solution of the present invention, the mass ratio of Cur:GDNs used in the co-incubation method for loading Cur is 8:1, 4:1 and 1:1 (GDNs are measured by protein mass), the incubation temperature is 4℃, 37℃ and 55℃, and the incubation time is 2h, 4h, 8h and 24h.

[0021] As a further improved technical solution of the present invention, the radiation intestinal injury protection based on oral GDN-Cur is provided. The timing of GDN-Cur administration is from one week before the body receives a large dose of ionizing radiation to immediately after irradiation. The administration method is oral (gavage), and the dosage is 5-20 mg / kg (measured by the concentration of Cur in GDN-Cur), 1-2 times / day.

[0022] As a further improvement of the present invention, the ionizing radiation exposure dose is 1-10 Gy in a single exposure or several exposures in a short period of time, the dose rate is 0.1-2 Gy / min, and the site is whole-body exposure or local exposure in the abdominal and pelvic cavities.

[0023] Compared with existing radiation-induced intestinal injury protection technologies, this invention has significant originality and remarkable technological advancements:

[0024] This invention utilizes next-generation pharmacological carriers, GDNs, to optimize oral administration of Curcuma longa (Cur), opening up a new application area for plant exovesicles. The GDNs drug carrier used in this invention are derived from ginger, a common spice in daily life. Compared to synthetic carriers, they are widely available, environmentally friendly, simple to prepare, and pose no immunogenicity or safety risks. Firstly, as a drug carrier, GDNs exhibit high gastrointestinal stability, preventing the oral drug Curcuma longa from being degraded by gastric acid and digestive enzymes. Inflammation-induced intestinal mucosal inflammation is accompanied by the in-situ aggregation of positively charged proteins, which provide anchoring sites for negatively charged GDNs through electrostatic interactions, promoting specific drug accumulation in the intestine and prolonging drug retention time. Secondly, GDNs themselves carry a large number of plant active ingredients and possess intestinal flora regulation functions. Loading Curcuma longa into GDNs not only improves intestinal retention and targeted accumulation but also exerts intestinal flora regulation and synergistic anti-radiation efficacy. By loading different types of radiation protectants into GDNs, it is possible to prevent and control various radiation injuries based on different mechanisms. This has great application prospects for developing new and efficient radiation intestinal injury protection technologies. It solves the problems of radiation intestinal injury caused by ionizing radiation, the potential biotoxicity of artificially synthesized radiation protectants, and the limited protective effect of single radiation protectants. Attached Figure Description

[0025] Figure 1 (A) is a schematic diagram of the GDN-Cur preparation process.

[0026] Figure 1 Image B shows transmission electron microscopy (TEM) images of GDNs and GDN-Cur.

[0027] Figure 1 (C) shows an atomic force microscope image of GDNs and GDN-Cur.

[0028] Figure 1 The middle (D) diagram shows the hydrated particle size distribution of GDNs and GDN-Cur.

[0029] Figure 1 (E) shows the Zeta potential diagram of GDNs and GDN-Cur.

[0030] Figure 1 The middle (F) diagram shows the lipid distribution of GDNs.

[0031] Figure 1 The middle (G) diagram shows the distribution of protein types in GDNs.

[0032] Figure 1 The middle (H) image is a confocal microscope image of PKH67 fluorescently labeled GDN-Cur.

[0033] Figure 1Figure (I) shows the particle size variation of GDN-Cur at different pH values.

[0034] Figure 1 The graph in (J) shows the encapsulation efficiency of GDNs on Cur under different incubation times.

[0035] Figure 1 The graph in (K) shows the encapsulation efficiency of GDNs on Cur at different incubation temperatures.

[0036] Figure 1 The graph in (L) shows the encapsulation efficiency of GDNs on Cur under different mass ratios.

[0037] Figure 1 The middle (M) image shows in vivo imaging of Cur fluorescence in the gastrointestinal tract and major organs of mice after oral administration of GDN-Cur or Cur for different time periods.

[0038] Figure 1 The graph in the middle (N) is a quantitative analysis diagram of the fluorescence intensity in the gastrointestinal tract.

[0039] Figure 2 The diagram in (A) is a schematic diagram of the process of constructing a gut microbiota clearance mouse and the fecal microbiota transplantation (FMT) experiment.

[0040] Figure 2 The middle (B) diagram shows the effect of gut microbiota clearance on the survival of irradiated mice.

[0041] Figure 2 The middle (C) diagram shows the effect of FMT on the survival of irradiated mice.

[0042] Figure 2 The middle (D) graph shows the effect of FMT on the clinical scores of irradiated mice.

[0043] Figure 2 The middle (E) diagram shows the abdominal anatomy of FMT and irradiated mice.

[0044] Figure 2 The middle (F) diagram shows the length of the colorectal region in FMT and irradiated mice.

[0045] Figure 2 (G) is a schematic diagram of FMT and H&E staining of irradiated mouse tissue pathology.

[0046] Figure 2 Sequencing analysis of fecal 16S rRNA of mice irradiated with (H)GDN-Cur, and relative abundance of bacterial flora.

[0047] Figure 2 (I) is a comparison of the Shannon α diversity index of 16S rRNA sequencing.

[0048] Figure 2The middle (J) plot is the principal coordinate analysis (PCoA) diagram of the genus-level composition of 16S rRNA sequencing.

[0049] Figure 2 The middle (K) diagram is a LefSe analysis of 16S rRNA sequencing.

[0050] Figure 2 The middle (L) plot shows the relative abundance of Akkermansia, Alistipes, and Parabacteroides in 16S rRNA sequencing.

[0051] Figure 3 (A) is a schematic diagram of oral administration and irradiation treatment in mice.

[0052] Figure 3 (B) is a schematic diagram of the survival curves of irradiated mice.

[0053] Figure 3 The middle (C) graph shows the score of the irradiated mice as assessed by clinical indicators.

[0054] Figure 3 The middle (D) graph shows the average feed intake of mice (10 mice / cage) during the observation period.

[0055] Figure 3 Image (E) shows H&E staining of irradiated mouse tissue.

[0056] Figure 3 The middle (F) image shows the immunohistochemical staining of Ki67 and Caspase-3 in the spleen of a mouse.

[0057] Figure 3 The middle (G) plot shows the percentage of Ki67 and Caspase-3 immunohistochemically positive cells in the mouse spleen.

[0058] Figure 3 The diagram in H shows the serum superoxide dismutase (SOD) level in mice.

[0059] Figure 3 (I) is a schematic diagram of mouse serum catalase (CAT) levels.

[0060] Figure 3 The diagram in (J) shows the serum interleukin-1β (IL-1β) level in mice.

[0061] Figure 3 The diagram in K shows the serum interleukin-6 (IL-6) level in mice.

[0062] Figure 4 (A) is an anatomical diagram of the abdomen of an irradiated mouse.

[0063] Figure 4(B) shows a photograph and length measurement of the mouse colon and rectum.

[0064] Figure 4 The middle (C) image shows the H&E staining of mouse colon tissue.

[0065] Figure 4 The middle (D) image shows the H&E staining of mouse small intestinal tissue.

[0066] Figure 4 The middle (E) diagram shows the Western blotting analysis of mouse colon apoptosis protein expression.

[0067] Figure 4 The graph in Figure FI shows the relative expression levels of apoptosis proteins in the mouse colon.

[0068] Figure 4 The figure in the middle (J) is a Western blotting analysis of mouse colon tight junction protein expression.

[0069] Figure 4 The graph in the middle (KM) shows the relative expression levels of tight junction protein in the mouse colon.

[0070] Figure 4 The middle (N) diagram shows the expression of mucin in colon tissue observed by AB / PAS immunostaining.

[0071] Figure 5 The middle (A) is a graph showing the mRNA levels of A. muciniphila in GDN-Cur pretreated mouse fecal samples analyzed by RT-qPCR.

[0072] Figure 5 (B) is a schematic diagram of A. muciniphila gavage and mouse irradiation.

[0073] Figure 5 The middle (C) diagram shows the effect of gavage on the survival of irradiated mice.

[0074] Figure 5 The middle (D) diagram shows the abdominal anatomy of an A. muciniphila-treated irradiated mouse.

[0075] Figure 5 Image (E) shows the colon and rectum of mice treated with A. muciniphila.

[0076] Figure 5 The middle (F) diagram shows a comparison of colorectal length in mice treated with A. muciniphila.

[0077] Figure 5(G) is a schematic diagram of serum IL-1β levels in A. muciniphila-treated irradiated mice.

[0078] Figure 5 The diagram in H shows the serum IL-6 levels in irradiated mice treated with A. muciniphila.

[0079] Figure 5 (I) is a schematic diagram of serum TNF-α levels in irradiated mice treated with A. muciniphila.

[0080] Figure 5 The middle (J) graph shows the clinical symptom scores of mice after irradiation, obtained by treating mice with A. muciniphila cells (pA. muciniphila) or supernatant (sA. muciniphila).

[0081] Figure 5 The diagram in the middle (K) shows the amount of food consumed by mice irradiated by gavage with pA. muciniphila or sA. muciniphila.

[0082] Figure 5 The middle (L) diagram shows the serum SOD level of mice after gavage administration of pA. muciniphila or sA. muciniphila.

[0083] Figure 5 The middle (M) diagram shows the serum CAT levels of mice after gavage administration of pA. muciniphila or sA. muciniphila.

[0084] Figure 5 The diagram in (N) shows the serum MDA level of mice irradiated by gavage with pA. muciniphila or sA. muciniphila.

[0085] Figure 5 (O) is a schematic diagram of H&E staining of mouse tissue after gavage administration of pA. muciniphila or sA. muciniphila.

[0086] Figure 5 The middle (P) diagram shows the H&E staining of mouse colon tissue after gavage with pA. muciniphila or sA. muciniphila.

[0087] Figure 5 The diagram in (Q) shows the H&E staining of mouse small intestinal tissue after gavage administration of pA. muciniphila or sA. muciniphila.

[0088] Figure 5 The diagram in (R) shows a comparison of the length of the small intestinal villi in mice after gavage with pA. muciniphila or sA. muciniphila.

[0089] Figure 6 The image in the middle (AE) is a schematic diagram of the expression and relative quantification of apoptosis-related proteins in the colon tissue of irradiated mice by gavage with *S. muciniphila* and Western blot analysis.

[0090] Figure 6 The middle (F) diagram shows the expression and relative quantification of tight junction protein in the colon tissue of irradiated mice by immunohistochemical analysis using sA. muciniphila via gavage.

[0091] Figure 6 The image in the middle (GH) is a schematic diagram of the observation of irradiated mouse colon goblet cells by gavage administration of *S. muciniphila* and mucin immunostaining.

[0092] Figure 6 The diagram in IK shows the expression of CD86 / CD206 in the colon tissue of irradiated mice by gavage with sA. muciniphila and immunofluorescence analysis.

[0093] Figure 6 The diagram in L shows the relative content of the 30 most expressed metabolites in *S. muciniphila*.

[0094] Figure 6 The diagram in the middle (M) is a schematic diagram of the chemical structure of tanspiramycin (17-AAG).

[0095] Figure 7 A schematic diagram of H&E staining of organ tissues after intraperitoneal injection of 17-AAG in mice.

[0096] Figure 8 (AB) is a schematic diagram of the relative expression and phosphorylation level of AKT in the colon tissue of irradiated mice after intraperitoneal injection of 17-AAG and Western blot analysis.

[0097] Figure 8 The middle (CG) diagram shows the relative expression and phosphorylation levels of IKK-α / β, IκBα, and NF-κB p65 in the colon tissue of irradiated mice after intraperitoneal injection of 17-AAG using Western blot analysis.

[0098] Figure 8The diagram in (HI) shows the relative expression and phosphorylation level of AKT in the colon tissue of irradiated mice after gavage with *S. muciniphila* and Western blot analysis.

[0099] Figure 8 The image (JN) is a schematic diagram showing the relative expression and phosphorylation levels of IKK-α / β, IκBα, and NF-κB p65 in the colon tissue of irradiated mice after gavage with *S. muciniphila* and Western blot analysis.

[0100] Figure 9 This is a schematic diagram of a radiation protection strategy based on ginger exovesicles loaded with curcumin. Detailed Implementation

[0101] The present invention will be further described in detail below with reference to the accompanying drawings and the following embodiments, but this should not limit the scope of the invention. Simple modifications to the present invention under the premise of the present invention's concept are all within the scope of protection claimed by the present invention.

[0102] This invention provides a method for protecting against radiation-induced intestinal injury based on plant active ingredients. Using ginger as the raw material, ginger exovesicles (GDNs) are isolated and purified. Then, the plant active compound curcumin (Cur) is loaded onto these vesicles to form a ginger exovesicle complex (GDN-Cur). Before the body receives high-dose ionizing radiation, this preparation is taken orally prophylactically to reduce radiation-induced intestinal injury. Specifically, this is achieved by regulating the intestinal flora and its metabolites to alleviate ionizing radiation damage, thus achieving the purpose of preventing and reducing radiation-induced intestinal injury. Figure 9 This diagram illustrates a radiation protection strategy based on ginger exovesicles loaded with curcumin. Specifically, curcumin (Cur) is loaded into ginger exovesicles (GDNs) to form composite nanovesicles (GDN-Cur). In addition to its antioxidant, anti-apoptotic, and anti-inflammatory effects, oral administration of this complex can also exert a synergistic anti-radiation effect by regulating the gut microbiota. Furthermore, GDN-Cur can increase the abundance of *A. muciniphila* bacteria in the gut. These bacteria promote macrophage polarization towards M2 by secreting the metabolite 17-AAG and reduce inflammatory responses by inhibiting the activation of the AKT / NF-κB signaling pathway, thereby alleviating ionizing radiation damage.

[0103] Example 1: The above-mentioned ginger exovesicle complex (GDN-Cur) is a radiation protectant based on plant active ingredients, and its preparation method is as follows:

[0104] Step 1: GDNs isolation and extraction:

[0105] As attached Figure 1(A) shows a schematic diagram of the GDN-Cur preparation process. Fresh ginger was purchased from a farmers' market, thoroughly washed with pure water, and soaked in PBS for 2 minutes to remove surface residue. The treated ginger was peeled, and a homogenate of the ginger tissue was extracted using a juicer. The tissue fluid was filtered through a 0.7 μm filter to remove residue. The filtered tissue fluid was transferred to a 50 mL centrifuge tube and centrifuged at 4°C for 1000 g × 10 min, 3000 g × 20 min, and 6000 g × 40 min, respectively. The supernatant was collected to remove residue, cell debris, and large particles from the precipitate. The obtained supernatant was transferred to a 40 mL ultracentrifuge tube and centrifuged at 4°C for 140000 g × 150 min. After centrifugation, the supernatant was carefully discarded, and the precipitate was resuspended in 1.5–2 mL of PBS to obtain GDNs.

[0106] Step 2: GDN purification:

[0107] As attached Figure 1 The schematic diagram of the GDN-Cur preparation process shown in Figure (A) illustrates the purification of GDNs using a sucrose density gradient centrifugation method: Sucrose solutions with concentrations of 60%, 45%, 30%, 10%, and 5% were prepared, and 5 mL of each concentration solution was slowly added to a 40 mL ultracentrifuge tube to create a density gradient. The GDNs obtained in step 1 were slowly added to the top of the density gradient sucrose solution, and the tube was centrifuged at 4°C for 100,000 g × 180 min. After centrifugation, clear stratification of the solutions at different concentration gradients was observed. The sample from the brightest sucrose band (30-45%) was transferred to a new 40 mL ultracentrifuge tube, 20 mL of PBS was added, and the tube was centrifuged at 4°C for 100,000 g × 70 min. The supernatant was discarded, and the precipitate was resuspended in 1.5-2 mL of PBS. The sample was filtered using a 0.22 μm syringe filter to obtain purified GDNs. Protein concentration determination showed that this method can obtain GDNs containing 2 mg of protein from 1 kg of ginger.

[0108] Step 3: Curve loading:

[0109] As attached Figure 1 The schematic diagram of the GDN-Cur preparation process shown in Figure (A) illustrates the co-incubation method for loading Cur: Cur is dissolved in DMSO and diluted to the corresponding concentration with PBS. A certain concentration of Cur is added to the GDNs obtained in step 2 (Cur mass to GDN protein mass ratio 4:1, where GDNs are measured by protein mass), and the mixture is vortexed briefly to mix. The mixture is placed in a 37°C water bath and incubated for 4 hours. The sample is transferred to a 40mL ultracentrifuge tube and centrifuged at 100,000g × 70min at 4°C. The supernatant is discarded, and the precipitate is resuspended in 1.5–2mL of PBS to obtain the Cur-loaded exovesicular complex GDN-Cur.

[0110] Example 2: The preparation method of ginger exovesicle complex (GDN-Cur) is as follows:

[0111] Step 1: GDNs isolation and extraction:

[0112] As attached Figure 1 (A) shows a schematic diagram of the GDN-Cur preparation process. Fresh ginger was purchased from a farmers' market, thoroughly washed with pure water, and soaked in PBS for 2 minutes to remove surface residue. The treated ginger was peeled, and a homogenate of the ginger tissue was extracted using a juicer. The tissue fluid was filtered through a 0.7 μm filter to remove residue. The filtered tissue fluid was transferred to a 50 mL centrifuge tube and centrifuged at 4°C for 1000 g × 10 min, 3000 g × 20 min, and 6000 g × 40 min, respectively. The supernatant was collected to remove residue, cell debris, and large particles from the precipitate. The obtained supernatant was transferred to a 40 mL ultracentrifuge tube and centrifuged at 4°C for 140000 g × 150 min. After centrifugation, the supernatant was carefully discarded, and the precipitate was resuspended in 1.5–2 mL of PBS to obtain GDNs.

[0113] Step 2: GDN purification:

[0114] As attached Figure 1 The schematic diagram of the GDN-Cur preparation process shown in Figure (A) illustrates the purification of GDNs using a sucrose density gradient centrifugation method: Sucrose solutions with concentrations of 60%, 45%, 30%, 10%, and 5% were prepared, and 5 mL of each concentration solution was slowly added to a 40 mL ultracentrifuge tube to create a density gradient. The GDNs obtained in step 1 were slowly added to the top of the density gradient sucrose solution, and the tube was centrifuged at 4°C for 100,000 g × 180 min. After centrifugation, clear stratification of the solutions at different concentration gradients was observed. The sample from the brightest sucrose band (30-45%) was transferred to a new 40 mL ultracentrifuge tube, 20 mL of PBS was added, and the tube was centrifuged at 4°C for 100,000 g × 70 min. The supernatant was discarded, and the precipitate was resuspended in 1.5-2 mL of PBS. The sample was filtered using a 0.22 μm syringe filter to obtain purified GDNs. Protein concentration determination showed that this method can obtain GDNs containing 2 mg of protein from 1 kg of ginger.

[0115] Step 3: Curve loading:

[0116] As attached Figure 1The schematic diagram of the GDN-Cur preparation process shown in Figure (A) illustrates the co-incubation method for loading Cur: Cur is dissolved in DMSO and diluted to the corresponding concentration with PBS. A certain concentration of Cur is added to the GDNs obtained in step 2 (Cur mass to GDN protein mass ratio 8:1, where GDNs are measured by protein mass), and the mixture is vortexed briefly to mix. The mixture is placed in a 4℃ water bath and incubated for 2 hours. The sample is transferred to a 40mL ultracentrifuge tube and centrifuged at 100,000g × 70min at 4℃. The supernatant is discarded, and the precipitate is resuspended in 1.5-2mL of PBS to obtain the Cur-loaded exovesicular complex GDN-Cur.

[0117] Example 3: The preparation method of ginger exovesicle complex (GDN-Cur) is as follows:

[0118] Step 1: GDNs isolation and extraction:

[0119] As attached Figure 1 (A) shows a schematic diagram of the GDN-Cur preparation process. Fresh ginger was purchased from a farmers' market, thoroughly washed with pure water, and soaked in PBS for 2 minutes to remove surface residue. The treated ginger was peeled, and a homogenate of the ginger tissue was extracted using a juicer. The tissue fluid was filtered through a 0.7 μm filter to remove residue. The filtered tissue fluid was transferred to a 50 mL centrifuge tube and centrifuged at 4°C for 1000 g × 10 min, 3000 g × 20 min, and 6000 g × 40 min, respectively. The supernatant was collected to remove residue, cell debris, and large particles from the precipitate. The obtained supernatant was transferred to a 40 mL ultracentrifuge tube and centrifuged at 4°C for 140000 g × 150 min. After centrifugation, the supernatant was carefully discarded, and the precipitate was resuspended in 1.5–2 mL of PBS to obtain GDNs.

[0120] Step 2: GDN purification:

[0121] As attached Figure 1The schematic diagram of the GDN-Cur preparation process shown in Figure (A) illustrates the purification of GDNs using a sucrose density gradient centrifugation method: Sucrose solutions with concentrations of 60%, 45%, 30%, 10%, and 5% were prepared, and 5 mL of each concentration solution was slowly added to a 40 mL ultracentrifuge tube to create a density gradient. The GDNs obtained in step 1 were slowly added to the top of the density gradient sucrose solution, and the tube was centrifuged at 4°C for 100,000 g × 180 min. After centrifugation, clear stratification of the solutions at different concentration gradients was observed. The sample from the brightest sucrose band (30-45%) was transferred to a new 40 mL ultracentrifuge tube, 20 mL of PBS was added, and the tube was centrifuged at 4°C for 100,000 g × 70 min. The supernatant was discarded, and the precipitate was resuspended in 1.5-2 mL of PBS. The sample was filtered using a 0.22 μm syringe filter to obtain purified GDNs. Protein concentration determination showed that this method can obtain GDNs containing 2 mg of protein from 1 kg of ginger.

[0122] Step 3: Curve loading:

[0123] As attached Figure 1 The schematic diagram of the GDN-Cur preparation process shown in Figure (A) illustrates the co-incubation method for loading Cur: Cur is dissolved in DMSO and diluted to the corresponding concentration with PBS. A certain concentration of Cur is added to the GDNs obtained in step 2 (Cur mass to GDN protein mass ratio 1:1, where GDNs are measured by protein mass), and the mixture is vortexed briefly to mix. The mixture is placed in a 55℃ water bath and incubated for 24 h. The sample is transferred to a 40 mL ultracentrifuge tube and centrifuged at 4℃ for 100,000 g × 70 min. The supernatant is discarded, and the precipitate is resuspended in 1.5–2 mL of PBS to obtain the Cur-loaded exovesicular complex GDN-Cur.

[0124] Example 4: The preparation method of ginger exovesicle complex (GDN-Cur) is as follows:

[0125] Step 1: GDNs isolation and extraction:

[0126] As attached Figure 1(A) shows a schematic diagram of the GDN-Cur preparation process. Fresh ginger was purchased from a farmers' market, thoroughly washed with pure water, and soaked in PBS for 2 minutes to remove surface residue. The treated ginger was peeled, and a homogenate of the ginger tissue was extracted using a juicer. The tissue fluid was filtered through a 0.7 μm filter to remove residue. The filtered tissue fluid was transferred to a 50 mL centrifuge tube and centrifuged at 4°C for 1000 g × 10 min, 3000 g × 20 min, and 6000 g × 40 min, respectively. The supernatant was collected to remove residue, cell debris, and large particles from the precipitate. The obtained supernatant was transferred to a 40 mL ultracentrifuge tube and centrifuged at 4°C for 140000 g × 150 min. After centrifugation, the supernatant was carefully discarded, and the precipitate was resuspended in 1.5–2 mL of PBS to obtain GDNs.

[0127] Step 2: GDN purification:

[0128] As attached Figure 1 The schematic diagram of the GDN-Cur preparation process shown in Figure (A) illustrates the purification of GDNs using a sucrose density gradient centrifugation method: Sucrose solutions with concentrations of 60%, 45%, 30%, 10%, and 5% were prepared, and 5 mL of each concentration solution was slowly added to a 40 mL ultracentrifuge tube to create a density gradient. The GDNs obtained in step 1 were slowly added to the top of the density gradient sucrose solution, and the tube was centrifuged at 4°C for 100,000 g × 180 min. After centrifugation, clear stratification of the solutions at different concentration gradients was observed. The sample from the brightest sucrose band (30-45%) was transferred to a new 40 mL ultracentrifuge tube, 20 mL of PBS was added, and the tube was centrifuged at 4°C for 100,000 g × 70 min. The supernatant was discarded, and the precipitate was resuspended in 1.5-2 mL of PBS. The sample was filtered using a 0.22 μm syringe filter to obtain purified GDNs. Protein concentration determination showed that this method can obtain GDNs containing 2 mg of protein from 1 kg of ginger.

[0129] Step 3: Curve loading:

[0130] As attached Figure 1 The schematic diagram of the GDN-Cur preparation process shown in Figure (A) illustrates the co-incubation method for loading Cur: Cur is dissolved in DMSO and diluted to the corresponding concentration with PBS. A certain concentration of Cur is added to the GDNs obtained in step 2 (Cur mass to GDN protein mass ratio 4:1, where GDNs are measured by protein mass), and the mixture is vortexed briefly to mix. The mixture is placed in a 37°C water bath and incubated for 8 hours. The sample is transferred to a 40mL ultracentrifuge tube and centrifuged at 100,000g × 70min at 4°C. The supernatant is discarded, and the precipitate is resuspended in 1.5–2mL of PBS to obtain the Cur-loaded exovesicular complex GDN-Cur.

[0131] The various characteristics of GDN-Cur in Example 1 are analyzed below.

[0132] Analysis Example 1: Physicochemical Properties Analysis of Vesicles:

[0133] Transmission electron microscopy (TEM) and atomic force microscopy (AFM) analyses showed that the isolated and purified GDNs possessed a typical spherical membrane structure. Dynamic light scattering (DLS) analysis revealed a hydrated particle size of 168.2 ± 9.0 nm (PDI = 0.21) and a negative charge (-17.6 ± 0.67 mV). Cur loading did not alter the morphology and size of the vesicles, but due to the inherent negative charge of Cur, the zeta potential of GDN-Cur increased (-25.6 ± 1.02 mV). The TEM, AFM, hydrated particle size, and zeta potential diagrams are attached. Figure 1 As shown in (B)-(E).

[0134] Analysis Example 2: Analysis of the bioactive components of GDNs:

[0135] The protein concentration of GDNs was determined using a BCA protein assay kit. For lipidomics analysis, total lipids were extracted from GDNs using a mixture of methyl tert-butyl ether (MTBE) and methanol (5:1, v / v). The lipid composition of the samples was determined by liquid chromatography-tandem mass spectrometry (HPLC-MS / MS), and the data are expressed as a percentage of the total signal of each lipid relative to the identified molecular species. The results showed that GDNs were rich in fatty acyl groups (FA, ~26%) and glycerophospholipids (GP, ~20%) associated with the lipid bilayer structure. For proteomics analysis, the extracted protein samples were analyzed by HPLC-MS / MS, and the results were compared with the COG protein database. The results showed that GDNs had low protein content and were mainly composed of proteins related to translation, biosynthesis, post-translational modification, and protein metabolism. Vesicles contained approximately 36% uncharacterized proteins, indicating that they may have undiscovered functions. The distribution map of lipid and protein species in GDNs is attached. Figure 1 As shown in (F)-(G).

[0136] Analysis Example 3: Vesicle Stability Analysis:

[0137] GDNs were suspended in PBS at pH 1.2 or 7.2 at 37°C to simulate an acidic environment. Results showed that the vesicles exhibited high stability in the acidic environment (pH 1.2), with minimal particle size change after 8 hours (281.2 nm) and no significant aggregation (PDI = 0.22). The particle size change of the vesicles over incubation time is shown in the attached figure. Figure 1 As shown in (I).

[0138] Analysis of Example 4: Loading Efficiency Analysis

[0139] GDNs were labeled with PKH67 fluorescently. Observation using confocal laser scanning microscopy (CLSM) showed that the green fluorescence of PKH and the red fluorescence of Cur co-localized, resulting in an orange color, indicating successful Cur loading. The Cur loading conditions were adjusted and optimized. Free Cur was removed by ultracentrifugation. GDN-Cur was lysed using protein lysis buffer (4°C, 10 min). The Cur content was determined by UV-Vis spectrophotometry at 450 nm, and the Cur concentration in GDN-Cur was calculated based on the standard curve. The results showed that an incubation at 37°C for 4 hours with a GDN to Cur mass ratio of 1:4 was the optimal loading strategy, achieving an encapsulation efficiency of approximately 30% for Cur. The confocal fluorescence scan of GDN-Cur is attached. Figure 1 As shown in Figure (H), the comparison of Cur encapsulation efficiency under different loading conditions is attached. Figure 1 As shown in (J)-(L).

[0140] Analysis Example 5: Organizational Distribution Analysis:

[0141] Adult male Balb / c mice (6 weeks old) were fasted for 4 hours and then orally administered 200 μL of Cur (1.2 mg / mL) or GDN-Cur (protein content 1 mg / mL) by gavage. Mice were sacrificed at different time points, and the digestive tract and major organs were collected for imaging using the IVIS Spectrum imaging system. Results showed that GDN-Cur was mainly distributed in the gastrointestinal tract after gavage, accumulating in the stomach, small intestine, ileum, and colon, and could cross the intestinal barrier into the circulatory system, reaching the liver and kidneys. Using GDNs as drug carriers significantly prolonged the accumulation and retention time of Cur in the intestine; Cur fluorescence in the intestines of GDN-Cur group mice was still detectable 24 hours later. In vivo imaging images of Cur fluorescence in organs at different time points after gavage and quantitative analysis of gastrointestinal fluorescence intensity are attached. Figure 1 As shown in (M)-(N).

[0142] Analysis of Example 6: Analysis of the regulatory effect of GDN-Cur on gut microbiota in IR-exposed mice:

[0143] First, the relationship between IR damage and gut microbiota was verified using a gut microbiota clearance mouse model, as shown in the attached figure. Figure 2The diagram in Figure (A) illustrates the process of constructing a gut microbiota-clearing mouse model. SPF-grade adult male Balb / c mice (6 weeks old) were administered 100 μL of a quadruple antibiotic regimen (ABX, penicillin 200 mg / kg, neomycin 200 mg / kg, metronidazole 200 mg / kg, vancomycin 100 mg / kg) daily by gavage for 5 consecutive days to clear the gut microbiota. Twelve hours after the last gavage, the mice were irradiated with a single dose of 4.0 Gy (108.6 cGy / min) of X-rays, and the 30-day survival rate was observed. The results showed that gut microbiota clearance significantly reduced the survival rate of irradiated mice, leading to the death of all mice within 9 days of IR exposure (see attached diagram). Figure 2 B) indicates that gut microbiota is closely related to IR damage.

[0144] Secondly, the effects of GDN-Cur on gut microbiota regulation and radiation protection were evaluated using a fecal microbiota transplantation (FMT) experiment, as shown in the attached figure. Figure 2 The FMT workflow is shown in Figure (A). Donor mice (SPF-grade adult male Balb / c mice, 6 weeks old) were administered PBS or GDN-Cur via gavage daily for one week (Cur concentration: 12 mg / kg, GDNs: 10 mg / kg, measured by vesicle protein content), and their fresh feces were collected on day 7. The feces were suspended in physiological saline (40 mg feces / mL), filtered through a 0.25 mm sieve to remove residues, and then mixed with 10% autoclaved glycerol. Recipient mice were gut microbiota-cleared mice and were administered 200 μL of the above donor fecal mixture via gavage daily for 5 consecutive days. 24 hours after the last gavage, the FMT recipient mice were subjected to whole-body X-ray irradiation (4.0 Gy). The survival of the mice was tracked for 30 days after radiation exposure. Seven physiological indicators of the mice were monitored, and the clinical score of the mice was calculated. Experimental mice were sacrificed on day 7 post-irradiation or when in a critical condition. Organ samples were collected, weighed, fixed in 10% paraformaldehyde, and sectioned in paraffin for H&E histopathological staining. Results showed that, after irradiation exposure, recipient mice receiving GDN-Cur-treated feces (GDN-Cur-Recip) had a significantly higher survival rate (70% vs. 50%) compared to recipient mice receiving PBS gavage (NC-Recip). Figure 2 Medium (C) and lower clinical scores (with) Figure 2 (D)). GDN-Cur-FMT significantly prevented IR-induced intestinal bleeding and edema (see appendix). Figure 2 (E)), to alleviate the shortening of colon length caused by IR (Appendix) Figure 2 (F)), and reduced pathological damage to the colon, liver, spleen and bone marrow tissues (Appendix) Figure 2 (G)). This suggests a potential role for gut microbiota in GDN-Cur-mediated radioprotection.

[0145] Finally, the differential gut microbiota induced by GDN-Cur in irradiated mice was analyzed. Fresh feces were collected from GDN-Cur-pretreated mice on day 5 post-irradiation. The variable region of the bacterial 16S rRNA gene in the fecal DNA was amplified by PCR. Sequencing was performed using an Illumina NovaSeq sequencer, and bacterial sequence fragments were analyzed using QIIME 1.8.0 software. The data were processed using t-tests, Wilcoxon rank-sum tests, and Tukey tests to analyze the differences between α-diversity indices and β-diversity indices. Histograms showed significant differences in gut microbiota abundance (see attached). Figure 2 (H)). The Shannon index indicates that the IR-induced decrease in α-diversity was significantly reversed by GDN-Cur (see appendix). Figure 2 (I)). PCoA analysis showed significant differences in bacterial community structure among the three groups of mice (see appendix). Figure 2 (J)). Specifically, the abundance of bacteria such as Akkermansia, Alistipes, and Parabacteroides was significantly higher in the GDN-Cur+IR group than in the IR group (see appendix). Figure 2 (K), (L)). This illustrates the crucial role of GDN-Cur in regulating IR exposure-induced gut microbiota homeostasis.

[0146] This invention also provides a radiation protection technology based on GDN-Cur, and the radiation protection applications of GDN-Cur are described below.

[0147] Application Example 1: Application of GDN-Cur in whole-body radiation damage protection:

[0148] As attached Figure 3The diagram in Figure (A) illustrates the oral administration and irradiation treatment of mice. Adult male Balb / c mice (6 weeks old) were administered Cur (12 mg / kg), GDNs (10 mg / kg, measured by vesicle protein content), or an equivalent dose of GDN-Cur via gavage before irradiation, once daily for one week. Subsequently, the mice were irradiated with a single dose of 4.0 Gy (108.6 cGy / min) of X-rays to establish an acute radiation syndrome (ARS) animal model. Mice survival was monitored for 30 days post-irradiation. Food intake and other physiological parameters were monitored using metabolic cages, and clinical scores were calculated using seven physiological indicators. Mice were euthanized on day 7 post-irradiation or when in a critical condition, and ocular blood and organ samples were collected. Blood was centrifuged at 3,000 g for 20 minutes at 4°C, and the supernatant was collected. Serum antioxidant enzyme levels were assessed using SOD and CAT detection kits. For tissue samples, mouse organs were rinsed with physiological saline, weighed, fixed with 10% paraformaldehyde, and sectioned in paraffin for H&E histopathological staining or Ki67 and Caspase-3 immunohistochemical staining. Results showed that, compared with the irradiation-only group (IR) (60% survival rate), prophylactic intervention significantly improved mouse survival, especially since almost all mice pretreated with GDN-Cur (19 out of 20) survived within 30 days after irradiation, highlighting its synergistic protective potential (see appendix). Figure 3 (B)). Pretreated GDN-Cur mice showed significantly lower clinical scores than the IR, GDNs, or Cur groups (see attached). Figure 3 (C)). GDN-Cur preventive intervention can significantly alleviate IR-induced reduction in dietary intake (see appendix). Figure 3 (D) IR exposure can cause colon cell necrosis and crypt epithelial cell shedding, spleen atrophy and lymphopenia, hepatocyte swelling and diffuse vacuolar degeneration, bone marrow interstitial damage and nucleated cell reduction in mice. Prophylactic gavage with GDN-Cur effectively alleviates the damage to these organs and tissues, and its protective effect is stronger than that of GDNs or Cur alone, suggesting a synergistic anti-radiation effect (see appendix). Figure 3 (E)). GDN-Cur treatment of mice reduced the expression of the apoptosis protein Caspase-3 in spleen tissue, while enhancing the proliferation-related Ki67 staining, further indicating reduced spleen damage and maintenance of hematopoietic function (see appendix). Figure 3 (F) and (G)). Serum enzyme activity levels were measured using commercially available diagnostic kits. The results showed that IR-induced SOD and CAT depletion was significantly alleviated under preventative intervention, especially in the GDN-Cur group, where levels approached normal. (See attached image). Figure 3 (H), (I)). Serum inflammatory factor analysis showed that GDN-Cur significantly reduced IR exposure-induced IL-6 and IL-1β levels (see appendix). Figure 3(J), (K)). Therefore, Cur-loaded GDNs exhibit excellent synergistic anti-inflammatory and antioxidant properties, protecting mice from IR-induced systemic damage. Survival curves, clinical scores, intake levels, tissue H&E staining, immunohistochemical staining, and serum antioxidant enzyme and inflammatory factor levels in irradiated mice are shown in the attached figures. Figure 3 As shown in (B)-(K).

[0149] Application Example 2: Application of GDN-Cur in Radiation Intestinal Injury Protection

[0150] As attached Figure 3 The diagram in Figure (A) illustrates the oral administration and irradiation treatment of mice, evaluating the protective effect of GDN-Cur against radiation-induced intestinal injury. For colonic mucin immunostaining, frozen sections of colonic tissue were stained using the AB / PAS kit. Administration of GDN-Cur significantly prevented intestinal bleeding and edema induced by whole-body irradiation (see attached diagram). Figure 4 (A) reduced IR-induced colon length shortening (Appendix) Figure 4 In the middle (B) group, the effect was better than that of the GDNs or Cur groups alone. Pathological analysis showed that the colonic tissue structure of mice pretreated with GDN-Cur was almost normal, while mice in other groups showed severe tortuosity and crypt degeneration, and there was a significant gap between the base and the muscularis mucosae (see appendix). Figure 4 (C)). Furthermore, GDN-Cur pretreatment significantly alleviated IR-induced small intestinal villus flattening and atrophy (see appendix). Figure 4 (D) indicates the synergistic protective effect of GDN-Cur. Studies on colonic tissue apoptosis cascade proteins revealed that prophylactic interventions, particularly prophylactic gavage with GDN-Cur, effectively reversed IR-induced depletion of the anti-apoptotic protein BCL-2, while simultaneously reducing the expression of pro-apoptotic proteins (BAX, PARP, Caspase-3) (see Appendix). Figure 4 (E)-(I). Intestinal barrier function analysis results showed that IR exposure damaged the intestinal mucosal barrier function, leading to a significant downregulation of tight junction proteins (Occludin, Claudin-1, ZO-1) expression in colonic tissue. However, GDN-Cur prophylactic intervention significantly alleviated this damage (see appendix). Figure 4 (J)-(M)). Furthermore, goblet cells, as the main secretory cells of the intestinal mucus layer, can protect the intestine by resisting the invasion of intestinal microorganisms. GDN-Cur can effectively alleviate the reduction of colonic goblet cells caused by IR exposure (see appendix). Figure 4 (N). Anatomical diagrams of the irradiated mouse abdomen, colorectal photographs, histopathological H&E staining, Western blot analysis of apoptosis protein and colonic tight junction protein expression, and immunostaining images of mucin proteins in colonic tissue are attached. Figure 4 As shown.

[0151] Application Example 3: Application of GDN-Cur-associated specific bacteria A. muciniphila in radiation-induced intestinal injury protection:

[0152] Based on the results of Example 6 above, GDN-Cur has a significant regulatory effect on the intestinal flora of IR-exposed mice. Compared with the irradiation group alone, the abundance of Akkermansia bacteria in irradiated mice receiving prophylactic GDN-Cur gavage was significantly increased, suggesting that this genus may have a protective effect against radiation-induced intestinal injury. We plan to select *A. muciniphila* from this genus as the research subject to explore its application in the protection against radiation-induced intestinal injury. On the 5th day after irradiation, fresh feces were collected from mice pretreated with GDN-Cur. Fecal RNA was extracted using the RNeasy mini kit, cDNA was synthesized using PrimeScript™ RT Master Mix, and the expression level of *A. muciniphila* in feces was evaluated using RT-qPCR. Primers: 5'-CAGCACGTGAAGGTGGGGAC-3' (forward) and 5'-CCTTGCGGTTGGCTTCAGAT-3' (reverse). The results showed that the abundance of *A. muciniphila* in the feces of irradiated mice receiving prophylactic GDN-Cur intervention was significantly increased (see attached). Figure 5 (A) indicates that GDN-Cur can promote the growth of this bacterium.

[0153] The following describes the radiation protection effects of A. muciniphila: (see attached image) Figure 5 The diagram in (B) shows the gavage administration of A. muciniphila and the irradiation of mice. Adult male Balb / c mice (6 weeks old) were administered A. muciniphila (1×10⁻⁶) by gavage before irradiation. 9Mice were administered 200 μL of pasteurized bacterial cells (pA. muciniphila) or equivalent doses of bacterial culture supernatant (sA. muciniphila) via gavage for one week. Following this, mice were irradiated with a single dose of 4.0 Gy (108.6 cGy / min) of X-rays. Mice survival was monitored for 30 days post-irradiation. Food intake and other physiological parameters were monitored using metabolic cages, and clinical scores were calculated using seven physiological indicators. Mice were euthanized on day 7 post-irradiation or when in critical condition, and ocular blood and organ samples were collected. Blood samples were centrifuged at 3,000 g for 20 minutes at 4°C, and the supernatant was collected. Serum antioxidant enzyme levels were assessed using SOD, CAT, and MDA assay kits, and serum IL-1β, IL-6, and TNF-α levels were detected using ELISA. For tissue samples, mouse organs were rinsed with physiological saline, weighed, fixed with 10% paraformaldehyde, and sectioned in paraffin for H&E histopathological staining. Results showed that prophylactic gavage of *A. muciniphila* significantly improved the survival rate of irradiated mice (80% vs. 60%). Figure 5 (C) ), to reduce intestinal bleeding and edema (attached) Figure 5 (D) ), restore colon and rectum length (attached) Figure 5 (E), (F)), reduce serum inflammatory factor levels (IL-1β, IL-6 and TNF-α, appendix) Figure 5 The values ​​(G)-(I) indicate that *A. muciniphila*, as a specific strain regulated by GDN-Cur, exhibits protective efficacy against radiation-induced intestinal injury. The effects of *A. muciniphila* gavage on the survival of irradiated mice, abdominal anatomy, comparison of colorectal length, and serum IL-1β, IL-6, and TNF-α levels are shown in the attached figures. Figure 5 As shown in (C)-(I).

[0154] The following analysis distinguishes whether the radiation-protective effect of *A. muciniphila* is mediated by the bacterial cells themselves or by its secreted active metabolites: Compared to pasteurized bacterial cells (p. muciniphila), pretreatment of mice with the supernatant of *A. muciniphila* (sA. muciniphila) was more effective in reducing the clinical scores of irradiated mice (see appendix). Figure 5 (J)), increase dietary intake (attached) Figure 5 (K) increases serum levels of antioxidant enzymes SOD and CAT (see appendix) Figure 5 (L), (M)), reduce lipid peroxidation product MDA (attached) Figure 5 (N)), to reduce pathological damage to liver, spleen, and bone marrow tissues (attached) Figure 5In addition, the degree of colonic epithelial cell necrosis and mucosal damage in the sA. muciniphila+IR group was significantly lower than that in the pA. muciniphila+IR group (see appendix). Figure 5 (P) and can significantly alleviate IR-induced small intestinal villus atrophy (see appendix) Figure 5 (Q) and (R)). Therefore, the above analysis demonstrates that the protective activity of *A. muciniphila* against radiation-induced intestinal injury may mainly derive from its secreted active substances. Clinical symptom scores, food intake, serum SOD, CAT, MDA, histopathological H&E staining, and small intestinal villus length comparisons of mice after irradiation are shown in the attached figure. Figure 5 As shown in (J)-(R).

[0155] The following describes the mechanism of action of *A. muciniphila* bacterial culture supernatant (sA. muciniphila) in protecting against radiation-induced intestinal injury: Prophylactic gavage of sA. muciniphila significantly alleviated the IR-induced increase in the expression of apoptotic proteins (Caspase-3, BAX, PARP) and the depletion of the anti-apoptotic protein BCL-2 (see appendix). Figure 6 (A)-(E)), reversing IR-induced downregulation of key tight junction proteins (ZO-1, Claudin-1, Occludin) in colonic tissue (see appendix) Figure 6 (F)), to maintain the number of colonic goblet cells in irradiated mice (attached) Figure 6 (G) and (H) showed that *S. muciniphila* effectively protected the intestinal barrier function of irradiated mice. Furthermore, the effect of *S. muciniphila* on intestinal macrophage polarization was investigated using immunofluorescence. The results showed that the colon tissue of the IR group had a higher number of CD86-positive cells, while a significant increase in CD206 expression was observed in the *S. muciniphila* pretreated group (see appendix). Figure 6 The expression (I)-(K) indicates that *A. muciniphila* promotes the transformation of macrophages from M1 to M2 polarization. Therefore, the active substances secreted by *A. muciniphila* mainly exert their protective effect against radiation-induced intestinal injury through antioxidant, anti-inflammatory, and anti-apoptotic effects, as well as maintaining the intestinal mucosal barrier. The expression of apoptosis-related proteins, tight junction proteins, goblet cells, and CD86 / CD206 in the colonic tissue of irradiated mice is shown in the attached figure. Figure 6 As shown in (A)-(K).

[0156] The following analysis examines the bioactive substances secreted by *A. muciniphila*. Non-targeted metabolomics analysis of the culture supernatant of *A. muciniphila* identified 2426 metabolites. Figure 6The middle (L) section shows the top 10 metabolites by relative abundance. For subsequent single association analysis, metabolite selection was primarily based on: ① abundant expression in *S. muciniphila*; ② biological annotation indicating a possible radiation-induced reaction; and ③ availability of standard substances for functional analysis. The results revealed an enrichment of a novel macrolide compound, 17-AAG, in *S. muciniphila*. 17-AAG is a chemical analog of gledycin (see appendix). Figure 6 M), an HSP90 inhibitor, has shown broad antitumor activity in clinical trials. Studies have shown that 17-AAG can enhance the sensitivity of tumor cells to radiotherapy (IR), thus providing potential for targeted radiotherapy of cancer. The relative amounts of the 30 most expressed metabolites in *S. muciniphila* and the chemical structure of 17-AAG are attached. Figure 6 As shown in (L) and (M).

[0157] In summary, the above studies demonstrate that the specific GDN-Cur-associated bacterium *A. muciniphila* has a protective effect against radiation-induced intestinal injury. Its protective effect may be achieved by secreting metabolites (such as 17-AAG) and acting on intestinal cells (such as intestinal macrophages), indicating the potential application of *A. muciniphila* in radiation protection.

[0158] Application Example 4: Application of 17-AAG, a metabolite of GDN-Cur-associated specific bacterium *A. muciniphila*, in radiation protection:

[0159] Based on the analysis of application example 3 above, 17-AAG is likely a functional metabolite secreted by *A. muciniphila*. The following analysis examines the anti-radiation activity of 17-AAG and evaluates its application in radiation protection. Mice were intraperitoneally injected with 17-AAG (50 mg / kg, once daily) for three consecutive days prior to IR exposure. Histopathological analysis showed that prophylactic administration of 17-AAG significantly reduced IR-induced tissue damage to the colon, small intestine, liver, and spleen (see attached). Figure 7 This demonstrates its radiation protection function.

[0160] The following analysis examines the signaling mechanism mediated by the protective effect of 17-AAG: 17-AAG is known to inhibit the function of molecular chaperones involved in signal transduction (including PI3K / AKT, MEK / ERK, and NF-κB pathway proteins) by binding to the conserved domain of HSP90. Studies have shown that 17-AAG inhibits the pro-inflammatory activity of M1 macrophages by suppressing the NF-κB and MAPK pathways. On the other hand, the AKT / NF-κB pathway is crucial for regulating cellular radiation-induced responses. IR exposure leads to potent AKT activation, which subsequently phosphorylates the key downstream substrate IKK complex, prompting the release of NF-κB from its inhibitors (IκBα and IκBβ) and regulating target genes. To determine whether the radioprotective effect of 17-AAG is related to the regulation of the AKT / NF-κB pathway, mice were pretreated with 17-AAG, and their colon tissue was analyzed by Western blot analysis. The results showed that 17-AAG had no effect on AKT expression, but significantly reduced IR-induced AKT phosphorylation levels (p-AKT) (see appendix). Figure 8 (A) and (B) suggest that the binding of 17-AAG to HSP90 may lead to AKT instability or dephosphorylation, resulting in AKT activation inhibition. Subsequently, by inhibiting the phosphorylation levels of the IKK-α and IKK-β subunits (p-IKK-α / β), IR-induced activation of the downstream IKK complex was inhibited (see appendix). Figure 8 (C)-(E)). This further inhibits downstream IκBα phosphorylation, maintains its stability and prevents degradation, and limits the release and nuclear translocation of NF-κB (see appendix). Figure 8 (C), (F), (G)). Inhibition of NF-κB activation (p-NF-κB p65) reduces its transcriptional activity in the nucleus, ultimately regulating cellular stress responses associated with inflammation, apoptosis, and / or oxidative stress. Therefore, the active metabolite 17-AAG secreted by *A. muciniphila* protects mice from radiation-induced inflammatory damage by inhibiting radiation-induced AKT / NF-κB pathway activation.

[0161] Further investigation was conducted to determine whether 17-AAG-mediated signaling pathway regulation is involved in the radioprotective effects of *S. muciniphila*. A similar trend was observed in the colonic tissue of mice orally administered *S. muciniphila*: IR-induced phosphorylation of AKT, IKK-α / β, IκBα, and NF-κB p65 was inhibited (see appendix). Figure 8 (H)-(N)). This indicates that *S. muciniphila* can also downregulate IR-induced AKT / NF-κB pathway activation. The relative expression and phosphorylation levels of AKT, IKK-α / β, IκBα, and NF-κBp65 in irradiated mouse colon tissue are shown in the attached figure. Figure 8 As shown.

[0162] In summary, GDN-Cur exerts a protective effect against radiation-induced intestinal injury by increasing the abundance of A. muciniphila, promoting the secretion of 17-AAG, and thereby regulating the AKT / NF-κB signaling pathway.

[0163] The scope of protection of this invention includes, but is not limited to, the above embodiments. The scope of protection of this invention is defined by the claims. Any substitutions, modifications, or improvements to this technology that are easily conceived by those skilled in the art fall within the scope of protection of this invention.

Claims

1. A method for protecting against radiation-induced intestinal injury based on plant active ingredients, characterized in that, include: Using ginger as raw material, ginger exovesicles (GDNs) were isolated and purified; on this basis, the plant-active compound curcumin Cur was loaded to form the ginger exovesicle complex GDN-Cur. This preparation can be taken orally as a preventative measure before the body is exposed to high doses of ionizing radiation to reduce radiation-induced intestinal damage.

2. The method for protecting against radiation-induced intestinal injury based on plant active ingredients according to claim 1, characterized in that, The preparation method of the GDN-Cur includes the following steps: GDNs were separated and extracted using tissue disruption and differential ultracentrifugation. GDNs were purified using sucrose density gradient centrifugation. GDN-Cur was prepared by loading Cur using a co-incubation method.

3. The method for protecting against radiation-induced intestinal injury based on plant active ingredients according to claim 2, characterized in that, in, GDNs were separated and extracted using tissue disruption and differential ultracentrifugation, specifically as follows: Step 1.1: Wash fresh ginger thoroughly with pure water and soak it in PBS solution for 2 minutes to remove surface residue; Step 1.2: Peel the processed ginger, use a juicer to extract the ginger tissue homogenate, and filter the tissue liquid through a 0.7μm filter membrane to remove residue; Step 1.3: Transfer the filtered tissue fluid to a 50mL centrifuge tube and centrifuge at 4℃ for 1000g×10min, 3000g×20min and 6000g×40min respectively. Collect the supernatant and remove the residue, cell debris and large particles from the precipitate. Step 1.4: Transfer the obtained supernatant to a 40mL ultracentrifuge tube and centrifuge at 4℃ for 140000g × 150min. After centrifugation, carefully discard the supernatant and resuspend the precipitate in 1.5-2mL of PBS to obtain GDNs.

4. The method for protecting against radiation-induced intestinal injury based on plant active ingredients according to claim 2, characterized in that, in, GDNs were purified using sucrose density gradient centrifugation, specifically as follows: Step 2.1: Prepare sucrose solutions with concentrations of 60%, 45%, 30%, 10% and 5% respectively. Take 5 mL of each sucrose solution of different concentrations and slowly add it to a 40 mL ultracentrifuge tube to form a density gradient. Step 2.2: Slowly add the GDNs obtained in Step 1 to the top of the density gradient sucrose solution and centrifuge at 4°C for 100,000g × 180min. Step 2.3: After centrifugation, solutions of different concentration gradients show clear stratification. Step 2.4: Take the sample from the brightest sucrose band, transfer it to a new 40mL ultracentrifuge tube, add 20mL PBS, centrifuge at 4℃ for 100000g×70min, discard the supernatant, and resuspend the precipitate in 1.5-2mL PBS. Step 2.5: Filter the sample obtained in step 2.4 using a 0.22μm needle filter to obtain purified GDNs.

5. The method for protecting against radiation-induced intestinal injury based on plant active ingredients according to claim 2, characterized in that, in, GDN-Cur was prepared by loading Cur using a co-incubation method, specifically as follows: Step 3.1: Dissolve Cur in DMSO and dilute with PBS to the corresponding concentration; Step 3.2: Add a certain concentration of Cur to the purified GDNs obtained in Step 2, and vortex briefly to mix; place the mixture in a water bath at a certain temperature and incubate for a period of time. Step 3.3: The final sample obtained in step 3.2 is transferred to a 40mL ultracentrifuge tube and centrifuged at 4℃ for 100,000g × 70min. The supernatant is discarded, and the precipitate is resuspended in 1.5-2mL of PBS to obtain the Cur-loaded exovesicle complex GDN-Cur.

6. The method for protecting against radiation-induced intestinal injury based on plant active ingredients according to claim 5, characterized in that: In step 3.2, the mass ratio of Cur to GDNs is 8:1 to 1:1; the incubation temperature is 4 to 55°C; and the incubation time is 2 to 24 hours.

7. The method for protecting against radiation-induced intestinal injury based on plant active ingredients according to claim 1, characterized in that: The timing of GDN-Cur administration is from one week before the body receives high-dose ionizing radiation to immediately after irradiation. The administration method is oral, with a dose of 5-20 mg / kg, where 5-20 mg / kg represents the Cur concentration in GDN-Cur, 1-2 times / day.

8. The method for protecting against radiation-induced intestinal injury based on plant active ingredients according to claim 4, characterized in that: The ionizing radiation exposure dose is 1-10 Gy for a single exposure or several exposures in a short period of time, with a dose rate of 0.1-2 Gy / min, and the site of exposure is either whole-body exposure or local exposure in the abdominal and pelvic cavities.

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