Preparation method of polygonatum odoratum exosome and anti-radiation damage application of polygonatum odoratum exosome
The exosomes of Polygonatum odoratum prepared by ultracentrifugation and sucrose gradient purification have solved the problems of high toxicity and poor efficacy of existing anti-radiation drugs, and achieved safe and efficient radiation protection, significantly improving radiation-induced weight loss, immune dysfunction and tissue damage.
Patent Information
- Application Number
- CN202511395203.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-09-28
- Publication Date
- 2025-10-31
AI Technical Summary
Existing anti-radiation drugs have drawbacks such as strong side effects, high toxicity, and short half-life, and their protective effect against radiation damage is limited. Traditional Chinese medicine is also insufficient in the application of radiation protection.
Exosomes of Polygonatum odoratum were prepared by ultracentrifugation and purified by sucrose gradient concentration to obtain saucer-shaped exosomes for radiation protection. The specific steps included washing fresh Polygonatum odoratum, multi-stage centrifugation, filtration and purification with sucrose solution to prepare high-purity exosomes with a particle size of 92.4-178.3 nm.
The prepared Polygonatum odoratum exosomes have high safety, stable active pharmaceutical ingredients and low toxicity. They can effectively reduce radiation-induced weight loss, inflammatory response and immune damage, restore spleen and thymus indices, reduce serum TNF-α and IL-6 levels, and significantly improve pathological damage to the spleen and thymus.
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Figure CN120860153A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of exosome preparation technology, specifically a method for preparing Polygonatum odoratum exosomes and their application in resisting radiation damage. Background Technology
[0002] With the rapid development of my country's economy, radiation is widely used in various fields. While this benefits humanity, it also brings unavoidable harm and poses a significant threat to human health. In recent years, the number of workplaces involving radiation has gradually increased, and more and more working groups are susceptible to potential mild radiation damage, such as aerospace workers, researchers, and medical workers.
[0003] Radiation not only causes significant damage to healthy tissues by destroying DNA and generating reactive oxygen species, but it also disrupts the body's immune function through immune stimulation and suppression, leading to cancer, physical deformities, and even death. In traditional Chinese medicine theory, radiation is considered a "hot and toxic evil," invading the blood vessels, affecting the internal organs, depleting qi and yin, and potentially causing dysfunction of related organs and an imbalance of yin and yang in the body. Therefore, it is essential to develop traditional Chinese medicine to combat radiation damage and balance immune function. Currently, the number of drugs developed for radiation protection is very limited, and they suffer from drawbacks such as strong side effects, high toxicity, and short half-lives. Designing safe and effective radiation protectants is an urgent problem to be solved.
[0004] Polygonatum odoratum (Mill.) Druce, a plant in the Liliaceae family, is a dried rhizome with a sweet taste and slightly cold properties. It is believed to nourish yin, moisten dryness, promote fluid production, and quench thirst. It was among the first medicinal materials included in the list of foods and medicines with similar properties. Modern pharmacological studies have found that Polygonatum odoratum possesses excellent antioxidant stress and immunomodulatory activities, increasing the spleen and thymus indices in immunosuppressed mice and alleviating the decline in immune cell counts.
[0005] Plant-derived exosome-like nanovesicles (PDELNs) are nanoscale vesicles secreted by plant cells. They are saucer-shaped or cup-shaped, composed of abundant lipids, nucleic acids, and proteins, and possess a phospholipid bilayer structure, with a diameter of 30–300 nm. Studies have shown that the bioactive molecules such as lipids, proteins, and nucleic acids contained in PDERNs can transmit information. These components can be absorbed by human cells and exert therapeutic effects. They act as extracellular messengers in intercellular communication, delivering biomolecules, and may also serve as potential therapeutic agents for various diseases.
[0006] Therefore, in view of the above situation, there is an urgent need to develop a method for preparing Polygonatum odoratum exosomes and its application in resisting radiation damage, so as to overcome the shortcomings in current practical applications. Summary of the Invention
[0007] The purpose of this invention is to provide a method for preparing Polygonatum odoratum exosomes and their application in resisting radiation damage, so as to solve the problems mentioned in the background art.
[0008] To achieve the above objectives, the present invention provides the following technical solution: A method for preparing Polygonatum odoratum exosomes includes the following steps: After washing the fresh Solomon's Seal, add it to pre-cooled PBS buffer and homogenize. Filter to obtain the filtrate. The filtrate was subjected to multi-stage centrifugation at 4°C: centrifuged sequentially at speeds from 1200×g to 150000×g for 10-90 min. (3) Take the precipitate from the last centrifugation, resuspend it in PBS buffer, and filter it through 0.45μm and 0.22μm filters in sequence to obtain a suspension; (4) The suspension was further purified using sucrose solutions of different concentrations. After centrifugation, the target band was aspirated and then diluted with PBS. (5) Centrifugation was used to remove sucrose and high-purity exosomes derived from Solomon's seal were obtained.
[0009] As a further aspect of the present invention: in step (1), the amount of PBS buffer added is 3 times the weight of fresh Solomon's seal.
[0010] As a further aspect of the present invention: in step (2), the multi-stage centrifugation extraction of exosome-like nanovesicle precipitate includes: Take the filtered homogenate and centrifuge it at 1200×g for 10-20 minutes, and take the supernatant. Centrifuge the first supernatant at 3000×g for 20-60 minutes and take the second supernatant; Centrifuge the second supernatant at 10000×g for 60 minutes and take the third supernatant; The supernatant was centrifuged at 100,000 × g to 150,000 × g for 60 to 90 minutes to obtain exosome precipitate.
[0011] As a further aspect of the present invention: in step (4), the mass concentration of the sucrose solution is 8%, 15%, 30%, 45%, and 60%, wherein the intermediate sample bands with concentrations of 30% and 45% are the target bands.
[0012] As a further aspect of the present invention: in step (5), the centrifugation conditions are 4°C, 100000×g, and 60 min.
[0013] As a further aspect of the present invention: In step (5), the prepared Polygonatum odoratum exosomes are tea tray-shaped, have a double-membrane structure, a particle size of 92.4-178.3 nm, and a concentration of 1×10⁻⁶. 7 Up to 1.4×10 7 Particles / mL.
[0014] Application of Polygonatum odoratum exosomes prepared by the method described above in the preparation of anti-radiation damage drugs.
[0015] As a further aspect of the present invention: the radiation damage is caused by 60 Damage caused by Co gamma rays.
[0016] As a further aspect of the present invention: the radiation damage resistance includes at least one of the following: It can reduce weight loss; increase the number of peripheral blood leukocytes and platelets; restore spleen and thymus indices; reduce serum TNF-α and IL-6 levels; and improve pathological damage to the spleen and thymus.
[0017] An anti-radiation damage drug comprising Polygonatum odoratum exosomes prepared by the above-described method as an active ingredient.
[0018] Compared with the prior art, the beneficial effects of the present invention are: First, the Polygonatum odoratum exosomes in this invention are derived from the natural Chinese herb Polygonatum odoratum, and the extraction process is simple, safe, environmentally friendly and pollution-free. Secondly, the Polygonatum odoratum exosomes prepared by this invention can be well integrated into the field of traditional Chinese medicine; it has high safety, stability of effective drug components, low toxicity and good solubility, and these advantages together give it a wide range of application prospects. Finally, the Polygonatum odoratum exosomes in this invention have a good effect on reducing radiation-induced damage, which refers to damage in mice exposed to radiation. 60 Short-term weight loss, inflammatory response, and immune damage were observed after exposure to Co gamma rays; this application represents an innovative expansion of traditional Chinese medicine treatment for radiation damage; it possesses many excellent characteristics, such as high safety, stability of effective drug components, extremely low toxicity, and good solubility, making Polygonatum odoratum exosomes a promising candidate for application in related therapeutic fields. Attached Figure Description
[0019] Figure 1 This is a flowchart of the experiment on the radiation damage resistance of Solomon's seal exosomes in an embodiment of the present invention.
[0020] Figure 2 This is a transmission electron microscope image of purified Polygonatum odoratum exosomes in an embodiment of the present invention.
[0021] Figure 3 This is a particle size distribution diagram of Polygonatum odoratum exosomes before and after purification in an embodiment of the present invention.
[0022] Figure 4 This represents the weight gain rate of mice in each group in this embodiment of the invention. Among them, compared with the model group: ** (P<0.01); compared with the medium-dose administration group: ns (P > 0.05), ## (P<0.01).
[0023] Figure 5 This is a schematic diagram showing the comparison of white blood cell counts in different groups of mice in an embodiment of the present invention.
[0024] Figure 6 This is a schematic diagram showing the comparison of platelet counts in different groups of mice in an embodiment of the present invention.
[0025] Figure 7 This is a schematic diagram comparing the organ indices of mice in each group in this embodiment of the invention.
[0026] Figure 8 This is a schematic diagram showing the comparison of serum IL-6 levels in mice in each group in this embodiment of the invention.
[0027] Figure 9 This is a schematic diagram showing the comparison of TNF-α levels in the serum of mice in each group in this embodiment of the invention.
[0028] Figure 10 This is a schematic diagram of the pathological morphological observation (H&E staining) of the spleen and thymus of mice in each group in the embodiments of the present invention. Detailed Implementation
[0029] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. 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.
[0030] The specific implementation of the present invention will be described in detail below with reference to specific embodiments.
[0031] Please see Figures 1-10 This invention provides a method for preparing Polygonatum odoratum exosomes and their application in resisting radiation damage. First, Polygonatum odoratum exosomes are prepared by ultracentrifugation, purified using a sucrose gradient concentration method, and their morphology observed by transmission electron microscopy. Second, a 6 Gy... 60A mouse model of mild radiation injury induced by Co-γ rays was established, using Polygonatum odoratum exosomes as a therapeutic agent. The effectiveness of Polygonatum odoratum exosomes in treating radiation injury was evaluated by observing changes in mouse body weight, detecting peripheral blood cell counts, calculating organ indices, and detecting changes in serum inflammatory factor levels. Details are as follows: (1) The preparation steps of Polygonatum odoratum exosomes are as follows: 1. Instruments and reagents: juicer, high-speed refrigerated centrifuge, ultra-high-speed refrigerated centrifuge, vacuum freeze dryer, Malvern particle size analyzer, and PBS buffer.
[0032] 2. The experimental method is as follows: Preparation of Solomon's Seal Exosomes by Ultracentrifugation: Weigh an appropriate amount of fresh Solomon's Seal, wash it clean, add pre-cooled PBS homogenate equivalent to 3 times the weight of the fresh Solomon's Seal to a juicer, and filter. The filtrate was centrifuged sequentially at 4°C: centrifuged at 1200×g for 10-20 minutes, and supernatant one was collected; supernatant one was centrifuged at 3000×g for 20-60 minutes, and supernatant two was collected; supernatant two was centrifuged at 10000×g for 60 minutes, and supernatant three was collected; supernatant three was centrifuged at 100000×g to 150000×g for 60-90 minutes. The precipitate was diluted with PBS and purified using 8%, 15%, 30%, 45%, and 60% sucrose solutions. Intermediate sample bands of 30% and 45% were centrifuged at 4°C and 100000×g for 60 minutes to remove sucrose, yielding high-purity Polygonatum odoratum-derived exosomes. The precipitate was resuspended in an appropriate amount of PBS and filtered through a 0.45 µm filter membrane. The particle size was measured using a Malvern particle size analyzer, the morphology was observed using a transmission electron microscope, and the concentration was determined using a NanoSight instrument.
[0033] (2) Application of Polygonatum odoratum exosomes in radiation damage protection: 1. Irradiation parameters: using 60 Mice were subjected to a single whole-body irradiation with Co γ rays at a distance of 3.75 m, a dose rate of 1.2 Gy / min, an irradiation dose of 6 Gy, an irradiation time of 5 min, and a projection area of 10 cm × 10 cm.
[0034] 2. Animal Grouping: SPF-grade female Kunming (KM) mice, 4 weeks old and weighing 25±4 g, were randomly divided into four groups: a blank control group, a model group, a low-dose administration group (1.36 mg fresh drug / g), a medium-dose administration group (2.73 mg fresh drug / g), and a high-dose administration group (5.46 mg fresh drug / g), with 6 mice in each group. The low, medium, and high-dose administration groups received Polygonatum odoratum exosomes via gavage daily for 7 days prior to irradiation, while the model and control groups received physiological saline via gavage. The single gavage volume was 0.2 mL, once daily. Administration continued for 13 days after irradiation, and samples were collected on the 14th day post-irradiation for parameter determination.
[0035] 3. The experimental content is as follows: 3.1 Preparation of Polygonatum odoratum exosomes: Weigh an appropriate amount of fresh Polygonatum odoratum, wash it clean, add pre-cooled PBS (3 times the weight of the fresh Polygonatum odoratum) to a juicer for homogenization, and filter. Centrifuge the filtrate several times at 1200 ×g to 150000 ×g at 4℃, 10-90 min each time. The precipitate from the last centrifugation is the Polygonatum odoratum exosome. Resuspend the precipitate with an appropriate amount of PBS and filter it sequentially through 0.45μm and 0.22μm filters. Purify it using 8%, 15%, 30%, 45%, and 60% sucrose solutions. Take the intermediate sample bands of 30% and 45% and centrifuge at 100000×g for 60 min at 4℃ to remove sucrose, obtaining high-purity Polygonatum odoratum-derived exosomes. The particle size was determined using a Malvern particle size analyzer, and the morphology was observed using a transmission electron microscope. The concentration was determined using a NanoSight instrument.
[0036] 3.2 Observation of weight gain changes: The dose-response relationship of Polygonatum odoratum exosomes was evaluated using the weight gain rate as an indicator. Mice were weighed on day 2 after radiation and again on day 14, and the weight gain rate was calculated. Weight gain rate = (mouse weight on day 14 - mouse weight on day 1) / mouse weight on day 1 × 100%.
[0037] 3.3 Blood routine analysis: On day 14 after radiation, mice in each group were weighed, and blood was collected by enucleation. Approximately 100 μL of blood was collected into a 1.5 mL anticoagulated centrifuge tube coated with EDTA, and the remainder was placed in a 1.5 mL ordinary centrifuge tube and stored at 4°C for later use. The number of white blood cells (WBC) and platelets (PLT) in peripheral blood was determined using a blood cell analyzer.
[0038] 3.4 Organ Index: Mice were euthanized by cervical dislocation, the thymus and spleen were separated, fat was removed, blood was washed away with pre-cooled physiological saline, and the surface moisture of the organs was blotted dry with filter paper. The mice were weighed, and the organ index was calculated. Organ Index = Organ weight (mg) / Body weight (g).
[0039] 3.5 Serum levels of tumor necrosis factor (TNF-α) and interleukin-6 (IL-6): Blood was collected from the eyeballs of mice and placed in 1.5 mL centrifuge tubes. The tubes were allowed to stand at room temperature for 2 h, then centrifuged at 1000 × g for 10 min at 4 °C to separate the serum. The levels were detected using an ELISA kit for mouse tumor necrosis factor (TNF-α) and interleukin-6 (IL-6) according to the instructions of the kit.
[0040] 3.6 Pathological changes in spleen and thymus: The spleen and thymus of mice were fixed in 4% paraformaldehyde for 24 h, rinsed and soaked thoroughly under tap water, dehydrated using an automatic tissue dehydration machine, cleared, embedded in paraffin, sectioned and stained with hematoxylin and eosin to obtain pathological sections, which were then observed under a microscope.
[0041] Example 1: Preparation of Polygonatum odoratum exosomes by ultracentrifugation; Example 1.1: Take 20g of fresh Polygonatum odoratum rhizome, wash it clean, cut it into small pieces, add an appropriate amount of PBS to a juicer, homogenize it in an ice bath, and filter it. Centrifuge the filtrate at 1200×g for 10 minutes to remove residual plant tissue and collect the first supernatant. Centrifuge the first supernatant at 3000×g for 20 minutes and collect the second supernatant. Centrifuge the second supernatant at 10000×g for 60 minutes to remove large particles such as cell debris and collect the third supernatant. Centrifuge the third supernatant at 100000×g for 60 minutes, discard the supernatant, dilute the precipitate with PBS, and purify it with 8%, 15%, 30%, 45%, and 60% sucrose solutions. Take the intermediate sample bands of 30% and 45% and centrifuge them at 100000×g for 60 minutes at 4℃ to remove sucrose, and obtain high-purity Polygonatum odoratum-derived exosomes.
[0042] Example 1.2: Take 20g of fresh Polygonatum odoratum rhizome, wash it clean, cut it into small pieces, add an appropriate amount of PBS to a juicer, homogenize it in an ice bath, and filter it. Centrifuge the filtrate at 1200×g for 20 minutes to remove residual plant tissue and collect the first supernatant. Centrifuge the first supernatant at 3000×g for 40 minutes and collect the second supernatant. Centrifuge the second supernatant at 10000×g for 60 minutes to remove large particles such as cell debris and collect the third supernatant. Centrifuge the third supernatant at 150000×g for 60 minutes, discard the supernatant, dilute the precipitate with PBS, and purify it with 8%, 15%, 30%, 45%, and 60% sucrose solutions. Take the intermediate sample bands of 30% and 45% and centrifuge them at 100000×g for 60 minutes at 4℃ to remove sucrose, and obtain high-purity Polygonatum odoratum-derived exosomes.
[0043] Example 1.3: Take 20g of fresh Polygonatum odoratum rhizome, wash it clean, cut it into small pieces, add an appropriate amount of PBS to a juicer, homogenize it in an ice bath, and filter it. Centrifuge the filtrate at 2000×g for 20 minutes to remove residual plant tissue and collect the first supernatant. Centrifuge the first supernatant at 3000×g for 60 minutes and collect the second supernatant. Centrifuge the second supernatant at 10000×g for 60 minutes to remove large particles such as cell debris and collect the third supernatant. Centrifuge the third supernatant at 150000×g for 90 minutes, discard the supernatant, dilute the precipitate with PBS, and purify it with 8%, 15%, 30%, 45%, and 60% sucrose solutions. Take the intermediate sample bands of 30% and 45% and centrifuge them at 100000×g for 60 minutes at 4℃ to remove sucrose, and obtain high-purity Polygonatum odoratum-derived exosomes.
[0044] Example 2: Identification of Polygonatum odoratum exosomes; 1. Transmission electron microscopy (TEM) observation: 10 μL of Polygonatum odoratum exosomes were added to a copper grid and precipitated for 1 min. The floating liquid was then absorbed with filter paper. 15 μL of 2% uranium acetate staining agent was added to the copper grid and precipitated for 1 min. The floating liquid was absorbed with filter paper, and deionized water was added. The mixture was immediately blotted dry to remove excess staining agent. After drying at room temperature for several minutes, electron microscopy was performed at 100 kV to obtain TEM images, confirming that it possesses a typical double-membrane, saucer-like structure. Figure 2 As shown; 2. Particle Size Distribution Detection: 100 μL of unpurified and purified Polygonatum odoratum exosome suspension was diluted to the optimal detection concentration range of the instrument, and the particle size distribution was detected using a nanoparticle tracking analyzer (ZetaView). According to the Malvern particle size analyzer results, the unpurified Polygonatum odoratum exosomes contained impurities; the purified particle size was 92.4-178.3 nm. Figure 3 As shown.
[0045] 3. Exosome Concentration Detection: The purified exosomes were appropriately diluted with PBS, and their concentration was detected using nanoparticle tracking analysis (NTA). According to the NanoSight instrument results, the concentration of high-purity Polygonatum odoratum exosomes was 1×10⁻⁶. 7 Up to 1.4×10 7 Particles / mL.
[0046] Example 3: Effects of Polygonatum odoratum exosomes on body weight changes in mice; SPF-grade female Kunming (KM) mice, 4 weeks old and weighing 25±4 g, were randomly divided into four groups: a blank control group, a model group, a low-dose administration group (1.36 mg fresh drug / g), a medium-dose administration group (2.73 mg fresh drug / g), and a high-dose administration group (5.46 mg fresh drug / g), with 6 mice in each group. The low, medium, and high-dose administration groups received Polygonatum odoratum exosomes via gavage daily for 7 days prior to irradiation, while the model and control groups received physiological saline via gavage. The gavage volume was 0.2 mL once daily. The mice were observed daily after irradiation, and the administration continued for 13 days. Body weight was measured on day 1 and day 14 post-irradiation, and the weight gain rate was calculated. Weight gain rate = (body weight on day 14 - body weight on day 1) / body weight on day 1 × 100%. Results are as follows: Figure 4 As shown.
[0047] No mice died after irradiation, and there were no significant differences in appearance, diet, or water intake. The model group showed a lower weight gain rate, while the low, medium, and high dose groups all showed significantly higher weight gain rates than the model group (P<0.05; P<0.01; P<0.01). The medium dose group showed a significantly higher weight gain rate than the low dose group (P<0.01), but not significantly different from the high dose group (P>0.05). These results indicate that radiation slows weight gain in mice. The Polygonatum odoratum exosomes prepared in this application can effectively avoid the effect of irradiation on slowed weight gain in mice and exhibit dose-related behavior. However, the effect on weight gain decreases when the dose is too high (≥5.46 mg / g of fresh drug).
[0048] Considering dose-effect relationship and cost, subsequent experiments will mainly focus on the anti-radiation damage effect of medium-dose Polygonatum odoratum exosomes.
[0049] Example 4: Effect on peripheral blood cell levels in mice; Radiation can adversely affect the hematopoietic system, causing varying degrees of blood cell reduction, thereby lowering the body's immune function. This embodiment aims to observe the effect of *Polygonum multiflorum* extract on radiation-induced reduction in blood immune function in mice by detecting the number of white blood cells (WBC) and platelets (PLT) in the blood of mice.
[0050] On day 14 post-irradiation, mice in each group were weighed, and blood was collected by enucleation. The blood samples were placed in 1.5 mL centrifuge tubes containing EDTA, and the white blood cell (WBC) and platelet (PLT) counts in the whole blood of each group of mice were measured using a complete blood count analyzer. The results are as follows: Figure 5 and Figure 6As shown, the levels of WBC and PLT in the blood of mice exposed to radiation but not treated decreased, while the treated group showed the opposite trend and the differences were significant (P<0.01; P<0.01). This indicates that gamma rays cause a decrease in blood WBC and PLT, which reduces the immune function of mice. The Polygonatum odoratum exosomes prepared in this application can reverse the imbalance of immune cell levels caused by radiation, thereby promoting the recovery of blood immune function.
[0051] Example 5: Effects on organ indices in mice; Irradiation typically causes damage to bodily organs, resulting in corresponding changes in their weight. The spleen and thymus are important immune organs. The spleen is the primary site of the immune response; a decrease in the spleen index affects humoral and cellular immunity, leading to decreased immunity. The thymus's main function is to cultivate various T lymphocytes and regulate immune function by secreting thymic hormones and cytokines through stromal cells, differentiating mature T cells. Therefore, the weight of the thymus can, to some extent, reflect the overall immune function. This embodiment aims to observe the effect of Polygonatum odoratum exosomes on radiation-induced immune function decline in mice by calculating the organ indices of these two immune organs.
[0052] On day 14 post-irradiation, mice were euthanized by cervical dislocation. The thymus was dissected, fat removed, and blood was washed away with pre-cooled saline. The organs were dried with filter paper, weighed, and the organ index was calculated. Organ index = organ weight (mg) / body weight (g). Results are as follows: Figure 7 As shown, the spleen index and thymus index of the model group mice were lower than those of the control group (P<0.01), indicating that radiation damages immune organs, leading to decreased immunity. For irradiated immune organ damage, the spleen index and thymus index of the treatment group were significantly increased (P<0.05). This indicates that the Polygonatum odoratum exosomes prepared in this invention can significantly promote the repair of immune organs and promote the recovery of immune function in mice.
[0053] Example 6: Effect on serum TNF-α levels in mice; TNF-α is mainly produced by activated immune cells such as macrophages, T cells, and natural killer cells (NK cells). During infection or tissue damage, TNF-α can activate the inflammatory response, promote the release of other inflammatory factors (such as IL-1 and IL-6), enhance vascular permeability, and recruit immune cells to the site of inflammation. This embodiment aims to observe the alleviating effect of Polygonatum odoratum exosomes on radiation-induced inflammatory responses by measuring serum TNF-α levels in mice.
[0054] Blood was collected from the eyeballs of mice and placed in 1.5 mL centrifuge tubes. The tubes were incubated at room temperature for 2 hours, then centrifuged at 1000 xg for 10 minutes at 4°C to separate the serum. Serum was detected using an ELISA kit for mouse tumor necrosis factor (TNF-α) according to the manufacturer's instructions. Results are as follows: Figure 8 As shown, compared with the control group, the serum TNF-α level in the model group mice was higher (P<0.01), indicating that radiation induced an inflammatory response. In contrast, the serum TNF-α level in the drug-treated group mice was significantly lower than that in the model group (P<0.05). This indicates that the Polygonatum odoratum exosomes prepared in this invention can effectively alleviate inflammation in the body.
[0055] Example 7: Effect on serum IL-6 levels in mice; IL-6 is mainly produced by T cells, B cells, macrophages, fibroblasts, and endothelial cells. It can promote acute inflammatory responses and stimulate the release of other inflammatory factors. This embodiment aims to further observe the alleviating effect of Polygonatum odoratum exosomes on radiation-induced inflammatory responses by measuring serum IL-6 levels in mice.
[0056] Blood was collected from the eyeballs of mice and placed in 1.5 mL centrifuge tubes. The tubes were incubated at room temperature for 2 hours, then centrifuged at 1000 xg for 10 minutes at 4°C to separate the serum. Serum was detected using an ELISA kit for mouse interleukin-6 (IL-6) according to the manufacturer's instructions. Results are as follows: Figure 9 As shown, compared with the control group, the serum IL-6 level in the model group mice was significantly increased (P<0.01), indicating that radiation induced an inflammatory response in the body. In contrast, the serum IL-6 level in the drug-treated group mice was significantly decreased compared with the model group (P<0.05). This further confirms that the Polygonatum odoratum exosomes prepared in this invention can effectively alleviate the inflammatory response in the body.
[0057] Example 8: Effects on the pathology of the spleen and thymus in mice; The spleen and thymus are important immune organs in the body. This embodiment aims to visually evaluate the alleviating effect of Polygonatum odoratum exosomes on radiation-induced immune organ damage by observing H&E-stained sections of the spleen and thymus of mice in each group.
[0058] Mouse spleens and thymus were fixed in 4% paraformaldehyde for 24 h, thoroughly rinsed and soaked under tap water, dehydrated using an automated tissue dehydrator, cleared, embedded in paraffin, sectioned, and stained with hematoxylin and eosin to obtain pathological sections, which were then observed under a microscope. Results are as follows: Figure 10 As shown, in the model group mice, the splenic trabeculae were fractured and dissolved, with significant inflammatory cell infiltration and indistinct boundaries between the white and red pulp. In the drug-treated group mice, the spleen boundaries were clear and the structure was intact, with significantly reduced inflammation. The model group mice showed medullary thickening and cortical thinning accompanied by inflammatory infiltration, while the drug-treated group mice had a normal ratio of thymic medulla to cortex and reduced inflammation. This indicates that the Polygonatum odoratum exosomes prepared in this invention can effectively alleviate radiation-induced tissue damage and structural changes.
[0059] It should be noted that, in this invention, although the specification describes the embodiments, not every embodiment contains only one independent technical solution. This way of describing the specification is only for clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.
Claims
1. A method for preparing Polygonatum odoratum exosomes, characterized in that, Includes the following steps: (1) After washing the fresh Solomon's Seal, add pre-cooled PBS buffer to homogenize and filter to obtain the filtrate; (2) Centrifuge the filtrate at 4℃ in multiple stages: centrifuge at speeds from 1200×g to 150000×g for 10-90 min in sequence; (3) Take the precipitate from the last centrifugation, resuspend it in PBS buffer, and filter it through 0.45μm and 0.22μm filters in sequence to obtain a suspension; (4) The suspension was further purified using sucrose solutions of different concentrations. After centrifugation, the target band was aspirated and then diluted with PBS. (5) Centrifugation was used to remove sucrose and high-purity exosomes derived from Solomon's seal were obtained.
2. The method for preparing Polygonatum odoratum exosomes according to claim 1, characterized in that, In step (1), the amount of PBS buffer added is 3 times the weight of fresh Solomon's seal.
3. The method for preparing Polygonatum odoratum exosomes according to claim 1, characterized in that, In step (2), the multi-stage centrifugation extraction of exosome-like nanovesicle precipitates includes: Take the filtered homogenate and centrifuge it at 1200×g for 10-20 minutes, and take the supernatant. Centrifuge the first supernatant at 3000×g for 20-60 minutes and take the second supernatant; Centrifuge the second supernatant at 10000×g for 60 minutes and take the third supernatant; The supernatant was centrifuged at 100,000 × g to 15,000 × g for 60 to 90 minutes to obtain exosome precipitate.
4. The method for preparing Polygonatum odoratum exosomes according to claim 1, characterized in that, In step (4), the sucrose solution mass concentrations are 8%, 15%, 30%, 45%, and 60%, with the intermediate sample bands at concentrations of 30% and 45% being the target bands.
5. The method for preparing Polygonatum odoratum exosomes according to claim 1, characterized in that, In step (5), the centrifugation conditions are 4℃, 100000×g, 60 min.
6. The method for preparing Polygonatum odoratum exosomes according to claim 5, characterized in that, In step (5), the prepared Polygonatum odoratum exosomes are saucer-shaped, have a double membrane structure, a particle size of 92.4-178.3 nm, and a concentration of 1×10⁻⁶. 7 Up to 1.4×10 7 Particles / mL.
7. The application of Polygonatum odoratum exosomes prepared by the method described in any one of claims 1-6 in the preparation of anti-radiation damage drugs.
8. The application of Polygonatum odoratum exosomes according to claim 7 in the preparation of anti-radiation damage drugs, characterized in that, The radiation damage is caused by 60 Damage caused by Co gamma rays.
9. The application of Polygonatum odoratum exosomes according to claim 7 in the preparation of anti-radiation damage drugs, characterized in that, The radiation damage resistance includes at least one of the following: It can reduce weight loss; increase the number of peripheral blood leukocytes and platelets; restore spleen and thymus indices; reduce serum TNF-α and IL-6 levels; and improve pathological damage to the spleen and thymus.
10. A radiation damage protection drug, characterized in that: The active ingredient is the Polygonatum odoratum exosome prepared by the method described in any one of claims 1-6.