Application of a strain of family trichocellaceae and / or daidzein in radiation-induced intestinal injury

Abstract

The application discloses an application of Lachnospiraceae bacterium and / or daidzein in radiation-induced intestinal injury. The study finds that the intestinal flora-derived Lachnospiraceae bacterium (Lachnospiraceae bacterium BNCC354474) has a significant protective effect on radiation-induced intestinal injury, can remodel the intestinal flora structure after irradiation, and restore the flora homeostasis. Meanwhile, through the target regulation of the inflammation pathway by the metabolite daidzein, the radiation-induced intestinal inflammation is relieved. The application clarifies the role and application potential of Lachnospiraceae bacterium and its metabolite in the regulation of radiation-induced intestinal injury, and provides a new microecological strategy for the intervention treatment of patients with radiation-induced intestinal injury.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to the application of a strain of Trichophyton spp. and / or daidzein in radiation-induced intestinal injury. Background Technology

[0002] Radiation-induced bowel injury (RH) is a type of intestinal lesion caused by abdominal and pelvic radiotherapy, manifesting as abdominal pain, diarrhea, tenesmus, and intestinal obstruction. The etiology of RH is complex, involving intestinal mucosal inflammation, intestinal epithelial cell necrosis, and inflammatory cell infiltration of the lamina propria, but the mechanisms are not fully understood. The gut microbiota and its metabolites are closely related to RH. Currently, clinical treatments for radiation proctitis mainly include anti-inflammatory drugs, mucosal protectants, endoscopic treatment, and surgery. However, these treatments all produce varying degrees of adverse reactions. Therefore, increasing research is shifting towards intervention strategies that balance safety and efficacy, such as probiotics and fecal microbiota transplantation (FMT), to improve radiation damage. A small-scale case report showed that FMT safely and effectively improved intestinal symptoms and mucosal damage in patients with radiation enteritis over a period of time. Further research indicates that washed microbiota transplantation demonstrates more significant clinical value in the treatment of different stages of radiation enteritis.

[0003] In recent years, an increasing number of clinical studies have found that the abundance of Lachnospiraceae bacteria is low in patients with radiation-induced intestinal injury, while the abundance of this bacterium is significantly increased in patients who received the same dose of radiotherapy but did not experience gastrointestinal reactions. Summary of the Invention

[0004] Therefore, this invention provides the application of a strain of *Trichophyton spp.* and / or daidzein in the prevention and treatment of radiation-induced intestinal injury. The applicant's research found that colonization of an irradiation model with *Trichophyton spp.* BNCC354474 altered the diversity, composition, and relative abundance of the gut microbiota, alleviating intestinal damage and highlighting its important role in radiation-induced intestinal injury. Based on this, this invention further utilizes transcriptomics, metabolomics, and bioinformatics techniques to screen for its related metabolite, daidzein, revealing its anti-inflammatory and intestinal barrier protective functions, providing a new approach for microbial prevention and treatment of radiation-induced intestinal injury.

[0005] To achieve the above objectives, the embodiments of the present invention provide the following technical solutions:

[0006] According to a first aspect of the present invention, the present invention provides the use of Trichophyton spp. and / or daidzein in the preparation of products for the prevention and treatment of radiation-induced intestinal injury.

[0007] Furthermore, the Lachnospiraceae bacterium is Lachnospiraceae bacterium BNCC354474.

[0008] Furthermore, the radiation-induced intestinal injury includes radiation enteritis caused by X-rays.

[0009] Furthermore, the application is as follows: Trichophyton and / or daidzein achieve the prevention and treatment of radiation-induced intestinal injury by downregulating CLEC4D expression, inhibiting SYK activity, and stabilizing IκB protein levels.

[0010] According to a second aspect of the present invention, the present invention provides a product for the prevention and treatment of radiation-induced intestinal injury, wherein the above-mentioned Hericium micranthum and / or daidzein are active ingredients.

[0011] Furthermore, the products include health foods and medicines.

[0012] The embodiments of the present invention have the following advantages:

[0013] This study found that *Lachnospiraceae bacterium* (BNCC354474), derived from gut microbiota, has a significant protective effect against radiation-induced intestinal injury, reshaping the gut microbiota structure and restoring homeostasis after irradiation. Simultaneously, it alleviates radiation-induced intestinal inflammation by targeting and regulating inflammatory pathways through its metabolite daidzein. This invention clarifies the role and application potential of *Lachnospiraceae bacterium* and its metabolites in regulating radiation-induced intestinal injury, providing a novel microecological strategy for the intervention and treatment of patients with radiation-induced intestinal injury. Attached Figure Description

[0014] To more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are merely exemplary, and those skilled in the art can derive other embodiments based on the provided drawings without creative effort.

[0015] Figure 1To investigate the role of Lach. colonization in alleviating radiation-induced gastrointestinal damage in mice and its radiation protection effect. The study included: (A) Lach. colonization experimental design; (B) Changes in body weight in mice after 10 Gy irradiation and Lach. colonization (n=6 mice / group, # / *p<0.05, ## / **p<0.01, ### / ***p<0.001; no statistically significant difference in ns); (C) Changes in survival rate in mice after 10 Gy irradiation and Lach. colonization (n=6 mice / group); (D) FISH staining verification of successful Lach. colonization in the mouse intestine; (E) HE staining of colonic tissue and radiation damage scoring (n=6 mice / group); (F) ELISA detection of changes in inflammatory factors (TNFα, IL-1β, IL-10) levels in each group of mice (n=6 mice / group, # / *p<0.05, ## / **p<0.01, ### / ***p<0.001; no statistically significant difference in ns). (G) Western blot analysis was performed on the expression levels of intestinal mucosal barrier proteins in each group of mice (n=6 mice / group, # / *p<0.05, ## / **p<0.01, ### / ***p<0.001; no statistical difference in ns). Control: control group; X-Ray: irradiated group; Lach.: Lachnospiraceae colonization group.

[0016] Figure 2 The study investigated the changes in the gut microbiota structure of mice after irradiation and colonization with *Lach.*. The results included: (A) Venn diagram of ASV classification; (B) ASV classification at the phylum, class, order, family, genus, and species levels; (C) Comparison of α-diversity indices of gut microbiota among the groups; (D) NMDS analysis of gut microbiota in each group (based on the Unweighted Unifrac algorithm); (E) PCA analysis (PC1 and PC2 explained 32.5% and 27.1% of the variance, respectively); (F) Statistical analysis of β-diversity indices of gut microbiota in each group (n=6 mice / group, p<0.05, p<0.01, p<0.001; no statistically significant difference in ns); and (G) Abundance of gut microbiota at the phylum, class, order, family, genus, and species levels in each group.

[0017] Figure 3This study aimed to analyze the changes in serum metabolites and their functions in mice after irradiation and colonization with *Lach.* bacteria. The results included: (A) PLS-DA analysis of samples from each group under positive ion mode; (B) PLS-DA analysis of samples from each group under negative ion mode; (C) Venn diagram showing changes in serum metabolites in each group; (D) Trend-correlated differential metabolite expression heatmap (n=6 mice / group, # / *p<0.05, ## / **p<0.01, ### / ***p<0.001); (E) Enrichment analysis of trend-correlated differential metabolites; (F) Correlation analysis of trend-correlated differential metabolite pathways and inflammatory factors (TNFα, IL-1β, IL-10) (*p<0.05, **p<0.01); (G) Correlation analysis of trend-correlated differential metabolites and gut bacteria.

[0018] Figure 4 This study analyzed the changes in intestinal metabolic profiles and functions in mice after irradiation and colonization with *Lach.* bacteria. The results included: (A) PLS-DA analysis of samples from each group under positive ion mode; (B) PLS-DA analysis of samples from each group under negative ion mode; (C) Venn diagram showing changes in serum metabolites in each group; (D) KEGG pathway analysis; (E) Venn diagram of differentially expressed metabolites in serum and intestine; (F) Statistical analysis of the intersection of differentially expressed metabolites in serum and intestine (# / *p<0.05; no statistical difference in ns); (G) Schematic diagram of the potential mechanism of the intersection metabolite Daidzein mediating radiation protection; (H) Correlation analysis between Daidzein and the top 10 intestinal bacteria at the family level; (I) Co-occurrence network analysis of Daidzein-related bacteria.

[0019] Figure 5 This study analyzed the changes and functions of intestinal gene expression profiles after irradiation and colonization by *Lach.* bacteria. The results included: (A) Volcano plot of differentially expressed genes in the intestine (p<0.05; |Log2 FC|>0.58). (B) Statistical analysis and intersection analysis of differentially expressed genes in different groups. (C) KEGG pathway analysis. (DE) Heatmaps of differentially expressed genes related to inflammation in the intestinal tissue of mice in each group. (F) Molecular docking results of Daidzein with proteins related to the NF-κB signaling pathway. (G) Schematic diagram of the anti-inflammatory mechanism of Daidzein through the NF-κB signaling pathway.

[0020] Figure 6To establish an irradiated intestinal organoid model and screen for the protective effect of Daidzein concentrations. The results include: (A) Schematic diagram of the irradiated intestinal organoid model. (B) Morphology of intestinal organoids and fluorescence intensity of inflammation-related proteins. (C) Statistical analysis of fluorescence intensity of inflammation-related proteins (n=6, *p<0.05, **p<0.01, ***p<0.001). (D) RT-qPCR detection of syk and nfkb expression in intestinal organoids (n=3). (E) Statistical analysis of germination rate of intestinal organoids under different irradiation intensities and Daidzein concentrations. (F) Morphology of intestinal organoids under different irradiation intensities and Daidzein concentrations.

[0021] Figure 7 To verify the mechanism by which Lachnospiraceae and its metabolite Daidzein alleviate radiation damage. The study included: (A) Protein expression analysis of CLEC4D, SYK, IκB, and NF-κB in intestinal tissues of mice in each group. (B) Quantitative analysis of protein expression in intestinal tissues of mice in each group (n=3, # / *p<0.05, ## / **p<0.01, ### / ***p<0.001; no statistically significant difference in ns). (C) Immunofluorescence staining of NF-κB in intestinal organoids under different concentrations of Daidzein intervention. (D) Immunofluorescence staining of SYK in intestinal organoids under different concentrations of Daidzein intervention. (E) Quantitative analysis of fluorescence intensity in intestinal organoids of mice in each group (**p<0.01, ***p<0.001). (F) RT-qPCR detection of syk, nfkb, and clec4d expression in intestinal organoids (n=3). Detailed Implementation

[0022] The following specific embodiments illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0023] Example 1

[0024] 1. Method

[0025] (1) Laboratory animals and grouping

[0026] SPF-grade C57BL / 6J mice (male, 6-8 weeks old, provided by Hangzhou Ziyuan Experimental Animal Technology Co., Ltd.) were divided into a control group, an irradiation group, and a Lach. group, with 6 mice in each group. The animal experiments were approved by the Animal Experiment Ethics Committee of Jiangnan University (ethics number: JN.No20241230t0180715

[713] ). All mice were housed in a 12-hour light-dark cycle environment at 20-24℃ and 50% relative humidity.

[0027] (2) Preparation of bacterial suspension of Lachnospiraceae BNCC354474

[0028] The Lachnospiraceae bacterium (BNCC354474) used in the experiment was purchased from Beina Biotechnology. The bacterial strain was inoculated onto Columbia blood agar plates and activated by anaerobic incubation at 37°C for 48-72 h. Single colonies were picked and inoculated into anaerobic liquid medium, and cultured further until the logarithmic growth phase. The bacterial suspension was collected under anaerobic conditions, centrifuged at 5000×g for 10 min at 4°C, washed twice with pre-cooled, deoxygenated sterile PBS, resuspended, and the bacterial concentration adjusted to 1×10⁻⁶. 9 CFU / mL.

[0029] (3) Experimental intervention

[0030] All mice underwent a 10-day environmental acclimatization period. Mice in the irradiation group received a 10 Gy dose of irradiation without intestinal colonization. Mice in the Lach. group were administered BNCC354474 bacterial suspension via gavage daily for one week, followed by 10 Gy X-ray irradiation. Gavage was suspended on the day of irradiation and resumed after irradiation, with colonization continuing for one week. The single gavage dose for each mouse was 200 μL. The control group was fed normally without any treatment. All mice were euthanized after the irradiation, and relevant samples were collected for subsequent testing.

[0031] (4) Detection of inflammation-related indicators

[0032] After modeling and intervention, the colon tissue was anesthetized with isoflurane using a small animal ventilator (Shenzhen Ruiwode Life Science & Technology, model: f8821-010). After homogenization with 1% PMSF-NP40 and centrifugation at 10,000 rpm for 15 minutes at 4℃, the levels of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and interleukin-10 (IL-10) in the colon tissue supernatant were measured using a species-specific ELISA kit (produced by China Enzyme Immunosorbent Assay Corporation).

[0033] (5) Fluorescence in situ hybridization (FISH) staining of colon tissue

[0034] Mouse colon tissue was fixed in 4% paraformaldehyde, embedded in OCT, and then frozen into 5 μm sections. Hybridization was performed overnight at 46°C using a Lachnospiraceae-specific fluorescent probe (FITC-labeled). After washing, cell nuclei were counterstained with DAPI, mounted, and observed under a fluorescence microscope. Three fields of view were randomly selected from each section to count the number and colonization status of Lachnospiraceae-positive bacteria.

[0035] (6) Western blot detection and quantitative analysis of colon tissue proteins

[0036] Total protein was extracted from colon tissue and its concentration was determined. 10 μg of protein was subjected to SDS-PAGE gel electrophoresis, followed by membrane transfer and blocking. Colon tissue was incubated with primary antibody (Abcam, diluted according to the manufacturer's instructions) containing Claudin-1, Occludin, p65 NF-κB, p-p65 NF-κB, IKBα, SYK, CLEC4D, β-actin, and GADPH. Incubation was then performed with HRP-labeled secondary antibody (dilution ratio 1:2500). After ECL chemiluminescence, the bands were scanned and analyzed. The relative expression level of the protein was represented by the ratio of the gray value of the target protein to the gray value of the corresponding internal control.

[0037] (7) Histopathological observation of colon tissue (HE staining)

[0038] Colon tissue from one mouse was randomly selected in each group, fixed overnight at room temperature in 4% paraformaldehyde, and then routinely dehydrated, embedded, and prepared for HE and Masson staining. The tissue was observed under an optical microscope (Nikon Eclipse ci, imaging system: Nikon DS-FI2). Three fields of view were photographed from each group of slides, and Image-Pro Plus 6.0 software was used to ensure consistent background lighting in all images.

[0039] (8) 16S rRNA sequencing

[0040] Sample pretreatment and DNA extraction: Fecal samples were collected and stored at -80℃. After homogenization, a suitable amount of sample was taken and total DNA was extracted using a microbial genomic DNA extraction kit. OD was measured using Nanodrop One. 260 / OD 280 The ratio was used to verify DNA integrity by 1% agarose gel electrophoresis.

[0041] 16S rRNA gene amplification: The V3-V4 region of the 16S rRNA gene was selected for PCR amplification. The reaction system was 20 μL, containing 10 μL of 2×Taq PCR Master Mix, 0.8 μL each of forward and reverse primers, 1 μL of template DNA, and 7.4 μL of sterile ddH2O. The amplification program was: 95℃ pre-denaturation for 3 min, 95℃ denaturation for 30 s, 55℃ annealing for 30 s, 72℃ extension for 45 s, for 30 cycles, followed by a final extension at 72℃ for 10 min. The amplified products were verified by 2% agarose gel electrophoresis.

[0042] Library construction and sequencing: After purifying the PCR products, end repair, adapter ligation, and PCR amplification were performed using a library construction kit to construct sequencing libraries. After verifying library quality using Qubit 4.0 and an Agilent 2100 Bioanalyzer, paired-end sequencing was performed using the Illumina MiSeq / HiSeq platform to obtain raw data.

[0043] Bioinformatics analysis: Trimmomatic software was used to filter the raw data to obtain Clean Reads; Uparse software was used to cluster OTUs with 97% sequence similarity and remove chimeras; species annotation was performed using the Silva database; Alpha diversity, Beta diversity and PCoA analysis were calculated, and methods such as LEfSe were used to screen for differentially expressed species.

[0044] (9) Metabolomics sequencing

[0045] Sample pretreatment: Serum samples: Take frozen serum, thaw at 4℃, add pre-cooled methanol, vortex mix, incubate at 4℃ to precipitate proteins, centrifuge at 12000 r / min for 15 min, collect the supernatant, freeze-dry under vacuum, reconstitute, and filter through a 0.22 μm membrane for later use. Intestinal tissue samples: Take an appropriate amount of colon tissue, homogenize with pre-cooled physiological saline, add methanol-acetonitrile mixture (1:1, v / v), vortex to extract metabolites, centrifuge at 4℃ for 15 min, collect the supernatant, freeze-dry, reconstitute, and filter through a membrane for later use.

[0046] Metabolomics detection: Detection was performed using an ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS / MS) platform. Chromatographic conditions: C18 column, mobile phase: acetonitrile-0.1% formic acid aqueous solution, gradient elution; Mass spectrometry conditions: electrospray ionization (ESI), positive and negative ion modes, acquiring metabolite mass spectrometry signals to obtain raw detection data.

[0047] Data preprocessing: Progenesis QI software was used to perform peak identification, peak alignment, noise reduction, and quantitative analysis on the raw data, removing low-quality peaks and interfering peaks from the blank control to obtain standardized metabolite peak intensity data.

[0048] Bioinformatics analysis: Multivariate statistical analysis was performed on the preprocessed data to screen for differentially expressed metabolites among groups; differentially expressed metabolites were annotated using the HMDB and KEGG databases, and metabolic pathways were analyzed.

[0049] (10) RNA-seq sequencing

[0050] RNA extraction: Total RNA was extracted from colon tissue using TRIzol® reagent (Invitrogen, USA), and genomic DNA was removed using DNase I (Takara). RNA quality was assessed using a 2100 Bioanalyzer (Agilent), and quantification was performed using an ND-2000 (NanoDrop Technologies). Only high-quality RNA samples (OD) were selected. 260 / 280 =1.8~2.2, OD 260 / 230 Construct a library with ≥2.0, RIN≥6.5, 28S:18S≥1.0, and >1μg.

[0051] Library preparation and Illumina sequencing: RNA-seq transcriptome libraries were constructed using the Illumina TruSeq™ RNA Sample Preparation Kit (San Diego, USA). After quantification with a TBS380, paired-end sequencing (2×150bp reads) was performed using an Illumina HiSeq xten / NovaSeq 6000 sequencer.

[0052] Differential expression analysis and functional enrichment: Transcript expression levels were calculated using the TPM method, and gene quantification was performed using RSEM (http: / / deweylab.biostat.wisc.edu / rsem / ). Metabolic pathways with significant enrichment of differentially expressed genes (DEGs) were screened using KEGG pathway analysis (Goatools and KOBAS tools).

[0053] (11) Real-time quantitative PCR (RT-qPCR)

[0054] Total RNA was extracted from tissues using TRIzol® reagent (Invitrogen, USA), and genomic DNA was removed using DNase I (Invitrogen, USA). Each group was divided into three replicates. Gene expression was detected using an ABI PRISM® 7500 system with a SYBR Green qPCR SuperMix Kit (Promega), with GAPDH as an internal control.

[0055] (12) Construction of intestinal organoids

[0056] C57BL / 6J mice aged 8 to 10 weeks were used to isolate small intestine, cecum, and colon tissues. The intestines were longitudinally dissected and rinsed with pre-chilled DPBS buffer to remove contents. The tissues were cut into small pieces and incubated in pre-chilled dissociation buffer (DPBS containing 30 mM EDTA). After incubation, the dissociation buffer was discarded, and the tissue fragments were washed with pre-chilled DPBS and transferred to 50 mL centrifuge tubes. The tubes were shaken vigorously for approximately 30-60 seconds to release the villous crypt units from the basement membrane. The mixed suspension was then filtered through a 70 μm cell sieve, and the filtrate was collected and centrifuged at 4°C to purify the crypts. The enriched intestinal crypts were mixed with Matrigel (RSQ-OSR006) and seeded into 24-well plates. The plates were incubated at 37°C for 20 minutes, followed by the addition of intact organoid culture medium (RSQ-OCM1102) and incubated at 37°C in a 5% CO2 incubator.

[0057] Twelve hours before X-ray irradiation, organoid culture media were pretreated with 0, 10, 30, 60, 120, and 180 μM Daidzein, respectively. After pretreatment, organoids were irradiated with 6 Gy, 8 Gy, and 10 Gy at a dose rate of 1.1 Gy / min using an X-ray irradiator, and a sham irradiation group (i.e., 0 Gy, 0 Daidzein) was set up as a control.

[0058] (13) Immunofluorescence staining

[0059] The constructed intestinal organoid models were collected and fixed. The organoids were resuspended in permeabilization reagent (Beyotime, P0115-100ML) and permeated at room temperature for 20 minutes. After washing with PBS, the organoids were blocked. The blocking solution was then discarded, and SYK and NF-κB primary antibodies (Abcam, dilution as recommended in the manufacturer's instructions) were added and incubated overnight. Subsequently, the corresponding fluorescein-labeled secondary antibody was added, and incubation was performed at room temperature in the dark. After incubation, the samples were washed again in the dark. The organoids were then co-incubated with DAPI solution at room temperature in the dark. After washing with PBS, an appropriate amount of organoid suspension was dropped onto a glass slide and mounted with an anti-fluorescence quenching mounting medium. The mounted samples were stored at 4℃ in the dark, and images were acquired using a confocal microscope.

[0060] Statistical analysis

[0061] Data are expressed as mean ± standard error (SEM). GraphPad Prism software was used for t-tests and graphing. A p-value < 0.05 was considered statistically significant.

[0062] 2. Results

[0063] (1) Lachnospiraceae colonization inhibits radiation-induced intestinal inflammation and barrier damage.

[0064] To investigate the protective effect of Lachnospiraceae colonization on the intestines of irradiated mice, we conducted the following experiments. We measured the body weight, survival rate, Lachnospiraceae colonization status, intestinal tissue radiation damage score, villous mucosal structure, colonic tissue inflammatory factors (TNF-α, IL-1β, IL-10), and expression of intestinal mucosal barrier proteins Claudin1 and Occludin in mice after a 10 Gy abdominal full-dose irradiation. Figure 1 A- Figure 1 G). The results showed that the average body weight of mice in the Lach. colonization group increased, and the survival rate was higher than that of the control group (G). Figure 1 B Figure 1 C); Colon FISH staining confirmed successful colonization of Lachnospiraceae in the mouse intestine. Figure 1 D). Histological and pathological analysis showed that Lachnospiraceae colonization significantly reduced radiation damage scores in mouse intestinal tissue and effectively restored irradiation-induced shortening of intestinal villi and mucosal structures. Figure 1 E). Inflammatory factor detection showed that Lachnospiraceae colonization significantly inhibited the upregulation of radiation-induced pro-inflammatory factors TNF-α and IL-1β, while restoring the decrease in the anti-inflammatory factor IL-10. Figure 1 F). Furthermore, Lachnospiraceae colonization can repair irradiation-induced damage to the intestinal mucosal barrier structure and upregulate the expression of Claudin1 and Occludin (F). Figure 1 G). The results showed that colonization of Lachnospiraceae effectively alleviated radiation-induced gastrointestinal damage in mice.

[0065] (2) Lachnospiraceae colonization improves radiation-induced gut microbiota dysbiosis at multiple taxonomic levels.

[0066] To investigate changes in the gut microbiome composition of irradiated and Lachnospiraceae-colonized mice, we examined the ASV count, α-diversity, β-diversity, and microbial composition at various taxonomic levels in the X-Ray group, Lach. group, and control group. Figure 2 A- Figure 2 G). The results showed that the bacterial flora of the X-Ray group was significantly different from that of the control group (G). Figure 2 A, Figure 2 B). α-diversity index analysis showed that the peak values ​​of the three sample groups exhibited a separation characteristic ( Figure 2C). β-diversity analysis showed that the bacterial community clustering regions of the Lach. group and the X-Ray group highly overlapped, while significantly separating from the control group (C). Figure 2 D- Figure 2 F). Further analysis showed significant differences between the Lach. group and the X-Ray group in Bray-Curtis and Unweighted Unifrac (F). Figure 2 (F) indicates that Lachnospiraceae can maintain the stability of the overall gut microbiota structure after irradiation, while altering species composition and evolutionary relationships, and enhancing the radiation resistance of the microbiota. Irradiation induces significant dysbiosis in the gut microbiota, while Lachnospiraceae colonization improves the dysbiosis at multiple taxonomic levels. Figure 2 (G) At the phylum level, Lachnospiraceae colonization restored the Firmicutes / Bacteroidetes ratio to the control group level; at the class level, irradiation increased the abundance of Bacteroidetes and decreased the abundance of Clostridium, but Lachnospiraceae colonization reversed these changes; at the order level, irradiation inhibited the Trichophyles and Lactobacilliles, but Lachnospiraceae colonization significantly restored the abundance of Trichophyles and partially restored the abundance of Lactobacilliles; at the family level, irradiation significantly depleted Trichophyceae, but Lachnospiraceae colonization significantly increased the abundance of Trichophyceae; at the genus level, irradiation decreased the abundance of related genera of Trichophyceae, Muribaculaceae, Lactobacillus, and Akkermansia, but Lachnospiraceae colonization reversed the decline of these bacteria, enriched related genera of Trichophyceae, and restored the abundance of beneficial genera such as Lactobacillus and Akkermansia. These findings suggest that Lachnospiraceae colonization can improve irradiation-induced gut microbiota dysbiosis and restore microbiota composition at multiple taxonomic levels.

[0067] (3) Lachnospiraceae colonization improves radiation-induced gut microbiota dysbiosis at multiple taxonomic levels.

[0068] To investigate the effect of Lachnospiraceae colonization on the remodeling of serum metabolic profiles in irradiated mice, the differences in serum metabolic profiles, the number of differentially expressed metabolites, the levels of key metabolites, pathway enrichment, and the correlation between metabolites and inflammatory factors and Lachnospiraceae were detected among the X-Ray group, the Lach. group, and the control group. Figure 3 A- Figure 3 G). PLS-DA analysis showed that Lachnospiraceae colonization caused the serum metabolic characteristics of radiation-damaged mice to tend towards those of the healthy control group (G). Figure 3 A, Figure 3B). Compared with the control group, irradiation led to a significant downregulation of 71 metabolites and a significant upregulation of 136 metabolites; while Lachnospiraceae colonization reduced the number of downregulated metabolites to 38 and increased the number of upregulated metabolites to 106. Figure 3 C). The heatmap confirmed that the gut microbiota is involved in the expression changes of specific metabolites in serum after Lachnospiraceae colonization, and 14 key metabolites were screened out to be involved in the gut microbiota dysbiosis caused by irradiation and Lachnospiraceae colonization. Figure 3 D). KEGG pathway analysis showed that key metabolites were significantly enriched in starch and sucrose metabolism, butyrate metabolism, and histidine metabolism pathways (D). Figure 3 E). Correlation analysis showed that butyrate metabolism was significantly negatively correlated with pro-inflammatory factors TNF-α and IL-1β, while histidine metabolism was positively correlated with pro-inflammatory factors, indicating that Lachnospiraceae colonization can maintain intestinal immune homeostasis by regulating key pathways such as butyrate metabolism. Figure 3 F). Furthermore, Lachnospiraceae showed a significant negative correlation with Pc(18:1e / 12-hete) and Daidzein (F). Figure 3 G). These findings confirm that Lachnospiraceae colonization may be associated with metabolites such as Pc(18:1e / 12-hete) and Daidzein in alleviating radiation-induced intestinal injury.

[0069] (4) Daidzein is the core effector molecule of Lachnospiraceae colonization-mediated radiation protection.

[0070] To further investigate differences in intestinal metabolites, we conducted the following experiments: We examined the differences in intestinal metabolic profiles between the X-Ray group, the Lach. group, and the control group; the number of differentially expressed metabolites; the enrichment of core metabolite pathways; the intersection of key metabolites in the gut and serum; changes in Daidzein levels; the correlation between microbiota and metabolites; and the microbiota interaction network. Figure 4 A- Figure 4 I). The results showed that Lachnospiraceae colonization restored irradiation-induced metabolic spectrum disorders ( Figure 4 A, Figure 4 B). Irradiation induced a significant downregulation of 572 metabolites and a significant upregulation of 237 metabolites; among them, 293 irradiation-downregulated metabolites were significantly upregulated after colonization, and 93 irradiation-upregulated metabolites were significantly downregulated after colonization. Figure 4 C). KEGG pathway analysis showed that differentially metabolites were mainly concentrated in pathways such as arginine and proline metabolism, amino sugar and nucleotide sugar metabolism, galactose metabolism, and glutathione metabolism. Figure 4D). By taking the intersection of key differentially expressed metabolites in the gut and serum, only daizein showed significant expression changes between the two groups, suggesting that daizein may be a core effector molecule across the gut-serum axis. Figure 4 E- Figure 4 G). Correlation analysis showed that Daidzein was significantly negatively correlated with Lachnospiraceae and Akkermansiaceae, and positively correlated with Bacteroidaceae. Microbiota interaction network analysis further indicated that Lachnospiraceae, along with other bacteria, constitutes a complex regulatory network, suggesting that the regulation of Daidzein by Lachnospiraceae is a synergistic effect of the gut microbiota. Figure 4 H, Figure 4 I). These findings suggest that Daidzein is a key effector molecule in the intestinal radioprotective function of Lachnospiraceae colonization.

[0071] (5) Daidzein directly targets CLEC4D to exert a regulatory effect.

[0072] To investigate the molecular mechanism by which Lachnospiraceae-mediated Daidzein alleviates intestinal radiation damage, we conducted the following experiments. We examined the transcriptomic changes, number of differentially expressed molecules, core differentially expressed molecules and their enrichment pathways, expression of NF-κB signaling pathway-related genes, CLEC4 family receptor expression, and molecular docking of Daidzein with CLEC4 family proteins in the X-Ray group, Lach. group, and control group. Figure 5 A- Figure 5 Transcriptomic analysis showed that irradiation induced the upregulation of 2002 molecules and the downregulation of 998 molecules; after Lachnospiraceae colonization, only 446 genes were significantly upregulated and 472 genes were significantly downregulated. Venn diagrams further identified 224 core differentially expressed molecules (G). Figure 5 A- Figure 5 B). Enrichment analysis of core differentially expressed molecules revealed that differentially expressed genes were highly concentrated in immune regulation-related pathways (B). Figure 5 C). Further mechanism-focused analysis revealed that most differentially expressed genes closely related to the inflammatory response significantly converged in the NF-κB signaling pathway (C). Figure 5 D、 Figure 5 E). Based on sequencing results, molecular docking of Daidzein with key CLEC4 family proteins CLEC4D and CLEC4E was verified. Molecular docking results showed that Daidzein can stably bind to CLEC4D and CLEC4E, suggesting that Daidzein may exert its regulatory role by directly targeting CLEC4D. Figure 5F). These findings suggest that Daidzein may directly target CLEC4D, thereby inhibiting the NF-κB pathway and downstream immune inflammatory responses, which is a key molecular mechanism by which Lachnospiraceae colonization alleviates radiation-induced intestinal injury. Figure 5 G).

[0073] (6) Intestinal organoid models confirmed that Daidzein alleviates radiation damage in a concentration-dependent manner.

[0074] To verify that Daidzein targets the CLEC4D / NF-κB pathway to alleviate radiation-induced intestinal injury, we constructed an irradiated intestinal organoid model. Figure 6 A). The morphology, budding rate, number, translucency, and structural integrity of organoids under different irradiation intensities (0, 6, 8, 10 Gy), as well as the fluorescence intensity and mRNA expression levels of NF-κB and SYK, were detected. Simultaneously, the morphology, budding rate, translucency, structural integrity, and toxic effects of organoids after intervention with different concentrations of Daidzein (0-180 μM) were also detected. Figure 6 BF). The results showed that irradiation intensity affected the morphology of intestinal organoids in a dose- and time-dependent manner: the 0 Gy group showed a stable increase in the number of organoids, good light transmittance, normal budding rate, and typical cystic growth; the 6 Gy group showed only mild damage, with a slight decrease in budding rate and number; the 8 Gy and 10 Gy groups showed almost zero budding rate, significantly reduced light transmittance, abnormal swelling, and a sharp decrease in number of organoids. Figure 6 B Figure 6 E). The fluorescence intensity of NF-κB and SYK increased with increasing irradiation intensity, and at the mRNA level, NF-κB and SYK showed the same trend (E). Figure 6 B- Figure 6 D). Since very few organoids survived after 10 Gy irradiation, and most were large vacuolated structures, 8 Gy was chosen for subsequent experiments. Daidzein intervention alleviated radiation damage in a concentration-dependent manner: a concentration of 60 μM showed the best protective effect, significantly restoring organoid translucency, budding rate, and structural integrity; while concentrations ≥120 μM led to vacuolated structures in the organoids, exhibiting toxic damage. Figure 6 E, Figure 6 F). Therefore, we set up a concentration gradient centered at 60 μM for subsequent experiments. These findings further validate the crucial role of Daidzein in radiation protection and its suitable concentration range.

[0075] (7) Lachnospiraceae and its metabolite Daidzein target the CLEC4 D-SYK-NF-κB axis to alleviate radiation-induced intestinal inflammation.

[0076] To further validate the protective effect of Lachnospiraceae against radiation enteritis at the overall level, we examined the protein expression and activity of CLEC4D, SYK, NF-κB, and IκB in the intestinal tissues of mice in the X-Ray group, Lach. group, and control group, as well as the effects of different concentrations of Daidzein (0-120 μM) on the expression of CLEC4D-SYK-NF-κB pathway-related proteins and mRNA induced by 8 Gy radiation in an in vitro intestinal organoid model. Figure 7 A- Figure 7 F). The results showed that irradiation significantly upregulated CLEC4D expression and activated downstream SYK and NF-κB inflammatory signaling pathways; while Lachnospiraceae colonization significantly inhibited the overactivation of these pathways, effectively alleviating radiation-induced intestinal inflammation by downregulating CLEC4D expression, inhibiting SYK activity, and stabilizing IκB protein levels. Figure 7 A, Figure 7 B). In an in vitro intestinal organoid model, daizein inhibited 8 Gy radiation-induced activation of the CLEC4D-SYK-NF-κB pathway in a concentration-dependent manner. Figure 7 C- Figure 7 E). At 60 μM, the fluorescence intensity of NF-κB and SYK, as well as their mRNA expression, decreased to levels close to those of the sham-irradiated group; while the inhibitory effect of 120 μM Daidzein was weakened. Figure 7 C- Figure 7 F). These findings collectively confirm that Lachnospiraceae and its metabolite Daidzein alleviate radiation-induced intestinal inflammation by targeting the CLEC4D-SYK-NF-κB axis.

[0077] In summary, this study found that Lachnospiraceae colonization remodels the gut microbiota structure after irradiation and restores microbiota homeostasis; the metabolite Daidzein targets the NF-κB inflammatory pathway and exerts a systemic protective effect against radiation-induced intestinal injury.

[0078] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. Application of a strain of Trichophyton spp. and / or daidzein in the preparation of products for the prevention and treatment of radiation-induced intestinal injury.

2. The application according to claim 1, characterized in that, The species described is Lachnospiraceaebacterium BNCC354474.

3. The application according to claim 1, characterized in that, The radiation-induced intestinal injury includes radiation enteritis caused by X-rays.

4. The application according to claim 1, characterized in that, The application is as follows: Trichophyton and / or daidzein achieve the prevention and treatment of radiation-induced intestinal injury by downregulating CLEC4D expression, inhibiting SYK activity, and stabilizing IκB protein levels.

5. A product for preventing and treating radiation-induced intestinal injury, characterized in that, The active ingredients are the Trichophyton spp. and / or daidzein as described in claim 1.

6. The radiation-induced intestinal injury drug product according to claim 5, characterized in that, The products include health foods and medicines.

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