Application of astragalus membranaceus exosome in cell protection in vascular endothelial cell apoptosis process

Abstract

The invention relates to the technical field of exosomes, in particular to application of astragalus membranaceus exosomes to cell protection in the vascular endothelial cell apoptosis process. According to the application, the astragalus membranaceus exosome and the abelmoschus esculentus exosome are extracted respectively, and the influence of the two exosomes on the vascular endothelial cell apoptosis process is compared, so that the result shows that the astragalus membranaceus exosome can protect cells in the vascular endothelial cell apoptosis process.

Description

Technical Field

[0001] The present application relates to the technical field of exosomes, and in particular to the application of astragalus exosomes in protecting cells during apoptosis of vascular endothelial cells. Background Art

[0002] Exosomes are multivesicular bodies produced in cells. They are membrane vesicles with a diameter of about 30-150nm and a density of 1.13-1.19g / ml. They have a typical "cup-and-disc" shape. Almost all types of cells in the human body can produce exosomes, with an average of 1,000-10,000 per human cell. Usually, there are 1×10 12 Exosomes are heterogeneous, and even exosomes secreted by the same cell type may have very different functions.

[0003] The main methods for isolating exosomes include differential centrifugation, density gradient centrifugation, size exclusion chromatography, filtration, polymer precipitation, immunoseparation, isolation and screening, etc. However, the mainstream separation method in the literature on exosomes in CNS is still ultracentrifugation. Ultracentrifugation can accurately and repeatedly obtain exosomes while minimizing the co-purification of protein aggregates and other membrane particles.

[0004] Exosomes exist in almost all tissues, intercellular spaces, and body fluids, including blood, saliva, urine, and breast milk. Exosomes carry proteins, miRNAs, lncRNAs, circRNAs, mRNAs, and their degradation fragments involved in intracellular signal transduction, and participate in the important regulation of cell activities; they have made their mark in the treatment of difficult and complicated diseases such as tumor metastasis, immune regulation mechanisms, disease occurrence and development, Alzheimer's disease, and immune diseases, and are expected to become early diagnostic markers for a variety of diseases. Summary of the invention

[0005] The present application provides an application of astragalus exosomes in protecting cells during vascular endothelial cell apoptosis.

[0006] Optionally, the vascular endothelial cells are HUVECs (human umbilical vein endothelial cells).

[0007] Optionally, the source of the astragalus exosomes is astragalus hydrolysate.

[0008] Optionally, the average particle size of the astragalus exosomes is 87.5 nm.

[0009] Optionally, the protein concentration of the astragalus exosomes is 1.94 μg / μL.

[0010] In summary, the present application includes at least one of the following beneficial technical effects:

[0011] In the present application, there is no obvious difference in appearance between the prepared Astragalus exosomes and the Astragalus exosomes; at the same time, it can be seen from the transmission electron microscopy images that the separation effects of the above two types of exosomes are good, both are typical exosomes, and the size is within the range of exosomes.

[0012] After experimental analysis, astragalus exosomes can repair the damage caused to vascular endothelial cells by H / R modeling, while sunflower exosomes cannot repair the damage caused to vascular endothelial cells by H / R modeling. Therefore, the astragalus exosomes provided in this application can protect cells during the apoptosis of vascular endothelial cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 This is a transmission electron micrograph of Astragalus exosomes.

[0014] Figure 2 This is a transmission electron micrograph of the exosomes of Hibiscus scabra.

[0015] Figure 3 Schematic diagram of the particle size and concentration of Astragalus exosomes.

[0016] Figure 4 Schematic diagram of the particle size and concentration of Hibiscus serrata exosomes.

[0017] Figure 5 This is a standard curve created using standard samples.

[0018] Figure 6 This is the state of laboratory HUVEC cells in the basic cell culture stage (normal culture).

[0019] Figure 7 This is the growth state of HUVEC cells after H / R modeling in the laboratory.

[0020] Figure 8 The results of CCK8 detection in HUVEC cells after treatment with Astragalus exosomes.

[0021] Fig. 9 The results of CCK8 detection in HUVEC cells after treatment with Amaranthus chinensis exosomes.

[0022] Fig.10 These are the results of apoptosis flow cytometry detection of HUVEC cells treated with Astragalus exosomes and HUVEC cells treated with Sunflower Acumin exosomes.

[0023] Fig.11 Statistical analysis results of HUVEC cell apoptosis after treatment with Astragalus exosomes.

[0024] Fig.12 Statistical analysis results of HUVEC cell apoptosis after treatment with Asparagus repens exosomes.

[0025] Fig.13 This is the status of laboratory HK-2 cells in the basic cell culture stage (normal culture).

[0026] Fig.14 This is the growth state of HK-2 cells after H / R modeling in the laboratory.

[0027] Fig.15 The results of CCK8 detection in HK-2 cells after treatment with Astragalus exosomes.

[0028] Fig.16 The results of CCK8 detection in HK-2 cells after treatment with Acuminata exosomes.

[0029] Fig.17 These are the results of apoptosis flow cytometry detection of HK-2 cells treated with Astragalus exosomes and Sunflower Acumin exosomes.

[0030] Fig.18 Statistical analysis results of apoptosis of HK-2 cells after treatment with Astragalus exosomes.

[0031] Fig.19 Statistical analysis results of apoptosis of HK-2 cells after treatment with Hibiscus scutellariae exosomes. DETAILED DESCRIPTION

[0032] Before describing the embodiments of the present application in detail, it should be understood that the terms used herein are only used for the purpose of describing specific embodiments. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by ordinary technicians in the field to which the terms belong.

[0033] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features. Therefore, the features defined as "first" and "second" may explicitly or implicitly include one or more of the features. Further, in the description of this application, unless otherwise specified, the meaning of "plurality" is two or more.

[0034] The endpoints and any values ​​of the ranges disclosed in this application are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of each range, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed in this article.

[0035] In the present application, the terms "comprise" or "include" are open expressions, that is, including the contents specified in the present application but not excluding other contents.

[0036] In order to make the purpose, technical scheme and advantages of the present application clearer, the technical scheme in the embodiments of the present application will be clearly and completely described below in conjunction with the accompanying drawings. Based on the embodiments of the present application, all other embodiments obtained by those skilled in the art without making creative work belong to the scope of protection of the present application. The embodiments described below are exemplary and are only used to explain the present application, and cannot be understood as limiting the present application.

[0037] If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the field or the product instructions are used. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased commercially.

[0038] In the following examples, some of the reagents used are as follows: PBS (E607008) was purchased from Sangon Biotech; 0.45 μm filter membrane (FPV403030) was purchased from Jie Te; 0.22 μm filter membrane (16541) was purchased from Sartorius; uranyl acetate (GZ02625-5) was purchased from Henan Xinrui; BCA protein concentration determination kit (P0011) was purchased from Biyuntian; pectinase (S10007) was purchased from Yuanye; cellulase (S10041) was purchased from Yuanye; D-mannitol (A600335) was purchased from Shanghai Shenggong.

[0039] In the following examples, some of the instruments used are as follows: a pipette (Research Plus) was purchased from Eppendorf; a small refrigerated centrifuge (Microfuge 20R) was purchased from Beckman; a refrigerated centrifuge (Neofuge1600R) was purchased from HealForce; an ultra-low temperature refrigerator (905) was purchased from Thermo; an ultracentrifuge (CP100MX) was purchased from Hitachi; a transmission electron microscope (HT-7700) was purchased from Hitachi; a particle size analyzer (N30E) was purchased from NanoFCM; and a multifunctional microplate reader (Varioskan LUX) was purchased from Thermo.

[0040] For cell detection, some of the reagents used are as follows: cell culture plates (430167) were purchased from Corning 430167; multi-well cell culture plates (150628) were purchased from Thermo; cell counting chambers (12-0005) were purchased from Shanghai Ruiyu; penicillin and streptomycin mixture (MP 1670249-M) was purchased from Gibco; DMEM (SH30243.1) was purchased from Hyclone; FBS (10099-141) was purchased from Gibco; 0.25% Trypsin (25200-072) was purchased from Gibco; CCK8 (Bs350A-100T) was purchased from Biosharp; apoptosis kit PI / FITC double staining method (E606336-0100) was purchased from Biotech; DEPC water was purchased from Tiangen; EP tubes were purchased from Axygen.

[0041] For cell detection, some of the instruments used are as follows: multifunctional microplate reader (VARIOSKAN LUX) purchased from Thermo; biological safety cabinet (AC2-4S1) purchased from ESCO; cell culture incubator (CCL-170B-8) purchased from ESCO; automatic cell counter (IC1000) purchased from Shanghai Ruiyu; pipette purchased from Rainin; liquid nitrogen tank (Cryosystem 2000) purchased from MVE; electric suction assist device (PX-100) purchased from RININ; centrifuge (Microfuge) purchased from Beckman; flow cytometer (GuavaeasyCyte) purchased from Merck; ultra-low temperature refrigerator (905) purchased from Thermo.

[0042] The present application is further described in detail below in conjunction with the embodiments, drawings and test results.

[0043] Example

[0044] This embodiment provides an astragalus exosome. The sample is astragalus, and the sample for extracting exosomes is astragalus enzymatic hydrolyzate.

[0045] The specific steps include:

[0046] (I) Sample pretreatment

[0047] (1) Cut the whole plant sample with a scalpel, wash it with distilled water, and absorb the surface moisture with filter paper.

[0048] (2) All sampling instruments were sterilized by high temperature and high pressure, and then the samples were cut into pieces (1 mm × 1 mm) and soaked in 75% alcohol for 1 min. The remaining alcohol was then washed with distilled water, and the samples were dried with sterile paper. 1500 mL of enzyme solution (4% cellulase, 2% pectinase, 0.6 mol / L D-mannitol, pH 5.8) was added and placed at 50°C for enzymatic hydrolysis for 6 h.

[0049] (3) After enzymatic hydrolysis, centrifuge at 10,000 rpm for 1 h to allow the enzymatic hydrolyzed tissue residue to settle, collect the supernatant, and store it at -80°C for later use.

[0050] (II) Extraction of exosomes

[0051] (1) Dissolve the Astragalus enzymatic hydrolysate at 37°C.

[0052] (2) Transfer the sample to a new centrifuge tube and centrifuge at 2000 × g and 4°C for 30 min.

[0053] (3) Transfer the supernatant to a new centrifuge tube and centrifuge again at 10,000 × g and 4°C for 45 min to remove larger vesicles.

[0054] (4) Take the supernatant, filter it through a 0.45 μm filter membrane, and collect the filtrate.

[0055] (5) Transfer the filtrate to a new centrifuge tube and centrifuge at 100,000 × g and 4°C for 70 min in an ultraspeed rotor.

[0056] (6) Remove the supernatant, resuspend with 10 mL of pre-cooled 1× PBS, select an ultraspeed rotor, filter through a 0.22 μm filter membrane, and centrifuge again at 100,000 × g and 4°C for 70 min.

[0057] (7) Remove the supernatant and resuspend with 1000 μL pre-cooled 1× PBS to obtain Astragalus exosomes, which are then stored at -80°C for later use.

[0058] Comparative Example

[0059] This comparative example provides an exosome of Asparagus corydalis. The difference between this comparative example and the embodiment is that the sample is Asparagus corydalis, and the sample for extracting exosomes is Asparagus corydalis hydrolysate. Asparagus corydalis hydrolysate is obtained by sample pretreatment, and exosomes in the Asparagus corydalis hydrolysate are extracted to obtain Asparagus corydalis exosomes. The rest are consistent with the steps of the embodiment.

[0060] Test 1

[0061] The following tests were performed on the Astragalus exosomes of the example and the Astragalus exosomes of the comparative example, respectively.

[0062] (1) Transmission electron microscopy observation

[0063] The method is as follows: take 10 μL of Astragalus exosomes or Hibiscus sibiricum exosomes respectively, add them on the copper grid to precipitate for 1 minute, and absorb the floating liquid with filter paper; continue to take 10 μL of uranyl acetate, add them on the copper grid to precipitate for 1 minute, and absorb the floating liquid with filter paper; dry at room temperature for several minutes, perform electron microscopy detection and imaging at 80 kV, and obtain transmission electron microscopy imaging results.

[0064] Test results such as Figure 1-Figure 2 As shown. Among them, Figure 1 This is a transmission electron microscopy image of Astragalus exosomes. Figure 2 This is a transmission electron micrograph of the exosomes of Hibiscus sibiricus.

[0065] Depend on Figure 1-Figure 2 It can be seen that there is no obvious difference in appearance between Astragalus exosomes and Sunflower sunflower exosomes; at the same time, it can be seen from the transmission electron microscopy images that the separation effects of the above two types of exosomes are good, both are typical exosomes, and the size is within the range of exosomes.

[0066] (2) Particle size analysis

[0067] The method is as follows: take 10 μL of Astragalus exosomes or Hibiscus sibiricum exosomes respectively, dilute to an appropriate multiple, and first use a standard to test the instrument performance before loading the exosome sample. Note that gradient dilution is required to avoid clogging the injection needle with the sample. After completing the sample test, the particle size and concentration information of the exosomes can be obtained.

[0068] The analysis results are shown in Table 1 and Figure 3-4 As shown. Among them, Figure 3 Schematic diagram of the particle size and concentration of Astragalus exosomes. Figure 4 This is a schematic diagram of the particle size and concentration of exosomes from Hibiscus serrata.

[0069] Table 1 Particle size analysis results of Astragalus exosomes and Sunflower sunflower exosomes

[0070] Serial number sample Average particle size (nm) Concentration (Particles / mL) Example Astragalus exosomes 87.5 9.9E+9 Comparative Example Exosomes from Astragalus membranaceus 87 2.18E+10

[0071] From Table 1 and Figure 3-Figure 4 It can be seen that there is no significant difference in particle size between Astragalus exosomes and Hibiscus sibiricum exosomes.

[0072] (3) Protein extraction and concentration determination

[0073] The method is as follows: quickly thaw Astragalus exosomes or Hibiscus sibiricum exosomes at 37°C, and quickly add 5× RIPA lysis buffer, mix well, and lyse on ice for 30 minutes, mixing well during the period. Prepare the standard sample for BCA method protein concentration, and take 20μL of the diluted sample and add it to the BCA mixture, mix well. Incubate at 37°C for 30 minutes, and read the OD value on the microplate reader. 562nm Detect the absorbance value and record it. Calculate the protein concentration of the sample to be tested according to the standard curve.

[0074] The standard curve is as follows Figure 5 As shown. 562nm The standard curves were used for calculation and the results are shown in Table 2.

[0075] Table 2 Analysis results of protein concentration of Astragalus exosomes and Astragalus membranaceus exosomes

[0076] Serial number sample <![CDATA[Optical Density (OD 562nm )]]> Protein concentration (diluted 4 times and then added to lysis buffer, μg / μL) Protein concentration (original concentration before dilution, μg / μL) Example Astragalus exosomes 0.237 0.193507 1.935073 Comparative Example Exosomes from Astragalus membranaceus 0.225 0.159153 1.591525

[0077] As shown in Table 2, the protein concentration of Astragalus exosomes is greater than that of Astragalus exosomes, and the protein concentration of Astragalus exosomes is 21.6% higher than that of Astragalus exosomes.

[0078] Test result 2

[0079] The exosomes of Astragalus membranaceus in the example and the exosomes of Astragalus membranaceus in the comparative example were used to perform cell function detection. The cells used were HUVEC cells (human umbilical vein endothelial cells) for laboratory use. The details are as follows:

[0080] (1) Basic cell culture

[0081] The cells were cultured in complete culture medium containing 1% double antibody and 10% fetal bovine serum at 37°C and 5% CO2 to maintain saturated humidity. When the cell density reached about 80-90%, routine subculture was performed.

[0082] (2) Cell modeling

[0083] HUVEC cells were treated with Astragalus exosomes or Hibiscus sibiricum exosomes at different concentrations (0, 5, 10, 20, 30, 40, 50 μg / ml), and then H / R model was established without changing the medium, with hypoxia for 8 h and reoxygenation for 16 h.

[0084] (3) CCK8 assay to detect cell proliferation

[0085] The cells were plated in a 96-well plate. After 24 hours, the old culture medium was discarded and CCK8 reagent was added for cell viability detection. The OD450 value was read within 2 hours.

[0086] (4) Flow cytometry double staining to detect cell apoptosis

[0087] Before use, all reagents in the kit should be centrifuged immediately. Use sterile water to prepare 1×Binding buffer from the 4×Binding buffer in the kit.

[0088] The cells were digested with trypsin and centrifuged at 1200 rpm / min for 5 min. The cells were collected and washed twice with 1× PBS. Each tube was suspended with 195 μL 1× Binding buffer (cell density was 2×10 5-5×10 5 cell / mL); add 5 μL Annexin V-FITC to each tube and incubate at 4°C in the dark for 10-15 min; add 200 μL 1×Binding buffer to wash the cells, centrifuge at 1200 rpm / min for 5 min, and remove the supernatant; use 190 μL 1×Binding buffer to suspend the cells in each tube; add 10 μL PI to each tube and mix well, then detect as soon as possible.

[0089] The test results are as follows:

[0090] The state of HUVEC cells used in the laboratory in the basic cell culture stage (normal culture) is as follows Figure 6 As shown. Figure 6 It can be seen that the HUVEC cells used in the laboratory are in good condition under normal culture conditions.

[0091] The HUVEC cells used in the laboratory were treated with H / R model, and the cell status was as follows Figure 7 shown.

[0092] Depend on Figure 7 It can be seen that the state of HUVEC cells was damaged after H / R modeling. Since there was no obvious difference in the cell state after H / R modeling of cells treated with Astragalus exosomes or Amur exosomes, only the cell growth rate was different, so only Figure 7 To reflect the growth state of HUVEC cells after H / R modeling.

[0093] After H / R modeling, CCK8 was detected in HUVEC cells used in the laboratory. The results are as follows Figure 8-Figure 9 As shown. Among them, Figure 8 For CCK8 detection of HUVEC cells treated with Astragalus exosomes, Fig. 9 This is the CCK8 detection test of HUVEC cells after treatment with Amaranthus chinensis exosomes.

[0094] By comparison Figure 8 and Fig. 9 The results showed that Astragalus exosomes can repair the damage caused by H / R modeling to HUVEC cells, while Sunflower Acumin exosomes cannot repair the damage caused by H / R modeling to HUVEC cells.

[0095] After H / R modeling, HUVEC cells were subjected to apoptosis flow cytometry. Fig.10 Statistical analysis of cell apoptosis was as shown in Figure 11-Figure 12 shown.

[0096] Depend on Figure 10-12It can be seen that it is further verified that Astragalus exosomes can repair the damage caused by H / R modeling to HUVEC cells, while Sunflower Acumin exosomes cannot repair the damage caused by H / R modeling to HUVEC cells.

[0097] Test result three

[0098] Cell function detection was performed using the Astragalus exosomes of the example and the Astragalus exosomes of the comparative example. The cells used were HK-2 cells (human renal tubular endothelial cells) for laboratory use. The details are as follows:

[0099] (1) Basic cell culture

[0100] The cells were cultured in complete culture medium containing 1% double antibody and 10% fetal bovine serum at 37°C and 5% CO2 to maintain saturated humidity. When the cell density reached about 80-90%, routine subculture was performed.

[0101] (2) Cell modeling

[0102] HK-2 cells were treated with Astragalus exosomes or Amur sunflower exosomes at different concentrations (0, 5, 10, 20, 30, 40, 50 μg / ml), and then H / R model was established without changing the medium, with hypoxia for 8 h and reoxygenation for 16 h.

[0103] (3) CCK8 assay to detect cell proliferation

[0104] The cells were plated in a 96-well plate. After 24 hours, the old culture medium was discarded and CCK8 reagent was added for cell viability detection. The OD450 value was read within 2 hours.

[0105] (4) Flow cytometry double staining to detect cell apoptosis

[0106] Before use, all reagents in the kit should be centrifuged immediately. Use sterile water to prepare 1×Binding buffer from the 4×Binding buffer in the kit.

[0107] The cells were digested with trypsin and centrifuged at 1200 rpm / min for 5 min. The cells were collected and washed twice with 1× PBS. Each tube was suspended with 195 μL 1× Binding buffer (cell density was 2×10 5 -5×10 5 cell / mL); add 5 μL Annexin V-FITC to each tube and incubate at 4°C in the dark for 10-15 min; add 200 μL 1×Binding buffer to wash the cells, centrifuge at 1200 rpm / min for 5 min, and remove the supernatant; use 190 μL 1×Binding buffer to suspend the cells in each tube; add 10 μL PI to each tube and mix well, then detect as soon as possible.

[0108] The test results are as follows:

[0109] The status of HK-2 cells used in the laboratory during the basic cell culture stage (normal culture) is as follows Fig.13 As shown. Fig.13 It can be seen that the HK-2 cells used in the laboratory are in good condition under normal culture conditions.

[0110] HK-2 cells were used in the laboratory for H / R modeling. The cell status was as follows Fig.14 shown.

[0111] Depend on Fig.14 It can be seen that the state of HK-2 cells was damaged after H / R modeling. Since there was no obvious difference in the cell state after H / R modeling of cells treated with Astragalus exosomes or Amur exosomes, only the cell growth rate was different, so only Fig.14 To reflect the growth status of HK-2 cells after H / R modeling.

[0112] After H / R modeling, HK-2 cells were tested for CCK8. The results are as follows Figure 15-16 As shown. Among them, Fig.15 For CCK8 detection of HK-2 cells treated with Astragalus exosomes, Fig.16 This is the CCK8 detection test of HK-2 cells after treatment with A. umbellata exosomes.

[0113] By comparison Fig.15 and Fig.16 The results showed that both Astragalus exosomes and Hibiscus sibiricum exosomes could not repair the damage caused by H / R model to HK-2 cells.

[0114] After H / R modeling, HK-2 cells were subjected to flow cytometry for apoptosis detection. Fig.17 Statistical analysis of cell apoptosis was as shown in Figure 18-19 shown.

[0115] Depend on Figure 17-Figure 19 It can be seen that it was further verified that both Astragalus exosomes and Hibiscus sibiricum exosomes could not repair the damage caused to HK-2 cells by H / R modeling.

[0116] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example" or "some examples" etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present application. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine the different embodiments or examples described in this specification and the features of the different embodiments or examples, without contradiction.

[0117] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present application, rather than to limit it. Although the present application has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present application.

Claims

1. Application of Astragalus exosomes in protecting cells during vascular endothelial cell apoptosis.

2. The use according to claim 1, characterized in that: The vascular endothelial cells are HUVECs.

3. The use according to claim 1, characterized in that: The source of the astragalus exosomes is astragalus hydrolysate.

4. The use according to claim 1, characterized in that: The average particle size of the astragalus exosomes is 87.5 nm.

5. The use according to claim 1, characterized in that: The protein concentration of the astragalus exosomes is 1.94 μg / μL.

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