Application of peony-derived exosome-like nano-vesicles in preparation of medicine for treating inflammatory bowel disease

Abstract

The invention discloses application of peony-derived exosome-like nano-vesicles in preparation of medicines for treating inflammatory bowel diseases, and particularly relates to application of peony-derived exosome-like nano-vesicles in preparation of medicines for treating inflammatory bowel diseases, which is characterized in that peony roots are used as raw materials, exosome-like nano-vesicles are extracted through a high-speed centrifugation method, and the form and size of POELNs are determined through an electron microscope and particle size detection; the action mechanism is analyzed through detection of a mouse in-vivo colitis model and an in-vitro cell inflammation model, miRNA sequencing and non-targeted metabolism. The method has the advantages of easily available raw materials, simple preparation, mild conditions and high production safety.

Description

Technical Field

[0001] This invention relates to the application of peony-derived exosome-like nanovesicles (POELNs) in the preparation of drugs for treating inflammatory bowel disease. Background Technology

[0002] The information disclosed in this background section is intended only to enhance some understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that the information constitutes prior art known to those skilled in the art.

[0003] Ulcerative colitis (UC) is a classic inflammatory bowel disease (IBD) affecting millions worldwide and placing a heavy burden on healthcare systems. Clinical manifestations of UC include diarrhea, rectal bleeding, abdominal pain, and weight loss. In UC cases, a persistent and diffuse inflammatory process originates confined to the colonic mucosa and extends sequentially from the rectum towards the proximal colon. The etiology of UC is complex, involving the interaction of multiple disease-related genetic variations, increased pro-inflammatory cytokines, disruption of the intestinal barrier function, and environmental triggers. Existing therapies, such as aminosalicylates and corticosteroids, have varying degrees of side effects and do not provide long-term relief for UC patients. Therefore, new therapies are urgently needed to overcome these limitations.

[0004] In recent years, exosomal nanotherapies targeting colitis tissue and colon tumors have been widely used. Exosomes are extracellular vesicles secreted by eukaryotic cells with a diameter of 30–150 nm, playing a crucial role in regulating intercellular communication and influencing the progression of various diseases. Naturally derived exosomes contain abundant biofunctional molecules, exhibit good biosafety, and have the potential for large-scale production. Studies have shown that plants, through exosome-like nanoparticles, act on mammalian cells (especially intestinal macrophages and stem cells), and different types of plant exosomes have different biological effects. For example, exosome-like nanoparticles derived from grapes, grapefruits, and citrus fruits have a targeting effect on intestinal cells and can play a role in protecting mice from sodium dextran sulfate-induced colitis. Research has shown that hydrophobic curcumin can be delivered to colon tumors and normal colon tissues via plant exosomes, and exosomes can serve as carriers for cellular processing of oncogenic miRNAs.

[0005] peony( Paeonia ostii(T. Hong et JX Zhang) Belongs to the genus Paeonia in the family Paeoniaceae, commonly known as Fengdan. Its root bark, called Mudanpi, is used medicinally and is a commonly used traditional Chinese medicine for cooling the blood and removing blood stasis. Mudanpi is slightly cold in nature and has a bitter and pungent taste. It enters the heart, liver, and kidney meridians. Modern research shows that Mudanpi has antibacterial, anti-inflammatory, anti-allergic, anti-tumor, hemostatic, blood stasis-removing, heat-clearing and detoxifying, sedative, analgesic, and antispasmodic activities. It can also promote phagocytic function of mononuclear cells, improve the body's specific immune function, and increase the weight of immune organs. The effects and mechanisms of Mudanpi on inflammatory bowel disease are currently unclear.

[0006] Similar examples to this invention include: Zhao et al. found that blueberry-derived exosome-like nanoparticles (BELNs) can alleviate rotenone-induced oxidative stress in HepG2 cells and C57BL / 6 mouse models, thereby effectively alleviating non-alcoholic fatty liver disease (Zhao et al., 2021). Liu et al. reported that oral administration of turmeric (turmeric) exosomes can effectively repair damaged intestinal barriers, regulate gut microbiota, remodel macrophage phenotype, and collectively exhibit strong anti-inflammatory effects (Liu et al., 2022). Citrus plant-derived exosome-like nanoparticles have pharmacological effects such as antitumor, antioxidant, and anti-inflammatory properties, and can also serve as drug delivery carriers, showing broad application prospects in disease treatment (Han Fei, 2024).

[0007] Currently, UC treatment is divided into two main approaches: surgery and medical intervention. The latter includes various pharmacological drugs, such as aminosalicylic acid (ASA) derivatives, glucocorticoids, immunosuppressants, and biological therapies. While these treatments can alleviate the symptoms of UC patients, they are often limited by drug resistance and adverse reactions, thus hindering complete disease control. Furthermore, different plant-derived exosome nanoparticles vary greatly in their biological functions, making it difficult to find nanoparticles that can effectively alleviate the symptoms of UC patients and have the potential to be widely used in edible plant-derived products. Summary of the Invention

[0008] Although existing technologies disclose various plant-derived exosome-like nanoparticles, exosomes from different species exhibit significant differences in miRNA composition, lipid structure, and biological function. This invention discovers that oral administration of peony-derived exosome-like nanovesicles (POELNs) can induce a series of specific miRNA expression profile changes in the intestinal tissue of diseased animals. These changes are closely related to inflammation relief, providing a novel mechanistic insight into the treatment of IBD with POELNs. Based on this, this invention proposes the application of peony-derived exosome-like nanovesicles (POELNs) in the preparation of drugs for treating inflammatory bowel disease, providing further evidence for the use of edible plant-derived nanoparticles in the treatment of inflammatory diseases such as UC.

[0009] The technical solution adopted in this invention is as follows: In a first aspect of the invention, the use of peony-derived exosome-like nanovesicles (POELNs) in the preparation of a medicament for treating inflammatory bowel disease is provided.

[0010] In one or more embodiments of the present invention, the peony-derived exosome-like nanovesicles are prepared by a method comprising the following steps: a) Extract juice from peony roots and collect the juice; b) Perform a first centrifugation on the juice and collect the first supernatant; c) Perform a second centrifugation on the first supernatant and collect the second supernatant; d) Perform a third centrifugation on the second supernatant and collect the third supernatant; e) The third supernatant is subjected to ultracentrifugation to collect the precipitate; f) Wash the precipitate with PBS buffer to obtain the peony-derived exosome-like nanovesicles.

[0011] Preferably, the centrifugal force of the first centrifugation treatment is 1000g and the time is 10 minutes; the centrifugal force of the second centrifugation treatment is 3000g and the time is 20 minutes; the centrifugal force of the third centrifugation treatment is 10000g and the time is 40 minutes; and the centrifugal force of the ultracentrifugation treatment is 100000g and the time is 90 minutes.

[0012] In one or more embodiments of the present invention, the average particle size of the peony-derived exosome-like nanovesicles is 50 nm to 300 nm, preferably 104.6 nm to 210.3 nm, and more preferably 115.7 nm.

[0013] In one or more embodiments of the present invention, the zeta potential of the peony-derived exosome-like nanovesicles is negative, preferably -29.0 mV.

[0014] In one or more embodiments of the present invention, the therapeutic effect of the peony-derived exosome-like nanovesicles is achieved at least in part by regulating the expression profile of miRNAs in the intestinal tissue of the recipient, wherein the regulated miRNAs are associated with the regulation of the NF-κB signaling pathway and the T-cell receptor signaling pathway.

[0015] In one or more embodiments of the present invention, the peony-derived exosome-like nanovesicles contain one or more lipid components, the lipid components being selected from glycerophosphocholines, glycerophosphoinositols, fatty acids, glycosphingolipids, etc.

[0016] In one or more embodiments of the present invention, the drug is formulated as an oral preparation, preferably granules, capsules, soft capsules, oral liquid preparations, injections, transdermal drug delivery preparations, etc.

[0017] In a second aspect of the invention, a pharmaceutical composition is provided comprising a therapeutically effective amount of the peony-derived exosome-like nanovesicles and a pharmaceutically acceptable carrier.

[0018] In one or more embodiments of the present invention, it is formulated for oral administration for the treatment of inflammatory bowel disease.

[0019] Compared with the related technologies known to the inventors, one of the technical solutions of the present invention has the following beneficial effects: (1) The materials are readily available, and the preparation and extraction process is simple, low-cost, efficient, environmentally friendly, and can be mass-produced; (2) POELNs have good biocompatibility, are non-toxic, have low immunogenicity, and possess specific targeting capabilities; (3) Peony exosomes can specifically regulate the expression of miRNA in intestinal tissues and have a significant immunomodulatory effect on UC. They can enhance the anti-inflammatory ability of intestinal immune cells without producing adverse reactions and can be used as an ideal choice for preparing drugs to treat colitis. Attached Figure Description

[0020] The accompanying drawings, which form part of this specification, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0021] Figure 1 Images of peony-derived exosome-like nanovesicles, where A, B, and C are transmission electron microscope images of peony exosome-like nanoparticles.

[0022] Figure 2 : Particle size statistics of peony-derived exosome-like nanovesicles, where A is the particle size statistics of nanovesicles and B is the zeta potential diagram of nanovesicles.

[0023] Figure 3: Peony-derived exosome-like nanovesicles near-infrared fluorescent dye DIR mouse imaging; A: in vivo mouse imaging detection at different time points; B: imaging detection of mouse heart, liver, spleen, lungs and kidneys at different time points; C: imaging detection of mouse colon at different time points; D: imaging detection of mouse cecum at different time points.

[0024] Figure 4 Safety evaluation of peony-derived exosome-like nanovesicles; AB represents cellular toxicity evaluation, CF represents acute toxicity test in mice, C represents body weight change, D represents peripheral blood, E represents liver and kidney function indicators, and F represents HE staining of major organs.

[0025] Figure 5 A. Four fluorescence micrographs of cells in different treatment groups: Zoom: magnified view; Merge: composite image, superimposing the fluorescence of cell nuclei (Hoechst, blue) and ROS (DCFH-DA, green); Hoechst: blue fluorescence, used to label the cell nuclei of all cells, showing the cell number; DCFH-DA: green fluorescence, the intensity of which represents the level of intracellular reactive oxygen species (ROS); Control (control group): normal cells, with the weakest green fluorescence, representing the baseline ROS level; 100 µM H2O2 (model group): treated with H2O2 alone, with extremely strong green fluorescence, indicating that the oxidative stress model was successfully constructed and intracellular ROS burst; POELNs (drug-treated group): treated with both H2O2 and peony exosomes, with significantly weaker green fluorescence than the model group, proving that POELNs can effectively scavenge ROS induced by H2O2; B. Quantitative statistical bar chart of fluorescence intensity, which quantifies the green fluorescence intensity of the fluorescence images in A.

[0026] Figure 6 A: Western Blot results: POELNs dose-dependently reduced the expression of three core inflammatory proteins (iNOS, NLRP3, COX-2); Groups: Ctrl: Normal control group, with very low basal expression of inflammatory proteins. LPS: Inflammation model group, stimulation with LPS alone resulted in a sharp increase in the expression of the three inflammatory proteins, indicating a successful inflammation model. LPS+POELNs (100, 200, 400 µg / mL): Treatment groups, with increasing POELNs concentration, the band color / thickness of the three inflammatory proteins gradually lightened, demonstrating that POELNs inhibited the expression of these proteins in a dose-dependent manner. B: NO concentration bar chart. NO is the final product of iNOS enzyme activity, and its concentration directly reflects the strength of the inflammatory response. C: Western Blot results: POELNs can regulate the cell's own antioxidant defense pathways (Keap1 / Nrf2 / HO-1).

[0027] Figure 7The efficacy of exosome therapy was evaluated using a DSS-induced ulcerative colitis mouse model. A: Mouse body weight changes; B: Mouse pathological scores; C: Colon length photographs; D: Colon length and weight statistics; E: Colon pathological staining; F: Immunohistochemical staining for colonic TNFα inflammatory factors to characterize inflammation; G: qPCR detection of colonic inflammatory factor expression in mice, further demonstrating the therapeutic effect.

[0028] Figure 8 Heatmap of differentially expressed miRNAs in groups G1 and G2. Red: upregulated expression, blue: downregulated expression. The darker the color, the greater the change in expression level.

[0029] Figure 9 Volcano plot of differential miRNA expression in groups G1 and G2, used to identify molecules with statistically significant and large variations.

[0030] Figure 10 Scatter plot showing the correlation between miRNA expression levels in groups G1 and G2, used to compare the correlation between miRNA expression levels in the two groups (G1 and G2).

[0031] Figure 11 :GO functional classification hierarchical bar chart, comparing the distribution of biological processes, cellular components and molecular functions.

[0032] Figure 12 Top 20 entries in G1 vs G2 biological process enrichment analysis, including plant hormone signals, leaf development and metabolic processes.

[0033] Figure 13 Top 20 entries in G1 vs G2 cell component enrichment analysis, including localization analysis of cell nuclei, chloroplasts, and related complexes.

[0034] Figure 14 Histogram of GO enrichment analysis of differentially expressed genes between G1 and G2 (Top 20), based on log-log 10 Ranking of enrichment significance based on (P-value).

[0035] Figure 15 The top 20 items in the G1 vs G2 molecular function enrichment analysis show key molecular functions such as abscisic acid binding and enzyme activity.

[0036] Figure 16 :GO enrichment analysis scatter plot, showing the three-dimensional relationship between enrichment factors, gene number, and significance.

[0037] Figure 17 Bar chart showing the enrichment hierarchy of the KEGG pathway from G1 to G2, categorized into five major areas: cellular processes, environmental information processing, genetic information processing, metabolism, and biological systems.

[0038] Figure 18 Top 20 KEGG pathway enrichment analysis of G1 vs G2, including key pathways such as carotenoid biosynthesis, oxidative phosphorylation, and plant-pathogen interactions.

[0039] Figure 19 Scatter plot of KEGG pathway enrichment analysis, showing the multidimensional relationship between enrichment factors, gene number, and statistical significance.

[0040] Figure 20 Statistical graph of lipid classification in peony exosomes based on lipidomics. Detailed Implementation

[0041] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration of the invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0042] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the exemplary embodiments of the present invention. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, and / or combinations thereof.

[0043] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0044] Example 1: 1. Extraction of peony exosomes Juice was extracted from freshly washed peony roots and the juice was collected. The collected juice was centrifuged at 1000g for 10 minutes. The supernatant from the first centrifugation was then centrifuged at 3000g for 20 minutes. The supernatant from the second centrifugation was then centrifuged at 10000g for 40 minutes to remove impurities. Finally, the supernatant from the third centrifugation was collected and centrifuged at 100000g for 90 minutes. The precipitate was washed three times with PBS to obtain peony exosome nanoparticles, which are peony-derived exosome-like nanovesicles (denoted as POELNs). They were stored at -80℃ for later use.

[0045] 2. Exosome characterization analysis 1) Transmission electron microscopy analysis Peony-derived exosome-like nanovesicle precipitates were fixed and examined by transmission electron microscopy (TEM) using standard procedures. Observation was performed using a Tecnai G2 20 S-TWIN TEM microscope (FEI, USA). The obtained TEM micrographs are shown below. Figure 1 As shown in A, B, and C.

[0046] 2) Particle size analysis The size of peony-derived exosome-like nanovesicles was measured using laser scattering spectroscopy; particle size was measured using dynamic and static light scattering spectroscopy, and the results were obtained as follows: Figure 2 The particle size distribution shown in Figure A ranges from 104.6 nm to 210.3 nm, with an average diameter of 115.7 nm. The surface potential was measured using the Zeta potential analysis function of the same instrument, and the results are as follows: Figure 2 As shown in Figure B, its Zeta potential value is -29.0 mV, indicating that POELNs carry a negative charge in solution and have excellent colloidal dispersion stability, which is beneficial for their long-term storage.

[0047] Example 2: Oral distribution and colonic targeting of DIR-labeled peony exosome-like nanovesicles (POELNs) I. Experimental Materials DIR-labeled POELNs: Peony exosome-like nanovesicles prepared in Example 1 were incubated with DIR fluorescent dye (final concentration 2.5 μM) at 37°C in the dark for 20 minutes. They were then purified using a Sephadex G-25 size exclusion column equilibrated with PBS to completely remove unbound free dye.

[0048] Laboratory animals: Male C57BL / 6J mice, SPF grade, weighing 20–22 g, purchased from Vital River Pharmaceuticals in Beijing and housed at the Barrier System of the Laboratory Animal Center, School of Pharmacy, Shandong First Medical University. The experimental protocol was approved by the Ethics Committee of Shandong First Medical University, and all procedures were strictly performed in accordance with relevant regulations. The mice were fasted (but allowed water) for 12 hours before the experiment.

[0049] Main instrument: Small animal live imaging system.

[0050] II. Experimental Methods Drug delivery and imaging: Administration: Each mouse was administered DIR-labeled POELNs (at a dose of 15 mg / kg POELNs) via oral gavage.

[0051] In vivo imaging: At 0, 1, 3, 6, 12 and 24 hours after drug administration, in vivo imaging system was used to capture images of fluorescence distribution throughout the mouse body to dynamically track the biodistribution of POELNs in vivo.

[0052] In vitro imaging: Mice were anesthetized and euthanized at 24 hours, and major organs such as heart, liver, spleen, lung, kidney, colon and cecum were quickly dissected. After being gently washed with PBS, in vitro organ fluorescence imaging was performed to accurately quantify the enrichment of POELNs in each organ.

[0053] III. Results and Analysis The biodistribution of POELNs in mice after oral administration is as follows: Figure 3 As shown.

[0054] Dynamic process of live imaging ( Figure 3 A): In vivo imaging results clearly demonstrate the entire gastrointestinal transit trajectory of POELNs after oral administration. Following administration, the fluorescence signal first appeared in the stomach (1 h), then moved downwards along the small intestine with digestive motility (3-12 h), finally exhibiting highly specific and strong enrichment in the lower abdominal colon region at 24 hours. This dynamic process visually demonstrates that POELNs can pass through the gastrointestinal tract intact and target the colon.

[0055] Quantitative verification of ex vivo imaging ( Figure 3 B~ Figure 3 D): Imaging results of ex vivo organs at 24 hours provide conclusive evidence for in vivo observation. For example... Figure 3 C and Figure 3 As shown in Figure D, strong fluorescence signals were specifically enriched in the cecum and intestinal tissues. In contrast, no significant fluorescence signals were detected in major organs such as the heart, liver, spleen, lungs, and kidneys. This result eliminates possible tissue overlap interference in in vivo imaging and directly confirms the superior colon-targeting efficiency of orally administered POELNs.

[0056] IV. Conclusion This embodiment, through a combination of in vivo and in vitro imaging, confirms that orally administered peony exosome-like nanovesicles (POELNs) can effectively cross the upper gastrointestinal tract and, as a highly efficient biodelivery system, specifically accumulate in the colon. This lays a solid experimental foundation for developing them as a delivery carrier for colon-targeted therapies or health products.

[0057] Example 3 Safety Evaluation Laboratory animals: Male C57BL / 6J mice, SPF grade, weighing 20–22 g, purchased from Vital River Pharmaceuticals in Beijing and housed at the Laboratory Animal Center Barrier System of the School of Pharmacy, Shandong First Medical University. The experimental formulation was approved by the Ethics Committee of Shandong First Medical University.

[0058] To assess the potential safety of oral POELNs, mice were administered 15 mg / kg of POELNs daily for 7 days. On day 8, mice were sacrificed and their hearts, livers, spleens, lungs, and kidneys were collected. Results showed that, compared to the control group, oral POELNs resulted in comparable cell viability of HUVECs and NCM460 cells, as well as comparable mouse body weight. Figure 4 A, Figure 4 B and Figure 4 C); Blood levels of WBC, Lymph, Gran, Mon, PLT, and RBC are all normal. Figure 4 D); The levels of enzymes such as ALT, AST, CK, CREAM, and BUN in the blood were comparable to those in the control group. Figure 4 E), HE staining of major organs also showed no abnormalities ( Figure 4 F); The above data indicate that no side effects were observed with oral POELNs.

[0059] Example 4: Protective effect of peony exosome-like nanovesicles (POELNs) against intracellular oxidative stress in HaCaT cells I. Experimental Materials Cell line: Human immortalized keratinocytes (HaCaT cells).

[0060] Main reagents: Peony exosome-like nanovesicles (POELNs, prepared by Example 1), hydrogen peroxide (H2O2), DCFH-DA fluorescent probe, Hoechst nuclear staining reagent, cell culture-related reagents.

[0061] Main instruments: CO2 incubator, fluorescence microscope, ImageJ image analysis software.

[0062] II. Experimental Methods Cell culture and grouping: HaCaT cells in good growth condition were seeded into cell culture plates and cultured at 37°C and 5% CO2 in an incubator to an appropriate density.

[0063] The experiment was divided into three groups: Control group: Replace with fresh complete culture medium.

[0064] Model group: Add culture medium containing 100 µM H2O2.

[0065] POELNs group: Add a culture medium containing 100 µM H2O2 and a certain concentration (50 µg / mL) of POELNs.

[0066] Each group of cells was cultured for another 24 hours.

[0067] Intracellular ROS level detection: After the culture is completed, discard the culture medium, dilute the DCFH-DA probe (1:1000) with serum-free culture medium according to the instructions, add it to each well to cover the cells, and incubate at 37°C in the dark for 20-30 minutes.

[0068] After incubation, the cells were washed three times with serum-free medium to thoroughly remove any probes that had not entered the cells.

[0069] Nuclear staining and image acquisition: After washing, Hoechst staining solution was added to stain the cell nuclei.

[0070] Using a fluorescence microscope, cell nuclei were imaged under the Hoechst channel (blue excitation) to locate cells, and the green fluorescence signal of DCFH-DA was imaged under the FITC channel (green excitation) to reflect ROS levels. Merge images and magnified local images were also captured simultaneously.

[0071] Image and Data Analysis: The average intensity of green fluorescence in each group of cell images was analyzed using ImageJ software.

[0072] Data are expressed as mean ± standard deviation and statistical analysis was performed.

[0073] III. Results and Analysis Intracellular ROS level detection results are as follows Figure 5 As shown.

[0074] Fluorescence image analysis ( Figure 5 A): Compared with the control group, cells in the 100 µM H2O2 model group showed extremely strong green fluorescence, indicating that the oxidative stress model was successfully established. However, compared with the model group, the green fluorescence intensity of the POELNs group was significantly reduced, and its intensity was similar to that of the control group.

[0075] Quantitative analysis of fluorescence intensity ( Figure 5 B): The statistical results were consistent with the image observations. The mean fluorescence intensity of the POELNs group was significantly different from that of the model group (p<0.001), and recovered to a level close to that of the normal control group.

[0076] IV. Conclusion The results of this embodiment show that peony exosome-like nanovesicles (POELNs) can significantly reduce the level of reactive oxygen species (ROS) in HaCaT cells induced by H2O2, effectively alleviate oxidative stress damage, and demonstrate that POELNs have excellent intracellular antioxidant activity.

[0077] Example 5: Study on the anti-inflammatory and antioxidant mechanisms of peony exosome-like nanovesicles (POELNs) on LPS-induced RAW264.7 cells. I. Experimental Materials Cell line: Mouse mononuclear macrophage leukemia cells (RAW264.7 cells).

[0078] Main reagents: Peony exosome-like nanovesicles (POELNs, prepared by Example 1), lipopolysaccharide (LPS), nitric oxide (NO) detection kit, Western blotting reagents and antibodies (anti-iNOS, anti-NLRP3, anti-COX-2, anti-Nrf2, anti-Keap1, anti-HO-1, anti-GAPDH).

[0079] Main instruments: CO2 incubator, protein electrophoresis system, chemiluminescence imaging system, ELISA reader.

[0080] II. Experimental Methods Cell culture and model establishment: RAW264.7 cells were seeded in cell culture plates.

[0081] Experimental groups: control group (Ctrl), model group (LPS), and POELNs treatment group (LPS + 100, 200, 400 μg / mL POELNs).

[0082] Except for the control group, all other groups were given LPS (100 ng / mL) to stimulate cells to establish an inflammation and oxidative stress model.

[0083] Sample collection: Anti-inflammatory marker samples: After 12 hours of co-treatment with the drug and LPS, cells were collected for protein extraction (to detect iNOS, NLRP3, and COX-2) and cell supernatant (to detect NO concentration).

[0084] Antioxidant index samples: Cells were collected for protein extraction (Nrf2, Keap1, HO-1) 3 hours after co-treatment with the drug and LPS.

[0085] Detection method: Western Blot: Performed according to standard procedures, using GAPDH as an internal control, to detect the expression levels of each target protein.

[0086] NO concentration detection: The concentration of nitrite in the cell supernatant was detected using the Griess method kit. The specific method included collecting the cell culture supernatant 12 hours after drug treatment.

[0087] Follow the instructions of the NO detection kit (such as the Griess method) to determine the concentration of nitrite (a stable end product of NO) in the supernatant of each group.

[0088] The absorbance was measured at 540 nm using an ELISA reader, and the actual concentration of NO was calculated based on the standard curve.

[0089] Data Analysis: ImageJ software was used to analyze the grayscale values ​​of Western Blot bands, and all data were statistically analyzed.

[0090] III. Results and Analysis The results are as follows Figure 6 As shown.

[0091] Anti-inflammatory effects of POELNs: Western blot results showed that ( Figure 6 (A) Compared with the model group, the POELNs treatment group was able to dose-dependently and significantly downregulate the high expression of LPS-induced iNOS, NLRP3 and COX-2 inflammatory proteins.

[0092] NO test results ( Figure 6 B) shows that POELNs significantly (p<0.001) inhibited LPS-induced excessive NO release. These results indicate that POELNs possess strong anti-inflammatory capabilities.

[0093] The antioxidant mechanism of POELNs: Western blot results showed that ( Figure 6 (C) Compared with the model group, POELN treatment upregulated the expression of the nuclear transcription factor Nrf2 and its downstream antioxidant protein HO-1. This indicates that POELNs activate the cellular Nrf2 / HO-1 antioxidant defense pathway, which is an important molecular mechanism for alleviating oxidative stress.

[0094] Meanwhile, the experiment showed that the expression regulation pattern of Keap1 protein by POELNs is not entirely consistent with the classical theory. This is a valuable discovery, suggesting that POELNs may regulate the Nrf2 pathway through an atypical pathway, and its specific mechanism deserves further investigation.

[0095] IV. Conclusion This embodiment comprehensively demonstrates that peony exosome-like nanovesicles (POELNs) can exert synergistic anti-inflammatory and antioxidant effects in an LPS-induced macrophage model by inhibiting the expression of key inflammatory mediators and activating the endogenous antioxidant signaling pathway (Nrf2 / HO-1). This provides a solid pharmacological basis for developing POELNs into a natural formulation for the treatment of inflammation-related diseases.

[0096] Example 6: Therapeutic effect of peony exosome-like nanovesicles (POELNs) on a mouse model of colitis. I. Experimental Materials Experimental animals: Seventy male C57BL / 6J mice, SPF grade, weighing 20–22 g, were purchased from Vital River Pharmaceuticals in Beijing and housed in the barrier system of the Experimental Animal Center, School of Pharmacy, Shandong First Medical University. The experimental protocol was approved by the Ethics Committee of Shandong First Medical University (Ethics No.: W202306190276), and all procedures were strictly performed in accordance with relevant regulations. Animal identification was performed using 5% picric acid labeling. Mice were housed in cages of 5 mice per cage, with bedding changed every 2 days. The temperature was 22±2℃, with a 12-hour light-dark cycle and a relative humidity of 55±15%.

[0097] Main reagents: Peony exosome-like nanovesicles (POELNs, prepared in Example 1), sodium dextran sulfate (DSS), TRIzol reagent, reverse transcription kit, real-time quantitative PCR (qPCR) kit and primers (targeting IL-1β, IFN-γ, IL-17, and IL-6 genes).

[0098] Main instruments: electronic balance, tissue embedding and sectioning system, optical microscope, real-time quantitative PCR instrument.

[0099] II. Experimental Methods Establishment and grouping of colitis model: Mice were randomly divided into a control group and a POELNs treatment group.

[0100] Both groups of mice were induced to have acute colitis by freely drinking a certain concentration (3%) of DSS solution.

[0101] Control group: PBS solution was administered by gavage daily during the modeling period.

[0102] POELNs treatment group: During the modeling period, a certain dose (15 mg / kg POELNs) of POELNs (dissolved in 200 μL PBS) was administered by gavage daily.

[0103] The experimental period is usually 7-10 days.

[0104] Indicator Testing: Weight monitoring: The weight of mice in each group was weighed and recorded at regular intervals every day, and the percentage change in weight was calculated.

[0105] Colon sample collection: At the end of the experiment, the mice were euthanized and the entire colon was separated.

[0106] Measure and record the length of the colon.

[0107] A portion of colon tissue was fixed with 4% paraformaldehyde, embedded in paraffin, sectioned, and stained with H&E for histopathological observation.

[0108] Another portion of colon tissue was frozen at -80°C for qPCR detection.

[0109] Detection of mRNA expression of inflammatory factors: Total RNA was extracted from colon tissue using the TRIzol method, reverse transcribed into cDNA, and the relative expression levels of mRNA of genes such as IL-1β, IFN-γ, IL-17, and IL-6 were detected by qPCR, with GAPDH or β-actin as internal reference genes.

[0110] III. Results and Analysis Treatment results as follows Figure 7 As shown.

[0111] The results are as follows Figure 7 As shown.

[0112] POELNs improve the disease activity of colitis: such as Figure 7 As shown in Figure A, compared with the normal control group, the DSS model control group mice showed a significant decrease in body weight, exhibiting typical disease progression. However, the POELNs treatment group mice showed a significant reduction in body weight loss, and their body weight change curve was consistently higher than that of the model control group, indicating that POELNs can effectively alleviate the systemic symptoms of the disease.

[0113] POELNs alleviate macroscopic pathological damage to the colon: Colon length: Shortened colon is one of the most prominent macroscopic pathological features of ulcerative colitis. For example... Figure 7 As shown in C and 7D, compared with the model control group, the colon length of mice in the POELNs treatment group was significantly increased (p<0.0001). Statistical results of colon weight also support this conclusion. These data macroscopically demonstrate the protective effect of POELNs on colonic tissue structure.

[0114] Disease pathology score: The Disease Activity Index (DAI), calculated based on a combination of weight, stool characteristics, and rectal bleeding, shows ( Figure 7 (B) The pathological score of the POELNs treatment group was significantly lower than that of the model control group, which confirmed its therapeutic effect from the perspective of comprehensive clinical indicators.

[0115] POELNs improve histopathological damage of the colon: histological assessment by H&E staining of colonic tissue ( Figure 7(E). In the model control group, severe inflammatory cell infiltration, crypt structure destruction, and even disappearance were observed in the colonic mucosa. In contrast, the POELNs treatment group showed significant histological improvement, including reduced inflammatory infiltration and better preservation of crypt structure. This result directly confirms the therapeutic efficacy of POELNs at the tissue and cellular level.

[0116] POELNs inhibit the expression of colonic inflammatory factors: Protein levels: The expression of the key pro-inflammatory factor TNF-α was detected by immunohistochemical staining. Figure 7 F). The results showed that TNF-α was highly expressed in the colon tissue of the model control group, while the positive signal of TNF-α was significantly weakened in the POELNs treatment group, indicating that POELNs can upregulate the production of pro-inflammatory mediators at the protein level.

[0117] At the gene level: the mRNA expression levels of a series of inflammatory factors (IL-1β, IFN-γ, IL-17, IL-6) in colon tissue were detected by qRT-PCR. Figure 7 (G). Compared with the model control group, the transcriptional levels of these key pro-inflammatory factors were significantly downregulated in the POELNs treatment group. This reveals the molecular mechanism by which POELNs exert their anti-inflammatory effects from the source of gene expression regulation.

[0118] IV. Conclusion This embodiment demonstrates through in vivo animal experiments that oral administration of peony exosome-like nanovesicles (POELNs) can significantly improve clinical symptoms (body weight, colon length) and histopathological damage in DSS-induced colitis in mice. Its mechanism of action is closely related to the inhibition of gene expression of pro-inflammatory cytokines in colonic tissue. This provides strong preclinical experimental evidence for developing POELNs into a natural agent for the prevention or treatment of inflammatory bowel disease (ulcerative colitis).

[0119] Example 7: Mechanism of miRNA Action of Peony Exosomes in Treating Colitis Based on High-Throughput Sequencing Technology I. Experimental Objective By comparing the microRNA (miRNA) expression profiles of intestinal tissues in colitis model mice and mice treated with peony exosomes (POELNs), key differentially expressed miRNAs were screened out and their functions were predicted, thereby elucidating the pharmacological mechanism of POELNs from an epigenetic perspective.

[0120] II. Experimental Materials and Methods Sample preparation: As described in Example 6, a DSS-induced mouse model of ulcerative colitis was established.

[0121] Two groups were set up: the DSS model group (G1) and the DSS+POELNs high-dose treatment group (G2).

[0122] At the end of the experiment, mouse colon tissue was taken and rapidly frozen at -80°C.

[0123] Total RNA extraction and quality control: Total RNA was extracted from two groups of colon tissues using the TRIzol method.

[0124] RNA was quality checked using an Agilent 2100 Bioanalyzer to ensure its integrity before use in subsequent library construction.

[0125] Small RNA library construction and sequencing: Small RNA libraries were constructed using Illumina's TruSeq Small RNA Sample Prep Kit.

[0126] High-throughput sequencing was performed on the Illumina HiSeq 2000 / 2500 sequencing platform to generate raw sequencing data.

[0127] Bioinformatics analysis: Data quality control and filtering: The raw data is assessed for quality, and low-quality reads, connector sequences, and contaminating sequences are removed to obtain high-quality Clean Data.

[0128] Sequence alignment and annotation: Clean Data is aligned with the miRBase database (a database of known miRNAs) to identify known miRNAs and calculate their expression levels. Simultaneously, unannotated sequences are aligned with a reference genome.

[0129] Novel miRNA prediction: For sequences that cannot be annotated with known miRNAs, novel miRNAs are predicted by analyzing their genomic location and whether they can form typical stem-loop secondary structures.

[0130] Differentially expressed miRNA screening: Statistical analysis of the expression levels of all known and novel miRNAs in the two groups of samples was performed to screen out differentially expressed miRNAs with significant differences in expression levels.

[0131] Target gene prediction and functional enrichment analysis: Software such as TargetScan and miRanda were used to predict potential target genes of differentially expressed miRNAs.

[0132] GO functional annotation was performed on the target gene set to analyze the biological processes, cellular components, and molecular functions in which they participate.

[0133] KEGG pathway enrichment analysis was performed on the target gene set to reveal the signaling pathways in which they may be involved.

[0134] III. Results and Analysis Identification of differentially expressed miRNAs: Comparative analysis revealed a series of significantly upregulated and downregulated miRNAs in the POELNs treatment group compared to the DSS model group. These differentially expressed miRNAs are considered potential key effector molecules for the therapeutic effect of POELNs. Figure 8 The heatmap visually shows that after POELNs treatment, most of the differentially detected miRNAs showed an upregulation trend in the G2 group. The significantly upregulated miRNAs included cca-miR319_R+2, mdm-MIR319b-p3_1ss9CG, ​​sly-miR319c-5p_L-1R-1, dpr-MIR156b-p3_2ss3TC22AG, stu-MIR399h-p5_2ss2CA17TA, ptc-MIR396c-p3_2ss12CT18AG, bna-MIR166b-p5_2ss3TA19GA, sly-MIR171c-p3_2ss13GA18TG, mes-miR159c_R-1, ahy-miR394, and mes-MIR398-p5_1ss9CT. Figure 9 Significantly upregulated miRNAs included PC-5p-50857_51, PC-3p-101004_26, PC-5p-79005_33, and gma-MIR6300-p5_2ss18AC24TA, while significantly downregulated miRNAs included gma-MIR6300-p3_2ss18AC24TA, pca-mir6478_R+3_2ss6CT21GA, PC-5p-144598_16, and PC-3p-193595_10. Comprehensive analysis of high-throughput sequencing data showed that peony-derived exosome-like nanovesicles (POELNs) significantly remodeled the miRNA expression profile of intestinal tissue in a colitis model mouse. Firstly, Figure 10 The correlation scatter plot showed a distribution deviation between the sample points in groups G1 and G2, confirming from a global perspective that POELN intervention triggered a systemic change in the miRNA expression profile. Furthermore, Figure 8 The heatmap shows that this change is manifested as an overall upregulation of a core miRNA cluster, represented by the miR319 / 156 family, by POELNs. Finally, Figure 9 The volcano plot statistically confirmed the individual molecules with the most significant changes. These three plots corroborate each other, revealing the profound regulatory role of POELNs on the gut miRNA genome.

[0135] Target gene function prediction reveals the mechanism of action: GO enrichment analysis showed that the target genes of differentially expressed miRNAs were significantly enriched in biological processes closely related to the pathology of colitis, such as inflammatory responses, regulation of apoptosis, and epithelial cell proliferation. Figures 11-16 .

[0136] KEGG pathway analysis further revealed that these target genes were significantly enriched in classic inflammatory and immune regulatory pathways such as the NF-κB signaling pathway, the T cell receptor signaling pathway, and cytokine-cytokine receptor interactions. Figures 17-19 .

[0137] IV. Conclusion This embodiment systematically revealed the dynamic changes in the miRNA expression profile of intestinal tissue during the treatment of colitis with peony exosomes (POELNs) through high-throughput sequencing and bioinformatics analysis. We found that POELNs exert a synergistic therapeutic effect by regulating a specific set of miRNAs, thereby affecting their downstream target genes and signaling pathways related to inflammation, immunity, and barrier repair. This provides important epigenetic evidence for elucidating the deep pharmacological mechanism of POELNs.

[0138] Example 8: Lipomics Characterization of Peony Exosomes I. Research Background Exosomes are nanoscale vesicles encapsulated in a lipid bilayer. Their lipid components not only constitute their physical structure but also participate in key biological processes such as targeted recognition, cellular uptake, and biological signal transduction. Plant-derived exosome-like nanoparticles have shown great potential in the pharmaceutical field due to their excellent biocompatibility and bioactivity. This study aims to systematically elucidate the lipid composition of peony exosomes, providing a material basis for their functional research and application development.

[0139] II. Experimental Design and Methods Lipid extraction: Total lipids were extracted from the exosomes of Example 1 using a chloroform-methanol mixed solvent.

[0140] Lipidomics analysis: Non-targeted lipidomics analysis was performed using liquid chromatography-mass spectrometry.

[0141] Data processing: The obtained mass spectrometry data are compared with the LIPID MAPS database to identify and classify lipid molecules, and a primary classification statistical chart of lipids is generated.

[0142] III. Results and Analysis like Figure 20As shown, the lipid metabolism components are mainly glycerophosphocholines, glycerophosphoinositols, fatty acids, and glycosphingolipids. Among them, glycerophosphocholines and glycosphingolipids, which account for a relatively high proportion, are known to help maintain the stability of vesicle membranes and may mediate recognition and fusion with intestinal cells. This provides a material basis for the colonic targeting and biological function of POELNs.

[0143] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. The application of a peony-derived exosome-like nanovesicle (POELNs) in the preparation of a drug for the treatment of inflammatory bowel disease.

2. The application as described in claim 1, characterized in that, The peony-derived exosome-like nanovesicles were prepared by a method comprising the following steps: a) Extract juice from peony roots and collect the juice; b) Perform a first centrifugation on the juice and collect the first supernatant; c) Perform a second centrifugation on the first supernatant and collect the second supernatant; d) Perform a third centrifugation on the second supernatant and collect the third supernatant; e) Centrifuge the third supernatant at high speed and collect the precipitate; f) Wash the precipitate with PBS buffer to obtain the peony-derived exosome-like nanovesicles.

3. The application according to claim 2, characterized in that, The centrifugal force for the first centrifugation treatment is 1000g, and the time is 10 minutes; the centrifugal force for the second centrifugation treatment is 3000g, and the time is 20 minutes; the centrifugal force for the third centrifugation treatment is 10000g, and the time is 40 minutes; the centrifugal force for the ultracentrifugation treatment is 100000g, and the time is 90 minutes.

4. The application as described in claim 1, characterized in that, The average particle size of the peony-derived exosome-like nanovesicles is 50 nm to 300 nm, preferably 104.6 nm to 210.3 nm, and more preferably 115.7 nm.

5. The application as described in claim 1, characterized in that, The zeta potential of the peony-derived exosome-like nanovesicles is negative, preferably -29.0 mV.

6. The application as described in claim 1, characterized in that, The therapeutic effect of the peony-derived exosome-like nanovesicles is achieved at least in part by regulating the expression profile of miRNAs in the receptor intestinal tissue, and the regulated miRNAs are associated with the regulation of the NF-κB signaling pathway and the T-cell receptor signaling pathway.

7. The application as described in claim 1, characterized in that, The peony-derived exosome-like nanovesicles contain one or more lipid components, which are selected from glycerophosphate choline, glycerophosphate inositol, fatty acids, glycosphingolipids, and combinations thereof.

8. The application as described in claim 1, characterized in that, The drug is formulated as an oral preparation, preferably in the form of granules, capsules, soft capsules, oral liquid preparations, injections, or transdermal preparations.

9. A pharmaceutical composition comprising a therapeutically effective amount of any one of claims 1-7 peony-derived exosome-like nanovesicles and a pharmaceutically acceptable carrier.

10. The pharmaceutical composition according to claim 9, characterized in that, It is formulated for oral administration to treat inflammatory bowel disease.

Images