Cistanche deserticola exosome as well as extraction method and application thereof

Abstract

The invention provides an exosome, the exosome is an exosome derived from cistanche deserticola, the cistanche deserticola is Cistanche deserticola Y.C. Ma and / or Cistanche tubulosa (Schenk) Light, and the Cistanche deserticola, the Cistanche deserticola, the Cistanche deserticola, the Cistanche tubulosa, the Cistanche deserticola, the Cistanche tubulosa, the Cistanche tubulosa and the Cistanche tubulosa and the Cistanche tubulosa are used for preparing the exosome. The invention also provides an extraction method and application of the exosome. The exosome disclosed by the invention is uniform in particle distribution, high in overall stability and good in cytocompatibility, and has an obvious improvement effect on various diseases including male reproductive injury and cancers.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine and / or food. Specifically, the present invention relates to Cistanche deserticola exosomes and an extraction method and application thereof. Background Art

[0002] Cistanche deserticola is a parasitic plant of the Orobanchaceae family. Its basal plants include Cistanche deserticola (Y. C. Ma) and Cistanche tubulosa (Schenk) Wight. Traditional Chinese medicine believes that Cistanche deserticola has the effects of tonifying the kidneys and enhancing yang, as well as moistening the intestines and promoting bowel movements. Modern research on Cistanche deserticola includes the study of its total glycosides, oligosaccharide alcohols, and polysaccharide extracts, with applications ranging from lowering blood sugar and lipids, delaying aging, and whitening skin. However, overall, current research has underexplored the potential applications of Cistanche deserticola, and these extracts suffer from low bioavailability.

[0003] Therefore, conducting in-depth research and development on Cistanche deserticola, finding other substances besides these known chemical components, and fully exploring its medicinal value are technical issues that urgently need to be solved. Summary of the Invention

[0004] In view of the above problems, the object of the present invention is to provide a Cistanche deserticola exosome and an extraction method and use thereof.

[0005] The above object of the present invention is achieved by providing the following technical solutions:

[0006] In a first aspect, the present invention provides exosomes, which are exosomes derived from Cistanche deserticola, wherein the Cistanche deserticola is Cistanche deserticola YC Ma and / or Cistanche tubulosa (Schenk) Wight.

[0007] According to some embodiments of the invention, the PDI (polydispersity index) of the exosomes is from 0.05 to 0.3, preferably from 0.15 to 0.3.

[0008] According to some embodiments of the present invention, the zeta potential of the exosomes is -5 mV to -30 mV, preferably -20 mV to -30 mV.

[0009] According to some embodiments of the present invention, the particle size distribution of the exosomes is 20-200 nm, preferably 30-120 nm.

[0010] In a second aspect, the present invention provides a method for extracting exosomes according to the first aspect of the present invention, comprising the following steps:

[0011] (1) Crush the Cistanche deserticola and extract the juice, subject the obtained Cistanche deserticola juice to differential centrifugation, and collect the supernatant;

[0012] (2) The supernatant is subjected to a first ultracentrifugation treatment, and the resulting precipitate is resuspended in PBS buffer to prepare a crude exosome suspension;

[0013] (3) The crude exosome suspension is subjected to sucrose density gradient centrifugation to collect components between adjacent sucrose concentrations and recover the exosomes.

[0014] According to some embodiments of the present invention, in step (1), the differential centrifugation treatment sequentially comprises:

[0015] ① Centrifugation at 700 g to 1200 g (e.g., 700 g, 800 g, 900 g, 1000 g, 1100 g or 1200 g) for 5 to 15 minutes (e.g., 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, 11 minutes, 12 minutes, 13 minutes, 14 minutes or 15 minutes) at 0°C to 10°C (e.g., 0°C, 1°C, 2°C, 3°C, 4°C, 5°C, 6°C, 7°C, 8°C, 9°C or 10°C);

[0016] ② centrifugation at 2500 g to 4000 g (e.g., 2500 g, 2700 g, 2900 g, 3000 g, 3200 g, 3400 g, 3600 g, 3800 g or 4000 g) for 15 to 30 minutes (e.g., 15 minutes, 17 minutes, 20 minutes, 22 minutes, 24 minutes, 26 minutes, 28 minutes or 30 minutes) at 0°C to 10°C (e.g., 0°C, 1°C, 2°C, 3°C, 4°C, 5°C, 6°C, 7°C, 8°C, 9°C or 10°C); and

[0017] ③ Centrifuge at 7000 g to 15000 g (for example, 7000 g, 8000 g, 10000 g, 12000 g, 14000 g or 15000 g) at 0°C to 10°C (for example, 0°C, 1°C, 2°C, 3°C, 4°C, 5°C, 6°C, 7°C, 8°C, 9°C or 10°C) for 25 to 40 minutes (for example, 25 minutes, 27 minutes, 30 minutes, 32 minutes, 34 minutes, 36 minutes, 38 minutes or 40 minutes).

[0018] According to some embodiments of the present invention, in step (2), the temperature of the first ultracentrifugation treatment is 0°C to 10°C, for example, 0°C, 1°C, 2°C, 3°C, 4°C, 5°C, 6°C, 7°C, 8°C, 9°C or 10°C.

[0019] According to some embodiments of the present invention, in step (2), the speed of the first ultracentrifugation treatment is 70,000 g to 150,000 g, for example, 70,000 g, 80,000 g, 100,000 g, 120,000 g, 140,000 g or 150,000 g.

[0020] According to some embodiments of the present invention, in step (2), the time of the first ultracentrifugation treatment is 0.5 hour to 1 hour, for example, 0.5 hour, 0.6 hour, 0.7 hour, 0.8 hour, 0.9 hour or 1 hour.

[0021] According to some embodiments of the present invention, in step (3), the sucrose density gradient centrifugation treatment uses sucrose solutions with concentrations of 30%, 45% and 60% by mass, i.e., concentration gradients of 30% to 45% and 45% to 60% by mass.

[0022] According to some embodiments of the present invention, in step (3), the temperature of the sucrose density gradient centrifugation treatment is 0°C to 10°C, for example, 0°C, 1°C, 2°C, 3°C, 4°C, 5°C, 6°C, 7°C, 8°C, 9°C or 10°C.

[0023] According to some embodiments of the present invention, in step (3), the sucrose density gradient centrifugation is performed at a speed of 100,000 g to 200,000 g, for example, 100,000 g, 110,000 g, 120,000 g, 130,000 g, 140,000 g, 150,000 g, 160,000 g, 170,000 g, 180,000 g, 190,000 g or 200,000 g.

[0024] According to some embodiments of the present invention, in step (3), the sucrose density gradient centrifugation treatment time is 0.5 to 3 hours, for example, 0.5 hours, 1.0 hours, 1.5 hours, 2.0 hours, 2.5 hours or 3.0 hours.

[0025] According to some embodiments of the present invention, in step (3), the recovery includes diluting the components, for example, by adding PBS buffer to the components for dilution, and then subjecting the resulting dilution to a second ultracentrifugation treatment. The specific degree of dilution of the components can be reasonably selected by those skilled in the art based on actual conditions and will not be described in detail here.

[0026] Preferably, in step (3), the temperature of the second ultracentrifugation treatment is 0°C to 10°C, for example, 0°C, 1°C, 2°C, 3°C, 4°C, 5°C, 6°C, 7°C, 8°C, 9°C or 10°C.

[0027] Preferably, in step (3), the speed of the second ultracentrifugation treatment is 100,000 g to 200,000 g, for example, 100,000 g, 110,000 g, 120,000 g, 130,000 g, 140,000 g, 150,000 g, 160,000 g, 170,000 g, 180,000 g, 190,000 g or 200,000 g.

[0028] Preferably, in step (3), the second ultracentrifugation treatment is performed for 0.5 to 3 hours, for example, 0.5 hours, 1.0 hours, 1.5 hours, 2.0 hours, 2.5 hours or 3.0 hours.

[0029] In a third aspect, the present invention provides use of the exosomes according to the first aspect of the present invention in preparing a product for one or more of the following uses:

[0030] (1) Improve normal male reproductive capacity;

[0031] (2) Prevent, improve and / or treat male reproductive impairment;

[0032] (3) Prevent, improve and / or treat inflammation;

[0033] (4) Prevent, improve and / or treat cancer;

[0034] (5) Prevent, improve and / or treat stroke;

[0035] (6) Immunomodulation and / or immunotherapy enhancement for subjects;

[0036] (7) Prevent, improve and / or treat liver damage;

[0037] (8) As a drug delivery carrier.

[0038] According to some embodiments of the present invention, the male reproductive capacity impairment is 4-Hydroperoxycyclophosphamide (4-HC), an active metabolite form of cyclophosphamide, and / or male reproductive capacity impairment caused by cyclophosphamide.

[0039] According to some embodiments of the present invention, the male reproductive capacity impairment is selected from one or more of oligospermia, asthenospermia and testicular abnormality.

[0040] According to some embodiments of the invention, the inflammation is neuritis and / or myocarditis.

[0041] According to some embodiments of the invention, the cancer is selected from one or more of skin cancer, lung cancer, liver cancer and breast cancer.

[0042] According to some embodiments of the present invention, the product is selected from one or more of medicines, medical products, cosmetics, foods and health products.

[0043] Preferably, the medical product is a cell therapy product.

[0044] Preferably, the food is a health food.

[0045] According to some embodiments of the present invention, the dosage form of the product is selected from one or more of powder, suppository, tablet, capsule, granule, oral solution, injection, emulsion and facial mask.

[0046] In a fourth aspect, the present invention provides a product comprising an effective amount of the exosomes according to the first aspect of the present invention and optionally one or more pharmaceutically, medically, food science or cosmetically acceptable carriers or excipients.

[0047] It should be noted that, in the present invention, an "effective amount" refers to a non-toxic amount of exosomes sufficient to provide the desired effect. Alternatively, an "effective amount" refers to an amount of exosomes effective to provide the desired effect when used in combination with other ingredients. The "effective amount" may vary depending on the subject, for example, age and general condition. Therefore, it is not always possible to specify a precise "effective amount." However, an appropriate "effective amount" for any individual can be determined by one of ordinary skill in the art using routine experimental methods.

[0048] According to some embodiments of the present invention, the product is selected from one or more of medicines, medical products, cosmetics, foods and health products.

[0049] Preferably, the medical product is a cell therapy product.

[0050] Preferably, the food is a health food.

[0051] According to some embodiments of the present invention, the dosage form of the product is selected from one or more of powder, suppository, tablet, capsule, granule, oral solution, injection, emulsion and facial mask.

[0052] The present invention has at least the following beneficial effects:

[0053] The exosomes of the present invention have high uniformity in particle distribution, strong overall stability of the system, good cell compatibility, and have a significant improvement effect on a variety of diseases. In vitro experiments have verified that the desert cistanche exosomes of the present invention have a proliferative effect on the four germ cell lines of the testicle (GC1, GC2, TM3 and TM4), and therefore can be used to improve normal male reproductive capacity; the desert cistanche exosomes of the present invention have a protective effect on 4-HC-induced testicular germ cell damage, and therefore can be used to prevent, improve and / or treat male reproductive capacity damage; the desert cistanche exosomes of the present invention have a protective effect on neuritis and myocarditis, and therefore can be used to prevent, improve and / or treat inflammation and related diseases; the desert cistanche exosomes of the present invention have a protective effect on the oxygen-glucose deprivation reperfusion nerve cell model, and therefore can be used to prevent, improve and / or treat brain The Cistanche deserticola exosomes of the present invention promote macrophage polarization from M2 to M1, and therefore can be used to regulate the immune system of subjects and enhance the immunotherapy of various diseases, including cancer. Furthermore, the Cistanche deserticola exosomes of the present invention have inhibitory effects on various tumor cell types, including skin cancer (B16F10), lung cancer (A549), liver cancer (HUH7), and breast cancer (4T1, MCF7, and MDA-MB-231), and therefore can be used to treat various cancers. The Cistanche tubulosa exosomes of the present invention have protective effects against CoCl2-induced PC12 hypoxic injury and macrophage-induced specific liver injury. Furthermore, animal experiments have confirmed that the Cistanche deserticola exosomes of the present invention have therapeutic effects on male reproductive damage and breast cancer.

[0054] The present invention uses a combination of ultracentrifugation and sucrose density gradient to extract exosomes. The exosome extraction method of the present invention has high extraction efficiency, low extraction cost, and the obtained product is of good quality and high purity. BRIEF DESCRIPTION OF THE DRAWINGS

[0055] Hereinafter, embodiments of the present invention will be described in detail with reference to the accompanying drawings, in which:

[0056] Figure 1 Characterization of exosomes 1# to 3# prepared in the examples of this application. (a)-(c) are transmission electron micrographs of exosomes 1#, 2#, and 3#, respectively; (d)-(f) show the nanoparticle tracking analysis results of exosomes 1#, 2#, and 3#, respectively; (g) shows the particle concentration of exosomes 1#, 2#, and 3#; (h) shows the polymer dispersibility index (PDI) of exosomes 1#, 2#, and 3#; and (i) shows the zeta potential values ​​of exosomes 1#, 2#, and 3#.

[0057] Figure 2Characterization of exosomes 4# to 6# prepared in the examples of this application. (a)-(c) are transmission electron micrographs of exosomes 4#, 5#, and 6#, respectively; (d) shows the polymer dispersity index (PDI) of exosomes 4#, 5#, and 6#; and (e) shows the zeta potential values ​​of exosomes 4#, 5#, and 6#.

[0058] Figure 3 Characterization of exosomes 7# and 8# prepared in the examples of this application. (a) and (b) are transmission electron micrographs of exosomes 7# and 8#, respectively; (c) and (d) show the particle size distribution of exosomes 7# and 8#, respectively; (e) shows the particle concentration of exosomes 7# and 8#; (f) shows the polymer dispersibility index (PDI) of exosomes 7# and 8#; and (g) shows the zeta potential of exosomes 7# and 8#.

[0059] Figure 4 Characterization of exosome 9# prepared in the examples of this application. (a) is a transmission electron microscopy image of exosome 9#; (b) is a particle distribution diagram obtained by nanoparticle tracking analysis (NTA); (c) shows the PDI of exosome 9#; and (d) shows the zeta potential of exosome 9#.

[0060] Figure 5 Shown are the effects of different concentrations of Cistanche deserticola exosomes on the cell viability of GC1 (a), GC2 (b), TM3 (c), and TM4 (d) cell lines.

[0061] Figure 6 The effect of different concentrations of Cistanche deserticola exosomes on the cell viability of GC1 (a), GC2 (b), TM3 (c), and TM4 (d) cell lines after 4-HC treatment is shown. N represents the control group, M represents the model group, and 12.5 and 25 μg / mL represent the drug-treated groups. Comparison between the model group and the control group: # p < 0.05, ## p < 0.01, ### p < 0.001; comparison between drug-treated group and model group: *p < 0.05, **p < 0.01, ***p < 0.001.

[0062] Figure 7This figure shows the effects of different concentrations of Cistanche deserticola exosomes on LPS-activated BV2 cells. (a) BV2 cell survival rate after drug treatment; (b) inhibition of NO production in BV2 cells by drugs. N represents the BV2 cell control group, M represents the model group, DXMS represents the dexamethasone-treated group, and 1.25, 2.5, 5, and 10 μg / mL represent different exosome-treated groups. Comparison between the drug-treated group and the model group: *p < 0.05, **p < 0.01, ***p < 0.001.

[0063] Figure 8 The effects of different concentrations of Cistanche deserticola exosomes on the H9c2 myocardial inflammation injury model established under different conditions are shown. (a) The effects of different concentrations of Cistanche deserticola exosomes on the myocardial inflammation injury model established with macrophage conditioned supernatant; (b) The effects of different concentrations of Cistanche deserticola exosomes on the myocardial inflammation injury model established based on co-culture microfluidic chip technology. N represents the control group, M represents the model group, and 1.25, 2.5, 5, and 10 μg / mL represent different concentrations of exosomes administered groups. Comparison between the model group and the control group: # p < 0.05, ## p < 0.01, ### p < 0.001; comparison between drug-treated group and model group: *p < 0.05, **p < 0.01, ***p < 0.001.

[0064] Figure 9 The effect of different concentrations of Cistanche deserticola exosomes on the oxygen-glucose deprivation-reperfusion model of PC12 neurons is shown. N represents the control group, M represents the model group, and 1.25, 2.5, 5, and 10 μg / mL represent different concentrations of exosomes administered groups. Comparison between the model group and the control group: # p < 0.05, ## p < 0.01, ### p < 0.001; comparison between drug-treated group and model group: *p < 0.05, **p < 0.01, ***p < 0.001.

[0065] Figure 10 Figure 3 shows flow cytometry analysis of the regulatory effects of different concentrations of Cistanche deserticola exosomes on macrophage polarization. NC refers to M0 macrophages, IL4+IL13 refers to IL4+IL13-induced M2 macrophages, and 1, 5, and 25 μg / mL refer to M2 macrophages treated with different concentrations of Cistanche deserticola exosomes.

[0066] Figure 11Shown are the effects of different concentrations of Cistanche deserticola exosomes on tumor cells. N represents tumor cells, and 6.25, 12.5, 25, and 50 μg / mL represent different exosome concentrations. *p < 0.05, **p < 0.01, ***p < 0.001.

[0067] Figure 12 Shown are the effects of different concentrations of Cistanche tubulosa exosomes on CoCl2-induced hypoxic injury in PC12 neurons. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

[0068] Figure 13 Shown are the effects of different concentrations of Cistanche tubulosa exosomes on a microarray model of macrophage-induced specific liver injury. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

[0069] Figure 14 The therapeutic effect of desert Cistanche deserticola exosomes on reproductive damage in male mice is shown. (a) shows body weight changes; (b) shows testicular index; (c) shows sperm concentration; (d) shows the percentage of grade A sperm in mice; (e) shows the percentage of total active sperm in mice; (f) shows the curve speed of sperm movement in mice; (g) shows the linear speed of sperm movement in mice; (h) shows the average path speed of sperm movement in mice; (i) shows the forward direction of sperm movement in mice; (j) shows the lateral swing amplitude of sperm movement in mice; (k) shows the whipping frequency of sperm movement in mice. N: control group; M: model group; TP: testosterone propionate positive drug group; EL: low-dose Cistanche deserticola exosome group; EM: medium-dose Cistanche deserticola exosome group; EH: high-dose Cistanche deserticola exosome group. Comparison between model group and control group: # p < 0.05, ## p < 0.01, ### p < 0.001; comparison between drug-treated group and model group: * p < 0.05, ** p < 0.01, *** p< 0.001.

[0070] Figure 15 This figure shows that exosomes from Cistanche deserticola inhibit tumor growth in mice bearing triple-negative breast cancer. (a) shows mouse weight monitoring, (b) shows mouse tumor photography, (c) shows statistical analysis of mouse tumor volume, and (d) shows statistical analysis of mouse tumor weight. N represents normal mice, M represents triple-negative breast cancer model mice, and EVs represents Cistanche deserticola exosomes injected intraperitoneally into triple-negative breast cancer mice. DETAILED DESCRIPTION

[0071] The present invention will be further described in detail below in conjunction with specific embodiments. The examples given are only for illustrating the present invention, not for limiting the scope of the present invention.

[0072] The Cistanche deserticola used in the following examples was purchased from the Cistanche deserticola cultivation base in Turpan, Xinjiang Uygur Autonomous Region, while the Cistanche tubulosa was purchased from the Cistanche tubulosa cultivation base in Yutian County, Hotan Prefecture, Xinjiang Uygur Autonomous Region. Professor Tu Pengfei of the Peking University School of Pharmacy identified the two species as Cistanche deserticola (Y.C. Ma) and Cistanche tubulosa (Schenk) Wight, both species of the genus Cistanche.

[0073] In the following examples, PBS solutions were prepared using deionized water, KH2PO4, and Na2HPO4·2H2O, and the following steps were followed:

[0074] (1) Prepare 1 / 15 mol / L KH2PO4 by dissolving 9.078 g KH2PO4 per liter of water;

[0075] (2) Prepare 1 / 15 mol / L Na2HPO4·2H2O, i.e. dissolve 11.876 g of Na2HPO4·2H2O per liter of water;

[0076] (3) Mix 18.2% (volume fraction) KH2PO4 solution and 81.8% Na2HPO4·2H2O.

[0077] Example 1: Preparation of exosomes from Cistanche deserticola

[0078] (1) Wash and dry 5 kg of fresh desert cistanche and squeeze the juice in a juicer. Centrifuge the juice at 1000 g for 10 min at 4°C in a low-speed centrifuge and retain the supernatant. Centrifuge the supernatant again at 3000 g for 20 min at 4°C in a low-speed centrifuge and retain the supernatant. Centrifuge the supernatant again at 10000 g for 30 min at 4°C in a low-speed centrifuge and retain the supernatant.

[0079] (2) The supernatant obtained in step (1) was centrifuged at 100,000 g for 30 minutes at 4°C using an ultracentrifuge, and the precipitate was retained; the precipitate was resuspended in 2 mL of PBS to obtain a crude Cistanche exosome suspension.

[0080] (3) Dissolve sucrose in PBS to prepare sucrose solutions with mass concentrations of 30%, 45%, and 60%. Add the sucrose solutions to the ultracentrifuge tube in descending order from the bottom of the tube to the top of the tube. Add the crude Cistanche deserticola exosome suspension obtained in step (2) to the ultracentrifuge tube, weigh and balance it, and centrifuge it at 150,000 g for 2 hours at 4°C. Remove the 30%-45% band and transfer it to a new ultracentrifuge tube. Add PBS to the ultracentrifuge tube to a distance of about 1-2 cm from the tube top. Centrifuge the ultracentrifuge tube at 150,000 g for 1 hour at 4°C, discard the supernatant, retain the precipitate, and resuspend the precipitate with PBS to obtain exosome 1#.

[0081] Example 2: Preparation of exosomes from Cistanche deserticola

[0082] The preparation process of the desert cistanche exosomes in this example is the same as that in Example 1, except that in step (2), the supernatant is centrifuged for 45 minutes.

[0083] The exosomes prepared in this example are recorded as exosome 2#.

[0084] Example 3: Preparation of exosomes from Cistanche deserticola

[0085] The preparation process of the desert cistanche exosomes in this example is the same as that in Example 1, except that in step (2), the supernatant is centrifuged for 60 minutes.

[0086] The exosomes prepared in this example are recorded as exosome 3#.

[0087] Example 4: Preparation of Exosomes from Cistanche deserticola

[0088] (1) Wash and dry 5 kg of fresh desert cistanche and squeeze the juice in a juicer. Centrifuge the juice at 800 g for 15 min at 6°C in a low-speed centrifuge and retain the supernatant. Centrifuge the supernatant again at 2800 g for 25 min at 6°C in a low-speed centrifuge and retain the supernatant. Centrifuge the supernatant again at 8000 g for 35 min at 6°C in a low-speed centrifuge and retain the supernatant.

[0089] (2) The supernatant obtained in step (1) was centrifuged at 120,000 g for 30 minutes at 4°C using an ultracentrifuge, and the precipitate was retained; the precipitate was resuspended in 2 mL of PBS to obtain a crude Cistanche exosome suspension.

[0090] (3) Dissolve sucrose in PBS to prepare sucrose solutions with mass concentrations of 30%, 45%, and 60%. Add the sucrose solutions to the ultracentrifuge tube in descending order from the bottom of the tube to the top of the tube. Add the crude Cistanche deserticola exosome suspension obtained in step (2) to the ultracentrifuge tube, weigh and balance it, and centrifuge it at 150,000 g for 1 hour at 4°C. Remove the 30%-45% band and transfer it to a new ultracentrifuge tube. Add PBS to the ultracentrifuge tube until it is about 1-2 cm away from the tube top. Centrifuge the ultracentrifuge tube at 150,000 g for 1 hour at 4°C, discard the supernatant, retain the precipitate, and resuspend the precipitate with PBS to obtain exosome 4#.

[0091] Example 5: Preparation of exosomes from Cistanche deserticola

[0092] The preparation process of the desert cistanche exosomes in this embodiment is the same as that in Example 4, except that: in step (3), the ultracentrifuge tube is weighed and balanced and then centrifuged for 1.5 hours.

[0093] The exosomes prepared in this example are recorded as exosome 5#.

[0094] Example 6: Preparation of Exosomes from Cistanche deserticola

[0095] The preparation process of the desert cistanche exosomes in this embodiment is the same as that in Example 4, with the only difference being that in step (3), the ultracentrifuge tube is weighed and balanced and then centrifuged for 2 hours.

[0096] The exosomes prepared in this example are recorded as exosome 6#.

[0097] Example 7: Preparation of Exosomes from Cistanche deserticola

[0098] (1) Wash and dry 5 kg of fresh desert cistanche and put it into a juicer to extract the juice. Centrifuge the juice at 1200 g for 8 min at 2°C in a low-speed centrifuge and retain the supernatant. Centrifuge the supernatant again at 3500 g for 16 min at 2°C in a low-speed centrifuge and retain the supernatant. Centrifuge the supernatant again at 12000 g for 28 min at 2°C in a low-speed centrifuge and retain the supernatant.

[0099] (2) The supernatant obtained in step (1) was centrifuged at 100,000 g for 30 minutes at 2°C using an ultracentrifuge, and the precipitate was retained; the precipitate was resuspended in 2 mL of PBS to obtain a crude Cistanche exosome suspension.

[0100] (3) Dissolve sucrose in PBS to prepare sucrose solutions with mass concentrations of 30%, 45%, and 60%. Add the sucrose solutions to the ultracentrifuge tube in descending order from the bottom of the tube to the top of the tube. Add the crude Cistanche deserticola exosome suspension obtained in step (2) to the ultracentrifuge tube, weigh and balance it, and centrifuge it at 180,000 g for 2 hours at 2°C. Remove the 30%-45% band and transfer it to a new ultracentrifuge tube. Add PBS to the ultracentrifuge tube until it is about 1-2 cm away from the tube top. Centrifuge the ultracentrifuge tube at 180,000 g for 1 hour at 2°C, discard the supernatant, retain the precipitate, and resuspend the precipitate with PBS to obtain exosome 7#.

[0101] Example 8: Preparation of Exosomes from Cistanche deserticola

[0102] The preparation process of the desert cistanche exosomes in this example is the same as that in Example 7, with the only difference being that in step (3), 45%-60 bands are taken out for subsequent operations.

[0103] The exosomes prepared in this example are recorded as exosome 8#.

[0104] Example 9: Preparation of Cistanche tubulosa exosomes

[0105] The preparation process of the exosomes of Cistanche tubulosa in this example is the same as that in Example 1, with the only difference being the raw materials. This example uses Cistanche tubulosa as the raw material.

[0106] The exosomes prepared in this example are recorded as exosome 9#.

[0107] Example 10: Characterization of Cistanche deserticola exosomes

[0108] The characterization method of exosomes in this example is as follows:

[0109] (1) Identification of the structure and morphology of exosomes: Characterization is performed using transmission electron microscopy (TEM). TEM is a commonly used exosome characterization technique with good resolution, which can be used to directly observe the structural and morphological characteristics of exosomes of different sizes.

[0110] (2) Exosome particle concentration, particle size, Zeta potential and polymer dispersion index (PDI): Nanoparticle tracking analysis (NTA) and dynamic light scattering (DLS) are used to evaluate whether the exosome particle concentration, particle size distribution, Zeta potential and polymer dispersion index are within the predetermined range.

[0111] Figure 1 It is a characterization of the performance of exosomes 1# to 3#. Figure 1It can be seen that when the time of the first ultracentrifugation is 30 to 60 minutes, the obtained exosomes 1# to 3# have good distribution uniformity and system stability, and a good yield is achieved.

[0112] Figure 2 It is a characterization of the performance of exosomes 4# to 6#. Figure 2 It can be seen that although the absolute values ​​of PDI and Zeta potential fluctuate with the increase of purification time, overall, the particle distribution of exosomes 4# to 6# is relatively uniform and the system is stable.

[0113] Figure 3 It is a characterization of the performance of exosomes 7# and 8#. Figure 3 As can be seen, sample 7# maintains relatively intact morphology, has low impurity content, and exhibits a relatively concentrated particle size distribution, primarily around 30 nm. Furthermore, sample 7# exhibits a higher exosome concentration and a lower PDI value, indicating a more consistent particle size distribution. Furthermore, the higher absolute value of the zeta potential reflects improved colloidal stability. These results indicate that a density gradient of 30%–45% is most conducive to efficient exosome separation and purification.

[0114] Figure 4 This is the performance characterization of exosome 9#, i.e., the exosomes of Cistanche tubulosa. Figure 4 It can be seen that the exosomes of Cistanche tubulosa present a typical cup-shaped structure, and the particle size is mainly concentrated around 70 nm. Figure 4 The results show that the present invention can obtain exosomes with high particle distribution uniformity and strong overall stability of the system by using different Cistanche deserticola raw materials.

[0115] Example 11: Pharmacological effects of Cistanche deserticola exosomes on various cells

[0116] (1) Experimental materials

[0117] The exosomes of Cistanche deserticola were exosomes 1# prepared in Example 1, and the exosomes of Cistanche tubulosa were exosomes 9# prepared in Example 4.

[0118] (2) Experimental methods

[0119] 1. Protective effect of desert cistanche exosomes on germ cells

[0120] 1.1 Cell proliferation experiments

[0121] 1.1.1 Detection of exosome protein concentration in Cistanche deserticola by BCA method

[0122] A 2 mg / mL protein standard solution from a BCA kit (Quanshijin, China) was diluted in equal volumes to create a series of protein standard solutions at 0, 0.04, 0.1, 0.16, 0.2, 0.4, 0.8, 1.2, and 1.6 mg / mL. Solution A and Solution B from the kit were mixed at a 50:1 ratio and stored in a dark place to ensure immediate use. In a 96-well plate, 25 μL of the prepared protein standard solutions of varying concentrations and the Cistanche deserticola exosome suspension were added to each well. Three replicates were set up for each solution to ensure accuracy. Subsequently, 100 μL of the mixture of Solution A and Solution B was added to each well. The 96-well plate was carefully wrapped with tin foil, also protected from light, and incubated in a 37°C incubator for 30 minutes. After incubation, the samples were detected using a microplate reader at a wavelength of 570 nm.

[0123] The detected desert exosome protein concentration was 1 mg / mL.

[0124] 1.1.2 MTT (thiazolyl blue) assay for cell viability

[0125] Cell culture dishes containing mouse spermatogonia (GC1), mouse spermatocytes (GC2), mouse Leydig cells (TM3), and mouse Sertoli cells (TM4) (purchased from the Cell Center of the Chinese Academy of Medical Sciences) were removed from the cell culture incubator and digested with trypsin. The cell suspension was then centrifuged, resuspended in cell culture medium, and counted. The cell population was adjusted to 2 × 10 4 Cells were then pipetted into 96-well plates, with 100 μL of the cell suspension added to each well in triplicate. After 16 hours, the culture medium was removed and 100 μL of PBS was added to each well for washing. The PBS was then removed and a gradient of exosome solutions from Cistanche deserticola (2.5, 5, 10, and 20 μg / mL) was added and incubated. After 48 hours, a 5 mg / mL MTT stock solution was diluted in a ratio of 10:1: DMEM medium: 5 mg / mL MTT. The culture medium in the 96-well plate was discarded, and 100 μL of the diluted MTT solution was added to each well. The cells were incubated for an additional 3 hours. After 3 hours, the cells were removed, the MTT solution discarded, and 100 μL of DMSO (dimethyl sulfoxide) solution was added to each well. After the reaction was complete, the cells were detected using a microplate reader at a wavelength of 570 nm.

[0126] The cell culture medium used in the above operation was DMEM medium supplemented with 10% fetal bovine serum (FBS) and 0.1% penicillin-streptomycin double antibody solution. DMEM medium was purchased from Thermo Fisher Scientific, FBS was purchased from Shanghai Nova Pharmaceutical Technology Co., Ltd., and penicillin-streptomycin double antibody solution was purchased from Zhongke Maichen (Beijing) Technology Co., Ltd.

[0127] MTT stock solution was purchased from Solebao Technology Co., Ltd. and DMSO solution was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.

[0128] 1.2 Effects of Cistanche deserticola exosomes on germ cell damage caused by 4-HC

[0129] 1.2.1 Detection of exosome protein concentration in Cistanche deserticola by BCA method

[0130] Same as 1.1.1.

[0131] 1.2.2 Preparation of 4-HC solution

[0132] Weigh 2.9308 mg of 4-hydroxycyclophosphamide (4-HC) into a 1.5 mL centrifuge tube. Dissolve the solution thoroughly in 100 μL of DMSO to prepare a 100 mM 4-HC stock solution. Wrap the solution in tinfoil, protect from light, and store at -80°C until needed.

[0133] Effects of different concentrations of Cistanche deserticola exosomes on the proliferation of germ cells damaged by 4-HC

[0134] The germ cell lines GC1, GC2, TM3 and TM4 were passaged at a rate of 4 × 10 4 Cells were seeded at a density of 1 μg / mL in 96-well plates, with three replicates per group. When the cell confluence area reached approximately 50%, the treatment group was treated with 4 μM 4-HC and exosome solutions of Cistanche deserticola at varying concentrations (12.5 and 25 μg / mL). The model group was treated with only 4 μM 4-HC, and the control group was treated with an equal volume of culture medium (DMEM). After 24 hours of culture, cell viability was assessed using the MTT assay (see 1.1.2 for specific methods).

[0135] 2. Anti-neuritis effects of Cistanche deserticola exosomes

[0136] 2.1 Effects of Cistanche deserticola exosomes on LPS-activated BV2

[0137] 2.1.1 Effects of different concentrations of Cistanche deserticola exosomes on cell viability of LPS-activated BV2 cells

[0138] Mouse microglial BV2 cells (Cell Center, Chinese Academy of Medical Sciences) in good condition were taken and prepared into cell suspension. 5 Cells were seeded at a density of 100 μg / mL in 48-well plates and divided into three replicates for each group. The cells were cultured overnight for approximately 12-16 hours. Microscopic observation revealed that the cells adhered well to the surface of the culture medium. A 1 μg / mL lipopolysaccharide (LPS) solution was prepared in DMEM / F12 medium. This solution was then used to prepare exosome solutions from Cistanche deserticola at concentrations of 1.25, 2.5, 5, and 10 μg / mL, as well as a 10 μM dexamethasone (DXMS) solution. After 24 hours of treatment, cell viability was assessed using the MTT assay (see 1.1.2 for specific methods).

[0139] During the above operation, the specific treatment of each group of cells is as follows:

[0140] Normal group: only culture medium was given; model group: only LPS was given; exosome group: exosome solution prepared with LPS solution was given; dexamethasone group: dexamethasone solution prepared with LPS solution was given.

[0141] 2.1.2 Effects of different concentrations of Cistanche deserticola exosomes on NO expression in LPS-activated BV2 cells

[0142] Take BV2 cells in good condition and prepare a cell suspension. 5 Cells were seeded at a density of 100 μg / mL in 48-well plates and divided into a model group, a dexamethasone-positive group, and an exosome-administered group (treatments were the same as in 2.1.2). Three replicates were set up in each group and cultured overnight for approximately 12-16 hours. Microscopic observation indicated that the cells adhered well to the culture medium and grew. A 1 μg / mL LPS solution was prepared using the culture medium. This solution was then used to prepare Cistanche deserticola exosome solutions at concentrations of 1.25, 2.5, 5, and 10 μg / mL, along with a 10 μM dexamethasone solution. After 24 hours of treatment, 300 μL of the supernatant was aspirated into a new 48-well plate and the supernatant NO content was assayed using a NO detection kit (APPLYGEN, Beijing Pulilai Gene Technology Co., Ltd.).

[0143] 3. Anti-myocarditis effect of Cistanche deserticola exosomes

[0144] 3.1 Effects of Cistanche deserticola exosomes on myocardial inflammatory injury model

[0145] 3.1.1 Construction of a macrophage-conditioned supernatant-induced myocardial inflammatory injury model and activity detection of Cistanche deserticola exosomes after administration

[0146] About 1×10 6RAW 264.7 macrophages (Cell Center, Chinese Academy of Medical Sciences) were seeded into 6-well plates at 1 μg / ml LPS for 6 hours (the control group was not stimulated with LPS), washed with PBS, and replaced with serum-free medium (DMEM, Gibco) for 24 hours. The cell culture medium was collected and centrifuged to obtain the supernatant. The NO content in the RAW264.7 cell supernatant was determined using a NO detection kit (APPLYGEN, Beijing Pulilai Gene Technology Co., Ltd.). The supernatant was diluted with DMEM (Gibco) to a NO concentration of 2 μM and set aside. Approximately 1×10 4 H9c2 cardiomyocytes (Cell Center, Chinese Academy of Medical Sciences) were seeded into 96-well plates at 100 μg / well. After cell attachment, the medium was replaced with serum-free DMEM (Gibco) and incubated for 12 hours. The cells were then divided into control, model, and drug-treated groups. Control cells were cultured in serum-free medium, while the model group was treated with conditioned supernatant containing 2 μM NO. Drug-treated groups were treated with exosomes from Cistanche deserticola diluted to 1.25, 2.5, 5, and 10 μg / mL using the conditioned supernatant. After 24 hours of treatment, cell viability was assessed using the MTT assay (see 1.1.2 for details).

[0147] 3.1.2 Construction of a myocardial inflammatory injury model based on microfluidic chip technology and activity detection of Cistanche deserticola exosomes after administration

[0148] Poly-lysine solution (1×) was added to the channel of a UV-treated PDMS chip (Dow Corning, USA) and incubated at 37°C overnight. The chip was then washed twice with PBS and DMEM medium (Gibco) was added to the side channel. Then, 5 μL of a 2×10 6 H9c2 cell suspension at a concentration of 1×10 cells / ml was added to the main channel. After overnight, RAW264.7 cells stimulated with LPS (1 μg / ml) digested with 0.25% trypsin for 6 hours or normal RAW264.7 cells (control group) were seeded into the side channel at a density of 1×10 7 cells / ml. After macrophages adhered, serum-free culture medium was added to the control and model groups. Treatment groups were treated with Cistanche deserticola exosome solutions diluted in serum-free culture medium to 1.25, 2.5, 5, and 10 μg / mL. The culture medium was changed every 8 hours. After 24 hours of culture, H9c2 cell viability was assessed using the LIVE / DEAD Cell Viability / Toxicity Assay Kit (Invitrogen, USA).

[0149] 4. Antioxidant activity of Cistanche deserticola exosomes against oxidative glucose deprivation / reperfusion (OGD / R)

[0150] 4.1 Effects of Cistanche deserticola exosomes on the OGD / R model of neural cells

[0151] 4.1.1 Establishment of the Oxygen-Glucose Deprivation-Reperfusion Model of Neuronal Cells and Post-drug Viability Detection

[0152] PC12 rat adrenal pheochromocytoma cells (Cell Center, Chinese Academy of Medical Sciences) in good condition were obtained and cultured at a rate of 8 × 10 4 Cells were seeded at a density of 100 μg / mL in 96-well plates, with six replicates per group. Cultures were performed overnight for approximately 12-16 hours. Microscopic observation revealed that the cells adhered well and grew well. The cells were then divided into a control group, a model group, a positive drug group, and a Cistanche deserticola exosome-treated group. The DMEM medium was discarded, and the control group was replaced with complete medium (i.e., DMEM, Gibco), while the model group was replaced with sugar-free medium (i.e., EBSS, Abcam). The positive drug group was treated with edaravone (Eda) diluted to 16 μM in sugar-free medium, while the Cistanche deserticola exosome-treated groups were treated with Cistanche deserticola exosome solutions diluted to 1.25, 2.5, 5, and 10 μg / mL in sugar-free medium. The cells were placed in a sealed box with a deoxygenated bag and incubated in a constant-temperature incubator for 6 hours. The original medium was then discarded and replaced with complete medium. The cells were then incubated in a constant-temperature incubator for an additional 24 hours. Cell viability was then assessed by MTT assay (see 1.1.2 for details).

[0153] 5. Reprogramming Effects of Desert Cistanche Exosomes on Macrophages

[0154] 5.1 Isolation and polarization of macrophages and the reprogramming effect of Cistanche deserticola exosomes on macrophages

[0155] 5.1.1 Isolation and polarization of macrophages

[0156] Primary mouse bone marrow-derived macrophages (BMDM) were isolated from the femur and tibia of 8-12 week-old C57BL / 6J mice (Experimental Animal Center, Peking University Health Science Center) using the following method:

[0157] (1) Prepare in advance ice plates, 10 cm culture dishes, 50 mL tubes, 6-well plates, syringes (1 mL and 10 mL / 20 mL), serum-free 1640 medium, and 1640 complete medium containing 10% FBS and MCSF induction factors.

[0158] (2) 6-8 week old C57BL / 6 male mice were killed by cervical dislocation and immersed in 75% alcohol.

[0159] (3) Under sterile conditions, separate the mouse lower limbs and place them in a 10 cm culture dish filled with alcohol.

[0160] (4) Expose the tibia and femur and remove soft tissues such as muscles and fascia. Place the bones in a 10 cm culture dish filled with alcohol.

[0161] (5) Transfer the bones to a 10 cm culture dish containing PBS, rinse the femur and tibia with PBS, and then cut off the epiphyses.

[0162] (6) Use a sterile syringe to draw serum-free 1640 culture medium to repeatedly flush the bone marrow cavity and collect the bone marrow in a 50 ml tube until there is no blood clot in the bone marrow cavity.

[0163] (7) Use a pipette to mix the cells and centrifuge at 1000 rpm / min for 5 min.

[0164] (8) Discard the supernatant, resuspend the cells in 1640 complete medium containing 10% FBS and MCSF induction factors, inoculate into 6-well plates, and culture in an incubator at 37°C and 5% CO2.

[0165] (9) Change the solution every 2 days.

[0166] Macrophages isolated by the above method were cultured in RPMI1640 medium (Gibco) supplemented with 10% fetal bovine serum (FBS) and 10 ng / ml recombinant macrophage colony-stimulating factor (M-CSF). The cells were then divided into three groups: NC group, IL4+IL13 group, and exosome-treated group. On day 7, cells in the IL4+IL13 and exosome-treated groups were stimulated with 100 ng / mL IL-4 and 100 ng / mL IL-13 for 24 hours to induce M2 macrophage differentiation. The NC group received only the corresponding culture medium (RPMI1640 medium supplemented with 10% fetal bovine serum (FBS) and 10 ng / ml recombinant macrophage colony-stimulating factor (M-CSF)).

[0167] 5.1.2 Reprogramming Effects of Cistanche Deserticola Exosomes on Macrophages

[0168] After 24 hours of IL-4 and IL-13 stimulation, the NC and IL4+IL13 groups received only the corresponding culture medium, while the exosome-treated group received 1, 5, or 25 μg / mL of Cistanche deserticola exosome solution. After 48 hours of stimulation, cells were harvested and stained with mouse monoclonal antibodies anti-F4 / 80 PE, anti-CD86 Brilliant Violet 421, and anti-CD206 APC (BioLegend). The staining method involved blocking with 0.5% BSA for half an hour and then staining with the staining solution for 0.5-1 hour. Flow cytometric analysis was performed using a four-laser flow cytometer (Beckman). Data were processed using FlowJo Version 10.8.1 (BD Biosciences, USA).

[0169] 6. Inhibitory Effects of Desert Cistanche Exosomes on Tumor Cells

[0170] 6.1 Effect of Cistanche deserticola exosomes on tumor cell viability

[0171] 6.1.1 Effects of different concentrations of Cistanche deserticola on tumor cell viability

[0172] Healthy mouse skin melanoma B16F10 cells, human non-small cell lung cancer A549 cells, human hepatocellular carcinoma HUH7 cells, mouse breast cancer 4T1 cells, human breast cancer MDA-MB-231 cells, and human breast cancer MCF-7 cells (Cell Center, Chinese Academy of Medical Sciences) were seeded at a density of 40,000 cells / mL in 96-well plates, with five replicates per group. The cells were cultured overnight for approximately 12-16 hours. Microscopic observation indicated that the cells adhered well to the plate and then treated with a solution of Cistanche deserticola exosomes at concentrations of 6.25, 12.5, 25, and 50 μg / mL in DMEM or 1640 medium (Gibco) for 24 hours. Cell viability was then assessed by MTT assay (see 1.1.2 for details).

[0173] 7. Protective Effect of Cistanche Tubulosa Exosomes on CoCl2-Induced PC12 Hypoxic Injury Model

[0174] PC12 rat adrenal pheochromocytoma cells (Cell Center, Chinese Academy of Medical Sciences) were passaged at a rate of 1 × 10 5Cells were seeded at a density of 100 μg / mL in 96-well plates in DMEM (Gibco) supplemented with 10% FBS and 0.1% penicillin-streptomycin solution. A control group, a model group, and an exosome-treated group were set up, with triplicate wells in each group. When the cell confluence reached approximately 50%, the model and exosome-treated groups were treated with 300 μM CoCl₂. After the treatment period, the plates were washed and the original medium was removed. Different concentrations of Cistanche deserticola exosome solution (0.01, 0.1, 5, 25, and 50 μg / mL) were then added and cultured in a cell culture incubator. After 24 hours, cell viability was assessed using the MTT assay (see 1.1.2 for details).

[0175] 8. Protective Effect of Cistanche Tubulosa Exosomes on Macrophage-Induced Specific Liver Injury Chip Model

[0176] Preparation of high-throughput biomimetic liver microtissue model: A 1.5 mL EP tube was placed on a 0°C metal bath, 3.26 μL of 0.5 M NaOH solution and 6.5 μL of 1 M HEPES buffer were added, mixed, and then 56 μL of 3.72 mg / mL type I rat tail collagen solution (Gibco) was added, and then 64 μL of 6.052×10 5 A HepG2 cell suspension (Cell Center, Chinese Academy of Medical Sciences) at 100 μg / mL was pipetted and mixed thoroughly. 2.5 μL of the suspension was then dripped onto the top of a high-throughput PDMS column chip and placed in a 37°C incubator to solidify for 10 min. Separately, a high-throughput microwell chip was prepared and 19 μL of complete EMEM (Gibco) medium was added to the microwells. The collagen-cured PDMS column chip was flipped over and inserted into the high-throughput microwell chip, ensuring that each hepatocyte collagen droplet was completely immersed in the medium. The microwell was then incubated in a 37°C, 5% CO2 incubator for 96 h, with the medium replaced every 48 h. This resulted in a high-throughput biomimetic liver microtissue model.

[0177] Preparation of a high-throughput biomimetic liver microtissue-macrophage interaction model: A mouse mononuclear macrophage leukemia cell suspension, RAW264.7, was prepared and diluted to 4.00 × 10 5 The high-throughput macrophage culture model was established by seeding 20 μL of the microparticles per well into a high-throughput microwell chip and culturing overnight in a 37°C, 5% CO2 incubator. Ninety-six hours after the liver microtissue model was inoculated, the PDMS column chip housing the high-throughput biomimetic liver microtissue model was inserted into the high-throughput microwell chip housing the macrophage culture model, resulting in a high-throughput biomimetic liver microtissue-macrophage interaction model.

[0178] Exosome administration and liver tissue activity detection: The wells on the macrophage chip were divided into four groups, namely, "nimesulide + LPS + exosomes", "exosomes", "control" and "nimesulide + LPS" groups. Complete mixed culture medium (DMEM medium (Gibco) containing 10% FBS and 0.1% penicillin-streptomycin solution) containing 400 μM nimesulide, 1 μg / mL LPS and different concentrations of exosomes (i.e., 5, 25, and 50 μg / mL), complete culture medium containing different concentrations of exosomes (i.e., 5, 25, and 50 μg / mL), blank complete mixed culture medium, and complete mixed culture medium containing 400 μM nimesulide and 1 μg / mL LPS were added to the high-throughput macrophage culture chip, respectively. The high-throughput biomimetic liver microtissue chip was then turned over and inserted into the high-throughput macrophage culture chip and incubated in an incubator at 37°C and 5% CO2 for 24 hours. After 4 h, the activity of liver microtissues was determined using the CellTiter Blue® Cell Viability Assay Kit (Promega).

[0179] Each experiment was performed in at least three replicate wells, with at least three replicates. All results are representative. Statistical analysis was performed using GraphPad Prism 8 software. Data are presented as mean ± standard deviation (SD). One-way ANOVA was used to compare data between groups. P < 0.05 was considered statistically significant, P < 0.01 was considered statistically significant, and P < 0.001 was considered extremely significant.

[0180] (3) Experimental results and discussion

[0181] 1. Proliferative effects of Cistanche deserticola exosomes on four testicular germ cell lines

[0182] GC1, GC2, TM3 and TM4 are four cell lines commonly used in reproductive biology and male reproductive system research. These cell lines represent different types of cells in the testis, including spermatogonia, spermatocytes, interstitial cells, and supporting cells, which play a key role in spermatogenesis and testicular function. Using these cell lines as models, it is possible to simulate and study the various stages of spermatogenesis under controlled conditions, as well as the molecular mechanisms and environmental factors that affect this process. Therefore, the present invention uses these four cell lines to preliminarily test the potential effects of Cistanche deserticola exosomes on the male reproductive system in vitro. Figure 5As shown in the figure, GC1, GC2, TM3, and TM4 normal cells were treated with 1.25, 2.5, 5, 10, and 20 μg / mL of Cistanche exosomes for 48 hours, and cell proliferation was measured using MTT. For GC1, GC2, and TM3 cell lines, Cistanche exosomes began to exert their efficacy at 2.5 or 5 μg / mL; for TM4 cell line, Cistanche exosomes took effect at a concentration of 10 μg / mL.

[0183] 2. Protective effect of Cistanche deserticola exosomes on 4-HC-induced germ cell damage

[0184] The results of the protective effect of Cistanche deserticola exosomes on 4-HC-induced germ cell damage are as follows Figure 6 As shown. Figure 6 It can be seen that when the concentration of Cistanche deserticola exosomes is 12.5 μg / mL, it has a protective effect on 4-HC-induced damage to GC1, GC2, TM3, and TM4 cells, and shows a certain dose-dependency in TM3 and TM4 cells.

[0185] 3. Protective Effects of Desert Cistanche Exosomes on Neuritis

[0186] The results of the protective effect of Cistanche deserticola exosomes on neuritis are as follows Figure 7 As shown. Figure 7 (a) It can be seen that when the concentration of Cistanche deserticola exosomes reaches 5 μg / mL, it has a toxic effect on BV2 cells activated by LPS. Figure 7 (b) It can be seen that Cistanche deserticola exosomes can inhibit the content of NO produced by activated BV2 cells.

[0187] 4. Protective Effect of Desert Cistanche Exosomes on Myocarditis

[0188] The results of the protective effect of Cistanche deserticola exosomes on myocarditis are as follows Figure 8 As shown. Figure 8 (a) It can be seen that the exosomes of Cistanche deserticola at various concentrations have a certain recovery effect on the myocardial inflammatory injury model constructed by macrophage conditioned supernatant. Figure 8 (b) It can be seen that when the exosomes of Cistanche deserticola increased to 2.5 μg / mL, it had a certain recovery effect on the myocardial inflammatory injury model constructed using co-culture microfluidic chip technology.

[0189] 5. Protective Effects of Desert Cistanche Exosomes on Oxygen-Glucose Deprivation-Reperfusion Model

[0190] The results of the protective effect of Cistanche deserticola exosomes on the oxygen-glucose deprivation-reperfusion model are as follows Figure 9 As shown. Figure 9It can be seen that the exosomes of Cistanche deserticola at various concentrations have a certain recovery effect on the oxygen-glucose deprivation-reperfusion model.

[0191] 6. Reprogramming Effects of Desert Cistanche Exosomes on Macrophages

[0192] The results of the reprogramming effect of Cistanche deserticola exosomes on macrophages are as follows Figure 10 As shown. Figure 10 It can be seen that the exosomes of Cistanche deserticola promote the polarization of macrophages from M2 to M1, and show a certain dose dependence.

[0193] 7. Inhibitory Effects of Desert Cistanche Exosomes on Tumor Cells

[0194] The results of the inhibitory effect of desert cistanche exosomes on tumor cells are as follows Figure 11 As shown. Figure 11 It can be seen that the exosomes of Cistanche deserticola have inhibitory effects on various types of tumor cells, including skin cancer (B16F10), lung cancer (A549), liver cancer (HUH7), breast cancer (4T1, MCF7, MDA-MB-231), and show a certain dose dependence.

[0195] 8. Protective Effect of Cistanche Tubulosa Exosomes on CoCl2-Induced PC12 Hypoxic Injury Model

[0196] The results of the protective effect of Cistanche tubulosa exosomes on the CoCl2-induced PC12 hypoxic injury model are as follows Figure 12 As shown. Figure 12 It can be seen that the exosomes of Cistanche tubulosa have a protective effect on the CoCl2-induced PC12 hypoxic injury model and show a certain concentration dependence.

[0197] 9. Protective Effect of Cistanche Tubulosa Exosomes on Macrophage-Induced Specific Liver Injury Chip Model

[0198] The results of the protective effect of Cistanche tubulosa exosomes on the macrophage-induced specific liver injury chip model are as follows Figure 13 As shown. Figure 13 It can be seen that Cistanche tubulosa exosomes have a protective effect on the macrophage-induced specific liver injury chip model at concentrations of 5, 25, and 50 μg / mL.

[0199] Example 12: Therapeutic effects of desert cistanche exosomes on male reproductive damage and breast cancer

[0200] (1) Experimental materials

[0201] Same as Example 11.

[0202] (2) Experimental animals

[0203] C57BL / 6J male mice (6-8 weeks old) were provided by the Laboratory Animal Center of Peking University Health Science Center. Standard animal welfare conditions were provided in accordance with the "Regulations of the People's Republic of China on the Administration of Laboratory Animals," the "Regulations of Beijing on the Administration of Laboratory Animals," and the "Implementation Rules for the Administration of Medical Laboratory Animals." They were housed in a standard environment with a temperature of 20 ± 2°C, a humidity of 60 ± 5%, a 12-h light-dark cycle, a standard diet, and free access to water. Animal Ethics Approval Number: LA2019099.

[0204] (3) Experimental methods

[0205] 1. Therapeutic effect of desert cistanche exosomes on reproductive damage in male mice

[0206] 1.1 Establishment of the mouse reproductive system injury model and its administration method

[0207] After three days of adaptive feeding in a clean animal laboratory environment, the animals were randomly divided into six groups, each consisting of eight animals: normal group (N), model group (M), positive drug group (TP), low-dose group (EL), medium-dose group (EM), and high-dose group (EH). Groups M, TP, EL, EM, and EH received intraperitoneal injections of cyclophosphamide (60 mg / kg) once daily for one week. During this period, animals in group N received an equivalent dose of normal saline once daily for one week. After model establishment, the TP group received an intraperitoneal injection of testosterone propionate (0.2 mg / kg) twice weekly for two weeks. The EL, EM, and EH groups received intraperitoneal injections of exosomes at three doses, low (1.25 mg / kg / d), medium (2.5 mg / kg / d), and high (5 mg / kg / d), respectively, once daily for two weeks. In addition, the M, TP, EL, EM, and EH groups received a supplemental injection of cyclophosphamide (60 mg / kg) once weekly for two weeks. During the remaining two weeks, animals received an intraperitoneal injection of an equivalent dose of normal saline daily. During this period, animals in the N group received an intraperitoneal injection of an equivalent dose of normal saline daily for two weeks. After 14 days of treatment, mice were weighed and blood was collected before being sacrificed. Both epididymal capitulums were weighed, and the left capitulum was used for sperm quality testing. Both testicles were weighed and preserved.

[0208] 1.2 Mouse sperm motility assay based on computer-assisted sperm analysis (CASA)

[0209] A single epididymal cauda from each mouse group was placed in 1 mL of 37°C preheated HTF IVF medium (Coolaber, Kangbo Benin), minced, and thoroughly shaken. The tube was then incubated in a 37°C waterbath for 10 minutes with the tube open to allow all sperm to migrate out of the epididymis. Using a pipette, 10 μL of HTF medium containing sperm was transferred to a slide and observed under a microscope. Four to five fields of view were selected for analysis of various mouse sperm parameters using computer-assisted sperm analysis (CASA). The solution was maintained at 37°C throughout the assay to avoid disrupting sperm motility or morphology.

[0210] Computer-assisted sperm analysis (CASA) was used to measure sperm count, sperm curve velocity (VCL), linear velocity (VSL), average path velocity (VAP), sperm progressiveness (STR), sperm whiplash frequency (BCF), and amplitude of lateral displacement of the sperm head (ALH) in order to evaluate and analyze mouse sperm count and sperm motility.

[0211] 1.3 Statistical analysis

[0212] Experimental data were statistically analyzed using GraphPad Prism 8 software. Data are expressed as mean ± standard deviation (mean ± SD). Data between groups were compared using one-way ANOVA. P < 0.05 was considered statistically different, p < 0.01 was considered statistically significant, and p < 0.001 was considered extremely significant.

[0213] 2. Therapeutic Effects of Desert Cistanche Exosomes on Triple-Negative Breast Cancer-Transplanted Mice

[0214] 4T1 cells were inoculated in the left axilla of BALB / c mice and the tumor volume reached 100 mm. 3 Around 60 days post-exposure, mice were intraperitoneally injected with 8 mg / kg of exosomes every two days for two consecutive weeks. During this time, mice in the normal and model groups received an equivalent dose of saline. The mice's body weight, activity, tumor growth, and survival were observed and recorded daily. Fourteen days after exosome administration, the mice were sacrificed, and tumor tissues were removed, weighed, and photographed.

[0215] (4) Experimental results

[0216] 1. Therapeutic effect of desert cistanche exosomes on reproductive damage in male mice

[0217] The results of the therapeutic effect of desert cistanche exosomes on reproductive damage in male mice are as follows Figure 14As shown. Figure 14 It can be seen that Cistanche deserticola exosomes showed a significant restorative effect on cyclophosphamide-induced reproductive system damage model mice. Specifically, Cistanche deserticola exosomes can effectively improve reproductive dysfunction caused by cyclophosphamide, such as decreased testicular index, decreased sperm concentration, and decreased sperm motility ( Figure 14 (b) to Figure 14 (e)). In addition, Cistanche deserticola exosomes showed better efficacy than the positive drug group in improving sperm motility ( Figure 14 (f) to Figure 14 (k)). These results indicate that Cistanche deserticola exosomes can improve cyclophosphamide-induced decrease in testicular index, sperm concentration, motility, and motility in mice.

[0218] 2. Therapeutic Effects of Desert Cistanche Exosomes on Triple-Negative Breast Cancer-Transplanted Mice

[0219] Figure 15 The tumor detection results are shown. Figure 15 It can be seen that the tumor volume and weight were significantly reduced after administration of Cistanche exosomes, proving that Cistanche exosomes effectively inhibit the tumor growth of triple-negative breast cancer.

[0220] The above descriptions are merely exemplary embodiments of the present invention and are not intended to limit the present invention in any way. Although the present invention is disclosed above with reference to preferred embodiments, they are not intended to limit the present invention. Any person skilled in the art who, without departing from the scope of the present invention, may make slight changes or modifications to the above-disclosed technical contents to obtain equivalent or equivalent embodiments falls within the scope of the present invention.

Claims

1. An exosome derived from Cistanche deserticola, wherein: The Cistanche deserticola is Cistanche deserticola YC Ma and / or Cistanche tubulosa (Schenk) Wight.

2. The exosome according to claim 1, wherein The PDI of the exosomes is 0.05 to 0.3, preferably 0.15 to 0.3; The zeta potential of the exosomes is -5 mV to -30 mV, preferably -20 mV to -30 mV; and / or The particle size distribution of the exosomes is 20 to 200 nm, preferably 30-120 nm.

3. The method for extracting exosomes according to claim 1 or 2, comprising the following steps: (1) Crush the Cistanche deserticola and extract the juice, subject the obtained Cistanche deserticola juice to differential centrifugation, and collect the supernatant; (2) The supernatant is subjected to a first ultracentrifugation treatment, and the resulting precipitate is resuspended in PBS buffer to prepare a crude exosome suspension; (3) The crude exosome suspension is subjected to sucrose density gradient centrifugation to collect components between adjacent sucrose concentrations and recover the exosomes.

4. The extraction method according to claim 3, wherein In step (1), the differential centrifugation treatment sequentially includes: ① Centrifugation at 700 g to 1200 g for 5 to 15 minutes at 0°C to 10°C; ② Centrifugation at 2500 g to 4000 g for 15 to 30 minutes at 0°C to 10°C; and ③ Centrifugation at 7000 g to 15000 g for 25 to 40 minutes at 0°C to 10°C.

5. The extraction method according to claim 3, wherein In step (2), the temperature of the first ultracentrifugation treatment is 0°C to 10°C; The speed of the first ultracentrifugation treatment is 70000 g to 150000 g; The time of the first ultracentrifugation treatment is 0.5 hour to 1 hour.

6. The extraction method according to claim 3, wherein In step (3), the sucrose density gradient centrifugation treatment adopts a concentration gradient of 30% to 45% and a concentration gradient of 45% to 60% by mass fraction. Preferably, the temperature of the sucrose density gradient centrifugation treatment is 0°C to 10°C; Preferably, the sucrose density gradient centrifugation speed is 100000 g to 200000 g; Preferably, the sucrose density gradient centrifugation treatment time is 0.5 to 3 hours.

7. The extraction method according to claim 3, wherein In step (3), the recovery comprises diluting the component and then subjecting the obtained dilution to a second ultracentrifugation treatment; Preferably, the temperature of the second ultracentrifugation treatment is 0°C to 10°C; Preferably, the speed of the second ultracentrifugation treatment is 100,000 g to 200,000 g; Preferably, the second ultracentrifugation treatment lasts for 0.5 to 3 hours.

8. Use of the exosomes according to claim 1 or 2 in the preparation of a product for one or more of the following uses: (1) Improve normal male reproductive capacity; (2) Prevent, improve and / or treat male reproductive impairment; (3) Prevent, improve and / or treat inflammation; (4) Prevent, improve and / or treat cancer; (5) Prevent, improve and / or treat stroke; (6) Immunomodulation and / or immunotherapy enhancement for subjects; (7) Prevent, improve and / or treat liver damage; (8) As a drug delivery carrier.

9. The use according to claim 8, wherein The male reproductive capacity damage is male reproductive capacity damage caused by 4-hydroxycyclophosphamide and / or cyclophosphamide; Preferably, the male reproductive impairment is selected from one or more of oligospermia, asthenospermia and testicular abnormality; Preferably, the inflammation is neuritis and / or myocarditis; Preferably, the cancer is selected from one or more of skin cancer, lung cancer, liver cancer and breast cancer.

10. A product comprising the exosomes according to claim 1 or 2 and optionally a carrier or excipient acceptable in pharmaceuticals, medicine, food science or cosmetic science; Preferably, the product is selected from one or more of medicines, medical products, cosmetics, foods and health products; more preferably, the medical product is a cell therapy product; more preferably, the food is a health food; Preferably, the dosage form of the product is selected from one or more of powder, suppository, tablet, capsule, granule, oral solution, injection, emulsion and facial mask.

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