Microglia-derived exosomes and preparation method and application thereof

Abstract

The present invention provides microglia-derived exosomes and a preparation method and application thereof. The present invention provides a preparation method of microglia-derived exosomes, the preparation method comprising: culturing microglia in a culture medium containing gardenia glycosides, ligustrazine and puerarin to secrete exosomes, thereby obtaining microglia-derived exosomes. The microglia-derived exosomes prepared by the present invention can reduce neuroinflammation, inhibit oxidative stress and ferroptosis, reduce cerebral infarction volume, improve ischemia-reperfusion neurological scores, and improve brain tissue pathological damage, and have a neuroprotective effect on cerebral ischemia-reperfusion injury.

Description

Technical Field

[0001] The present invention relates to microglial cell-derived exosomes and a preparation method and application thereof. Background Art

[0002] Microglia are the main immune cells in the central nervous system (CNS). Microglia-derived exosomes have been a hot topic in neuromedicine research in recent years. They are vesicles that are secreted into the extracellular matrix after the fusion of intracellular multivesicular bodies with the plasma membrane. Their contents are diverse and can carry RNA, miRNA, lipids, metabolites and proteins from the mother cells. They circulate to distant organs through paracrine secretion or blood flow, and deliver proteins and nucleic acids to receptor cells, thereby changing the immune response and cell state of the receptor cells, promoting or inhibiting the progression of related diseases, and producing biological effects throughout the body. As a communication medium between cells, tissues and even organs, microglia-derived exosomes play an important role in the development and treatment of diseases of the nervous system.

[0003] Cerebral ischemia-reperfusion injury (CIRI) is a critical disease caused by the aggravation of brain tissue damage caused by the restoration of blood perfusion after thrombolysis during the treatment of ischemic encephalopathy. It has a high morbidity and mortality rate. Its pathogenesis is complex, with multiple pathological processes such as oxidative stress and inflammatory damage intertwined and related to multiple cell death modes such as apoptosis, autophagy, and ferroptosis. It eventually develops into mitochondrial morphological changes, dysfunction, neuronal cell structural destruction, increased blood-brain barrier permeability, large-scale damage to microvessels and brain tissue, and severe cerebral edema. Recent studies have shown that ferroptosis and inflammatory cascade reactions are important pathological bases for cerebral ischemia-reperfusion injury.

[0004] In recent years, microglia-derived exosomes have attracted much attention for their role in improving CIRI due to their advantages such as low immunogenicity, high biocompatibility, and easy penetration of the blood-brain barrier, but the improvement effect needs to be further improved. The types of drugs currently available for clinical use to effectively improve CIRI are still severe. Finding new drugs that can simultaneously intervene in inflammatory responses and cell ferroptosis, perform multi-pathway, holistic regulation, and have high safety and good targeting is an urgent hope for the treatment of CIRI. Summary of the invention

[0005] One object of the present invention is to provide a method for preparing microglia-derived exosomes.

[0006] Another object of the present invention is to provide an improved microglia-derived exosome.

[0007] Another object of the present invention is to provide an application of the microglia-derived exosomes.

[0008] The inventors of this case found in their research that treating microglia with a composition containing gardenia glycoside, ligustrazine and puerarin can significantly increase the number of exosomes secreted by microglia, and the obtained exosomes contain a small amount of gardenia glycoside, ligustrazine and puerarin, have a higher expression of glutathione peroxidase 4 (GPX4), are more easily taken up by neuronal cells, have stronger targeting, and show a significant effect of improving cerebral ischemia-reperfusion injury (CIRI), thereby enhancing the therapeutic effect of microglia-derived exosomes on CIRI.

[0009] Thus, on the one hand, the present invention provides a method for preparing microglia-derived exosomes, the method comprising: culturing microglia in a culture medium containing gardenia glycoside, ligustrazine and puerarin to secrete exosomes, thereby obtaining microglia-derived exosomes.

[0010] According to a specific embodiment of the present invention, in the method for preparing microglia-derived exosomes of the present invention, the total amount of gardenia glycoside, ligustrazine and puerarin in the culture medium is: 50 μg / mL-800 μg / mL, and the mass ratio of gardenia glycoside, ligustrazine to puerarin is (20-250): (15-250): (15-300).

[0011] According to some specific embodiments of the present invention, in the method for preparing microglia-derived exosomes of the present invention, the total amount of geniposide, ligustrazine and puerarin in the culture medium is: 90 μg / mL-750 μg / mL.

[0012] According to some specific embodiments of the present invention, in the method for preparing microglia-derived exosomes of the present invention, the total amount of geniposide, ligustrazine and puerarin in the culture medium is: 100 μg / mL-700 μg / mL.

[0013] According to some specific embodiments of the present invention, in the method for preparing microglia-derived exosomes of the present invention, the total amount of geniposide, ligustrazine and puerarin in the culture medium is: 200 μg / mL-650 μg / mL.

[0014] According to some specific embodiments of the present invention, in the method for preparing microglia-derived exosomes of the present invention, the total amount of geniposide, ligustrazine and puerarin in the culture medium is: 300 μg / mL-600 μg / mL.

[0015] According to some specific embodiments of the present invention, in the method for preparing microglia-derived exosomes of the present invention, the total amount of geniposide, ligustrazine and puerarin in the culture medium is: 350 μg / mL-500 μg / mL.

[0016] According to some specific embodiments of the present invention, in the method for preparing microglia-derived exosomes of the present invention, the total amount of geniposide, ligustrazine and puerarin in the culture medium is 400 μg / mL.

[0017] According to some specific embodiments of the present invention, in the method for preparing microglia-derived exosomes of the present invention, the mass ratio of gardenia glycoside, ligustrazine and puerarin in the culture medium is (40-250): (25-200): (25-300).

[0018] According to some specific embodiments of the present invention, in the method for preparing microglia-derived exosomes of the present invention, in the culture medium, the mass ratio of geniposide, ligustrazine and puerarin is (20-200): (40-250): (40-250).

[0019] According to some specific embodiments of the present invention, in the method for preparing microglia-derived exosomes of the present invention, in the culture medium, the mass ratio of geniposide, ligustrazine and puerarin is (20-150): (90-250): (90-250).

[0020] According to some specific embodiments of the present invention, in the method for preparing microglia-derived exosomes of the present invention, in the culture medium, the mass ratio of gardenia glycoside, ligustrazine and puerarin is (30-100): (150-250): (120-250).

[0021] According to some specific embodiments of the present invention, in the method for preparing microglia-derived exosomes of the present invention, in the culture medium, the mass ratio of gardenia glycoside, ligustrazine and puerarin is (30-60): (170-220): (150-220).

[0022] According to some specific embodiments of the present invention, in the method for preparing microglia-derived exosomes of the present invention, the mass ratio of gardenia glycoside, ligustrazine and puerarin in the culture medium is 40:199:161.

[0023] According to some specific embodiments of the present invention, in the method for preparing microglia-derived exosomes of the present invention, the amounts of gardenia glycoside, ligustrazine and puerarin added to the culture medium of microglia are: gardenia glycoside 40 μg / mL-250 μg / mL, ligustrazine: 25 μg / mL-200 μg / mL, puerarin: 25 μg / mL-300 μg / mL, respectively.

[0024] According to some specific embodiments of the present invention, in the method for preparing microglia-derived exosomes of the present invention, the amounts of gardenia glycoside, ligustrazine and puerarin added to the culture medium of microglia are: gardenia glycoside 20 μg / mL-200 μg / mL, ligustrazine: 40 μg / mL-250 μg / mL, puerarin: 40 μg / mL-250 μg / mL, respectively.

[0025] According to some specific embodiments of the present invention, in the method for preparing microglia-derived exosomes of the present invention, the amounts of gardenia glycoside, ligustrazine and puerarin added to the culture medium of microglia are: gardenia glycoside 20 μg / mL-150 μg / mL, ligustrazine: 90 μg / mL-250 μg / mL, puerarin: 90 μg / mL-250 μg / mL, respectively.

[0026] According to some specific embodiments of the present invention, in the method for preparing microglia-derived exosomes of the present invention, the amounts of gardenia glycoside, ligustrazine and puerarin added to the culture medium of microglia are: gardenia glycoside 30 μg / mL-100 μg / mL, ligustrazine: 150 μg / mL-250 μg / mL, puerarin: 120 μg / mL-250 μg / mL.

[0027] According to some specific embodiments of the present invention, in the method for preparing microglia-derived exosomes of the present invention, the amounts of gardenia glycoside, ligustrazine and puerarin added to the culture medium of microglia are: gardenia glycoside 30 μg / mL-60 μg / mL, ligustrazine: 170 μg / mL-220 μg / mL, puerarin: 150 μg / mL-220 μg / mL.

[0028] According to some specific embodiments of the present invention, in the method for preparing microglia-derived exosomes of the present invention, the amounts of gardenia glycoside, ligustrazine, and puerarin added to the culture medium of microglia are 40 μg / mL, 199 μg / mL, and 161 μg / mL, respectively.

[0029] According to a specific embodiment of the present invention, in the method for preparing microglia-derived exosomes of the present invention, the culture medium of the microglia includes but is not limited to one or more of a complete culture medium and a high-glucose DMEM culture medium. In the present invention, the complete culture medium and the high-glucose DMEM culture medium can be a complete culture medium and a high-glucose DMEM culture medium conventional in the field of cell culture medium, wherein the sugar content of the high-glucose DMEM culture medium is generally 2 g / L-10 g / L. When culturing microglia to prepare exosomes, the present invention uses a complete culture medium and a high-glucose DMEM culture medium conventional in the field of cell culture medium as the base culture medium, and adds the gardenia glycoside, ligustrazine and puerarin to form the improved culture medium for culturing microglia of the present invention.

[0030] According to a specific embodiment of the present invention, in the method for preparing microglia-derived exosomes of the present invention, the microglia include but are not limited to one or more of BV2 cells, HMO6 cells, HMC3 cells, RHMC cells, HAPI cells and RMC cells.

[0031] According to a specific embodiment of the present invention, in the method for preparing microglia-derived exosomes of the present invention, the microglia culture conditions are: culture at 36.5° C.-37.5° C. for 12 h-72 h.

[0032] According to a specific embodiment of the present invention, in the method for preparing microglia-derived exosomes of the present invention, after the culture is completed, the culture supernatant can be taken, centrifuged, and filtered to obtain the exosomes.

[0033] According to a specific embodiment of the present invention, the method for preparing microglia-derived exosomes of the present invention comprises:

[0034] After the microglia are cultured in the complete medium for 12 h-48 h, the supernatant is replaced with a medium containing gardenia glycosides, ligustrazine, and puerarin (preferably a high-glucose DMEM medium containing gardenia glycosides, ligustrazine, and puerarin), and the culture is continued for 12 h-72 h;

[0035] The supernatant of the above culture system was centrifuged to remove dead cells and cell debris, and the supernatant was removed by ultra-high speed centrifugation. The bottom precipitate was resuspended with PBS and then ultra-high speed centrifuged again to remove the supernatant to obtain exosomes.

[0036] According to a specific embodiment of the present invention, in the method for preparing microglia-derived exosomes of the present invention, the ultrahigh-speed centrifugation condition is 80,000-200,000×g for 30-90 min.

[0037] On the other hand, the present invention also provides a microglia-derived exosome, which contains gardenia glycoside, ligustrazine and puerarin.

[0038] According to a specific embodiment of the present invention, the microglia-derived exosomes of the present invention contain 0.1-200 ng / 10 of jasminoides, ligustrazine, and puerarin. 9 Exosomes, ligustrazine 1-800 ng / 10 9 Exosomes, puerarin 1-600 ng / 10 9 According to a specific embodiment of the present invention, the microglia-derived exosomes of the present invention contain gardenia glycoside, ligustrazine, and puerarin in an amount of about 0.5 ng / 10 9 exosomes, ligustrazine about 2.1 ng / 10 9 exosomes, puerarin about 2.6 ng / 10 9 Exosomes.

[0039] According to a specific embodiment of the present invention, the microglia-derived exosomes of the present invention highly express glutathione peroxidase 4 (GPX4). The highly expressed glutathione peroxidase 4 described in the present invention refers to a higher expression of antioxidant glutathione peroxidase GPX4 protein compared to the exosomes obtained by conventional culture of microglia (i.e., wild-type microglia-derived exosomes). According to a specific embodiment of the present invention, the microglia-derived exosomes of the present invention have a glutathione peroxidase 4 (GPX4) expression level that is more than 3 times the GPX4 expression level of wild-type microglia-derived exosomes.

[0040] According to a specific embodiment of the present invention, the microglia-derived exosomes of the present invention are obtained according to the preparation method of the present invention described above.

[0041] On the other hand, the present invention also provides the use of the microglia-derived exosomes.

[0042] In some specific embodiments of the present invention, the present invention provides the use of the microglia-derived exosomes in the preparation of a medicament for improving and / or treating cerebral ischemia-reperfusion injury (CIRI).

[0043] According to a specific embodiment of the present invention, the improvement and / or treatment of cerebral ischemia-reperfusion injury (CIRI) includes inhibiting CIRI neuroinflammation and / or ferroptosis.

[0044] The microglia-derived exosomes of the present invention can effectively reduce the changes in ferroptosis-related indicators of cerebral ischemia-reperfusion injury (CIRI), reduce the total iron and MDA content in the infarct area, and upregulate the GSH level. It can reduce the volume of cerebral infarction, increase the ischemia-reperfusion neurological score, and improve brain tissue pathological damage by alleviating neuroinflammation and inhibiting oxidative stress and ferroptosis, and has a significant neuroprotective effect on CIRI.

[0045] On the other hand, the present invention also provides the use of a composition comprising gardenia glycoside, ligustrazine and puerarin in preparing microglia-derived exosomes having the effect of inhibiting cerebral ischemia-reperfusion injury.

[0046] According to a specific embodiment of the present invention, the composition comprising gardenia glycoside, ligustrazine and puerarin consists of gardenia glycoside, ligustrazine and puerarin.

[0047] According to a specific embodiment of the present invention, in the composition comprising gardenia glycoside, ligustrazine and puerarin, the mass ratio of gardenia glycoside, ligustrazine and puerarin is (20-250): (15-250): (15-300). According to some specific embodiments of the present invention, in the composition comprising gardenia glycoside, ligustrazine and puerarin, the mass ratio of gardenia glycoside, ligustrazine and puerarin is (40-250):(25-200):(25-300), preferably (20-200):(40-250):(40-250), further preferably (20-150):(90-250):(90-250), further preferably (30-100):(150-250):(120-250), further preferably (30-60):(170-220):(150-220), further preferably (30-60):(180-210):(150-200), further preferably 40:199:161.

[0048] According to a specific embodiment of the present invention, when a composition comprising gardenia glycoside, ligustrazine and puerarin is used to prepare microglia-derived exosomes that have the effect of inhibiting cerebral ischemia-reperfusion injury, the application method includes: adding the composition comprising gardenia glycoside, ligustrazine and puerarin to a culture medium of microglia, so that the microglia secrete exosomes when cultured in the culture medium. The specific method (including conditions such as the amount of gardenia glycoside, ligustrazine and puerarin added to the culture medium of microglia) can refer to the preparation method of microglia-derived exosomes of the present invention described above.

[0049] In summary, the present invention provides a technology for preparing exosomes by pre-treating microglia with gardenia glycosides, ligustrazine, and puerarin. The prepared exosomes can reduce the volume of cerebral infarction, improve the ischemia-reperfusion neurological score, improve brain tissue pathological damage, and have a significant neuroprotective effect on CIRI by alleviating neuroinflammation, inhibiting oxidative stress and ferroptosis. In vivo and in vitro studies have shown that gardenia glycosides, ligustrazine, and puerarin significantly increase the secretion of resting (i.e., wild-type) microglial exosomes and the expression of antioxidant protein GPX4 by stimulating microglia, and are more easily taken up by neuronal cells, suggesting that they have stronger targeting. Its mechanism is closely related to inhibiting the expression level of HMGB1 to reduce neuroinflammation and increasing the content of GPX4 to inhibit neuronal ferroptosis. In addition, only trace ineffective doses of gardenia glycoside, ligustrazine and puerarin components were found inside the exosomes prepared by the present invention, indicating that the effect of the microglial exosomes of the present invention on improving CIRI does not come from the trace amount of drugs carried by the exosomes, but the drugs significantly improve CIRI by increasing the number of microglial exosomes, the level of antioxidant proteins carried, and enhancing targeting, thereby enhancing the therapeutic effect of microglia-derived exosomes on CIRI. This is a new natural nanoparticle drug treatment idea for CIRI. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] Figure 1 The NTA detection results of exosomes PBS-EXO (left picture) and ZGC-EXO (right picture) prepared in a specific embodiment of the present invention are shown.

[0051] Figure 2 The transmission electron microscopy results of the exosomes PBS-EXO (left picture) and ZGC-EXO (right picture) prepared in a specific embodiment of the present invention are shown.

[0052] Figure 3 The results of marker protein detection of exosomes PBS-EXO and ZGC-EXO prepared in a specific embodiment of the present invention are shown.

[0053] Figure 4 The liquid chromatography-mass spectrometry conditions for detecting compound components contained in exosomes in one specific embodiment of the present invention are shown.

[0054] Figure 5 This is an MRM graph of the content of each compound in the exosome reference substance PBS-EXO prepared in a specific embodiment of the present invention.

[0055] Figure 6 This is an MRM graph of the content of each compound in the exosomes ZGC-EXO prepared in a specific embodiment of the present invention.

[0056] Figure 7The results of glutathione peroxidase 4 (GPX4) expression detection in exosomes ZGC-EXO prepared in a specific embodiment of the present invention are shown.

[0057] Figure 8 This is a fluorescence image of ZGC-EXO uptake by rat brain tissue in a specific embodiment of the present invention.

[0058] Fig. 9 It shows the neurological function score of MCAO / R rats intervened by ZGC-EXO in one specific embodiment of the present invention.

[0059] Fig.10 The results of TTC staining of brain slices of MCAO / R model with ZGC-EXO intervention in a specific embodiment of the present invention are shown.

[0060] Fig.11 The infarct area / whole brain volume ratio of the MCAO / R model intervened by ZGC-EXO in a specific embodiment of the present invention is shown.

[0061] Fig.12 Shown is the HE staining result (150×) of brain tissue in the MCAO / R model with ZGC-EXO intervention in a specific embodiment of the present invention.

[0062] Fig.13 It shows the total iron content, MDA and GSH levels of ZGC-EXO intervention in MCAO / R model in a specific embodiment of the present invention.

[0063] Fig.14 It shows that ZGC-EXO intervenes in the IL-6 level of MCAO / R model in a specific embodiment of the present invention.

[0064] Fig.15 It shows the detection of GPX4 and HMGB1 protein expression levels in the MCAO / R model intervened by ZGC-EXO in a specific embodiment of the present invention.

[0065] Fig.16 This is a fluorescence image of the PC12-Erastin cell model taking up exosomes derived from BV2 cells in a specific embodiment of the present invention.

[0066] Fig.17 The OD value of the cell proliferation experiment of the PC12-Erastin cell ferroptosis model intervened by exosomes in a specific embodiment of the present invention is shown.

[0067] Fig.18 It shows the GSH and MDA levels of exosomes intervening in the PC12-Erastin cell ferroptosis model in a specific embodiment of the present invention.

[0068] Fig.19It shows the detection of GPX4 / HMGB1 protein expression level in PC12-Erastin cell model intervened by ZGC-EXO in a specific embodiment of the present invention. DETAILED DESCRIPTION

[0069] In order to have a clearer understanding of the technical features, purposes and beneficial effects of the present invention, the technical scheme of the present invention is now described in detail below, but it should not be understood as limiting the scope of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in the field without creative work are within the scope of protection of the present invention. The experimental methods without specifying specific conditions in the embodiments are operated according to the conventional conditions of the field. The starting reagents and materials used in the embodiments can be commercially available or prepared with reference to the prior art in the field.

[0070] Example 1. Exosomes from BV2 cells and their preparation and identification

[0071] 1. Preparation of exosomes from BV2 cells pretreated with ZGC

[0072] Gardenia glycoside, ligustrazine and puerarin powder were dissolved in high-glucose DMEM with a glucose concentration of 4.5 g / L at a mass ratio of 40:199:161 to prepare a solution with a total concentration of 400 μg / mL of gardenia glycoside, ligustrazine and puerarin (named as ZGC solution in the present invention), which was dissolved by ultrasound and filtered with a 0.22 μm filter membrane for later use. BV2 cells (purchased from Concord Cell Center) were cultured at 5×10 5 The cells were inoculated with 4% paraformaldehyde and 4% paraformaldehyde at a concentration of 100 μg / mL. After culturing with complete medium for 24 h, the supernatant was replaced with 20 mL high-glucose DMEM (PBS-EXO group) or 20 mL ZGC solution (ZGC-EXO group, drug concentration 400 μg / mL). After pretreatment of BV2-M0 cells for 24 h, the supernatant of each group was collected and gradient centrifuged in sequence to remove dead cells and cell debris: 300 × g for 10 min, 3000 × g for 20 min, and 10000 × g for 30 min. The supernatant was filtered through a 0.22 μm filter for later use.

[0073] The ultracentrifuge bottles were sterilized and rinsed, and the supernatant after gradient centrifugation and filtration was transferred to the ultracentrifuge bottles. The liquid volume in the bottles was filled up and balanced to ensure that the mass difference of the opposite centrifuge bottles did not exceed 0.1g. The balanced ultracentrifuge bottles were placed in the ultracentrifuge rotor and the centrifugation conditions were set on the machine: 4℃, 100000×g centrifugation for 70 min, the supernatant was carefully removed, and the bottom precipitate was resuspended with 10 mL pre-cooled PBS and transferred to a new sterile ultracentrifuge tube. Centrifugation was performed at 4℃, 100000×g for 70 min, and the supernatant was removed. The tiny precipitate at the bottom of the bottle was the exosome. Each group of exosomes was resuspended in 100 μL PBS to prepare EXO solution (PBS-EXO sample solution and ZGC-EXO sample solution).

[0074] 2. Exosome identification

[0075] a) NTA2.

[0076] The freshly prepared exosome PBS-EXO sample solution and ZGC-EXO sample solution were diluted 1000-3000 times with deionized water, and the ZetaView nanoparticle tracking analyzer was used to detect the exosome particle size and particle concentration of each group.

[0077] NTA test results can be found in Figure 1 The experimental results of this example show that the particle size of the PBS-EXO sample is mainly distributed at 123 nm, and the concentration is 9.2×10 9 / mL, the particle size of ZGC-EXO sample is mainly distributed at 140 nm, and the concentration is 3.1×10 11 / mL, which is in line with the exosome particle size range (30-150 nm); and under the same cell culture density, ZGC incubation can significantly increase the number of exosomes secreted by BV2, which can reach about 34 times the number of exosomes secreted by conventional microglia.

[0078] b) Transmission electron microscopy

[0079] Take 5 μL of exosome sample and drop it on the copper grid, incubate it at room temperature for 5 min, absorb the excess liquid on one side with absorbent paper, then add a drop of 2% uranyl acetate to the copper grid and incubate it at room temperature for 1 min; dry it at room temperature for 20 min before observing it on the machine.

[0080] Transmission electron microscopy results can be found in Figure 2 Under the electron microscope, a clear double-layer capsule structure can be seen, which is saucer-shaped, and the size of the vesicles is consistent with the size range of exosomes.

[0081] c) Marker protein detection

[0082] The ZGC-EXO exosome sample solution of the present invention was diluted to the same exosome quantity as that of the PBS-EXO exosome sample solution of the control group, and marker protein detection was performed.

[0083] 40 μL of exosome sample was added with 20 μL of RIPA-PMSF lysis buffer and lysed on ice. Then, the exosome marker proteins CD9, TSG101 and negative protein Calnexin were detected in the exosome lysate sample and BV2 lysate using a fully automatic protein quantitative analysis system.

[0084] The results of marker protein detection can be found in Figure 3 The results showed that compared with BV2 cells, CD9 and TSG101 of PBS-EXO and ZGC-EXO were strongly positive, and Calnexin was negative, which met the detection standards of exosome marker proteins.

[0085] d) Quantitative detection of exosomes from BV2 cells pretreated with ZGC by liquid chromatography-mass spectrometry

[0086] In this experiment, the ZGC-EXO sample solution with the same exosome concentration (i.e., the diluted ZGC-EXO sample solution mentioned above) and the control group PBS-EXO sample solution were used for detection.

[0087] A 20 μL exosome sample was frozen and thawed three times, and 20 μL methanol (containing 0.1% formic acid) was added, vortexed for 5 min, sonicated for 10 min, centrifuged at 12000 rpm at 4°C for 10 min, and the supernatant was injected for analysis.

[0088] Weigh 1 mg of each standard accurately, dilute to 1 mL with methanol, prepare 1.00 mg / mL reference stock solution, dilute with methanol to a series of standard solutions containing gardenia glycosides, ligustrazine, and puerarin, and inject for determination. Figure 4 The peak area (Y) and the standard concentration (X) were used for linear regression, and the measured value of the PBS-EXO blank control group was used as the baseline to obtain the content of each ZGC compound in ZGC-EXO.

[0089] The test results are shown in Table 1. Figure 5 and Figure 6 The results of the detection of compounds in exosomes showed that the concentrations of gardenia glycoside, ligustrazine and puerarin in ZGC-EXO were converted to 10 9 The contents in the exosome particles were 0.53 ng, 2.11 ng, and 2.56 ng, respectively. It can be seen that the concentrations of gardenia glycoside, ligustrazine, and puerarin in the exosomes of the present invention are much lower than the conventional effective dosages of gardenia glycoside, ligustrazine, and puerarin, and do not produce relevant pharmacodynamic effects.

[0090] Table 1. Contents of Gardenia glycoside, Ligustrazine and Puerarin in ZGC-EXO

[0091]

[0092] e) Detection of glutathione peroxidase 4 (GPX4) expression in exosomes derived from BV2 cells pretreated with ZGC

[0093] ProteinSimple Abby fully automated protein quantitative analysis system was used to detect the protein expression level of glutathione peroxidase 4 (GPX4) in exosomes ZGC-EXO and PBS-EXO. The operation was performed according to the instructions of the analysis system. The expression quantification of the target protein was the Area value under the target protein peak, expressed as Area.

[0094] Results Figure 7 As shown in the figure, compared with the other groups, **: P The results showed that the expression level of glutathione peroxidase 4 (GPX4) in the microglia-derived exosomes ZGC-EXO of the present invention was more than 3 times that of the wild-type microglia-derived exosomes GPX4.

[0095] Example 2: In vivo efficacy experiment of BV2 cell-derived exosomes in improving CIRI

[0096] In this example, the following experiments were performed using the ZGC-EXO sample solution with the same exosome concentration prepared in Example 1 (i.e., the diluted ZGC-EXO sample solution) and the control group PBS-EXO sample solution.

[0097] 1. In vivo tracing experiment of BV2 cell-derived exosomes

[0098] The PKH26 dye was mixed with the exosome sample solution prepared in Example 1 in the diluent for staining for 5 min (the specific method is: take 10 μL of PBS-EXO or ZGC-EXO sample solution and add 1 mL of diluent, 10 μL of PKH26 dye was added to 1 mL of diluent, and the diluted EXO solution and dye solution were completely mixed). 20% exosome-free serum (fetal bovine serum was centrifuged at 4°C, 120,000×g for 18 h to remove exosomes in the serum) was mixed with high-glucose DMEM, and the staining system was added to bind excess dye to terminate the staining, and incubated at room temperature for 1 min. After dilution with PBS, ultracentrifugation was performed at 4°C, 120,000×g for 70 min, the supernatant was discarded, and the exosome precipitate was resuspended with PBS to obtain PKH26 fluorescently labeled exosomes. 5 μL of fluorescently labeled exosomes were injected into rats, and brain frozen sections were taken 1 hour later to observe the fluorescence intensity. In the field of fluorescence microscopy, red is the fluorescence signal of exosomes labeled with PKH26 dye, and blue is the fluorescence signal of the nucleus of PC12 cells labeled with DAPI.

[0099] Results Figure 8 . Among them, a: PBS-EXO; b: ZGC-EXO; c: PBS control. Through fluorescence microscopy observation, it was found that the PKH26 dye-labeled exosomes fluorescence signal distribution in the cerebral cortex and the brain tissue around the lateral ventricle in the Merge image, indicating that the BV2 cell-derived exosomes prepared by the present invention can be taken up by rat brain tissue, and the PKH26 dye-labeled exosomes of ZGC-EXO have a stronger fluorescence signal, indicating that its uptake rate is higher than that of the control group PBS-EXO.

[0100] 2. MCAO / R modeling

[0101] According to the rat body weight, the rats were randomly divided into sham operation group (Sham), MCAO / R model group, PBS-EXO+MCAO / R group, and ZGC-EXO+MCAO / R group. Particles: 5×10 8 Pieces / mL.

[0102] Before MCAO / R, 5 μL of corresponding exosome solution was injected into the PBS-EXO+MCAO / R group and the ZGC-EXO+MCAO / R group, and 5 μL of normal saline was injected into the sham operation group and the model group. After the injection, MCAO / R model was established, and the drug was injected again after the suture was removed 4 hours later. Blood samples were collected 20 hours after reperfusion.

[0103] 3. Detection of drug efficacy indicators

[0104] (1) Neurological function score

[0105] The four-point Bederson neurological score was used. Normal: 0 points, no obvious symptoms; mild: 1 point, forelimb flexion; severe: 2 points, decreased resistance to lateral push, accompanied by forelimb flexion; 3 points, decreased resistance to lateral push, accompanied by forelimb flexion and spontaneous rotation. The higher the score, the more severe the animal's behavioral disorder.

[0106] The experimental results are shown in Table 2 and Fig. 9 . Fig. 9 In, n=8, compared between groups, *: P <0.05;**: P <0.01;***: P <0.001; ****: P <0.0001. The results showed that ZGC-EXO injection reduced the neurological function score 4h after cerebral infarction and 20h after reperfusion to a greater extent than PBS-EXO injection, and the results were significantly different ( P <0.05), indicating that ZGC-EXO can significantly enhance the mobility of rats and significantly improve cerebral ischemia-reperfusion injury.

[0107] Table 2. Neurological function scores of MCAO / R rats treated with ZGC-EXO

[0108]

[0109] Note: Compared with the model group: *: P <0.05;**: P <0.01;***: P <0.001; ****: P <0.0001;

[0110] ZGC-EXO group compared with PBS-EXO group: #: P <0.05;##: P <0.01.

[0111] (2) Infarct volume

[0112] TTC staining of brain slices to observe the volume of the infarct area: Prepare 2% TTC solution: weigh 2,3,5-triphenyl tetrazonium chloride powder and dissolve it in physiological saline to prepare 2% TTC solution, prepare it before use and store it in the dark. After anesthesia, the rats in each group were cut open on the chest skin and sternum to expose the chest cavity. A small incision was made in the right atrial appendage with a scissor, and the whole brain tissue was taken after the heart was perfused at a uniform speed with physiological saline from the left apex. After freezing and fixing, the brain tissue was cut into five uniform slices along the coronal plane with a tissue blade, immersed in 2% TTC solution, and stained at 37°C for half an hour to one hour. After staining, it was fixed with 4% cell tissue fixative and photographed. ImageJ software was used to analyze the infarct area / whole brain volume ratio.

[0113] The results of TTC staining and analysis are shown in Tables 3 and Fig.10 , Fig.11 . Fig.10 Middle, a: Sham; b: MCAO / R; c: PBS-EXO; d: ZGC-EXO. Fig.11 In, n=4, compared between groups, *: P <0.05;**: P <0.01;***: P <0.001; ****: P <0.0001. The results showed that injection of ZGC-EXO could significantly reduce the volume of cerebral infarction area ( P <0.0001), and the effect was significantly better than PBS-EXO ( P <0.05), indicating that ZGC-EXO showed a more obvious protective effect on cerebral ischemic tissue.

[0114] Table 3. Ratio of infarct area / whole brain volume in MCAO / R model treated with ZGC-EXO

[0115]

[0116] Note: Compared with the model group: *: P <0.05;**: P <0.01;***: P <0.001; ****: P <0.0001; ZGC-EXO group compared with PBS-EXO group: #: P <0.05;##: P <0.01.

[0117] (3) Morphological changes in brain tissue

[0118] Hematoxylin-eosin (HE) staining was used to observe the morphological changes of brain tissue: the rat brain tissue was removed, fixed with 4% cell tissue fixative, dehydrated, embedded in paraffin, sliced, baked at 65°C, and stained with HE. The digital slice panoramic scanning system was used to observe and analyze the staining results.

[0119] HE staining results are shown in Fig.12 . Among them, a: Sham; b: MCAO / R; c: PBS-EXO; d: ZGC-EXO. The results showed that compared with the model group, brain tissue edema was alleviated after PBS-EXO intervention, and round regular nuclei were scattered; the brain tissue of the ZGC-EXO group was denser than that of the model group and PBS-EXO group, the degree of edema was greatly improved, the neuronal damage was milder, and the number of round regular nuclei was significantly increased.

[0120] (4) Total iron content, GSH and MDA detection

[0121] Colorimetric method was used to detect the total iron content, GSH and MDA levels in the cerebral cortex of the infarcted area: a certain mass of frozen cerebral cortex samples from the infarcted area of ​​each group were placed in a grinding tube, and the corresponding volume of homogenate medium and zirconium oxide grinding beads were added. Dry ice was used to cool the sample, and a 10% tissue homogenate (tissue mass g: homogenate medium volume mL = 1:9) was prepared in a Cryolys® Evolution tissue grinder at a low temperature. After centrifugation, the supernatant was collected and the total iron content (model E-BC-K772-M) of Wuhan Elabscience was calculated.

[0122] ), MDA (model E-BC-K028-M-96), GSH (model E-BC-K030-M-96T) according to the kit instructions to detect total iron content, GSH and MDA levels.

[0123] The experimental results are shown in Fig.13 . Among them, n=6, compared between groups, *: P <0.05;**: P <0.01;***: P <0.001; ****: P <0.0001. The results showed that injection of ZGC-EXO could effectively reduce the total iron and MDA content in the cerebral infarction area of ​​MCAO / R rats, and upregulate the GSH level ( P <0.01), the improvement effect of ZGC-EXO group on the above indicators was significantly better than that of PBS-EXO ( P The results showed that ZGC-EXO had a stronger inhibitory effect on oxidative stress and ferroptosis in brain tissue induced by ischemia-reperfusion.

[0124] (5) IL-6 detection

[0125] The ELISA method was used to detect the IL-6 level in the homogenate of cortical tissue in the infarcted area of ​​rats. The IL-6 level in the homogenate of cortical tissue was detected according to the instructions of the ELISA kit of Wuhan Elabscience (specification model E-EL-M0044c-96).

[0126] The experimental results are shown in Fig.14 . Among them, n=6, compared between groups, *: P <0.05;**: P <0.01;***: P <0.001; ****: P <0.0001. The results showed that injection of ZGC-EXO could effectively reduce the level of IL-6 in the cortical tissue of cerebral infarction area of ​​MCAO / R rats ( P<0.01). Compared with the PBS-EXO group, ZGC-EXO had a better inhibitory effect on IL-6 secretion, and the difference was statistically significant ( P <0.05). This indicates that ZGC-EXO has a stronger inhibitory effect on brain neuroinflammation caused by ischemia-reperfusion.

[0127] 4. Detection of key pathway proteins of neuroinflammation and ferroptosis

[0128] ProteinSimple Abby fully automated protein quantitative analysis system was used to detect the protein expression levels of HMGB1 and GPX4, key proteins regulating neuroinflammation and ferroptosis pathways, in the cortical tissue of rat cerebral infarction area. The primary antibodies HMGB1 (model ab79823) and GPX4 (model ab125066) from ABCAM were used. ProteinSimple's WB anti-rabbit secondary antibody detection kit (model DM001), separation module kit (model SM-W002), universal standard package kit (model PS-ST01EZ-8), TP total protein labeling kit (model 042-973) and TP detection kit (model DM-TP01-1) were used.

[0129] (1) Sample processing

[0130] Take appropriate tissue samples, mix with the corresponding volume of RIPA lysis buffer (containing 1mM PMSF), add zirconium oxide grinding beads, cool with dry ice, and fully lyse in a low-temperature environment of a Cryolys® Evolution tissue grinder. After centrifugation, take the supernatant to test the protein concentration, aliquot the supernatant and freeze it at -80℃ for testing.

[0131] (2) Sample protein quantitative detection

[0132] Take tissue lysate, dilute and balance to the same loading concentration between groups, mix with fluorescent master solution and heat at 95℃ for 5min. Add samples, chemiluminescent reagent, primary antibody (HMGB1, 1:100 dilution; GPX4, 1:50 dilution), secondary antibody and washing buffer into the microplate in the kit, and perform capillary electrophoresis analysis with ProteinSimple Abby instrument, setting the corresponding machine program: 12-230kda. Use built-in Compass software to acquire and analyze data. Molecular weight is approximated by peak integration using Compass software, and the Area value under each peak generated by the software is the absolute quantitative value of protein blot.

[0133] (3) Sample protein normalization

[0134] The total protein amount of the sample was determined using the ProteinSimple TP (Total Protein) module kit. The sample lysate was diluted and balanced to the same loading concentration between groups, mixed with the fluorescent master solution and heated at 95°C for 5 min. The sample, chemiluminescent reagent, TP solution, and washing buffer were placed in a microplate, and capillary electrophoresis analysis was performed using the ProteinSimple Abby instrument, and the corresponding machine program was set: 12-230kda. The sum of the Area values ​​under all peaks in each lane was analyzed using Compass software, which is the total protein amount (TP) loaded on the sample. Total protein quantification can replace the role of traditional internal reference protein and make more accurate corrections to the quantification of target protein. The expression quantification of the target protein is the Area value under the target protein peak / total protein TP value, expressed as Corr.Area.

[0135] The results of fully automated protein quantification analysis are shown in Fig.15 . Among them, n=3, compared between groups, *: P <0.05;**: P <0.01;***: P <0.001; ****: P <0.0001. The results showed that ZGC-EXO could significantly downregulate the expression level of HMGB1 ( P <0.01), increased the antioxidant GPX4 content ( P <0.05); the effect was better than that of PBS-EXO group, with significant difference ( P <0.05).

[0136] Example 3: In vitro experiment on improving neuronal ferroptosis by exosomes derived from BV2 cells

[0137] In this example, the following experiments were performed using the ZGC-EXO sample solution with the same exosome concentration prepared in Example 1 (i.e., the diluted ZGC-EXO sample solution) and the control group PBS-EXO sample solution.

[0138] 1. PC12 cell erastin model for BV2 cell-derived exosome uptake experiment

[0139] Neuronal PC12 cells (purchased from Concordia Cell Center) were divided into normal control group (CON), Erastin ferroptosis model group (10 μmol / L), PBS-EXO+ Erastin control group and ZGC-EXO+ Erastin group. 5The cells were inoculated into a 96-well black light-proof cell culture plate at a concentration of 100 μL per well, and 100 μL of high-glucose DMEM was added to the normal control group and cultured at 37°C for 24 h. The original culture medium was discarded, and 100 μL of high-glucose DMEM was added to the normal control group and the model group, respectively. 100 μL of the fluorescently labeled exosome-DMEM mixture was added to the PBS-EXO+Erastin group and the ZGC-EXO+Erastin group. The final exosome particle concentration in the system was 5×10 8 / mL, incubated at 37℃ for 4h, 5.5 μL 0.192 mmol / L Erastin diluted solution (final system contains 0.3% DMSO) was added to the model group, PBS-EXO+ Erastin group and ZGC-EXO+ Erastin group, respectively, and the normal control group was added with the same volume of high-glucose DMEM containing 0.3% DMSO. After 20 hours, each well of the 96-well plate was rinsed twice with PBS, fixed with 4% cell tissue fixative for 15 min, and then rinsed twice with PBS solution. 100 μL DAPI (nuclear dye, blue fluorescence) was added to each well to cover the sample, and stained at room temperature for 8 minutes. After the dye incubation was completed, 1mL PBS was added to shake and rinse twice, and an appropriate amount of basal culture medium was added, and the photos were observed under a fluorescence microscope. For the pictures of each group of cells taken under different filters in the same field of view, ImageJ software was used to integrate and obtain Merge pictures. Fluorescence microscopy observation found that red was the fluorescence signal of exosomes labeled with PKH26 dye, and blue was the fluorescence signal of the cell nucleus of PC12 cells labeled with DAPI.

[0140] Results Fig.16 . Through fluorescence microscopy, it was found that the fluorescence signal of exosomes labeled with PKH26 dye in the Merge image was distributed around the nucleus of PC12 cells. This shows that exosomes derived from BV2 cells can be taken up by the Erastin-induced PC12 ferroptosis cell model, and the fluorescence signal of exosomes labeled with PKH26 dye in the ZGC-EXO group is stronger, indicating that its uptake rate is higher than that of the control group PBS-EXO.

[0141] 2. CCK-8 assay to detect the intervention effect of ZGC-EXO on the Erastin ferroptosis model of PC12 cells

[0142] PC12 cells were divided into normal control group (C), Erastin ferroptosis model group (10 μmol / L), PBS-EXO-L+Erastin low-dose group, PBS-EXO-M+Erastin medium-dose group, PBS-EXO-H+Erastin high-dose group, ZGC-EXO-L+Erastin low-dose group, ZGC-EXO-M+Erastin medium-dose group, and ZGC-EXO-H+Erastin high-dose group. The cells were divided into normal control group (C), Erastin ferroptosis model group (10 μmol / L), PBS-EXO-L+Erastin low-dose group, PBS-EXO-M+Erastin medium-dose group, and ZGC-EXO-H+Erastin high-dose group. The cells were divided into 2×10 5 Cells were inoculated into 96-well cell culture plates at a concentration of 100 μL per well, and 100 μL of high-glucose DMEM was added to the normal control group and cultured at 37°C for 24 h. The original culture medium was discarded, and 100 μL of high-glucose DMEM was added to the normal control group and the model group, respectively. 100 μL of the exosome-DMEM mixture was added to each exosome dose group. In the system, the final exosome particle size of the low-dose group was 2.5×10 8 / mL, and the final exosome particles in the medium-dose group were 5×10 8 / mL, and the final exosome particles in the high-dose group were 1×10 9 After incubation at 37°C for 4 h, 5.5 μL of 0.192 mmol / L Erastin diluted solution (the final system contained 0.3% DMSO) was added to the model group and each dose group of exosomes, and the same volume of high-glucose DMEM containing 0.3% DMSO was added to the normal control group. After 20 hours, the supernatant was discarded and 100 μL of high-glucose DMEM containing 10% CCK-8 was added. After incubation at 37°C for 0.5 h, the absorbance (OD) value was detected and the cell proliferation inhibition rate was calculated.

[0143] The results of the CCK-8 assay are shown in Tables 4 and Fig.17 . Among them, n=8, compared between groups, *: P <0.05;**: P <0.01;***: P <0.001; ****: P <0.0001. The results showed that the low, medium and high dose groups of ZGC-EXO could significantly reduce the mortality rate of PC12-Erastin model ( P All were <0.01), but PBS-EXO only showed an improvement effect on the mortality of the PC12-Erastin model at a high dose; the effects of each dose group of ZGC-EXO were better than those of the PBS-EXO group at the same dose, and the difference was extremely significant ( P (all <0.001).

[0144] Table 4. Cell proliferation experimental data of exosome intervention in PC12-Erastin cell ferroptosis model

[0145]

[0146] Note: Compared between groups, *: P <0.05;**: P <0.01;***: P <0.001; ****: P <0.0001.

[0147] 3. Detection of GSH and MDA content in Erastin ferroptosis model of PC12 cells

[0148] Referring to the previous step, the reaction was carried out in a 6-well plate, and PC12 cells were divided into a normal control group (C), an Erastin ferroptosis model group (10 μmol / L), and a PBS-EXO+Erastin group (particles: 5×10 8 / mL), ZGC-EXO + Erastin group (particles: 5×10 8 / mL), 4h exosome pretreatment, and cells were collected 20h after modeling for later use.

[0149] According to the instructions of the MDA (model E-BC-K028-M-96) and GSH (model E-BC-K030-M-96T) kits produced by Wuhan Elabscience, the contents of MDA and GSH in each group of PC12 cell samples were detected.

[0150] Test results see Fig.18 . Among them, n=6, compared between groups, *: P <0.05;**: P <0.01;***: P <0.001; ****: P <0.0001. The results showed that compared with the normal group, the GSH content of the PC12-Erastin cell ferroptosis model was significantly reduced, and the MDA content was significantly increased ( P ZGC-EXO can significantly increase the content of GSH and reduce the content of MDA ( P All < 0.01); ZGC-EXO has stronger efficacy than PBS-EXO group, and the difference between the two groups is statistically significant ( P <0.01).

[0151] 4. Detection of key pathway proteins

[0152] ProteinSimple Abby fully automated protein quantitative analysis system was used to detect the expression levels of HMGB1 / GPX4 pathway proteins in PC12 cells. Primary antibodies HMGB1 (model ab79823) and GPX4 (model ab125066) from ABCAM were used. ProteinSimple's WB anti-rabbit secondary antibody detection kit (model DM001), separation module kit (model SM-W002), universal standard package kit (model PS-ST01EZ-8), TP total protein labeling kit (model 042-973) and TP detection kit (model DM-TP01-1) were used.

[0153] Cell sample processing, protein quantification and protein normalization methods were the same as before.

[0154] The results of fully automated protein quantification analysis are shown in Fig.19 . Among them, n=3, compared between groups, *: P <0.05;**: P <0.01;***: P <0.001; ****: P <0.0001. The results showed that ZGC-EXO intervention could effectively downregulate the expression level of HMGB1 in the Erastin-PC12 cell ferroptosis model ( P <0.05), increased GPX4 expression level ( P <0.05). However, the effect of PBS-EXO on the expression level of GPX4 did not show any difference from that of the model group.

[0155] Although the present invention has been described in detail above with general descriptions and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements may be made to the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection claimed by the present invention.

Claims

1. A method for preparing microglia-derived exosomes, characterized in that: The preparation method comprises: Microglia are cultured in a culture medium containing geniposide, ligustrazine and puerarin to secrete exosomes, thereby obtaining microglia-derived exosomes; In the culture medium, the total amount of gardenia glycoside, ligustrazine and puerarin is 50 μg / mL-800 μg / mL, and the mass ratio of gardenia glycoside, ligustrazine to puerarin is (20-250): (15-250): (15-300).

2. The preparation method according to claim 1, characterized in that The addition amounts of gardenia glycoside, ligustrazine and puerarin in the culture medium of microglia were: gardenia glycoside 40 μg / mL-250 μg / mL, ligustrazine: 25 μg / mL-200 μg / mL, puerarin: 25 μg / mL-300 μg / mL.

3. The preparation method according to claim 1, characterized in that: The culture medium of the microglia includes one or more of a complete culture medium and a high-glucose DMEM culture medium; The microglial cells include one or more of BV2 cells, HMO6 cells, HMC3 cells, RHMC cells, HAPI cells and RMC cells.

4. The preparation method according to claim 1, characterized in that: The culture conditions for microglia are: 36.5℃-37.5℃ for 12h-72h.

5. The preparation method according to any one of claims 1 to 4, characterized in that: The preparation method comprises: After microglia were cultured in complete medium for 12 h-48 h, the supernatant was replaced with medium containing gardenia glycoside, ligustrazine, and puerarin, and culture was continued for 12 h-72 h; The supernatant of the above culture system was centrifuged to remove dead cells and cell debris, and the supernatant was removed by ultra-high speed centrifugation. The bottom precipitate was resuspended with PBS and then ultra-high speed centrifuged again to remove the supernatant to obtain exosomes.

6. A microglia-derived exosome, characterized in that: The exosomes contain gardenia glycoside, ligustrazine and puerarin, and are obtained according to the preparation method according to any one of claims 1 to 5.

7. Use of the microglia-derived exosomes according to claim 6 in the preparation of a medicament for improving and / or treating cerebral ischemia-reperfusion injury.

Images