Method for separating extracellular vesicles from populus diversifolia alkali and application of extracellular vesicles to nerve cell injury
By isolating and preparing extracellular vesicles with clear structure and high concentration from the poplar alkaloid, the application problem of the lack of poplar alkaloid in the prior art in the treatment of neurological diseases is solved, and the protection effect on nerve cells is achieved, and the damage induced by hydrogen peroxide is significantly inhibited.
Patent Information
- Application Number
- CN202510234800.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-02-28
- Publication Date
- 2025-07-08
AI Technical Summary
The prior art lacks research and application of extracellular vesicles of poplar alkali in the treatment of neurological diseases, especially effective prevention and treatment methods for neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, Huntington's disease, etc.
A method for isolating extracellular vesicles from poplar alkaloid is provided, including crushing, shaker oscillation, crude filtration, centrifugation and ultracentrifugation, and the extracellular vesicles with clear structure and high concentration are prepared for nerve cell protection.
The prepared extracellular vesicles of poplar alkali can significantly inhibit the apoptosis and reactive oxygen production of human SHSY5Y nerve cells induced by hydrogen peroxide, have broad application prospects for nerve cell damage protection, and promote the development of drugs for the prevention and treatment of neurodegenerative diseases.
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Figure CN120272399A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the field of biomedicine, and specifically to a method for separating extracellular vesicles from poplar alkali and its application in treating nerve cell damage. Background Art
[0002] Neurological diseases affect more than one-third of the people globally and are the main causes of morbidity and disability. Among them, Alzheimer's disease (AD), which is caused by nerve cell damage and leads to memory loss, behavioral retardation, and cognitive dysfunction, mostly occurs in the middle-aged and elderly population. It is the main cause of the loss of ability in the elderly globally and is also one of the neurodegenerative diseases with the highest mortality rate globally. Therefore, it is particularly important to develop a drug that can safely and effectively prevent and treat Alzheimer's disease.
[0003] Extracellular Vesicles (EVs) are bilayer membrane nanostructures secreted by cells and rich in biomolecules such as nucleic acids, lipids, and proteins. They participate in intercellular signal transduction, immune regulation, and the occurrence and development of diseases. Research shows that plant-derived extracellular vesicles not only have high safety and good biocompatibility but also show very significant effects in promoting wound healing, treating osteoporosis, and promoting angiogenesis.
[0004] Poplar is a perennial tall tree that is salt-tolerant, heat-tolerant, and drought-tolerant. It is mainly distributed in arid desert areas such as Xinjiang, Inner Mongolia, and Gansu. The crystals formed by the natural resin secreted by it on the bark are called "poplar alkali" and "poplar tears". "Newly Revised Materia Medica" once recorded that "poplar tears are salty and bitter in taste, cold in nature, and can detoxify and clear heat and relieve pain". In Xinjiang, poplar alkali is commonly used as medicine and has the effects of detumescence and pain relief. It is often used to treat stomachaches, waist pains, leg pains, and joint pains. There is a lack of research content and related applications of poplar alkali extracellular vesicles in the field of treating neurological diseases in the prior art. Summary of the Invention
[0005] Aiming at the deficiencies of the prior art, the purpose of the present invention is to provide a method for separating extracellular vesicles from poplar alkali and its application in treating nerve cell damage, so as to solve the problems raised in the above background art. The poplar alkali extracellular vesicles prepared by the present invention not only have clear structures and high concentrations but also can significantly inhibit the apoptosis ratio of human SHSY5Y nerve cells induced by hydrogen peroxide and reduce the production of intracellular reactive oxygen species after being phagocytosed by cells.
[0006] To achieve the above purpose, the present invention is realized through the following technical solutions: A method for separating extracellular vesicles from poplar alkali, comprising the following steps:
[0007] Step 1: Crush euzylogen into powder, add it to a balanced salt solution, and place it on a shaker to oscillate overnight.
[0008] Step 2: Coarsely filter, centrifuge, and finely filter the solution obtained in Step 1 to remove large particle impurities in the solution.
[0009] Step 3: Separate extracellular vesicles from the filtrate obtained in Step 2 by ultracentrifugation.
[0010] Furthermore, the balanced salt solution in Step 1 is a phosphate solution, and the phosphate solution is composed of 5 - 15 mM Na2HPO4, 135 - 145 mM NaCl, and 1 - 5 mM NaH2PO4, with a pH of 7.2 - 7.6.
[0011] Furthermore, the formula of the phosphate solution is 8 mM Na2HPO4, 137 mM NaCl, 2 mM NaH2PO4, with a pH of 7.4.
[0012] Furthermore, the coarse filtration method in Step 2 includes gauze filtration, screen filtration, and vacuum filtration, and the coarse filtration is used to preliminarily remove large particle impurities in the solution.
[0013] Furthermore, the centrifugation in Step 2 removes impurities through multiple operations with gradually increasing centrifugal force, and the centrifugation conditions include centrifuging at 500 - 1500 g for 5 - 20 min; centrifuging at 2000 - 4000 g for 25 - 45 min; centrifuging at 10000 - 15000 g for 45 - 120 min.
[0014] Furthermore, the ultracentrifugation is carried out twice at 4°C at 100000 - 150000 g for 60 - 120 min.
[0015] An application of extracellular vesicles prepared by the above separation method in nerve cell injury, and euzylogen extracellular vesicles are used to provide neuroprotection for SHSY5Y cells.
[0016] Furthermore, the protective effect is used to achieve the prevention and treatment of Alzheimer's disease, Parkinson's disease, and Huntington's disease, neurodegenerative diseases, by euzylogen extracellular vesicles.
[0017] Furthermore, the neuroprotection includes inhibiting the oxidative damage of human SHSY5Y nerve cells induced by hydrogen peroxide.
[0018] The beneficial effects of the present invention:
[0019] 1. The present invention successfully isolates extracellular vesicles from euphorbine, and the isolated extracellular vesicles of euphorbine can significantly inhibit hydrogen peroxide-induced apoptosis of SHSY5Y neurons and the production of reactive oxygen species in cells after being phagocytosed by cells, which can promote the rapid development of drug research and development for preventing and treating neurodegenerative diseases.
[0020] 2. The extracellular vesicles of euphorbine prepared in the present invention not only have a clear structure and high concentration, but also can significantly inhibit the apoptosis ratio of human SHSY5Y neurons induced by hydrogen peroxide and reduce the production of intracellular reactive oxygen species after being phagocytosed by cells. The extracellular vesicles of euphorbine have very broad application and transformation prospects in protecting nerve cell damage. BRIEF DESCRIPTION OF THE DRAWINGS
[0021] Figure 1 is a transmission electron microscope image of extracellular vesicles of euphorbine;
[0022] Figure 2 is the NTA particle size distribution map of extracellular vesicles of euphorbine;
[0023] Figure 3 is the laser confocal image of extracellular vesicles of euphorbine being phagocytosed by SHSY5Y cells in this embodiment;
[0024] Figure 4 is the cell proliferation analysis chart of SHSY5Y treated with extracellular vesicles of euphorbine in this embodiment of the present invention;
[0025] Figure 5 is the cell apoptosis ratio chart of SHSY5Y treated with extracellular vesicles of euphorbine in this embodiment of the present invention;
[0026] Figure 6 is the positive ratio chart of reactive oxygen species in SHSY5Y cells treated with extracellular vesicles of euphorbine in this embodiment of the present invention. DETAILED DESCRIPTION OF THE INVENTION
[0027] In order to make the technical means, creative features, achieved purposes and effects of the present invention easy to understand, the present invention will be further described below in conjunction with specific embodiments.
[0028] Please refer to Figures 1 to 6 , the present invention provides the following technical solutions: A method for isolating extracellular vesicles from euphorbine and its application in nerve cell damage. The method for isolating extracellular vesicles of euphorbine provided in this embodiment is as follows:
[0029] S1. Crush the populus euphratica alkali into powder, add it to the balanced salt solution, and place it on a shaker to oscillate overnight. The balanced salt solution is a phosphate solution composed of 5-15 mM Na2HPO4, 135-145 mM NaCl, and 1-5 mM NaH2PO4, with a pH of 7.2-7.6;
[0030] S2. Coarsely filter, centrifuge, and finely filter the solution obtained in S1 to remove large particle impurities in the solution. Coarse filtration includes, but is not limited to, gauze filtration, screen filtration, and vacuum filtration to preliminarily remove large particle impurities in the solution. Gauze filtration is the filtration through a degreased cotton gauze funnel. The conditions for centrifugation are centrifugation at 500-1500 g for 5-20 min, centrifugation at 2000-4000 g for 25-45 min, and centrifugation at 10000-15000 g for 45-120 min. The impurities in the sample solution are gradually removed by differential centrifugation. Among them, fine filtration is membrane filtration, and the pore size of the filter membrane is 0.1-0.45 μm;
[0031] S3. Separate the extracellular vesicles from the filtrate obtained in S2 by ultracentrifugation. Ultracentrifugation includes the following operations: Transfer the sample solution after fine filtration to an ultracentrifugation tube, centrifuge at 4°C at 100000-150000 g for 60-120 min, pour out the supernatant, add the phosphate solution, resuspend the precipitate, and centrifuge again at 4°C at 100000-150000 g for 60-120 min. The obtained precipitate is the extracellular vesicles.
[0032] The extracellular vesicles of populus euphratica alkali prepared by the above method not only have clear structure and high concentration, but also can significantly inhibit the apoptosis ratio of human SHSY5Y nerve cells induced by hydrogen peroxide and reduce the production of intracellular reactive oxygen species after being phagocytosed by cells. The extracellular vesicles of populus euphratica alkali have very broad application and transformation prospects in the protection of nerve cell injury.
[0033] The present invention also provides the following specific embodiments for further detailed description to enable those skilled in the art to understand the present invention more clearly.
[0034] Example 1: Preparation of extracellular vesicles
[0035] In this example, the extracellular vesicles of populus euphratica alkali are prepared by differential ultracentrifugation. The specific operation steps are as follows:
[0036] S1. Crush 20 g of populus euphratica alkali into powder, add it to the phosphate solution, and place it on a shaker to oscillate overnight;
[0037] S2. Filter with a cotton gauze funnel, transfer the filtrate to a 50 mL centrifuge tube, centrifuge at 800 g for 10 min, and take the supernatant; centrifuge the supernatant at 3000 g for 30 min, and take the supernatant; centrifuge the supernatant at 12000 g for 60 min, and take the supernatant; filter the supernatant through a 0.22 μm filter membrane to remove impurities precisely;
[0038] S3. Transfer the sample solution after centrifugation to remove impurities to an ultracentrifuge tube, centrifuge at 100000 g for 90 min at 4 °C, and pour out the supernatant; add the pre-cooled phosphate solution to the ultracentrifuge tube to resuspend the precipitate; centrifuge again at 100000 g for 60 min, and pour out the supernatant in a laminar flow hood; add the pre-cooled phosphate solution to resuspend the precipitate, and the resulting solution is the extracellular vesicles of populus euphratica alkali.
[0039] The formula of the phosphate solution is 8 mM Na2HPO4, 137 mM NaCl, 2 mM NaH2PO4, pH 7.4.
[0040] Comparative Example 1: Preparation of the extract
[0041] In this comparative example, the extract of populus euphratica alkali was prepared by differential centrifugation. The specific operation steps are as follows:
[0042] S1. Crush 20 g of populus euphratica alkali into powder, add it to the phosphate solution, and place it on a shaker and shake overnight;
[0043] S2. Filter with a cotton gauze funnel, transfer the filtrate to a 50 mL centrifuge tube, centrifuge at 800 g for 10 min, and take the supernatant; centrifuge the supernatant at 3000 g for 30 min, and take the supernatant; centrifuge the supernatant at 12000 g for 60 min, and take the supernatant; filter the supernatant through a 0.22 μm filter membrane to remove impurities precisely;
[0044] The formula of the phosphate solution is 8 mM Na2HPO4, 137 mM NaCl, 2 mM NaH2PO4, pH 7.4.
[0045] Example 2: Characterization of extracellular vesicles
[0046] 1. Transmission electron microscopy analysis
[0047] Take 10 μL of the solution obtained in Example 1 and drop it onto a copper grid, then add 10 μL of uranyl acetate to stain the exosomes, blot the floating liquid with filter paper and dry it at room temperature; finally, place the copper grid in a transmission electron microscope for imaging.
[0048] The electron microscopy results are as Figure 1 , and a hemispherical bilayer membrane nanostructure with a middle depression in the solution was observed, which was consistent with the results reported in the literature.
[0049] 2. NTA particle size analysis
[0050] Take 10 μL of the solution obtained in Example 1, dilute it with phosphate solution, and then place it in a nanoparticle tracking analyzer (NTA) for detection.
[0051] The detection results are as Figure 2 shown. The average particle size of the particles in the solution is 184.8 nm, mainly distributed in the range of 80 - 220 nm, and the concentration is 1.2E+11 Particles / mL, which is in line with the reports in relevant literature.
[0052] Example 3: Functional verification of extracellular vesicles
[0053] Induce damage to human SHSY5Y nerve cells by adding hydrogen peroxide to further verify the biological function of poplarine extracellular vesicles.
[0054] The grouping of cell function experiments is as follows:
[0055] (1) Normal control group (SHSY5Y + PBS);
[0056] (2) Hydrogen peroxide-induced injury model group (SHSY5Y + H2O2);
[0057] (3) Extract experimental group (SHSY5Y + H2O2 + T);
[0058] (4) Extracellular vesicle experimental group (SHSY5Y + H2O2 + EVs).
[0059] 1. Cell phagocytosis experiment
[0060] Mix the poplarine extracellular vesicles obtained in Example 1 with PKH26 ethanol solution (PKH26 Red Fluorescent Cell Linker Kit, Sigma), incubate for 5 min, and then add serum to terminate the reaction. Subsequently, transfer the reaction solution to an ultracentrifuge tube, centrifuge at 100,000 g for 90 min at 4 °C, and then pour out the supernatant in a laminar flow hood. Add pre-cooled phosphate solution to resuspend the precipitate, which is the PKH26-labeled poplarine extracellular vesicles.
[0061] Seed human SHSY5Y cells into a 24-well plate with cell slides, add PBS and PKH26-labeled (red fluorescence) poplarine extracellular vesicles, mix well, and then culture overnight in a 37 °C, 5% CO2 cell incubator. Discard the medium, add 4% paraformaldehyde and let it stand for 15 min, then discard. Wash with PBS, add nuclear dye DAPI (blue fluorescence), let it stand for 15 min, then discard. Wash with PBS, invert and seal the slides. Finally, observe the cell phagocytosis situation through a laser confocal microscope.
[0062] The phagocytosis of cells is as follows Figure 3 In the PBS group, blue fluorescent spots were shown. In the juglone extracellular vesicles group, many small red fluorescent spots appeared around the blue fluorescent spots. Combining with the BF (bright field) view, it could be seen that the red fluorescent spots were all inside the cells.
[0063] 2. Cell proliferation experiment
[0064] Human SHSY5Y cells were inoculated into 96-well plates. PBS, hydrogen peroxide, juglone extract, and juglone extracellular vesicles were added successively according to the grouping. After mixing, they were cultured in a 37°C, 5% CO2 cell incubator for 0, 24, 48, and 72 h. Then, 10 μL of CCK-8 reagent (CCK-8 cell proliferation and toxicity detection kit, Solarbio) was added to each well, and they were continued to be incubated at 37°C for 1 h. Finally, the absorbance value (OD value) of each well was detected by an enzyme-linked immunosorbent assay (ELISA) reader.
[0065] The results of cell proliferation detection are as follows Figure 4 Compared with the SHSY5Y+PBS group, the proliferation ability of SHSY5Y cells after hydrogen peroxide treatment decreased. On this basis, after adding juglone extract, the proliferation ability of SHSY5Y cells was significantly inhibited. After adding juglone extracellular vesicles, the proliferation ability of SHSY5Y cells increased significantly, even significantly better than that of the normal control group.
[0066] 3. Cell apoptosis experiment
[0067] SHSY5Y cells were inoculated into 6-well plates. PBS, hydrogen peroxide, juglone extract, and juglone extracellular vesicles were added successively according to the grouping. After mixing, they were cultured overnight in a 37°C, 5% CO2 cell incubator. The medium was discarded, and the cells were digested, collected, and resuspended. 5 μL of FITC and PI dyes were added successively, and they were incubated at room temperature for 30 min. Finally, the cell apoptosis ratio was detected by a flow cytometer.
[0068] The detection results are as follows Figure 5 Compared with the SHSY5Y+PBS group, the apoptosis ratio of SHSY5Y cells after hydrogen peroxide treatment increased (6.1%), indicating that the cell oxidative damage model was successfully established. After adding juglone extract, the apoptosis ratio increased. After treatment with juglone extracellular vesicles, the apoptosis ratio of SHSY5Y cells decreased significantly (2.1%), even significantly lower than that of the normal control group (2.5%).
[0069] 4. Cell reactive oxygen species detection experiment
[0070] SHSY5Y cells were seeded into 6-well plates. PBS, hydrogen peroxide, populuscine extract, and populuscine extracellular vesicles were added successively according to the groups. After mixing, the plates were placed in a 37°C, 5% CO2 cell culture incubator and cultured overnight. The culture medium was discarded, and 1 mL of diluted DCFH-DA (Reactive oxygen species assay kit, Solarbio) was added. The plates were incubated at 37°C for an additional 60 min and then discarded. The cells were washed three times with basal medium to remove the unentered DCFH-DA. Finally, the cells were digested and collected, and the positive rate of intracellular reactive oxygen species in each group was detected by flow cytometry.
[0071] The detection results are as Figure 6 , the positive rate of intracellular reactive oxygen species significantly increased (26.2%) after hydrogen peroxide-induced modeling. The positive rate of reactive oxygen species further increased after the addition of populuscine extract. After treatment with populuscine extracellular vesicles, the positive rate of intracellular reactive oxygen species in SHSY5Y cells decreased significantly (12.6%), even lower than that of the normal control group (17.2%).
[0072] In this example, the above data indicate that the extracellular vesicles were successfully isolated from populuscine by the method provided by the present invention. After being phagocytosed by cells, the isolated populuscine extracellular vesicles can significantly inhibit hydrogen peroxide-induced apoptosis of SHSY5Y neurons and the production of reactive oxygen species in cells, showing great application transformation prospects in neuroprotection and is expected to promote the rapid development of drug research and development for preventing and treating neurodegenerative diseases.
[0073] The above shows and describes the basic principles, main features, and advantages of the present invention. For those skilled in the art, it is obvious that the present invention is not limited to the details of the above exemplary embodiments, and without departing from the spirit or basic features of the present invention, the present invention can be implemented in other specific forms.
[0074] In addition, it should be understood that although this specification is described according to embodiments, not every embodiment only contains an independent technical solution. This narrative way of the 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. A method for isolating extracellular vesicles from euphorbium, characterized in that, The method for separating extracellular vesicles from euphorbine includes the following steps: Step 1: Crush euphorbine into powder, add it to a balanced salt solution, and place it on a shaker to oscillate overnight; Step 2: Coarsely filter, centrifuge, and finely filter the solution obtained in Step 1 to remove large particle impurities in the solution; Step 3: Separate extracellular vesicles from the filtrate obtained in Step 2 by ultracentrifugation.
2. The method for separating extracellular vesicles from euphorbium alkali according to claim 1, wherein: The balanced salt solution in Step 1 is a phosphate solution, and the phosphate solution is composed of 5-15 mM Na2HPO4, 135-145 mM NaCl, and 1-5 mM NaH2PO4, with a pH of 7.2-7.
6.
3. The method for separating extracellular vesicles from poplar alkali according to claim 2, wherein: The formula of the phosphate solution is 8 mM Na2HPO4, 137 mM NaCl, and 2 mM NaH2PO4, with a pH of 7.
4.
4. A method for isolating extracellular vesicles from euphorbine, as claimed in claim 1, wherein: The coarse filtration method in Step 2 includes gauze filtration, screen filtration, and vacuum filtration, and the coarse filtration is used to preliminarily remove large particle impurities in the solution.
5. A method for isolating extracellular vesicles from euphorbia alkali according to claim 1, characterized in that: The centrifugation in Step 2 removes impurities through multiple operations with gradually increasing centrifugal force. The centrifugation conditions include centrifuging at 500-1500 g for 5-20 min; centrifuging at 2000-4000 g for 25-45 min; centrifuging at 10000-15000 g for 45-120 min.
6. The method for separating extracellular vesicles from euphorbine according to claim 5, wherein: The ultracentrifugation is carried out twice at 4°C at 100000-150000 g for 60-120 min.
7. Use of extracellular vesicles prepared by the separation method as described in claim 1 for nerve cell injury, characterized in that: Euphorbine extracellular vesicles are used to provide neuroprotection to SHSY5Y cells.
8. Use of the extracellular vesicles for nerve cell injury according to claim 7, characterized in that: The protective effect is used to achieve the prevention and treatment of Alzheimer's disease, Parkinson's disease, and Huntington's disease, neurodegenerative diseases, by euphorbine extracellular vesicles.
9. Use of extracellular vesicles for nerve cell injury according to claim 8, characterized in that: The neuroprotection includes inhibiting the oxidative damage of human SHSY5Y nerve cells induced by hydrogen peroxide.