Use of parabacteroides distasonis and succinic acid in the treatment of radiation hematopoietic injury exacerbated by microplastics

Abstract

The application discloses application of Bacteroides dorei and succinic acid in treatment of radiohematopoietic injury aggravated by microplastics. The application explores the influence of polystyrene particles, which are representative of microplastics, on hematopoietic injury induced by ionizing radiation, identifies beneficial intestinal microorganisms and metabolic products playing a key role therein, and verifies the replenishment effect, thereby screening the key intestinal microorganism Bacteroides dorei and the metabolic product succinic acid and its salts having the effect of relieving radiohematopoietic injury aggravated by microplastics. The application provides intervention materials and thought strategies for radiohematopoietic injury aggravated by microplastics, provides a basis and application basis for tumor radiotherapy, and has important practical significance and clinical transformation value.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, and in particular to the application of *Pseudomonas diffusa* and succinic acid in the treatment of radiation-induced hematopoietic damage exacerbated by microplastics. Background Technology

[0002] Since mass production began in the 1950s, plastics have been widely used in various industries, including packaging materials, clothing and textiles, medical devices, the automotive industry, and daily necessities. However, while ubiquitous plastic products have greatly facilitated human life, they have also brought serious environmental and health challenges. Since the concept of microplastics (MPs) (defining plastic particles or fragments with a diameter of less than 5 mm as microplastics) was first proposed in Science in 2004, it has received widespread public attention. Human MP exposure pathways mainly include three types: first, daily environmental exposure, i.e., ingestion (MPs in food), inhalation (MPs in the air), or skin absorption (MPs in cosmetics) through contact with everyday plastic products; second, medical exposure, i.e., exposure to pharmaceutical packaging, the use of disposable plastic instruments (such as IV tubing, blood bags, syringes, and masks), and the use of medical polymer biomaterials (such as artificial joint replacement, cardiac / vascular stent placement, denture fitting, and kidney dialysis); and third, occupational exposure, with workers in the textile industry, 3D printing, plastic production, and waste management and recycling facing higher daily exposure frequencies. Polystyrene (PS) is one of the most widely used and important plastic components. After entering the body, polystyrene (MP) can cross physiological barriers and distribute in various organs, tissues, and body fluids. Different types of MP have been found in human blood, feces, brain, lungs, placenta / feces, liver, kidneys, and semen. MP exposure can cause biological effects such as inflammation, oxidative stress, metabolic and immune dysregulation in target organs. Clinical studies show that MP accumulation in the body is closely related to the development of reproductive disorders, cardiovascular diseases, inflammatory bowel disease, nervous system diseases, and even cancer. Notably, in the field of radiation medicine, a large proportion of patients undergoing tumor radiotherapy or nuclear medicine-related examinations use medical devices derived from plastics during their medical procedures. Furthermore, they frequently ingest food and beverages packaged in plastic containers during hospitalization and daily life, thus suffering from the dual harm of MP and radiation exposure. MP has become a "hidden killer" of patient prognosis and health. Therefore, identifying intervention substances targeting the combined toxicity of MP and radiation is of great significance for enhancing the efficacy and reducing the toxicity of tumor radiotherapy and for the treatment of nuclear / radiation accidents.

[0003] The hematopoietic system is highly sensitive to external stimuli. When microplastics (MPs) are ingested, they can disrupt the intestinal barrier, enter the bloodstream, and accumulate in the bone marrow. Exposure alone can disrupt the gut microbiota-hypoxanthine-Wnt pathway, inhibiting hematopoietic stem cell self-renewal and hematopoietic reconstitution, leading to disruption of the "gut-blood axis." Furthermore, ionizing radiation (IR) is a classic cause of hematopoietic injury. Current research is limited to single-radiation hematopoietic injury or single MP hematopoietic injury, and has not yet explored whether MP affects or exacerbates IR-induced hematopoietic injury. There are also no specific therapeutic drugs for radiation-induced hematopoietic injury exacerbated by microplastics. Summary of the Invention

[0004] In order to address the technical problem of the lack of drugs for treating radiation-induced hematopoietic damage exacerbated by microplastics, this invention provides the application of *Pseudomonas difficile* and succinic acid in the treatment of radiation-induced hematopoietic damage exacerbated by microplastics.

[0005] In a first aspect, the present invention provides a use of succinic acid and its salts, which is achieved through the following technical solution.

[0006] Application of succinic acid and its salts in the preparation of drugs for treating radiation-induced hematopoietic damage exacerbated by microplastics.

[0007] Furthermore, succinates include disodium succinate.

[0008] Furthermore, compared with ordinary radiation-induced hematopoietic damage, radiation-induced hematopoietic damage exacerbated by microplastics is manifested in a further decrease in the weight of some important blood cell indicators and hematopoietic organs such as the spleen and thymus.

[0009] Secondly, the present invention provides a first drug for treating radiation-induced hematopoietic damage exacerbated by microplastics, which is achieved through the following technical solution.

[0010] A drug for treating radiation-induced hematopoietic damage exacerbated by microplastics includes succinic acid and its salts.

[0011] Thirdly, the present invention provides a use of *Pseudomonas dignitaria*, which is achieved through the following technical solution.

[0012] Application of *Pseudomonas dilatatus* in the preparation of drugs for treating radiation-induced hematopoietic damage exacerbated by microplastics.

[0013] Fourthly, the present invention provides a second medicament for treating radiation-induced hematopoietic damage exacerbated by microplastics, which is achieved through the following technical solution.

[0014] A drug for treating radiation-induced hematopoietic damage exacerbated by microplastics, including *Pseudomonas difficile*.

[0015] Fifthly, the present invention provides a use of succinic acid and its salts in combination with *Pseudomonas difficile*, which is achieved through the following technical solution.

[0016] Application of succinic acid and its salts in combination with *Pseudomonas difficile* in the preparation of a drug for treating radiation-induced hematopoietic damage exacerbated by microplastics.

[0017] In a sixth aspect, the present invention provides a pharmaceutical composition for treating radiation-induced hematopoietic damage exacerbated by microplastics, which is achieved through the following technical solution.

[0018] A pharmaceutical composition for treating radiation-induced hematopoietic damage exacerbated by microplastics, comprising succinic acid and its salts and *Pseudomonas difficile*.

[0019] This application has the following beneficial effects: Based on a clear understanding of the effects of microplastics (MP) on radiation-induced hematopoietic damage, this invention precisely identified key beneficial gut bacteria (Peribrobacterium digilei) and its metabolite (succinic acid) that mediate the radiation-induced hematopoietic damage aggravated by microplastics, and clarified their changing trends. Simultaneously, it demonstrated the alleviating effect of Peribrobacterium digilei or / and sodium succinate on the radiation-induced hematopoietic damage aggravated by microplastics, providing a highly effective probiotic or / and post-biotic substance for the treatment of radiation-induced hematopoietic damage aggravated by microplastics. Attached Figure Description

[0020] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0021] Figure 1 This is a graph showing the effect of 5 μm PS of the present invention on ionizing radiation-induced damage to hematopoiesis and intestinal integrity in mice; Figure 2 This is a graph showing the effect of 5 μm PS of the present invention on the growth viability and inflammatory factor secretion level of the mouse small intestinal epithelial cell line MODE-K; Figure 3 This is a diagram showing the effect of PS and / or IR treatment of the present invention on the structure of mouse gut microbiota; Figure 4 This is a diagram showing the effect of PS on radiation-induced hematopoietic damage after FMT treatment according to the present invention. Figure 5 This is a graph showing the effect of PS and / or IR treatment of the present invention on the abundance of key intestinal flora and the content of metabolites in mice; Figure 6 This is a supplement to the invention. P. distasonis Or the effect of disodium succinate hexahydrate on the reduction of blood count indicators induced by ionizing radiation in mice; Figure 7 This is a supplement to the invention. P. distasonis Or the effect of disodium succinate hexahydrate on ionizing radiation-induced spleen and thymus atrophy in mice; Figure 8 This is a supplement to the invention. P. distasonis Effects of sodium succinate hexahydrate and / or disodium succinate hexahydrate on the viability, inflammation, apoptosis, and oxidative stress levels of bone marrow cells in irradiated mice. Detailed Implementation

[0022] Preferred embodiments of the present disclosure will be described in detail below with reference to the accompanying drawings. Before description, it should be understood that the terminology used in the specification and appended claims should not be construed as limited to its general and dictionary meaning, but rather should be interpreted based on the principle of allowing the inventors to appropriately define the terminology for the best interpretation, and based on its meaning and concept corresponding to the technical level of the invention. Therefore, the description herein is merely a preferred example for illustrative purposes and is not intended to limit the scope of the invention; thus, it should be understood that other equivalent implementations and modifications can be made without departing from the spirit and scope of the invention.

[0023] This invention selects representative microplastic particles (PS) as the research object; uses C57BL / 6J mice irradiated with gamma rays as a model; employs omics technologies such as 16S rRNA sequencing and mass spectrometry-based targeted metabolic analysis; combines fecal microbiota transplantation (FMT), key single-bacterial and metabolite recovery, and various animal and cell molecular biology experimental techniques to study the specific effects of PS particles on radiation-induced hematopoietic damage (mainly including the effects on peripheral blood cell count and thymus and spleen weight), and identifies key beneficial gut microbes and metabolites that play a crucial role and verifies the recovery effect [mainly including the detection of recovery of peripheral blood cell count, thymus and spleen weight, bone marrow cell viability (number, viable cell ratio, and granulocyte-macrophage clonogenic capacity), and assessment of damage relief (inflammation, apoptosis, and oxidative stress levels). The aim is to screen key gut microbes and metabolites that can alleviate radiation-induced hematopoietic damage exacerbated by microplastics, providing interventional substances and strategies for radiation-induced hematopoietic damage exacerbated by microplastics, and providing a basis and application for enhancing the efficacy and reducing the toxicity of tumor radiotherapy and for the treatment of nuclear / radiation accidents.

[0024] I. Materials and Methods Example 1: Effects of MP PS on ionizing radiation-induced damage to hematopoiesis and intestinal integrity This application selects representative MP particles (PS) as the research object. Previous studies on MP hazards have primarily focused on nanoplastics, while micron-sized plastics are more widely distributed in real life, but their toxic effects are often overlooked to some extent. Therefore, we selected micron-sized (5 μm) PS particles, which have received little attention, as the research object, and set up high and low treatment concentrations to compare the differences between treatments. The groups were set as follows: normal control group (Ctrl), low-concentration (0.1 mg / day) PS treatment group, high-concentration (1.5 mg / day) PS treatment group, IR treatment group alone, low-concentration PS+IR treatment group, and high-concentration PS+IR treatment group, for a total of 6 groups. To simulate long-term MP exposure, mice were subjected to 8 weeks of gavage treatment: first, 2 weeks of gavage; mice requiring IR treatment received a single 15 Gy local abdominal irradiation, followed by 6 weeks of gavage treatment with the corresponding concentration of PS. Control group mice received sham irradiation or gavage treatment with an equal volume of ddH2O (200 μL) under the same conditions. Mice in each group were weighed every 3 days, and peripheral blood cells were counted by tail blood collection every 2 weeks. At the end of the experiment, 2-3 feces were collected per mouse, and blood was collected by enucleation of the eyeballs. The mice were then euthanized and their small intestine, spleen and thymus were dissected.

[0025] Detection indicators: a) Body weight of mice in each group. b) Peripheral blood cell count of mice in each group. c) Photographs and weight statistics of representative spleen and thymus of mice in each group. d) Distribution of PS in the intestine: Nanoscale hyperspectral technology was used to detect the differences in the distribution of PS in normal and radiation-damaged intestines and the differences in the integrity of small intestinal tissue in each group. e) Small intestinal tissue integrity: FITC-dextran gavage was used to detect the effect of different concentrations of PS / IR alone / in combination on the intestinal permeability of small intestinal tissue in each group; immunofluorescence (IF) staining was used to detect the expression level of the intestinal integrity marker ZO-1.

[0026] Peripheral blood cell counts were performed using an automated blood cell counter (Nihon Kohden, Tokyo, Japan); nano-hyperspectral analysis was performed by CytoViva; FITC-dextran intestinal permeability assays were performed using Sigma-Aldrich FD4-1G; and ZO-1 IF staining was performed using primary antibody sc-33725.

[0027] The above experiments were used to preliminarily assess the effect of PS on IR-induced hematopoietic damage, clarify its specific impact, and define the role of PS in IR-induced intestinal integrity impairment. This serves as a warning of the severity of the dual toxicity of MP and radioactivity in tumor radiotherapy and nuclear / radiation accident treatment. Based on these results, a suitable experimental concentration was selected to establish a mouse model of PS's effect on radiation-induced hematopoietic damage. To simulate the low-concentration exposure state of MP in the environment as closely as possible, a low PS concentration that did not cause weight change but only caused / exacerbated the hematopoietic damage effect was ultimately selected for subsequent experiments.

[0028] Example 2: Study on the mechanism by which gut microbiota (gut microorganisms and their metabolites) mediates the exacerbation of radiation-induced hematopoietic damage by PS 1. In vitro detection of the effects of PS on the viability and inflammation level of small intestinal epithelial cell lines To investigate the specific mechanism of PS-induced enterotoxicity in vitro, 5 μm PS was added to the culture supernatant of the mouse small intestinal epithelial cell line MODE-K at a series of working concentrations of 0, 0.000025, 0.00025, 0.0025, 0.025, 0.25, 2.5, 25 and 250 μg / mL and cultured for 72 h or 48 h. The effects on cell viability and inflammation were then examined.

[0029] Detection indicators: a) CCK-8 assay (using APExBIO K1018-5 mL kit): The effect of PS on MODE-K cell viability at 0, 24, 48, and 72 h was detected. Specifically, cells were seeded in 96-well plates at a density of 1000 cells / 100 μL / well. After 12 h, different concentrations of PS were added to the corresponding wells for co-culture with the cells, and CCK-8 cell viability was detected at the corresponding time points. b) ELISA method: The effect of 48 h PS on the protein expression levels of inflammatory factors IL-6, TNF-α, and IL-1β in the supernatant of MODE-K cell culture was detected. IL-6 (F2163-A), TNF-α (F2132-A), and IL-1β (F2040-A) were detected using a kit from Shanghai Vanco Biotechnology Co., Ltd.

[0030] Experimental results showed that direct treatment of MODE-K cells with PS at a series of concentration gradients in vitro had no significant effect on their viability or the level of inflammatory factors secreted in the supernatant. This indicates that 5 μm PS cannot directly cause damage to small intestinal epithelial cells, possibly due to the limited ability of micron-sized PS particles to penetrate the cell membrane and enter the cell.

[0031] This part of the study rules out that the toxic effects of PS are caused by direct action on intestinal cells. Furthermore, MP accumulates after oral ingestion and acts on the digestive system; the gut microbiota is the first barrier affected by MP, and MP, after ingestion, can disrupt the hematopoietic system through the gut microbiota-bone marrow axis, forming a "gut-blood axis." The above experimental and theoretical background lays the foundation for further elucidating the mechanism by which PS exacerbates radiation-induced hematopoietic damage from the perspective of gut microbiota.

[0032] 2. Research on the key role of gut microbiota in PS-induced radiation-induced hematopoietic injury (1) Effects of PS and / or IR treatment on the structure of mouse gut microbiota Based on the above in vitro experimental results, and considering that MP mainly accumulates and acts on the digestive system after oral ingestion, and that the gut microbiota can mediate the interaction between the external environment and the host, this study aimed to explore whether PS induces downstream damage effects by disrupting the gut microbiota structure in mice. Therefore, four groups were set up: Ctrl, PS (low concentration) alone, IR alone, and PS (low concentration) + IR combined treatment group. The treatment methods and cycles for each group were the same as in Example 1.

[0033] Detection indicators: At the end of the experiment, fecal samples from each group of mice were collected for 16S rRNA sequencing to analyze the effects of PS and / or IR treatment on the gut microbiota structure of each group of mice, mainly including the changing trends of α-diversity, β-diversity, and abundance of some bacterial species among the groups. 16S rRNA sequencing was performed by Beijing Novogene Technology Co., Ltd.

[0034] This study preliminarily demonstrates that PS and / or IR can indeed cause dysbiosis in the gut microbiota of mice, suggesting a key role of the gut microbiota in mediating the exacerbation of radiation-induced hematopoietic damage by PS, and providing an omics data basis for subsequent identification of key bacterial species.

[0035] (2) FMT technology verifies the key role of gut microbiota in mediating PS-induced radiation-induced hematopoietic damage. Three groups were set up: a Ctrl and IR treatment group, an IR treatment group, and a PS+IR combined treatment group. The treatment methods and cycles for each group of mice were the same as in Section (1) of Part 2 of Example 2. Fecal samples from each group of mice were collected daily to prepare donor samples. FMT receptor (FR) mice were also divided into three groups: normal fecal microbiota treatment group (FR-Ctrl), ionizing radiation fecal microbiota treatment group (FR-IR), and polystyrene + ionizing radiation fecal microbiota treatment group (FR-PS+IR). Recipient mice were given antibiotic ABX by gavage for one week before the start of FMT to clear the original microbiota, and the 6-week FMT treatment began 2 weeks after the donor mice received ionizing radiation treatment.

[0036] ABX formulation: 1 g / L ampicillin (MCE, HY-B0522), 0.5 g / L neomycin (MCE, HY-B0470), 0.5 g / L vancomycin (MCE, HY-B0671) and 1 g / L metronidazole (MCE, HY-B0318).

[0037] FMT experiment: Fresh feces were aseptically collected from donor mice in each group daily. Sterile PBS was added at a ratio of 120 mg feces / mL PBS, and the mixture was soaked for 15 min. After vortexing, the mixture was centrifuged at 75×g and 4℃ for 3 min, and the supernatant was collected. 100 μL was administered to each recipient mouse by gavage. One week before transplantation, antibiotic ABX was administered to drinking water to clear the original intestinal flora.

[0038] Technical indicators: a) Peripheral blood cell counts of mice in each group. b) Photographs and weight statistics of representative spleen and thymus of mice in each group.

[0039] Compared to FR-Ctrl, FR-IR and FR-PS+IR mice showed similar damage effects to the donor mice, and FR-PS+IR mice showed a more pronounced decrease in blood cell counts and a tendency for spleen and thymus atrophy compared to FR-IR mice alone.

[0040] By examining the effects of different fecal FMT treatments on peripheral blood cell counts and thymus and spleen weights in FR mice, this study verified that gut microbiota can indeed mediate the aggravating effect of PS on radiation-induced hematopoietic damage.

[0041] 3. Identification of key single bacteria and metabolites mediating PS-induced radiation-induced hematopoietic damage Based on the 16S rRNA sequencing results in Section (1) of Part 2 of Example 2, statistical analysis was performed on the abundance changes of different genera of gut bacteria in the four groups to screen out genera whose abundance was significantly lower in the PS+IR group compared to the ionizing radiation group. qRT-PCR experiments were then used to detect the abundance changes of each species within these genera, further identifying key gut bacteria species. Simultaneously, mass spectrometry-based targeted metabolomics analysis was performed on the four fecal samples from the same batch to screen out gut microbial metabolites whose content was significantly lower in the PS+IR group compared to the IR group, completing the screening of key gut microbial metabolites. Further abundance-content correlation analysis was performed on the selected genera and metabolites to clarify the correlation between their levels.

[0042] Detection indicators: a) Based on omics and experimental results, key bacterial species and metabolites most significantly affected by PS and / or IR were screened, with a focus on gut microbiota and metabolites whose abundance / content was significantly reduced in feces in the PS+IR group compared to the IR group. b) Correlation analysis was performed on the screened bacterial genera and metabolites that showed changes, and key single bacteria and metabolites mediating PS-induced radiation-induced hematopoietic damage were further screened to identify them and clarify the correlation between the two. 16S rRNA sequencing, fecal targeted metabolomics detection, and data analysis were conducted by Beijing Novogene Technology Co., Ltd.

[0043] This part of the study identified key single bacteria and metabolites that mediate PS-induced radiation-induced hematopoietic damage [namely, *Pseudomonas difficile* (…). Parabacteroides distasonis , P. distasonis [and succinic acid], used to verify the efficacy of subsequent treatments.

[0044] Example 3: Effect of reintroduction of key single bacteria and / or metabolites on hematopoietic damage induced by PS and / or IR treatment Purchase the model strains of the key single bacteria identified above ( P. distasonis The key single bacteria and its metabolite sodium salt (disodium succinate hexahydrate) were used to prepare seven treatment groups: Ctrl, PS, IR, PS+IR, PS+IR+key bacteria, PS+IR+key metabolite, and PS+IR+key bacteria+key metabolite. Key single bacteria / metabolite replenishment experiments were conducted. The PS and / or IR treatment methods and cycles were the same as in Section (1) of Part 2 of Example 2. After purchasing the key single bacteria, they were cultured in an anaerobic environment. The key single bacteria and metabolites were prepared at 1×10⁻⁶ mg / L. 8 Mice requiring treatment were administered CFUs / 100 μL / day and 300 mg / kg / day via gavage. Mice in the recovery group (the latter three groups) began a 6-week recovery treatment on the day of IR, and each group was treated with ABX for 1 week prior to recovery. Peripheral blood cell counts were performed on mice in each group every 2 weeks. At the end of the experiment, the spleen and thymus were dissected, photographed, and weighed. Bone marrow cells were collected for bone marrow cell counting, viable cell ratio analysis, and detection of bone marrow cell damage effects [including granulocyte-macrophage clonogenic capacity, apoptosis (detecting the levels of pro-apoptotic factors Caspase-3 and anti-apoptotic factors Bcl-2), inflammation (detecting the levels of pro-inflammatory factors TNF-α and IL-6), and oxidative stress (detecting the levels of oxidative damage factor ROS and the activity of antioxidant factor SOD) using ELISA].

[0045] P. distasonisThe bacteria (ATCC 8503) were purchased from BNCC (BNCC 364349) and cultured in an anaerobic incubator (Jiangsu Zhengji Instrument Co., Ltd., YQX-Ⅱ) filled with a mixture of 90% N2 + 5% H2 + 5% CO2. Disodium succinate hexahydrate was purchased from MCE (HY-W094516), and a 10× stock solution was prepared using ddH2O as solvent and stored at 4℃. Before use, the solution was diluted and administered to mice by gavage at a dose of 300 mg / kg / day.

[0046] Detection indicators: a) Peripheral blood cell count. b) Spleen and thymus, photographed and weighed. c) Bone marrow cell count. d) Bone marrow cell viability. e) Bone marrow cell granulocyte-macrophage clonogenic capacity. f) Bone marrow cell inflammation level. g) Bone marrow cell apoptosis level. h) Bone marrow cell oxidative stress level.

[0047] Bone marrow cell count and viable cell ratio analysis were performed using a Countstar automated cell counter (KEYENCE, Osaka, Japan); Caspase-3 (F2749-A), Bcl-2 (F2306-A), TNF-α (F2132-A), and IL-6 (F2163-A) were detected using kits from Shanghai Vancowell Co., Ltd.; ROS levels (GY0163W) and SOD activity (G0101W) were detected using kits from Suzhou Greens Biotechnology Co., Ltd.

[0048] Bone marrow cell isolation and counting: (1) Wear sterile lab coats, masks and latex gloves, open the laminar flow hood and prepare the necessary materials for the experiment. (2) After euthanizing the mouse by dislocation and disinfecting it by soaking it in 75% alcohol, place it on sterile tin foil in the laminar flow hood. (3) Cut open the skin at the ankle joint of the mouse, cut the skin along the midline of the inner and outer sides of the longitudinal axis of the lower leg, and bluntly separate the skin to the abdomen to fully expose the surgical field. (4) Cut off the end of the tibia above the ankle joint and peel off the tibia muscle and fibula proximally. (5) Fold the tibia away from the knee joint in the opposite direction of the knee joint movement. Continue to peel off the femoral muscle from the distal end to the proximal end, and then cut the femur at the iliac joint. (6) Use a 1 mL syringe to draw pre-cooled physiological saline and insert it into the tibia and femur respectively, repeatedly rinse out the bone marrow cells in the bone, and use a syringe to blow into a single cell suspension, and then filter the cell suspension through a 50 μm filter membrane into a 15 mL centrifuge tube. Bone marrow cell counts were performed using a Countstar automated cell counter (KEYENCE, Osaka, Japan) with 20 μL of cell suspension aspirated by a pipette.

[0049] Granulocyte-macrophage clonogenesis assay: (1) Based on the bone marrow cell count data, the cell concentration of each group was adjusted with PBS to: 3.5 × 10⁻⁶ cells in the unirradiated group.4 Cells / mL, irradiated group 3.5×10 5 Cells / mL. (2) Take 0.2 mL of cell suspension from each tube and add it to 3.5 mL of methylcellulose semi-solid culture medium (STEMCELL, M3234). Vortex thoroughly to mix, and let stand for 5-10 min until the bubbles disappear. (3) Connect a flat-tipped needle to a 2.5 mL syringe, take 500 μL of cell suspension, and add it to a 24-well plate, with 5 parallel wells per group. (4) Place the culture plate in a 37℃, 5% CO2 cell culture incubator for culture. (5) After one week, observe the colony formation in the culture medium under a low-power inverted microscope. A positive colony is counted if the number of cells is ≥30. Count the number of colonies formed in each well. (6) Convert the results to 10 cells per 10 wells. 5 The number of granulocyte-macrophage clone-forming units per bone marrow cell.

[0050] This part of the study verifies the important role of this key bacterial species and its metabolites in mediating radiation-induced hematopoietic damage exacerbated by microplastics. It also evaluates the therapeutic effect of this key bacterial species and its metabolites on radiation-induced hematopoietic damage exacerbated by microplastics from three aspects: peripheral blood cell count, spleen / thymus weight and bone marrow cell count, viable cell ratio and damage effects (granulocyte-macrophage clonogenic capacity, inflammation, apoptosis and oxidative stress). In this way, the screening and identification of intestinal bacterial species and metabolites that can alleviate radiation-induced hematopoietic damage exacerbated by microplastics can be completed.

[0051] II. Experimental Results 1. PS / IR treatment methods for mice are as follows: Figure 1 As shown in Figure A, IR treatment can lead to a decrease in mouse body weight. Low-concentration PS treatment has no effect on the body weight of mice in the normal control group / IR-treated group, while high-concentration PS treatment can cause a slight decrease in body weight in mice in the normal control group / IR-treated group. Figure 1 B).

[0052] Infrared irradiation (IR) treatment can lead to a decrease in the levels of multiple blood markers (such as white blood cells, lymphocytes, platelets, and red blood cells) after 14 days, while combined treatment with low concentrations of PS and IR can further reduce the levels of these markers. Figure 1 CF). 42 days after IR, the spleen and thymus weights of mice in each group showed a similar trend to the aforementioned blood count indicators (CF). Figure 1 (GJ). These results indicate that even low concentrations of PS can exacerbate IR-induced hematopoietic damage in mice.

[0053] After ingestion, MP mainly accumulates in the intestinal system. To further investigate whether the aggravating effect of PS on hematopoietic damage is related to the intestinal system, this application first examined the effect of PS on the integrity of the irradiated small intestine. The results showed that PS accumulated in the irradiated intestine more than in the normal intestine. Figure 1 K). Both PS and IR treatment alone disrupt the integrity of small intestinal tissue, and PS treatment further exacerbates the decline in small intestinal tissue integrity in the IR group. Specifically, the PS-low concentration + IR group showed more lesions on intestinal tissue imaging compared to the IR-only group. Figure 1 K, the area circled in white), FITC-glucan showed higher permeability in the intestines of the PS-low concentration + IR group compared to the IR-only group. Figure 1 L), and the expression level of the intestinal integrity marker ZO-1 in the intestinal tissue of the PS-low concentration + IR group was further reduced compared with the IR group alone (L), and the expression level of ZO-1, a marker of intestinal integrity, was further reduced in the intestinal tissue of the PS-low concentration + IR group compared with the IR group alone (L). Figure 1 The above studies indicate that long-term exposure to 5 μmPS can exacerbate the decline in intestinal integrity in irradiated mice.

[0054] 2. The growth viability of mouse small intestinal epithelial cell line MODE-K was investigated by directly adding different concentration gradients of PS to the culture medium. Figure 2 A) and inflammatory factors TNF-α, IL-6 and IL-1β in the supernatant Figure 2 BD (digestive tract infection) secretion levels had no significant effect. This result suggests that in vitro PS exposure does not directly induce damage to small intestinal epithelial cell lines.

[0055] 3. 16S rRNA sequencing technology was used to detect differences in the gut microbiota structure among the mouse groups. Venn diagrams showed that a certain number of unique and common bacterial species existed in the feces of mice in the Ctrl, PS, IR, and PS+IR groups. Figure 3 A); Principal coordinates analysis (pCoA) showed a clear separation of the bacterial community structure in each group ( Figure 3 B); PS or IR treatment alone did not significantly alter the β-diversity of the gut microbiota in mice, while the PS+IR treatment group caused significant changes. Figure 3 C); PS and / or IR treatment slightly reduced the Chao 1 richness index, observed species index, and Shannon diversity index in bacterial community α-diversity, with the most significant reduction observed in the PS+IR combined treatment group. Figure 3 (DF). The above results indicate that PS treatment can alter the structure of the gut microbiota and further exacerbate IR-induced changes in the microbiota structure, suggesting an important role of the gut microbiota in mediating the effects of PS on radiation-induced hematopoietic damage.

[0056] 4. Feces from recipient mice treated with normal mice, IR, and PS+IR were used for fecal microscopy (FMT). Results showed that, compared to FR-Ctrl, FR-IR and FR-PS+IR mice exhibited similar lesioning effects to the donor mice, as evidenced by lower white blood cell counts at 14 days after FMT in the FR-IR and FR-PS+IR groups. Figure 4 A) and lymphocytes ( Figure 4 B)] level and spleen at 42 days FMT ( Figure 4 C and D) and thymus ( Figure 4 Both E and F weights showed a decreasing trend, and the decrease was more significant in the FR-PS+IR group than in the FR-IR group. Figure 4 The above results indicate that the gut microbiota does indeed mediate the exacerbation of radiation-induced hematopoietic damage by PS.

[0057] 5. Based on the 16S rRNA sequencing results, Kruskal-Wallis H test statistical analysis screened out a group of bacterial genera that showed significant abundance changes in the Ctrl, PS, IR, and PS+IR groups, among which... Parabacteroides Abundance did not change significantly after PS or IR treatment alone, but decreased sharply in the PS+IR combined treatment group. Figure 5 A). RT-qPCR experiments showed that this genus of bacteria... P. distasonis The decrease in *Pseudomonas dell's bacillus* was most significant in the PS+IR combined treatment group. Figure 5 B). Targeted metabolomics data from the feces of mice in each group were analyzed. Heatmap analysis showed the types and changes in intestinal microbial metabolites that exhibited significant alterations in content among the four groups. Figure 5 C). The succinic acid content showed the most significant decreasing trend in PS+IR vs IR, and also exhibited a larger decreasing trend compared to the PS and TAI groups. Figure 5 D). Combined analysis of bacterial abundance and metabolite content revealed that in PS+IR vs IR... Parabacteroides There is a positive correlation between abundance and succinic acid content. Figure 7 E). Furthermore, literature reports indicate that succinic acid is... P. distasonis The metabolites [DOI: 10.1093 / femsle / fnac072] further support the finding that there is a positive correlation between the two. Based on the above results, the initial identification of the metabolites is... P. distasonis Succinic acid and succinic acid are key strains and metabolites that mediate the exacerbation of radiation-induced hematopoietic damage by PS.

[0058] 6. Key strains ( P. distasonis The recombinant experiment of sodium salt (disodium succinate hexahydrate) and key metabolites showed that recombinant... P. distasonisBoth sodium succinate hexahydrate and sodium succinate hexahydrate can significantly alleviate the decrease in mouse blood counts caused by PS+IR dual toxicity, including the decrease in white blood cell, lymphocyte, and red blood cell levels 14 days after irradiation. Figure 6 AC) and the decrease in white blood cell and lymphocyte levels 42 days after irradiation ( Figure 6 Damage effects such as D and E). The above results preliminarily indicate that... P. distasonis Both sodium succinate hexahydrate and disodium succinate hexahydrate have a mitigating effect on hematopoietic damage caused by the dual toxicity of MP-radioactive substances. These results further validate... P. distasonis The conclusion that succinic acid is a key bacterial species and metabolite mediating the exacerbation of radiation-induced hematopoietic damage by PS, and also indicates that... P. distasonis Potential of sodium succinate as a probiotic and a postbiotic in treating radiation-induced hematopoietic damage exacerbated by microplastics.

[0059] 7. In addition, replenishment P. distasonis Both sodium succinate hexahydrate and sodium succinate hexahydrate can significantly alleviate spleen damage in mice 42 days after irradiation. Figure 7 A and B) and thymus ( Figure 7 The decrease in weight of C and D) again indicates that P. distasonis Both sodium succinate hexahydrate and disodium succinate hexahydrate have a mitigating effect on hematopoietic damage caused by the dual toxicity of MP-radioactive substances. These results further validate... P. distasonis The conclusion that succinic acid is a key bacterial species and metabolite mediating PS's exacerbation of radiation-induced hematopoietic damage further demonstrates... P. distasonis Potential of sodium succinate as a probiotic and a postbiotic in treating radiation-induced hematopoietic damage exacerbated by microplastics.

[0060] 8. Bone marrow is the body's most essential hematopoietic organ, therefore this application further investigated... P. distasonis Effects of or / and disodium succinate hexahydrate on PS+IR-induced bone marrow cell damage. Results showed that exogenous complementation... P. distasonis Both or / and disodium succinate hexahydrate can reverse the decrease in total bone marrow cell count, viability, and granulocyte-macrophage clonogenic capacity caused by combined PS+IR treatment, demonstrating a protective effect on bone marrow cells. Figure 8 (AC). Inflammation, apoptosis, and oxidative stress are important biological effects of bone marrow cell damage. ELISA experiments revealed that PS treatment further increased the levels of pro-inflammatory factors TNF-α and IL-6 in irradiated mouse bone marrow cells, while rehydration... P. distasonis Alternatively, disodium succinate hexahydrate can lower its levels, and P. distasonis Combined supplementation with disodium succinate hexahydrate further reduced the levels of the above factors, indicating a synergistic effect of the two on relieving inflammation. Figure 8 DE). Caspase-3, a pro-apoptotic factor, was present in each group. Figure 8 F) and important markers of oxidative damage ROS (F) Figure 8 The levels of H) showed a consistent trend with the levels of the aforementioned pro-inflammatory factors, while the levels of the anti-apoptotic factor Bcl-2 (H) Figure 8 G) and the activity of SOD, a biomarker of antioxidant damage (G) Figure 8 I) The level shows the opposite trend, and P. distasonis Like disodium succinate hexahydrate, it exhibited a synergistic alleviating effect on bone marrow cell apoptosis and oxidative stress levels. These results once again validated... P. distasonis The conclusion that succinic acid is a key bacterial species and metabolite mediating PS's exacerbation of radiation-induced hematopoietic damage also once again demonstrates... P. distasonis It exhibits good efficacy, along with sodium succinate, as a probiotic and a postbiotic, in treating radiation-induced hematopoietic damage exacerbated by microplastics.

[0061] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. The use of succinic acid and its salts in the preparation of drugs for treating radiation-induced hematopoietic damage exacerbated by microplastics.

2. The application according to claim 1, characterized in that: Succinates include disodium succinate.

3. The application according to claim 1, characterized in that: Compared with ordinary radiation-induced hematopoietic damage, radiation-induced hematopoietic damage exacerbated by microplastics is manifested in a further decrease in the weight of some important blood cell counts and hematopoietic organs such as the spleen and thymus.

4. A drug for treating radiation-induced hematopoietic damage exacerbated by microplastics, characterized in that: This includes succinic acid and its salts.

5. Application of *Pseudomonas dilatatus* in the preparation of drugs for treating radiation-induced hematopoietic damage exacerbated by microplastics.

6. A drug for treating radiation-induced hematopoietic damage exacerbated by microplastics, characterized in that: Including *Pseudomonas dignitaria*.

7. The application of succinic acid and its salts in combination with *Pseudomonas difficile* in the preparation of a drug for treating radiation-induced hematopoietic damage exacerbated by microplastics.

8. A pharmaceutical composition for treating radiation-induced hematopoietic damage exacerbated by microplastics, characterized in that: This includes succinic acid and its salts, and *Pseudomonas dignitaria*.

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