Application of Flammulina velutipes-derived exosome-like nanovesicles in radiation protection

Abstract

Application of exosome-like nanovesicles derived from Flammulina velutipes in radiation protection, belonging to the technical field of radiation protectants. The specific scheme includes the following steps: Step 1: Cut off the roots of the Flammulina velutipes fruiting bodies, cut the remaining parts into pieces and add an extraction solvent; Step 2: After breaking the cell walls and pulverizing, obtain a filtrate by suction filtration, obtain a supernatant by differential centrifugation, and obtain a precipitate by ultra-high-speed centrifugation, namely exosome-like nanovesicles derived from Flammulina velutipes; Step 3: Use the exosome-like nanovesicles derived from Flammulina velutipes to resist radiation damage. The present invention first proves that animals after oral ingestion of exosome-like nanovesicles derived from Biyang Flammulina velutipes have the effect of resisting ionizing radiation-induced damage to the body; and first proves that exosome-like nanovesicles derived from Biyang Flammulina velutipes combined with Flammulina velutipes polysaccharide have the effect of synergistically reducing ionizing radiation-induced cell damage.

Description

Technical Field

[0001] The present invention belongs to the technical field of radiation protection, and particularly relates to the application of exosome-like nanovesicles derived from Flammulina velutipes in radiation protection. Background Art

[0002] Ionizing radiation is the general term for radiation that can cause ionization of substances and is a high-energy physical damage factor that seriously endangers human health. It can act on biological molecules such as nucleic acids and proteins by generating excessive reactive oxygen free radicals in the body, resulting in varying degrees of damage to the body's hematopoietic system, reproductive system, immune system, etc., and even canceration. Radiation protectants are one of the important means to prevent oxidative damage caused by ionizing radiation. At present, the developed radiation protection preparations mainly rely on chemical drugs, including sulfur-containing compounds, cytokines, hormones, etc., but their costs are relatively high, and they have certain toxic and side effects and are not suitable for long-term use. Radiation protection functional factors derived from natural products can improve the radiation tolerance of the body and have special advantages in terms of efficacy, cost, and safety, and can be used in fields such as pharmaceuticals and health foods. Currently, the mainly screened natural-source radiation protection functional factors are: small biological molecule polyphenols such as resveratrol, curcumin, and tea polyphenols; large biological molecule polysaccharides such as poria cocos polysaccharide and yam polysaccharide. However, both of these two types of natural radiation protection functional factors have certain limitations in practical applications: such as low bioavailability, high cost, and limitations in the application process, etc. Therefore, it is still necessary to search for and develop new natural-source radiation protection functional factors.

[0003] Extracellular vesicles (EVs) refer to extracellular structures enclosed by a lipid bilayer membrane, containing bioactive substances such as proteins, lipids, and nucleic acids, and playing an important role in cell - cell communication. EVs can be released by all types of cells (including animals, plants, and microorganisms) and have various sizes (100 - 1000 nm in diameter), and are usually referred to as microvesicles, exosomes, or microparticles in the reported literature. Due to the ideal characteristics of EVs themselves, such as small size, high biocompatibility, and high stability, they have received extensive attention. Dietary exosome - like nanovesicles (DELNs) are obtained from food, usually EVs with a size below 300 nm, and also contain various active substances such as proteins, lipids, and nucleic acids, showing various biological functions, such as anti - inflammatory, antioxidant, and anti - cancer functions. DELNs have the advantages of low safety risk, simple preparation and processing, nano - structure stability, and high bioavailability for absorption, and have a good foundation for development and application. Flammulina velutipes is a variant of the common edible mushroom Lentinula edodes, which is cultivated by controlling growth conditions and changing the development process of the fruiting body. Exosome - like nanovesicles derived from Flammulina velutipes also belong to the category of DELNs, but it is not yet clear whether they have radiation protection effects. Studying the radiation protection effects of exosome - like nanovesicles derived from Flammulina velutipes to develop new natural radiation protectants is of great significance. At the same time, due to the excellent biocompatibility and inherent stability shown by the characteristics of DELNs themselves. Therefore, it is also of great significance to explore whether there is a synergistic effect between exosome - like nanovesicles derived from Flammulina velutipes and other substances. Summary of the Invention

[0004] In order to solve the problems existing in the background technology, the present invention provides an application of exosome - like nanovesicles derived from Flammulina velutipes in radiation protection, and designs a synergistic combination formula of exosome - like nanovesicles derived from Flammulina velutipes and Flammulina velutipes polysaccharide in radiation protection.

[0005] In order to achieve the above - mentioned purpose, the present invention adopts the following technical solutions:

[0006] An application of exosome - like nanovesicles derived from Flammulina velutipes in radiation protection, including the following steps:

[0007] Step 1: After cutting off the roots of fresh Flammulina velutipes fruiting bodies, cut the remaining part into pieces and add an extraction solvent;

[0008] Step 2: After breaking the cell wall and pulverizing, obtain a filtrate by suction filtration, obtain a supernatant by differential centrifugation, and obtain a precipitate by ultra - high - speed centrifugation, which is the exosome - like nanovesicles derived from Flammulina velutipes;

[0009] Step 3: Use the exosome - like nanovesicles derived from Flammulina velutipes as a radiation protectant to resist radiation damage.

[0010] Further, in step one, the extraction solvent is 1×PBS buffer solution.

[0011] Further, in step two, the differential centrifugation is carried out at a centrifugal force of 800 - 1200 ×g for 20 - 30 min; 2000 - 3000 ×g for 20 - 30 min; 4000 - 5000 ×g for 30 - 50 min; 8000 - 10000 ×g for 60 - 120 min. After each centrifugation, the precipitate is removed and the supernatant is collected. The centrifugal force in the ultra-high speed centrifugation is 120000 - 180000 ×g, and the centrifugation time is 90 - 150 min. The supernatant is discarded to obtain the precipitate.

[0012] Further, in step three, the exosome-like nanovesicle solution from Flammulina velutipes and the polysaccharide solution from Flammulina velutipes are mixed to form a mixed solution, which is used as a radiation protection agent to resist radiation damage.

[0013] Further, the concentration ratio of exosome-like nanovesicles from Flammulina velutipes to polysaccharide from Flammulina velutipes in the mixed solution is 1:10 - 1:40.

[0014] Further, the preparation method of the polysaccharide from Flammulina velutipes includes the following steps:

[0015] S1. The dried Flammulina velutipes is crushed, mixed with ultrapure water, and subjected to constant temperature water bath extraction at 85 - 95 °C. The extract is centrifuged and separated, and the supernatant is taken. After suction filtration, the filtrate is obtained.

[0016] S2. The filtrate is rotary evaporated to 1 / 3 of its volume, 95% ethanol or absolute ethanol is added, and it is left to stand at 2 - 4 °C. After precipitation, it is centrifuged and separated. The precipitate is taken, and ultrapure water is added to redissolve the polysaccharide. It is dialyzed through a 7000 Da dialysis bag for 36 h, and finally freeze-dried to obtain the polysaccharide from Flammulina velutipes.

[0017] Further, in step S1, the material-liquid ratio of Flammulina velutipes powder to ultrapure water is 1:20 - 1:40 mg / mL; the time of constant temperature water bath extraction is 1 - 4 h.

[0018] Further, in step S2, the volume of 95% ethanol or absolute ethanol is 4 times the volume of the concentrated solution after rotary evaporation, and the standing time is 10 - 15 h.

[0019] Further, the radiation damage includes the decrease in the viability of in vitro cells or the damage of multiple organs in animals caused by direct or indirect exposure to ionizing radiation. Such as hematopoietic function damage and liver damage, which are mainly manifested as abnormal blood cell counts in peripheral blood, decreased spleen index, oxidative stress in the body, and abnormal liver function.

[0020] Further, the radiation protectant is used for preparing medicines or health foods; the types include oral solutions, buccal tablets, chewable tablets, hard capsules, soft capsules, and powders.

[0021] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0022] 1. The present invention proves for the first time that exosome-like nanovesicles derived from Flammulina velutipes have good gastrointestinal digestion stability and good cell absorption characteristics;

[0023] 2. The present invention proves for the first time that animals after oral ingestion of Biyang Flammulina velutipes exosome-like nanovesicles (BFMELNs) have the effect of resisting ionizing radiation-induced damage to the body;

[0024] 3. The present invention proves for the first time that BFMELNs combined with crude Biyang Flammulina velutipes polysaccharide (CBFMP) have the effect of synergistically reducing ionizing radiation-induced cell damage. Description of the Drawings

[0025] Figure 1 It is a flow chart for the extraction of BFMELNs;

[0026] Figure 2 It is a transmission electron micrograph of BFMELNs;

[0027] Figure 3 It is the particle size distribution, polydispersity index (PDI), and zeta potential of BFMELNs;

[0028] Figure 4 It is a figure for the identification of substances of BFMELNs, where (A) is a Coomassie brilliant blue staining map of proteins in BFMELNs, (B) is an agarose gel electrophoresis map of BFMELNs, and (C) is a thin layer chromatography map of BFMELNs;

[0029] Figure 5 It is a figure for the cell uptake results of BFMELNs, where (A) is a representative fluorescence image of HL-7702 cells treated with Dio-labeled BFMELNs, and (B) is a representative fluorescence image of Caco-2 cells treated with Dio-labeled BFMELNs;

[0030] Figure 6Results of gastrointestinal digestion stability of BFMELNs, where (A) shows the particle size change of BFMELNs in different simulated digestive fluids, and (B) shows the Zeta potential change of BFMELNs in different simulated digestive fluids;

[0031] Figure 7 Radiation protection effect of BFMELNs on HL-7702 cells;

[0032] Figure 8 Effects of BFMELNs on the body weight (A) and spleen index (B) of irradiated mice;

[0033] Figure 9 Effects of BFMELNs on the number of white blood cells (WBC) (A) and platelets (PLT) (B) in the peripheral blood of irradiated mice;

[0034] Figure 10 Effects of BFMELNs on the activity of superoxide dismutase (SOD) (A) and the content of malondialdehyde (MDA) (B) in the serum of irradiated mice;

[0035] Figure 11 Effects of BFMELNs on the activity of alanine aminotransferase (ALT) (A) and aspartate aminotransferase (AST) (B) in the serum of irradiated mice;

[0036] Figure 12 Results of the radiation protection synergistic effect of BFMELNs and CBFMP, where (A) shows the relative cell viability map after treating cells with BFMELNs and CBFMP alone and the BFMELNs+CBFMP composition and then irradiating, and (B) shows the combination index map of BFMELNs and CBFMP. Detailed implementation mode

[0037] The technical solutions in the present invention will be clearly and completely described below in conjunction with the accompanying drawings and embodiments. Obviously, the described embodiments are only a part of the embodiments of the invention, rather than all of the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0038] Example 1: Preparation and characterization of BFMELNs

[0039] Experimental methods: (1) Preparation of BFMELNs: Fresh raw materials of shiitake mushrooms were purchased from Miyang County, Henan Province. The extraction process of shiitake mushroom-derived exosome-like nanovesicles was as follows: Figure 1 As shown. First, the fresh fruiting bodies of Pleurotus eryngii were gently washed with deionized water and the roots were cut off. The remaining parts were cut into pieces and 1×PBS buffer with 3 times the volume of the fruiting bodies was added as the extraction solvent. The fruiting bodies were then crushed by a juicer or broken by a wall breaker, and then filtered through qualitative filter paper with the assistance of a vacuum pump to obtain the filtrate. The obtained filtrate was further subjected to differential centrifugation at centrifugal forces of 1000 × g, 20 min; 2000 × g, 20 min; 4000 × g, 40 min; 10000 × g, 60 min. After each centrifugation, the precipitate was removed and the supernatant was collected. The filtrate obtained for the last time was filtered, and the filtrate was obtained by using a filter paper with a pore size of 0.2 μm and vacuum pump-assisted filtration. The filtrate was then centrifuged at 150000 × g for 120 min by ultra-high speed centrifugation, and the supernatant was discarded to obtain the precipitate, which was the Pleurotus eryngii exosome-like nanovesicles (BFMELNs) and stored at -80 ℃.

[0040] (2) Physical and chemical parameters and component identification of BFMELNs: The morphology of BFMELNs was observed using a transmission electron microscope. The Zeta potential, PDI and particle size distribution of BFMELNs were determined using a laser particle size analyzer. Polyacrylamide gel electrophoresis (PAGE), sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and silica gel thin layer chromatography (TLC) were used to detect proteins, RNA and lipids in BFMELNs, respectively.

[0041] Experimental results: Figure 1 The preparation process of BFMELNs is demonstrated. Figure 2 Transmission electron microscopy images of BFMELNs show that BFMELNs have a spherical or cup-shaped structure similar to exosomes. Figure 3 The hydrated particle size of BFMELNs was shown to be 111.94 ± 10.48 nm, the PDI was 0.251 ± 0.019, and the Zeta potential was -3.77 ± 0.52 mV. Figure 4 A shows that most proteins in BFMELNs are concentrated in the molecular weight range of 25-50 kDa. Figure 4 B shows that most of the RNA in BFMELNs are small RNAs. Figure 4 C shows that BFMELNs are rich in lipids.

[0042] This example proves that BFMELNs have a cup-shaped structure similar to exosomes and are nanoscale, so they are named as exosome-like nanovesicles derived from Biyang Lentinula edodes (BFMELNs). In addition, this example also proves that BFMELNs contain abundant active proteins, RNAs and lipids, which are the core substances for their functional activities. Due to the characteristics of their morphology and contents, they are different from traditional natural product active components (polyphenols, polysaccharides, terpenoids, etc.), and can be used as a new type of active ingredient for functional research and corresponding development and application.

[0043] Example 2: Cellular uptake and stability of BFMELNs

[0044] Experimental method: (1) Cellular uptake of BFMELNs: Add 30 μmol / L DiO fluorescent dye to the BFMELNs solution (BFMELNs dissolved in 1×PBS buffer), and incubate at 37 °C for 30 min. Subsequently, the free fluorescent dye was discarded after centrifugation at 150000×g for 2 h, and the precipitate was resuspended in PBS. The Dio-labeled BFMELNs were co-incubated with human hepatocyte cell line (HL-7702) and human colorectal adenocarcinoma cell (Caco-2) for 12 h, and image acquisition was performed using an inverted microscope at 3, 6, and 12 h of incubation. After washing the cells twice with PBS, the nuclei were stained with Hoechst 33342.

[0045] (2) Stability of BFMELNs: Add 1.34 μL of 18.5% w / v HCI and 24 μL of pepsin solution (80 mg / mL, dissolved in 0.1 M HCI, pH 2.0) to 1 mL of BFMELNs, and incubate in a shaker at 37 °C for 1 h for simulated gastric digestion. Then, add 0.1 M NaHCO 3 and 80 μL of a mixture containing 24 mg / mL bile extract and 4 mg / mL trypsin, and adjust the pH to 6.5 with 1 M NaHCO 3 to form simulated intestinal fluid, and incubate in a shaker at 37 °C for 1 h for simulated intestinal digestion. After the simulated gastric digestion and simulated intestinal digestion were completed, the stability of BFMELNs was evaluated by measuring their particle size and Zeta potential, respectively.

[0046] Experimental results: Good cell absorption and uptake contribute to improving the bioavailability of exosome-like substances and further enhancing their biological activities in vivo. The experimental results are shown in Figure 5 A - B showing HL-7702 cells ( Figure 5 A) and Caco-2 cells ( Figure 5B) The uptake ability of Dio-labeled BFMELNs increased in a time-dependent manner. At 6 h of incubation, BFMELNs aggregated in HL-7702 and Caco-2 cells, and the green fluorescence intensity increased. As the culture time was extended to 12 h, the green fluorescence intensity gradually increased without obvious attenuation. The in vivo stability results of BFMELNs are as Figure 6 shown in A-B. After digestion in simulated gastric fluid for 1 h, BFMELNs showed a slight increase in particle size and surface charge inversion, which was due to the presence of a large amount of H + + in the simulated gastric fluid, which affected the surface charge of some BFMELNs, changing it from negative to positive, and thus causing the aggregation of nanovesicles. The particle size was still below 200 nm and the potential remained negative in simulated intestinal fluid. These results indicate that BFMELNs can remain stable under different temperatures and simulated gastrointestinal digestion. This example shows that the BFMELNs prepared by the present invention are easily absorbed by cells and have good digestive stability, and are suitable for the fields of pharmaceuticals or health foods.

[0047] Example 3: Radiation protection ability of BFMELNs

[0048] Experimental method: (1) In vitro radiation protection ability of BFMELNs: The HL-7702 cell suspension was added to a 96-well plate at 4×10 3 cells / well / 90 μL. After culturing at 37 °C and 5% CO 2 2 for 12 h, the groups were divided into a blank control group (NC group), a radiation control group (IR group), and a BFMELNs radiation group (BFMELNs group). 10 μL of 1×PBS buffer was added to each well in the NC group and the IR group. The BFMELNs group was divided into 5 concentration gradients, and 10 μL of the sample solution was added to each well: the final concentrations of the samples were 0.625, 1.25, 2.5, 5, and 10 μg / mL, respectively. After culturing for 12 h, the cells were irradiated with 60 60Co-γ rays. The radiation dose rate was 2 Gy / min, and the total dose was 10 Gy. After irradiation, the cells were continued to be cultured in the incubator for 24 h. Then, the supernatant of the cell culture medium was aspirated, 110 μL of CCK-8 working solution (CCK-8: serum: medium = 1:1:9) was added, and the cells were continued to be cultured in the incubator for 2 h. Then, the absorbance value of each well was measured at 450 nm with an enzyme-labeled instrument.

[0049] (2) In vivo radiation protection ability of BFMELNs: Specific pathogen free (SPF) male Kunming mice were randomly divided into 6 groups. The experiment set up a blank control group (NC group, intragastric administration of normal saline), an irradiated control group (IR group, intragastric administration of normal saline), a positive control group (PC group, Licojun tablets), and BFMELNs low-, medium-, and high-dose groups (1 mg / (kg·bw) / d, BFMELNs-L group; 2 mg / (kg·bw) / d, BFMELNs-M group; 4 mg / (kg·bw) / d, BFMELNs-H group). Each mouse was gavaged for 30 consecutive days. On the 31st day, all mice except the NC group were irradiated with the same dose of ionizing radiation. The irradiation conditions were as follows: 60 The mice were irradiated with Co-γ rays at a dose rate of 2 Gy / min. The total radiation dose of the mice was 6 Gy. The mice were killed after fasting for 1 day and various indicators were collected. The mice were weighed every day during the gavage period, and the spleen weight was weighed after the mice were killed. The spleen index was calculated by the spleen weight ratio of the mouse weight. The number of WBC and PLT in the peripheral blood of the mice were detected by a blood cell analyzer. The MDA content in the serum and the activity of SOD, ALT and AST were detected using the relevant kits of Nanjing Jiancheng Institute.

[0050] Experimental results: Relative cell viability can reflect the proliferation and growth status of cells, such as Figure 7 As shown, HL-7702 cells received 10 Gy 60 After Co-γ-ray irradiation for 24 h, the relative cell viability decreased significantly ( P <0.001), indicating that IR caused serious damage to cells, while pretreatment of cells with 0.625-10 μg / mL BFMELNs significantly increased cell viability ( P <0.05), proving that BFMELNs have a protective effect on IR-induced cell damage. Mouse weight is an important indicator of mouse growth status. Changes in weight can be used to preliminarily determine whether the experimental treatment has a negative impact on mouse growth. Figure 8 As shown in A, as the number of days of gavage increased, the mice in each group grew well, their weight continued to increase steadily, and there was no significant difference between the groups ( P >0.05), indicating that the maximum oral dose of BFMELNs had no acute toxic side effects on mice. Figure 8 B shows that compared with the NC group mice, the spleen organ index of the IR group mice was significantly decreased ( P <0.001). The BFMELNs-M and BFMELNs-H groups had a significant recovery effect on the spleen of irradiated mice ( P <0.05), indicating that 6 Gy60 Co-γ ray irradiation causes damage to the spleens of mice, and BFMELNs show a relatively obvious radiation protection effect on the spleens of mice. As shown by Figure 9 A, compared with the mice in the NC group, the WBC in the peripheral blood of the mice in the IR group decreased significantly from 4.53 ± 0.76×10 9 / L to 1.27 ± 0.28×10 9 / L ( P <0.001), while the WBC in the peripheral blood of the mice pretreated with BFMELNs was higher than that in the IR group; similarly, Figure 9 B shows that radiation causes a significant decrease in the number of PLTs in mice ( P <0.05). Compared with 780.83 ± 81.19×10 9 / L in the IR group, low, medium, and high doses of BFMELNs treatment all had a significant improvement effect, and the corresponding numbers were (976.83 ± 154.83×10 9 / L, P <0.05), (1039.83 ± 166.30×10 9 / L, P <0.01), (1024.50 ± 102.06×10 9 / L, P <0.01). Figure 10 A shows that the activity of SOD in the serum of mice after irradiation decreased significantly ( P <0.05), and compared with the SOD enzyme activity in the IR group, each BFMELNs pretreatment group had an obvious improvement effect. Among them, BFMELNs-M ( P <0.001) and BFMELNs-H ( P <0.001) had the best improvement effects. Figure 10 B shows that the content of MDA in the serum of the mice in the IR group increased significantly compared with that in the NC group ( P <0.001). The increase in MDA in the serum proves that radiation exacerbates the degree of peroxidation of the mouse body and causes damage. And BFMELNs pretreatment can significantly reduce the content of MDA in irradiated mice ( P <0.01), showing a good in vivo radiation protection effect. The excessive increase in the activities of ALT and AST in the serum indicates liver damage. As shown in Figure 11 A-B, compared with the NC group, the ALT activity ( P <0.01) and AST ( PThe vitality was significantly increased (<0.001), indicating that ionizing radiation could cause significant damage to the livers of mice, and groups of different doses of BFMELNs all had the effect of preventing liver injury to a certain extent ( P <0.05).

[0051] This example shows that BFMELNs have a protective effect on IR-induced cell damage in vitro; in animals, it has the effects of preventing 60 the body's hematopoietic function and liver injury induced by Co-γ rays and alleviating oxidative stress, and no oral toxic and side effects were shown within the effective range.

[0052] Example 4: Radiation protection combination formula of BFMELNs and CBFMP

[0053] Experimental method: (1) After the dried Flammulina velutipes was crushed and passed through a 40-mesh sieve to obtain Flammulina velutipes powder, it was mixed with ultrapure water at a solid-liquid ratio of 1:30 (g / mL), and then extracted in a constant temperature water bath at 90 °C for 2 h. The extract was centrifuged at 4000 r / min for 10 min and filtered to obtain a filtrate, which was rotary evaporated to 1 / 3 volume. Then, 4 times the volume of 95% ethanol or absolute ethanol of the concentrated solution after rotary evaporation was added and placed at 4 °C for 12 h. After the precipitate was precipitated, it was centrifuged at 4000 r / min for 10 min. Then, the precipitate was taken and added with ultrapure water to redissolve the polysaccharide, and dialyzed through a 7000 Da dialysis bag for 36 h, and finally freeze-dried to obtain crude polysaccharide from Biyang Flammulina velutipes. CBFMP was dissolved with 1×PBS and filtered through a 0.22 μm filter to prepare a CBFMP solution. The radiation protection combination formula of BFMELNs and CBFMP was named BFMELNs+CBFMP, and the preparation method was as follows: The BFMELNs solution and the CBFMP solution were mixed so that the concentration ratio of the two was 1:30. For example, 200 μg / mL of the BFMELNs solution was mixed with 6000 μg / mL of the CBFMP solution at a ratio of 1:1 to obtain a 100 + 3000 μg / mL BFMELNs+CBFMP solution.

[0054] (2)The usage method was the same as that in the in vitro radiation protection example of BFMELNs in Example 3. The specific grouping was the NC group, the IR group, the BFMELNs group, the CBFMP group, and the BFMELNs+CBFMP group. 10 μL of 1×PBS buffer was added to the NC group and the IR group. The sample groups were divided into 5 concentration gradients, and 10 μL of the sample solution was added to each well: the final concentrations of the samples in the BFMELNs group were 0.625, 1.25, 2.5, 5, and 10 μg / mL respectively; the final concentrations of the samples in the CBFMP group were 18.75, 37.5, 75, 150, and 300 μg / mL respectively; the final concentrations of the samples in the BFMELNs+CBFMP group were 0.625 + 18.75, 1.25 + 37.5, 2.5 + 75, 5 + 150, and 10 + 300 μg / mL respectively.

[0055] Experimental results: The Combination Index (CI) is a method for evaluating the combined effect of drugs, which was first proposed in 1984. This method is based on the dose-effect curve of the sample, and judges whether the combined use of the samples has a synergistic effect, an additive effect, or an antagonistic effect by calculating the ratio of the actual effect to the theoretical effect when the samples are used in combination. A CI value less than 1 indicates a synergistic effect, equal to 1 indicates an additive effect, and greater than 1 indicates an antagonistic effect. As Figure 12 A shows that the relative cell viability of HL-7702 cells decreased significantly to about 75% after being irradiated with 10 Gy of 60 Co-γ rays ( P <0.01). Both CBFMP and BFMELNs can effectively improve the decrease in the viability of HL-7702 cells caused by ionizing radiation ( P <0.05). The treatment of HL-7702 cells with BFMELNs alone, CBFMP alone, and BFMELNs+CBFMP combination all had a certain restorative effect on the decrease in cell viability induced by IR. According to the recovery rate of each treatment on the IR cell viability, the combination index (CI value) of the BFMELNs+CBFMP composition was calculated by CompuSyn software. As Figure 12 B shows that after treating HL-7702 cells with BFMELNs+CBFMP at the final concentrations of 0.625 + 18.75, 1.25 + 37.5, 2.5 + 75, 5 + 150 μg / mL for 10 Gy of 60After Co-γ ray irradiation, the CI values were calculated based on the relative cell viability at each concentration, and they were 0.12663, 0.10497, 0.22040, and 0.79311 respectively. The CI values were all less than 1, indicating that BFMELNs and CBFMP had a synergistic protective effect on ionizing radiation-induced cell damage within the above concentration range. The reason for the synergistic effect may be that the exosome-like nanovesicles from Flammulina velutipes act as carriers of polysaccharides and effectively deliver them to the target location; on the other hand, there may be an interaction between the exosome-like nanovesicles from Flammulina velutipes and polysaccharides, and this interaction can change their properties and enhance their radiation protection function. This example shows that BFMELNs and CBFMP have a synergistic effect on the protective effect against IR-induced damage at a certain concentration.

[0056] The results of the above 4 examples prove that the BFMELNs prepared by using the present invention are stable nano-sized particles, can be well taken up by cells, and have good radiation damage prevention (radiation protection) effects both in vitro and in vivo. At the same time, they have the potential to synergistically enhance the effect with other types of radiation protectants such as polysaccharides. BFMELNs are derived from the edible mushroom Flammulina velutipes, with natural components, and animal experiments have proved their safety. Therefore, BFMELNs can be applied to radiation protection (anti-radiation) drugs or health foods.

[0057] In addition, it should be understood that although this specification is described according to the embodiments, not every embodiment only contains an independent technical solution. The narrative way of this specification is only for clarity. Those skilled in the art should regard the specification as a whole, and 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. An application of exosome-like nanovesicles derived from Miyang shiitake mushroom in the preparation of ionizing radiation protection drugs, characterized in that: The method for preparing the Miyang shiitake mushroom-derived exosome-like nanovesicles comprises the following steps: Step 1: After cutting off the roots of the fresh shiitake mushroom fruiting bodies, the remaining parts are cut into pieces and an extraction solvent is added; the extraction solvent is 1×PBS buffer; Step 2: After the cell wall is broken and crushed, the filtrate is obtained by suction filtration, the supernatant is obtained by differential centrifugation, and the precipitate, i.e., the exosome-like nanovesicles derived from Miyang shimeji, is obtained by ultrahigh-speed centrifugation; The differential centrifugation is performed at a centrifugal force of 800-1200 × g, 20-30 min; 2000-3000 × g, 20-30 min; 4000-5000 × g, 30-50 min; 8000-10000 × g, 60-120 min, and the precipitate is removed and the supernatant is collected after each centrifugation; the centrifugal force in the ultra-high-speed centrifugation is 120000-180000 × g, and the centrifugation time is 90-150 min.

2. A use of the Miyang shiitake mushroom-derived exosome-like nanovesicles described in claim 1 in the preparation of a health food having an auxiliary protective effect against the hazards of ionizing radiation.

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