Preparation of cordate houttuynia-derived extracellular vesicles and application of cordate houttuynia-derived extracellular vesicles in radiation enteritis treatment

Abstract

The invention provides application of cordate houttuynia extracellular vesicles in preparation of a medicine for treating radiation enteritis. As a vesicle structure containing lipid, protein, miRNA and active small molecules, the exosome disclosed by the invention has a multi-target regulation effect; the cordate houttuynia is wide in source, high in yield, low in obtaining cost, high in biological safety and less in side effect; and the survival rate can be obviously improved under the condition of high radiation dose, and the intestinal tissues are protected.

Description

Technical Field

[0001] This invention relates to the use of Houttuynia cordata extracellular vesicles in the preparation of drugs for treating radiation enteritis, and belongs to the field of biomedicine. Background Technology

[0002] Radiation enteritis is a common complication after radiotherapy for patients with abdominal / pelvic tumors. It mainly manifests as abdominal pain, diarrhea, and burning sensation in the anus. In severe cases, it can lead to intestinal obstruction, perforation, or fistula, significantly reducing patients' quality of life. Currently, there is a lack of targeted, safe, and effective drug treatments for radiation enteritis. Treatment is mostly limited to symptomatic supportive care such as anti-infection, nutritional support, intestinal mucosal protection, antidiarrheal medication, and adjustment of the intestinal flora, sometimes even requiring surgical resection of the affected segment. Currently, there are no effective treatments for radiation-induced bowel injury, limited to symptomatic supportive care and surgical resection of the affected bowel segment. Therefore, there is an urgent need to develop more radiation protection drugs to prevent and / or treat radiation-induced bowel injury.

[0003] Houttuynia cordata Thunb. is a commonly used traditional Chinese medicine that is both food and medicine. It has the effects of clearing heat and detoxifying, and clearing heat and stopping dysentery. It is widely used in the treatment of diseases such as pneumonia and enteritis, but the active substance basis is unclear. Existing research and patents have focused on the application of Houttuynia cordata in the treatment of ulcerative colitis. Patent CN113940945A reported that polysaccharides isolated from Houttuynia cordata have a significant therapeutic effect on DSS-induced ulcerative colitis in mice. Its mechanism may include inhibiting inflammatory cell infiltration, reducing the secretion of inflammatory factors (such as TNF-α, IL-6, IL-1β), and inhibiting the NF-κB signaling pathway, thereby reducing intestinal damage, protecting the intestinal mucosa, and alleviating colonic shortening and weight loss.

[0004] In recent years, plant-derived extracellular vesicles (PDEVs) have attracted attention as a natural basis for bioactive nanostructures. These nanovesicles secreted by plant cells contain various bioactive substances such as lipids, proteins, and nucleic acids, exhibiting good biocompatibility, stability, and targeting properties. Studies have shown that PDEVs isolated from edible plants such as grapes, citrus fruits, ginger, and carrots possess natural anti-inflammatory effects, regulating intestinal immune responses and maintaining intestinal homeostasis. Literature (Int J Nanomedicine, 2024) and patent CN202410681155.X report that Houttuynia cordata-derived vesicles can significantly alleviate DSS-induced colitis in mice and promote intestinal mucosal repair, suggesting that Houttuynia cordata PDEVs have therapeutic potential for intestinal inflammation.

[0005] However, radiation enteritis and ulcerative colitis differ significantly in their etiology and pathogenesis: radiation enteritis is mainly caused by ionizing radiation damage from radiotherapy, where radiation directly induces DNA double-strand breaks and generates large amounts of reactive oxygen species, leading to acute necrosis and chronic fibrosis of intestinal epithelial cells; while ulcerative colitis is an autoimmune-mediated chronic inflammatory bowel disease, the exact cause of which is still unclear, but is believed to be closely related to abnormal immune responses to gut microbiota, genetic susceptibility, and gut microbiota dysbiosis. Based on these differences, there are currently no publicly available reports in the literature on the use of Houttuynia cordata-derived extracellular vesicles for the treatment of radiation enteritis. This largely reflects the different pathogenesis of the two diseases, and the research results on Houttuynia cordata vesicles in alleviating immune-mediated colitis cannot be directly extended to enteritis caused by radiation damage. Summary of the Invention

[0006] To address the aforementioned technical problems, this invention provides a novel use of extracellular vesicles derived from Houttuynia cordata in improving radiation enteritis.

[0007] Based on the medicinal properties of fresh Houttuynia cordata, this invention uses Houttuynia cordata vesicles as the research object to systematically analyze the characteristics of H-Exos components and anti-inflammatory mechanisms, and explore the composition, structure and anti-radiation enteritis effect of extracellular vesicles derived from fresh Houttuynia cordata.

[0008] This invention provides the use of Houttuynia cordata extracellular vesicles in the preparation of a medicament for treating radiation enteritis.

[0009] The extracellular vesicles of Houttuynia cordata are derived from fresh Houttuynia cordata and extracted using differential high-speed centrifugation.

[0010] The extracellular vesicles of *Houttuynia cordata* have an average particle size of 185.5 ± 5.1 nm, distributed between 96 and 406 nm. The particle concentration is 3.07 × 10⁻⁶. 11 ± 1.98 × 10 10 The protein concentration was 6.27 ± 0.16 mg / mL, and the molecular weight of the protein was 45–60 kDa.

[0011] The extracellular vesicles of Houttuynia cordata contain lipid components: cholesterol, sphingomyelin, glycosphingomyelin, and phosphatidylserine.

[0012] The method for preparing the extracellular vesicles of Houttuynia cordata is as follows: Weigh fresh whole Houttuynia cordata into a clean beaker, add PBS (pH=7.4), filter the residue with a filter cloth, and transfer the filtrate to a centrifuge tube; centrifuge at 2,000 g in a high-speed refrigerated centrifuge at 4 °C for 5 min; take the supernatant and centrifuge at 10,000 g for 1 h; combine the supernatants and transfer them to an ultracentrifuge tube, centrifuge at 100,000 g in an ultracentrifuge at 4 °C for 2 h, discard the supernatant, resuspend the precipitate in 0.5 mL of PBS, and filter through a 0.22 μm microporous membrane to obtain Houttuynia cordata exovesicles.

[0013] The pharmaceutical preparations mentioned above are oral preparations and colonic preparations.

[0014] The beneficial effects of this invention are: 1. Exosomes, as vesicle structures containing lipids, proteins, miRNAs, and active small molecules, have multi-target regulatory functions; 2. Houttuynia cordata has a wide range of sources, high yield, low acquisition cost, high biological safety, and fewer side effects; 3. Most existing drugs are symptomatic treatments, while plant exovesicles have preventive and protective effects, and can significantly improve survival rates and protect intestinal tissues under high doses of radiation. Attached Figure Description

[0015] Figure 1 Characterization and composition analysis of H-Exos (A) Schematic diagram of H-Exos preparation from Houttuynia cordata. (B) Transmission electron microscopy morphology of H-Exos and nanoparticle analysis (C). (D) Lipidomics analysis results of H-Exos. (EF) Quantitative distribution of H-Exos in the 18-25 nt range and small RNA classification statistics (unique). (G) Bubble diagram of KEGG pathway enrichment results. (G) Bubble diagram of miRNA enrichment results in the KEGG pathway. (H) Protein isolation and Coomassie brilliant blue staining in H-Exos. (I) Protein subcellular localization results and KEGG enrichment map (J)); Figure 2 Drug administration strategy for modeling 14 GY radiation enteritis; Figure 3 A modeling and dosing strategy for IR at a dose of 10 GY; Figure 4 Changes in body weight and survival rate of mice in each IR group at a dose of 14 GY; Figure 5 Representative images of the abdominal intestines of mice in each IR group receiving a 14 GY dose; Figure 6Representative images and statistical graphs of the colon of mice in each IR group at a dose of 14 GY; Figure 7 Representative H&E images and PAS images of the duodenum, jejunum, ileum, and colon of mice in each IR group at a dose of 14 GY; Figure 8 Representative images of the abdominal intestines of mice in each of the IR groups receiving a 10 GY dose; Figure 9 Representative images and statistical graphs of the colon of mice in each IR group at a dose of 10 GY; Figure 10 Representative images and statistical graphs showing the expression levels of CD4+ and CD8+ in the spleens of mice in each IR group at a dose of 10 GY. Detailed Implementation

[0016] Example 1: Preparation of Houttuynia cordata extracellular vesicles (H-Exos) according to the present invention Weigh 1000 g of fresh Houttuynia cordata whole herb into a clean beaker, add twice the amount of PBS (pH=7.4), filter the residue using a filter cloth, and transfer the filtrate to a 50 mL centrifuge tube. Centrifuge at 2,000 g in a high-speed refrigerated centrifuge at 4 °C for 5 min. Collect the supernatant and centrifuge at 10,000 g for 1 h. Combine the supernatants and transfer them to an ultracentrifuge tube. Centrifuge at 100,000 g in an ultracentrifuge at 4 °C for 2 h. Carefully discard the supernatant, resuspend the precipitate in 0.5 mL of PBS, and filter through a 0.22 μm microporous membrane to obtain H-Exos. Store at -80 °C for later use.

[0017] Example 2 Characterization of extracellular vesicles of Houttuynia cordata according to the present invention 1. Particle size determination of H-Exos Take 50 μL of sample, dilute to 1000 μL with deionized water, add to a cleaned Anton Paar particle size distribution cup, and measure three times consecutively. The concentration and size of H-Exos were analyzed using nanoparticle tracking analysis (NTA).

[0018] 2. Morphological characterization of H-Exos The vesicle structure of H-Exos was observed using transmission electron microscopy (TEM). A carbon-plated copper mesh support film was placed on a sealing film, and an appropriate amount of sample was applied to the support film. After about 10 minutes, excess solution was absorbed from the edges with filter paper, and the sample was placed on the filter paper for about 1 minute. The dried support film was then placed on the sealing film, and a drop of phosphotungstic acid stain was added. The sample was stained for 90 seconds, and excess stain was absorbed with pointed filter paper. The sample was then placed on the filter paper to dry for 3 hours. The H-Exos vesicle structure was observed and photographed under a TEM.

[0019] 3. Determination of H-Exos protein concentration The specific protein concentration of H-Exos was determined using a BCA protein kit, and the molecular weight of the protein was determined using an SDS protein gel. A 10% developing gel and a 180 kDa protein marker were selected for the experiment, and the molecular weight of the protein was obtained after comparison.

[0020] 4. H-Exos nanoflow cytometry After co-incubating the samples with the membrane dye antibody for 30 min, the free dye was removed by centrifugation at 12,000 g for 20 min. The purity of H-Exos was analyzed by nanoflow cytometry (NanoFCM).

[0021] 5. Lipid composition determination of H-Exos Lipids were extracted using the MTBE method. 800 µL of MTBE was then added, and the mixture was sonicated at 4 °C for 20 minutes, followed by standing at room temperature for 30 minutes. The solution was centrifuged at 10 °C and 14000 g for 15 minutes to obtain the upper organic solvent layer, which was then dried under nitrogen.

[0022] Reversed-phase chromatography was performed using a CSH C18 column (1.7 µm, 2.1 mm × 100 mm, Waters). The lipid extract was redissolved in 200 µL of 90% isopropanol / acetonitrile. After centrifugation at 14000 g for 15 min, a final 3 µL sample was injected. Solvent A was acetonitrile-water (6:4, v / v) containing 0.1% formic acid and 0.1 mM ammonium formate; solvent B was acetonitrile-isopropanol (1:9, v / v) containing 0.1% formic acid and 0.1 mM ammonium formate. The initial mobile phase was 30% solvent B at a flow rate of 300 μL / min. The flow rate was maintained for 2 min, then linearly increased to 100% solvent B over 23 min, followed by equilibration at 5% solvent B for 10 min. Mass spectrometry was performed using Q-Exactive Plus in both positive and negative ion modes. All measured ESI parameters have been optimized and preset as follows: ion source temperature 300 °C, capillary temperature 350 °C, ion spray voltage 3000 V, S-lens RF level 50%, and instrument scanning range 200-1800 m / z.

[0023] 6. H-Exos miRNA assay Total RNA was isolated using the NGB-58000 exosome RNA isolation kit (Norgen Biotek, Canada), and RNA concentration was determined using a NanoDrop spectrophotometer (Thermo Fisher Scientific, Massachusetts, USA). RNA profiling was performed using an Agilent 2100 bioanalyzer (Agilent, California, USA). Small RNA sequencing libraries were then created using the TruSeq Small RNA Sample Preparation Kit (Illumina, San Diego, USA) according to the manufacturer's protocol. After library preparation, single-end sequencing was performed using a HiSeq 2000 / 2500 next-generation sequencer (Illumina, San Diego, USA) at 1x50 bp read lengths. Raw data were analyzed using ACGT101-miR v4.2 (LC Sciences, Houston, USA). Clean data were obtained by removing 3' linkers and junk sequences, retaining sequences of 18–26 nt in length. Valid data were obtained by comparing with mRNA databases (https: / / www.ncbi.nlm.nih.gov / genome) and (http: / / rfam.janelia.org (http: / / www.girinst.org / repbase)). Finally, miRNAs were identified by comparing with precursors and the genome. TargetScan v5.0 and miRanda v3.3a were used to screen for miRNA target genes, with the screening criteria being a TargetScan score ≥ 50 and a miRanda energy < -10. The final miRNA target genes were the intersection of the above screening results. To further determine the relationship between miRNAs and their functions, miRNA target genes were annotated using the Genome Ontology (GO, http: / / www.geneontology.org) and the Kyoto Genome Encyclopedia (KEGG, http: / / www.genome.jp / kegg / ).

[0024] 7. Proteomics assay of H-Exos 7.1 Sample preparation First, remove the sample from the -80 °C freezer and add an appropriate amount of lysis buffer (8 M urea, 1 mM PMSF, and 2 mM EDTA). Sonicate the sample for 5 minutes, then centrifuge at 15000 g for 10 minutes at 4 °C and collect the supernatant. Finally, determine the protein concentration using a BCA assay kit.

[0025] 7.2 Protein hydrolysis and desalting First, take 100 μg of protein solution according to the protein concentration, adjust to 200 μL with 8 M urea, reduce with 5 mM DTT at 37 °C for 45 minutes, and alkylate with 11 mM iodoacetamide (IAM) at room temperature for 15 minutes. Then, add 800 μL of 25 mM ammonium bicarbonate solution and 2 μL of trypsin (Promega, V5280), and digest overnight at 37 °C. Adjust the pH of the digested peptides to 2-3 with 20% TFA, and then desalt using C18 (Millipore, Billerica, MA) resin. Finally, determine the peptide concentration using the Pierce™ Quantitative Peptide Assay Kit (including standards).

[0026] 7.3 LC-MS / MS Analysis Samples were separated using a Vanquish Neo UHPLC system. Mobile phase A was 0.1% formic acid aqueous solution, and mobile phase B was acetonitrile containing 0.1% formic acid. A trap-elution dual-column method was used, with a PepMap Neo TrapCartridge (300 μm × 5 mm, 5 μm) as the trap column and an Easy-Spray™ PepMap™ Neo UHPLC column (150 μm × 15 cm, 2 μm) as the analytical column. Column temperature was controlled at 55 ℃, injection volume was 200 ng, flow rate was 2.5 uL / min, effective gradient was 6.9 min, and total run time was 8 min.

[0027] Independent data acquisition (DIA) analysis was performed using a Vanquish Neo system (Thermo Fisher Scientific). Samples separated by nano-flow high-performance liquid chromatography (HPLC) were analyzed by DIA mass spectrometry. A Thermo Scientific mass spectrometer was used for DIA mass spectrometry analysis. The detection mode was positive ion mode, with a precursor ion scan range of 380–980 m / z, a principal mass resolution of 240,000 (200 m / z), a normalized AGC target of 500%, and a maximum IT of 5 ms. MS2 used DIA data acquisition mode with 299 scan windows, an isolation window of 2 Th, and an HCD collision energy of 25%. The normalized AGC target was 500%, and the maximum IT was 5 ms.

[0028] 8. Results and Analysis: Fresh houttuynia cordata Thunb was crushed and juiced, and H-Exos was collected by multiple differential ultracentrifugation methods. Figure 1A). The final precipitate was resuspended in PBS and purified by ultrafiltration through a 0.22 μm membrane. The collected H-Exos were stored at -80 °C until use. The morphology of H-Exos was observed by transmission electron microscopy (TEM) images. Figure 1 (B) The nanoparticles (NPs) exhibit a teacup-shaped support structure or a concave hemispherical shape. Nanoparticle tracking analysis (NTA) results show that the average particle size of H-Exos is 185.5 ± 5.1 nm, distributed between 96 and 406 nm. The particle concentration is 3.07 × 10¹¹ ± 1.98 × 10¹¹. 0 Particles / mL ( Figure 1 C). Liquid chromatography-mass spectrometry (LC-MS) identified 146 substances, including amino acids and their derivatives, lipids, terpenes, flavonoids, and coumarin derivatives (Table S1). Based on previous studies, plant-derived exosomes typically contain lipids, RNA, proteins, and some metabolites. The lipid, RNA, and protein composition of H-Exos was analyzed. Non-targeted lipidomics was used to analyze the lipid composition of H-Exos, identifying 30 lipid subclasses and 527 lipid species. Figure 1 As shown in Figure D, the three most abundant lipids in H-Exos are phosphatidic acid (PA, 24%), lysophosphatidylcholine (LPC, 17%), and diacylglycerol (DG, 8%). These lipids are significantly enriched in naturally derived extracellular vesicles, especially in plant-derived vesicle structures, in stark contrast to sphingomyelin, cholesterol, phosphatidylcholine, and related components commonly found in mammalian exosomes. This difference reflects the unique biosynthetic pathways and membrane properties of plant-derived vesicles.

[0029] Non-targeted miRNA sequencing analysis of H-Exos showed that the miRNA size distribution was mainly 18-25 nt, with peak abundance at 24 nt (4×10⁻⁶). 5 sequence, Figure 1 E). The top 10 miRNAs accounted for 86% of all miRNAs, exhibiting a typical "long-tailed distribution" characteristic, suggesting that key therapeutic effects may be mediated by a few core miRNAs. The miRNAs with the highest expression levels were ath-miR166g, ath-miR159a, and ath-miR156j (E). Figure 1 F). Functional prediction: KEGG pathway analysis of the identified miRNAs showed that H-Exos miRNAs are involved in the biosynthesis of monocyclic β-lactams, purine metabolism, and sulfur metabolism, as well as in the biosynthesis of unsaturated fatty acids, sesquiterpenes, and triterpenes, and the MAPK signaling pathway, etc. Figure 1 G). miRNA analysis suggests that H-Exos may exert therapeutic effects by participating in multiple metabolic pathways in vivo through miRNA delivery.

[0030] The molecular weight range of H-Exos protein is 50-60 kDa. Figure 1 H), proteomics results showed that a total of 4348 peptides were identified, of which 1370 proteins were identified and quantified, including 419 cytoplasmic-associated proteins, 381 chloroplast-associated proteins, and 238 nuclear-associated proteins. Figure 1 I). KEGG functional annotation results showed that H-Exos proteins are associated with cellular processes, environmental information processing, metabolism, and organic systems. Among them, 1489 proteins were associated with metabolic pathways. Proteomics analysis indicated that H-Exos may participate in tissue metabolic regulation and immune homeostasis restoration by delivering functional proteins. Figure 1 J).

[0031] Comprehensive evidence from LC-MS, lipidomics, miRNA, and proteomics analyses demonstrates that H-Exos is a unique nanoplatform. Its distinctive lipidomics profile, dominated by PA and LPC, enables its uptake by animal cells. It also exhibits abundant miRNA and protein expression, with particularly significant enrichment of miRNAs and proteins in metabolic pathways. In conclusion, H-Exos can be considered a natural drug carrier nanovesicle, demonstrating promising anti-inflammatory therapeutic potential without the need for additional drug loading.

[0032] The following efficacy tests demonstrate the beneficial effects of this invention.

[0033] Experimental Example 1: Efficacy Test of the Invention 1. Radiation enteritis model Six- to eight-week-old ICR mice were acclimatized for 3 days. The mice were then randomly divided into three groups: PBS, IR+PBS, and IR+H-Exos. Mice were orally administered 300 μL of PBS and H-Exos (200 μg / mL) daily for 3 days. The control group received no X-ray, while the IR+PBS and IR+H-Exos groups received 14 Gy of X-ray irradiation. Subsequently, mice were orally administered 300 μL of H-Exos or PBS daily. On day 8, the mice were sacrificed, and the duodenum, jejunum, ileum, and colon were fixed in 4% paraformaldehyde for histological evaluation (see [link to relevant documentation]). Figure 2 ).

[0034] 2. Survival rate test for 14 GY radiation enteritis Six- to eight-week-old ICR mice were acclimatized for 3 days. The mice were then randomly divided into three groups: PBS, IR+PBS, and IR+H-Exos, with 11 mice in each group. Mice were orally administered 300 μL of PBS and H-Exos (200 μg / mL) daily for 3 days. The control group received no X-ray, while the IR+PBS and IR+H-Exos groups received 14 Gy of X-ray irradiation. Subsequently, mice were orally administered 300 μL of H-Exos or PBS daily. Survival status of the mice in each group was recorded and plotted over 8 days.

[0035] 3. 10 GY Radiation-Induced Enteritis Model Six- to eight-week-old ICR mice were acclimatized for 3 days. The mice were then randomly divided into three groups: PBS, IR+PBS, and IR+H-Exos. Mice were orally administered 300 μL of PBS and H-Exos (200 μg / mL) daily for 7 days. The control group received no X-ray, while the IR+PBS and IR+H-Exos groups received 10 Gy of X-ray irradiation. Subsequently, mice were orally administered 300 μL of H-Exos or PBS daily. Mice were sacrificed on day 16 for histological evaluation (see [link to relevant documentation]). Figure 3 ).

[0036] 4. Changes in spleen T cell typing To determine the effects of H-Exos on the body's immune system, spleens were minced and filtered through a 70 mm cell sieve to prepare single-cell suspensions. These suspensions were then incubated with mouse antibodies CD3 (PE), CD4 (FITC), and CD8 (percp / cyanine 5.5). Cell analysis was then performed using FCM (Thermo Fisher Scientific).

[0037] 5. Test Results 5.1 The effect of H-Exos on radiation enteritis at a dose of 14 Gy Intestinal damage and inflammation directly affect the diet and digestive function of mice. From the start of modeling, mouse weight was recorded, and mice were fed a fixed amount of food daily. It was observed that ionizing radiation led to reduced food intake and a continuous decrease in weight in the mice. H-Exos administration mitigated these weight changes and reduced ionizing radiation-induced mortality and improved survival rates within five days (see [link to H-Exos]). Figure 4 ).

[0038] Directly observe the lesions and repair status of the entire abdominal intestine in mice of each group, such as Figure 5As shown in the IR+PBS group, ionizing radiation caused mucosal edema in the entire intestine of mice, watery stools, and thickened intestinal walls. In contrast, mice treated with H-Exos showed better intestinal repair. Although intestinal damage still existed, the mucosal edema was greatly improved.

[0039] H-Exos significantly reversed the shortening of the colon in mice, by Figure 6 It is known that ionizing radiation causes the colon of mice to become shorter and thinner, and the cecum and intestinal contents to become unformed, resulting in diarrhea. H-Exos reversed this damage caused by ionizing radiation, and the intestinal contents of mice became formed, and the colon was maintained at a normal length.

[0040] In the clinical setting of tumor radiotherapy, radiation can cause various abnormalities in different parts of the small intestine. Based on the time of occurrence after irradiation, these can be divided into early and delayed injuries. Therefore, we tested various representative indicators in the duodenum, jejunum, and ileum using an abdominal irradiation model. We performed pathological evaluations of all parts of the mouse intestine using H&E and PAS to evaluate the radioprotective effect of H-Exos on the entire intestine. The results showed that 14 GY of ionizing radiation caused significant damage to the duodenum, jejunum, and ileum of the mouse intestine, including a reduction in regenerating crypt cells and shortening of intestinal villi. The results also indicated that the H-Exos group showed a significant reduction in damage in all three parts of the small intestine. In contrast, the colon was less affected by ionizing radiation (H&E, PAS), but damage still occurred. However, the protective effect of H-Exos on the distal colon was still observed (see [link to relevant documentation]). Figure 7 ).

[0041] 5.2 The effect of H-Exos on radiation enteritis at a dose of 10 Gy Even with reduced radiation doses, ionizing radiation damage to the mouse intestines was still observed. Mice in the IR+PBS group still exhibited intestinal wall thickening and mucosal edema. Mice treated with H-Exos also showed mucosal edema, but it was significantly different from that in the IR+PBS group. (See...) Figure 8 ) 10 GY still induced inflammation in the intestines of mice, but H-Exos significantly reversed the shortening of the colon in mice (see [link to article]). Figure 9 ).

[0042] The IR+PBS group showed an increased CD8+ / CD4+ ratio and an increase in helper T cells (CD4+). + CD8 cells are less tolerant of ionizing radiation than cytotoxic T cells (CD8+). + (cells), radiation can directly induce CD4 + Apoptosis, and increased levels of IL-6 and IFN-γ after radiation, promoting CD8 cell proliferation. +Effector T cell expansion, coupled with intestinal barrier disruption leading to the entry of microbial products into circulation, activates CD8. + Response to pathogens. H-Exos can reverse this change (see...) Figure 10 ).

[0043] 6. Conclusion Extracellular vesicles of Houttuynia cordata were isolated and purified from fresh Houttuynia cordata rhizomes using differential centrifugation. These vesicles were characterized by particle size, number, morphology, lipid content, protein content, and miRNA activity. They were then applied to 14 Gy and 10 Gy radiation-induced enteritis mouse models.

[0044] The following conclusions were drawn: (1) H-Exos particles obtained by differential centrifugation using fresh Houttuynia cordata rhizomes had a hydrodynamic diameter of 173.27 ± 2.35 nm. NTA results showed that the main peak of H-Exos particles was around 133 nm, and the particle concentration was 3.07 × 10⁻⁶. 11 ± 1.98 × 10 10 Particles / mL, H-Exos observed by TEM showed a teacup-shaped or concave hemispherical vesicle structure with a distinct membrane and clear three-dimensional structure.

[0045] (2) H-Exos contains 30 lipid subclasses and 527 lipid types, among which PA (24%), LPC (17%), and DG (8%) are abundant, and 4 × 10 miRNAs have a sequence length of 24. 5 The top three H-Exos mRNAs expressed were ath-miR166g, ath-miR159a, and ath-miR156j. The molecular weight of H-Exos proteins ranges from 50 to 60 kDa. Proteomics results showed a total of 4348 peptides identified, of which 1370 were identified and quantified as proteins. GO and KEGG results indicated that H-Exos play a role at the cellular level, in metabolic processes, and in glycolysis.

[0046] (3) H-Exos can improve the survival rate of mice at modeling doses of 14 GY and 10 GY, alleviate symptoms such as intestinal mucosal edema, intestinal wall thickening and diarrhea, and reverse the shortening and thinning of the colon in mice, thus achieving the purpose of whole intestinal repair and anti-radiation enteritis.

Claims

1. The use of Houttuynia cordata extracellular vesicles in the preparation of drugs for treating radiation enteritis.

2. The use according to claim 1, characterized in that: The extracellular vesicles of Houttuynia cordata are derived from fresh Houttuynia cordata and extracted using differential high-speed centrifugation.

3. The use according to claim 2, characterized in that: The average size of the extracellular vesicles from *Houttuynia cordata* was 185.5 ± 5.1 nm, distributed between 96 and 406 nm. The particle concentration was 3.07 × 10⁻⁶. 11 ± 1.98 × 10 10 The protein concentration was 6.27 ± 0.16 mg / mL, and the molecular weight of the protein was 45–60 kDa.

4. The use according to claim 1 or 2, characterized in that: The extracellular vesicles of Houttuynia cordata contain lipid components: cholesterol, sphingomyelin, glycosphingolipids, and phosphatidylserine.

5. The use according to any one of claims 1-4, characterized in that: The method for preparing the extracellular vesicles of Houttuynia cordata is as follows: Weigh fresh whole Houttuynia cordata into a clean beaker, add PBS (pH=7.4), filter the residue with a filter cloth, and transfer the filtrate to a centrifuge tube; centrifuge at 2,000 g in a high-speed refrigerated centrifuge at 4 °C for 5 min; take the supernatant and centrifuge at 10,000 g for 1 h; combine the supernatants and transfer them to an ultracentrifuge tube, centrifuge at 100,000 g in an ultracentrifuge at 4 °C for 2 h, discard the supernatant, resuspend the precipitate in 0.5 mL of PBS, and filter through a 0.22 μm microporous membrane to obtain Houttuynia cordata exovesicles.

6. The use according to claim 1 or 2, characterized in that: The pharmaceutical preparations mentioned are oral preparations and colonic preparations.

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