CN111249293A - Application of astragaloside in preparation of medicine for promoting angiogenesis - Google Patents
Application of astragaloside in preparation of medicine for promoting angiogenesis Download PDFInfo
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- CN111249293A CN111249293A CN202010176674.2A CN202010176674A CN111249293A CN 111249293 A CN111249293 A CN 111249293A CN 202010176674 A CN202010176674 A CN 202010176674A CN 111249293 A CN111249293 A CN 111249293A
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Abstract
The invention provides application of Astragaloside IV (AS-IV) in preparation of a medicine for promoting angiogenesis, wherein the medicine for promoting angiogenesis is realized by promoting human Endothelial Progenitor Cells (EPCs) to secrete Exosomes (Exosomes) and angiogenesis factors, and the medicine for promoting angiogenesis is hydrogel containing the human Endothelial progenitor cell Exosomes (EPC-Exosomes) and the angiogenesis factors. The invention discloses the application of astragaloside IV in the preparation of medicaments for promoting angiogenesis for the first time, improves the cell activity of EPCs, and improves the biological functions of the EPCs such as proliferation, adhesion, migration, tube formation and the like; promotes EPCs to secrete exosomes and vascular growth factors, is beneficial to rapid regeneration and repair of blood vessels, can be applied to the field of repair of various vascular injuries, and has wide application range.
Description
Technical Field
The invention belongs to the technical field of medicines, and particularly relates to application of astragaloside in preparation of a medicine for promoting angiogenesis.
Background
With the change of life style and habits of human, disease spectrum changes silently, metabolic diseases increase, the incidence rate of skin ulcer rises continuously, and even chronic refractory wounds, such as diabetic feet, appear. The diabetic foot is a serious complication of diabetes, is persistent, has high morbidity and high repair difficulty, and seriously threatens human health. The related data show that the incidence rate of the diabetic foot is 15 percent at foreign countries, the incidence rate of the diabetic foot at home is 8.1 percent, and the disability rate and the fatality rate are higher. Current research indicates that angiogenesis disorders and inadequate blood supply in diabetic feet are the major causes of prolonged, prolonged and even aggravated wounds. Aiming at the metabolic diseases, the current clinically and conventionally used treatment methods are epidermal growth factor gel and fibroblast growth factor gel, but the problems cannot be solved fundamentally and the curative effect is limited.
Endothelial Progenitor Cells (EPCs) in the human body are precursor cells having migratory properties and capable of directionally proliferating and differentiating into mature vascular endothelial cells, mainly originating from bone marrow and existing in small amounts in adult peripheral blood. A large number of studies indicate that EPCs, on the one hand, promote angiogenesis of ischemic tissues by secreting vascular growth factors, and, on the other hand, participate in re-endothelialization and angiogenesis of damaged blood vessels by mobilizing, migrating, homing and differentiating vascular endothelial cells. At present, animal experimental research shows that the endothelial progenitor cells transplanted to a lesion area can obviously promote angiogenesis so as to achieve the purpose of repairing the lesion. Therefore, the development and application of the EPCs bring good news for the repair of chronic intractable wounds.
The research shows that the vascular growth factors secreted by the EPCs mainly comprise Vascular Endothelial Growth Factor (VEGF), Fibroblast Growth Factor (FGF) and angiopoietin-1 (Ang-1). Further intensive research shows that VEGF is a regulatory factor with highest specificity and strongest function for promoting angiogenesis, wherein VEGFa can regulate endothelial reaction, such as proliferation and migration of endothelial cells, vascular permeability and the like, and is the most main factor of a VEGF family; VEGFb has the potential of inducing coronary artery growth and myocardial hypertrophy, can regulate the growth and tissue metabolism of blood vessels and myocardial cells in the heart and improve the activity of the myocardial cells; VEGFc can promote endothelial cell proliferation and migration through the p38MAPK-CREB-DLL4/NOTCH1 axis, thereby participating in angiogenesis. FGF can promote the degradation of a basement membrane of a capillary vessel, the migration, the proliferation and the tube formation of endothelial cells; ang-1 can reduce endothelial cell permeability, regulate VEGF effect, maintain endothelial cell homeostasis, and promote angiogenesis during embryonic development and wound healing. The above 5 angiogenesis factors respectively play their roles in the angiogenesis process, regulate each link of angiogenesis, and protect the health of angiogenesis; in the absence of any factor, the angiogenesis function of the body is blocked or incomplete, and the angiogenesis function cannot be normally performed. However, due to the limited number of vascular endothelial progenitor cells in the human body, even when mobilized from the bone marrow to the peripheral blood circulation, their migration, number of entry into damaged tissues and their secreted vascular growth factors are far from adequate for clinical treatment.
The exosome (EPC-Exos) secreted by endothelial progenitor cells is a nanoscale extracellular membrane vesicle which has a small molecular structure and high biocompatibility, can be used as an important transfer vector for intercellular communication and genetic materials, carries various bioactive molecules (such as protein, mRNA, miRNAs and the like), and transfers into target cells so as to play a role in angiogenesis, wherein the miRNAs play a key role in the regulation of angiogenesis. However, the number of EPCs in humans is limited and the number of EPC-Exos that can function is also limited.
Therefore, under the premise of limited quantity of EPCs, the key point is to promote the secretion of exosomes and vascular growth factors by EPCs so that EPCs can better play a role in repairing damaged tissues and quickly promoting angiogenesis. The search for the component with the effect has important significance for treating ischemic diseases caused by insufficient or even lost of the neovascular capacity of the body, and is a problem to be solved urgently.
Astragaloside IV, also known AS Astragaloside IV, is a high-purity drug extracted from Astragalus membranaceus, is called super Astragalus polysaccharide, and is the main active component of Astragalus membranaceus AS traditional Chinese medicine. At present, the research on astragaloside IV mainly focuses on enhancing the immunity of the organism and improving the disease resistance of the organism, and the research on promoting angiogenesis is rarely reported at home and abroad.
Disclosure of Invention
In order to solve the problem of how to promote endothelial progenitor cells to secrete exosomes and angiogenesis factors in the prior art, the invention provides the application of astragaloside in preparing a medicament for promoting angiogenesis.
The technical scheme adopted by the invention is as follows:
application of astragaloside IV in preparing medicine for promoting angiogenesis; the angiogenesis promotion is realized by promoting the secretion of exosome and vascular growth factor by human endothelial progenitor cells.
The method for promoting the secretion of exosomes and angiogenic growth factors by human endothelial progenitor cells by astragaloside IV comprises the following steps:
s1, selecting P3-P6 generation human endothelial progenitor cells from a cell bank for resuscitation, culturing the recovered EPCs in a culture plate coated with human fibronectin, and pre-culturing the EPCs for 24h by adopting an M199 culture medium; m199 culture medium containing 20% fetal bovine serum, Vascular Endothelial Growth Factor (VEGF)10mg/L, penicillin 100kU/L and streptomycin 100 kU/L;
s2, taking adherent cells, digesting and centrifuging the adherent cells by digestive enzyme, then suspending the adherent cells, washing the cells by Phosphate Buffer Solution (PBS) after the EPCs are attached to the wall again, culturing the cells by adopting EGM-2MV medium without Fetal Bovine Serum (FBS) containing human serum substitute, and adding astragaloside IV and the EPCs into a culture bottle for combined culture for 48 hours;
s3, collecting cell culture supernatant, centrifuging for 1 min to remove residual cells, and centrifuging for 20min to remove cell debris;
s4, collecting the cell supernatant after centrifugation, and concentrating to obtain cell suspension rich in human endothelial progenitor cell exosomes and vascular growth factors.
Further limiting, the concentration of the astragaloside in the culture bottle in the step S2 is 100-400 mg/L.
According to the invention, the astragaloside IV and the EPCs are added for mixed culture, and when the concentration of the astragaloside IV in a culture bottle is 100mg/L, the EPCs secretion function reaches the best, and the quantity of the obtained EPC-Exos and the blood vessel growth factor reaches the maximum.
The digestive enzyme is trypsin.
The medicine for promoting angiogenesis is hydrogel containing human endothelial progenitor cell exosomes and vascular growth factors. The active ingredients in the hydrogel are that after the traditional Chinese medicine active ingredient astragaloside intervenes in the human endothelial progenitor cells, the active ingredients are collected to be rich in human endothelial progenitor cell exosomes and vascular growth factors, and miR-126-3p, miR-126-5p, miR-21, miR-146a, miR-210, miR-155 and miR-214 related to angiogenesis are loaded in the human endothelial progenitor cell exosomes; vascular growth factors include VEGFa, VEGFb, VEGFc, FGF and Ang-1.
The grain diameter of the human endothelial progenitor cell exosome is 88.7-123.5nm, and the concentration is 100-1000 mg/ml.
The hydrogel also comprises a stabilizing agent, a humectant, a hydrogel matrix, a pH regulator and an isotonic agent.
Preferably, the concentration of VEGFa in the hydrogel is 700-1400pg/ml, the concentration of VEGFb is 700-1400pg/ml, the concentration of VEGFc is 500-1200pg/ml, the concentration of FGF in the hydrogel is 50-400pg/ml, and the concentration of Ang-1 in the hydrogel is 50-400 pg/ml.
Preferably, the hydrogel has a pH of 6.2 to 7.8.
Preferably, the concentration of the astragaloside in the step S2 is 100-400 mg/L.
Preferably, the stabilizer is sodium hyaluronate or hydroxyethyl starch.
Preferably, the humectant is glycerol.
Preferably, the hydrogel matrix is carbomer.
Preferably, the isotonic agent is sodium chloride or PBS solution with the mass concentration of 0.9%.
At present, the main treatment methods for treating metabolic diseases such as diabetic foot and the like are epidermal growth factor gel and fibroblast growth factor gel, but the problems cannot be solved fundamentally and the curative effect is limited. It is found that the exosomes and the blood vessel growth factors secreted by the EPCs can promote angiogenesis, but the number of the EPCs is limited, and the exosomes and the blood vessel growth factors secreted by the EPCs are also limited, so that the EPCs cannot play a good role in repairing damaged tissues, and the effect of the EPCs on treating diseases caused by insufficient or even lost of the angiogenesis capacity of the body is limited, so that the secretion of the exosomes and the blood vessel growth factors by the EPCs is required to be improved. The search for a component with the effect has important significance for treating the diseases, and is a problem to be solved urgently.
The invention discovers the new application of astragaloside IV in promoting EPCs to secrete vascular growth factors (VEGFa, VEGFb, VEGFc, FGF and Ang-1) and exosomes through research, the secreted exosomes are rich in miRNAs molecules for promoting angiogenesis, and the vascular growth factors and EPC-Exos can enhance the tube forming function after being taken by endothelial cells.
The existing dressing for the wound surface mainly comprises the traditional dressing and a novel synthetic dressing, wherein the traditional dressing is wide in source, soft in texture, low in cost and widely used due to wide source, and the like. However, the traditional dressing has poor hemostatic effect and no moisturizing effect, and is easy to cause exogenous infection and secondary injury when the dressing is soaked; and the traditional dressing is easy to adhere to the wound surface, and the wound surface can be damaged again when the traditional dressing is removed. The novel synthetic dressing has good elasticity and air permeability, but poor hygroscopicity, and can cause the formation of hydrops at the wound surface to cause the propagation of bacteria. The tissue engineering products applied to clinic have certain rejection immune response and poor treatment effect.
The hydrogel prepared from the angiogenesis promoting drug has good biocompatibility, does not influence the metabolic process of a living body, can discharge metabolites through the hydrogel, and can overcome the defects of the traditional dressing and the novel synthetic dressing.
The invention has the beneficial effects that:
1. the invention discovers the new application of the astragaloside IV in promoting EPCs to secrete EPC-Exos and angiogenesis factors, the EPC-Exos and the angiogenesis factors have obvious effect of promoting angiogenesis, compared with the conventional treatment means at present, the astragaloside IV has the advantages of easily obtained raw materials, simple and safe administration method, low cost and the like, can fundamentally solve the problem of angiogenesis obstacle diseases, and has obvious treatment effect.
2. The invention discovers that the astragaloside IV can improve the cell activity of EPCs and improve the biological functions of the EPCs such as proliferation, adhesion, migration, tube formation and the like; the method is beneficial to the rapid regeneration and repair of blood vessels, can be applied to various fields requiring repair of blood vessels, and has wide application range.
3. The exosomes secreted by EPCs contain proteins, mRNA and miRNAs molecules, particularly miRNAs, which play a critical role in regulating angiogenesis; astragaloside IV plays a role in promoting angiogenesis by promoting EPC-Exos to secrete angiogenesis-related miRNAs, and does not cause deletion of other functions.
4. The hydrogel containing the human endothelial progenitor cell exosome and the blood vessel growth factor prepared by the invention is externally used for wounds/wound surfaces, can effectively solve the problem of wound/wound surface angiogenesis, improves the blood circulation of the wound surfaces, accelerates the healing of the wound surfaces, and is particularly suitable for chronic intractable wound surfaces.
Drawings
FIG. 1 is a graph of morphological features of primary human EPCs cultured under light microscopy at day 7 in exosome group;
FIG. 2 is a graph of the immunofluorescence staining identification results of primary human EPCs CD31 cells;
FIG. 3 is a graph of the results of bifluorescent staining identification of primary human EPCs;
FIG. 4 is a graph showing the expression of marker proteins CD9, CD63 and CD81 in human EPC-Exos (control group) and astragaloside intervening in human EPC-Exos (experimental group) under normal conditions by Western detection;
FIG. 5 is a graph comparing two sets of human EPC-Exos marker protein CD9 expression;
FIG. 6 is a graph comparing two sets of human EPC-Exos marker protein CD63 expression;
FIG. 7 is a graph comparing two sets of human EPC-Exos marker protein CD81 expression;
FIG. 8 is an electron microscope observation of EPC-Exos in the control group;
FIG. 9 is an electron microscope observation of EPC-Exos in experimental group;
FIG. 10 is a graph showing the results of particle size measurement of a control group EPC-Exos by NTA technique;
FIG. 11 is a graph showing the results of particle size measurements of EPC-Exos in experimental groups by NTA technique;
FIG. 12 is a curve of OD value detection standard concentration of EPC-Exo expression;
FIG. 13 is a graph of a concentration comparison study of two sets of EPC-Exos with the BCA kit;
FIG. 14 is two sets of EPC-Exos total RNA agarose gel electrophoresis images;
FIG. 15 is a graph of RT-PCR detection of angiogenesis-related miR-126-3p expression in two sets of EPC-Exos compared to study;
FIG. 16 is a graph of RT-PCR detection of angiogenesis-related miR-126-5p expression in two sets of EPC-Exos compared to study;
FIG. 17 is a graph of a comparative study of RT-PCR detection of miR-21 expression associated with angiogenesis in two sets of EPC-Exos;
FIG. 18 is a graph of a comparative study of RT-PCR detection of miR-210 expression associated with angiogenesis in two sets of EPC-Exos;
FIG. 19 is a graph of a comparative study of RT-PCR detection of miR-214 expression associated with angiogenesis in two sets of EPC-Exos;
FIG. 20 is a graph of a comparative study of RT-PCR detection of miR-155 expression associated with angiogenesis in two sets of EPC-Exos;
FIG. 21 is a graph of a comparative study of RT-PCR detection of miR-146a expression associated with angiogenesis in two sets of EPC-Exos;
FIG. 22 is a microscopic electron microscope image of cell supernatant formed by control group PBS liquid mediated EPCs interfering HUVECs in vitro tube formation;
FIG. 23 is a microscope electron micrograph of the cell supernatant formed by the astragaloside mediated EPCs in the experimental group interfering with the in vitro tube formation of HUVECs;
FIG. 24 is a graph showing a comparison study of the effects of cell supernatants formed by two groups of EPCs on the in vitro tube formation of HUVECs.
Detailed Description
The invention is further described with reference to the following figures and specific embodiments.
Reference will now be made in detail to embodiments of the present invention, examples of which are illustrated in the accompanying drawings, wherein like or similar reference numerals refer to the same or similar elements or elements having the same or similar function throughout. The embodiments described below with reference to the drawings are illustrative and intended to be illustrative of the invention and are not to be construed as limiting the invention.
In the description of the present invention, some structures or devices are not specifically described, and it is understood that there are structures or devices that can be implemented in the prior art.
Example 1
In vitro angiogenesis promoting experiment
1. Test reagent and test device
1.1 materials
Test reagents: astragaloside IV (Shanghai Ji Co.), fetal bovine serum (Gibco, USA), M199 culture medium, PBS (Gibco, USA), trypsin (Amresco, USA), cell culture box (Thermoscientific, USA), Anti-CD31 antibody (ab28364), goat Anti-rabbit IgG H&L (FITC) (ab6717), human VEGFAELISA kit (ab119566), human Anti-VEGFB antibody (ab233503), human VEGFC ELISA kit (ab100664), human FGF basic ELISA kit (FGF2) (ab99979), human Angiopoietin 1ELISA kit (ANG1) (ab99972), (Abcam, UK), DAPI solution (Kitay, Beijing), BCA protein concentration determination kit (Solebao, PC 0020); phosphotungstic acid negative dye liquor (solibao, G1872); Anti-CD9 antibody [ EPR2949 ]](Abcam,ab92726,1:2000)；Anti-CD63 antibody[TS63](Abcam,ab59479,1:1000)；Anti-CD81 antibody[M38](Abcam, ab79559,1: 1000); TRNzol total RNA extraction reagent (tiangen biochemical technology (beijing) ltd); PrimeScriptTM RT reagent Kit with gDNA Eraser TaKaRa,
Premix Ex TaqTMII (Tli RNaseH Plus), ROX Plus TaKaRa, DL2,000DNAmarker TaKaRa (Takara Bio); primer synthesis (Invitrogen); and (3) gene primers.
Test equipment: vortex oscillator QL-902 (Haiman, Linbel instruments, Inc.); centrifuge 5415d (eppendorf); spectrophotometer nano diagnostic 2000(Therno scientific); gel imaging system Tanon 1600 (Shanghai Nature technologies Co., Ltd.) fluorescence quantitative PCR instrument ABI7500(applied biosystems); fluorescence microscope (Olympus-BX51) (beijing samzhou kayod), Thermo-electric MK3 microplate reader (Thermo fisher Scientific, usa); an electron beam microscope (JEOL corporation, JEM1230, japan); zetaview (Brand: Particle metric)
2. Method of implementation
2.1 isolation and culture of EPCs
Collecting 10ml of umbilical cord blood of full-term newborn, obtaining mononuclear cells by density gradient centrifugation, spreading the mononuclear cells on culture plate coated with human fibronectin, and culturing with M199 culture medium (containing 20% fetal calf serum, VEGF 10mg/L, penicillin 100kU/L, streptomycin 100kU/L) at 37 deg.C and 5% CO2Culturing for 4 days in an incubator with saturated humidity, washing off non-adherent cells by PBS (phosphate buffer solution), continuously culturing for 7 days by changing culture solution, observing the morphology of the cells by using an optical microscope, and showing the result as shown in figure 1, wherein the cells in the logarithmic growth phase under the optical microscope can be observed, the edges of the cells are clear and are arranged like paving stones, the central cells are circular, and the peripheral cells are fusiform. Non-adherent cells were washed off again with PBS solution and adherent cells were used for administration. When the cell confluency reached 80%, passage was performed at a ratio of 1: 3.
2.2 identification of EPCs
Human endothelial progenitor cells are identified by referring to a CD31 immunomagnetic bead sorting method and a FITC-UEA-I and Dil-ac-LDL double fluorescence staining method together.
CD31 antibody in combination with DAPI nuclear staining: fixing the P3 generation cells with 4% paraformaldehyde for 15 min; 0.5% TritonX-100 (prepared by PBS) is transparent for 20min at room temperature; after PBS immersion washing, goat serum is dripped, and the mixture is sealed for 30min at room temperature; Anti-CD31 antibody was added dropwise and incubated in a wet box overnight at 4 ℃ and the image was collected by observation under a fluorescence microscope, as shown in FIG. 2A, in which the bright area is the stained area. After PBST is soaked, goat anti-rabbit IgG (H & L) antibody is added dropwise, and the mixture is incubated for 1H at the temperature of 20-37 ℃ in a wet box; soaking and washing with PBST, adding DAPI dropwise, incubating for 5min in dark place, staining nuclei, washing with PBST to remove excessive DAPI, adding 50% glycerol, and observing and collecting image under fluorescence microscope, as shown in figure C of figure 2, wherein the bright region is double staining region; DAPI was extracted from the above cells, and the collected images were observed under a fluorescence microscope, as shown in B-diagram in fig. 2, where the bright areas are stained areas.
FITC-UEA-I and Dil-ac-LDL dual-fluorescence staining identification: inoculating the P3 generation cells on a cover glass to make the cells slide, adding 2.4g/L Dil-ac-LDL, incubating at 37 ℃ in the dark for 1h, fixing for 10min by using 20g/L paraformaldehyde, and observing and collecting images under a fluorescence microscope, wherein a bright area in the image is a staining area as shown in a B picture in figure 3; adding 10g/L FITC-UEA-I into PBST after soaking, incubating for 1h at 37 ℃ in the dark, washing PBST, and observing and acquiring images under a fluorescence microscope, wherein a bright area in the images is a staining area as shown in an A picture in figure 3; cells were double stained after binding to FITC-UEA-I and uptake of Dil-ac-LDL and the images were collected by observation under a fluorescent microscope, as shown in FIG. 3C, where the bright areas are the stained areas. As can be seen from FIGS. 2-3, the cells were identified as EPCs.
2.3 administration of drugs
S1, selecting P3-P6 generation human Endothelial Progenitor Cells (EPCs) from a cell bank for resuscitation, culturing the recovered EPCs in a culture plate coated with human fibronectin, and pre-culturing EPCs24h by using an M199 culture medium; m199 culture medium containing 20% fetal bovine serum, Vascular Endothelial Growth Factor (VEGF)10mg/L, penicillin 100kU/L and streptomycin 100 kU/L;
s2, taking adherent cells, digesting and centrifuging by using digestive juice, then resuspending, after EPCs are attached to the wall again, washing the cells by using Phosphate Buffer Solution (PBS), culturing by using EGM-2MV culture medium without Fetal Bovine Serum (FBS) containing human serum substitute, randomly dividing EPCs obtained by culturing into an astragaloside group and a control group, adding astragaloside and EPCs into a culture bottle for combined culture for 48h, wherein the concentration of the astragaloside is 100 mg/L; treating a control group with an equal amount of PBS (phosphate buffer solution) for 48 h; control is a Control group; AS-IV is astragaloside IV group;
s3, collecting cell culture supernatant, centrifuging for 1 min to remove residual cells, and centrifuging for 20min to remove cell debris;
s4, collecting the cell supernatant after centrifugation, and concentrating to obtain two groups of cell suspensions containing the human endothelial progenitor cell exosomes and the vascular growth factors.
3. Results
3.1 Effect of Astragaloside IV on secretion of exosomes from endothelial progenitor cells
3.1.1 isolation and characterization of endothelial progenitor exosomes (EPC-Exos)
Extracting exosome, namely EPC-Exos, secreted by EPCs in an early culture stage (generation P3-P6) by adopting an ultracentrifugation method and an ultrafiltration method, transferring the extracted EPC-Exos into a sterile EP tube, storing the EPC-Exos in a refrigerator at the temperature of-80 ℃ for later use, and detecting characteristic markers CD9, CD63 and CD81 of the EPC-Exos by using a western technology, wherein the result is shown in figure 4; as can be seen from fig. 4: western immunoblot results showed that characteristic endothelial progenitor cell exosome marker proteins (CD9, CD63 and CD81) were positive, confirming that exosomes had been successfully extracted.
3.1.2 expression of marker protein Components in two sets of EPC-Exos
The expressions of EPC-Exos marker proteins CD9, CD63 and CD81 isolated from the two groups of EPCs were compared, and the results are shown in Table 1, and the comparative study graphs of the expressions of EPC-Exos marker proteins CD9, CD63 and CD81 in the two groups are shown in FIG. 5, FIG. 6 and FIG. 7, respectively.
TABLE 1 comparative study of expression of two sets of EPC-Exos marker proteins CD9, CD63 and CD81
| Group of | CD9 | CD63 | CD81 |
| AS-IV | 0.078±0.019 | 0.422±0.042 | 0.214±0.013 |
| Control | 0.050±0.002 | 0.097±0.004 | 0.098±0.006 |
| t value | 2.603 | 13.456 | 13.684 |
| P | 0.118 | 0.000 | 0.000 |
As can be seen from table 1 and fig. 5, fig. 6 and fig. 7, the relative expression of EPC-Exos marker proteins CD9, CD63 and CD81 in the astragaloside group is higher than that in the control group, and the difference between them indicates that astragaloside can promote EPCs to secrete exosomes containing CD9, CD63 and CD81 characteristics. And shows that the vesicle promoting the secretion of EPCs by astragaloside is EPC-Exos.
3.1.3 forms of two groups of EPC-Exos
Fixing a sample-carrying copper net with the diameter of 2nm on a bracket, dropwise adding 20 mu L of EPC-Exos suspension, and standing at room temperature for 3 min; sucking the liquid from the side of the copper mesh by using filter paper, adding 30 mu L of 3% phosphotungstic acid solution, and carrying out negative dyeing on EPC-Exos for 5min at room temperature; the negative staining solution was blotted with filter paper, the copper mesh was placed in the sample chamber of the transmission electron microscope, and the morphology of EPC-Exos was observed and the electron microscope photograph was taken, and as shown in fig. 8 and 9, it can be seen from fig. 8 and 9 that the separated and purified EPC-Exos of astragaloside group and control group are circular membrane vesicles, and the morphology of both groups has no significant difference, indicating that both groups of exosomes are derived from endothelial progenitor cells.
3.1.4 particle size of two sets of EPC-Exos
mu.L of EPC-Exos suspension was diluted 1000-fold with PBS, and the particle size of EPC-Exos in the astragaloside IV group and the control group was measured by the NTA technique, as shown in FIGS. 10 and 11, and as shown in FIGS. 10 and 11: 98.7% of human EPC-Exos in the astragaloside group have diameters of 84.7-143.1nm and slightly increased volume; 98% of human EPC-Exos in the control group had diameters between 88.7-123.5nm, were round vesicles, and had a slight increase in volume. The content (miRNAs, proteins and the like) in EPC-Exos after the astragaloside dry prediction is increased through two groups of particle size analysis.
3.1.5 concentration of EPC-Exos in two groups
The concentration of EPC-Exos in the astragaloside group and the control group was determined strictly according to the BCA protein quantification kit instructions. mu.L of a protein standard solution (BSA: 5mg/ml) was diluted to 100. mu.L with PBS to a final concentration of 0.5 mg/ml. BSA solution was stored for a long period at-20 ℃. 5.1ml of BCA working solution was prepared in a volume ratio of 50:1(BCA reagent A: BCA reagent B). The BCA working solution is stable at room temperature for 24 h. Adding BSA solution into A1-A5 wells of a 96-well plate in sequence according to 0, 2.5, 5, 10 and 20 mu L, and supplementing PBS solution to 20 mu L; sequentially adding 2 mu L of exosomes suspension into B1-B3 wells, and complementing 18 mu L of PBS solution to 20 mu L; adding 200 mu L of BCA working solution into each hole in sequence, and standing in an incubator at 37 ℃ for 30 min; the absorbance A562 nm was measured with a microplate reader, and the protein concentration was calculated from the standard curve as shown in FIG. 12. Independently repeating for 2 times, and averaging; the concentration of EPC-Exos detected in the two groups of cells is shown in Table 2, and the comparative study chart of the BCA kit for detecting the concentration of EPC-Exos in the two groups of cells is shown in FIG. 13.
TABLE 2 BCA kit for determining the concentration of EPC-Exos in two groups of cells
As can be seen from FIG. 12, Table 2 and FIG. 13, the mean concentration of EPC-Exos in the astragaloside group is (0.363. + -. 0.023) mg/ml, the mean concentration of EPC-Exos in the control group is (0.184. + -. 0.015) mg/ml and P <0.01, which has statistical significance, so that EPC-Exos produced by the astragaloside group is significantly more than that in the control group, further indicating that astragaloside can promote EPC secretion exosomes.
3.1.6 molecular analysis of miRNAs related to angiogenesis in two groups of EPC-Exos
Designing target gene primers (miR-126-3p, miR-126-5p, miR-21, miR-210, miR-214, miR-155 and miR-146a), carrying out agarose gel electrophoresis on two groups of EPC-Exos total RNA as shown in Table 3, and carrying out agarose gel electrophoresis on the results as shown in FIG. 14; RT-PCR detection shows that expressions of miR-126-3P, miR-126-5P, miR-21, miR-210, miR-214, miR-155 and miR-146a related to angiogenesis in EPC-Exos of AS-IV group are obviously increased compared with those of a control group, the expression difference is significant, P is less than 0.01, the statistical significance is achieved, and the astragaloside plays a role in promoting angiogenesis by promoting the EPC-Exos to secrete miRNA related to angiogenesis, AS shown in Table 4; expression comparison study graphs of miR-126-3p, miR-126-5p, miR-21, miR-210, miR-214, miR-155 and miR-146a are respectively shown in figures 15-21.
As can be seen from the data in Table 4 and FIGS. 15-21, the expression of each target gene of the astragaloside group is obviously increased compared with that of the control group, and the difference is significant, wherein P < 0.05 has statistical significance, which indicates that miRNAs molecules related to angiogenesis are loaded in exosomes secreted by endothelial progenitor cells mediated by astragaloside.
TABLE 3 primer sequences for molecular amplification reactions of miRNAs related to angiogenesis
TABLE 4 RT-PCR detection of expression of angiogenesis-related miRNAs molecules in two sets of EPC-Exos
3.2 Effect of Astragaloside IV on vascular growth factor secretion from endothelial progenitor cells
According to the operation of an ELISA kit specification, detecting the absorbance of Vascular Endothelial Growth Factor (VEGF), Fibroblast Growth Factor (FGF) and angiopoietin-1 (Ang-1) by the ELISA kit, respectively detecting the OD value of each vascular growth factor in a standard hole, zeroing the OD value of a blank hole to calculate the OD value difference, drawing a standard curve by taking the concentration (pg/ml) of the vascular growth factor as a horizontal coordinate and the OD value difference as a vertical coordinate, and finally substituting the actually measured OD value in a sample hole to be detected into calculation to obtain the concentration of each vascular growth factor; the concentration of the vascular growth factors secreted by the two groups of cells is shown in Table 5, and the concentration of VEGFa, VEGFb, VEGFc, FGF and Ang-1 in the astragaloside group is respectively higher than that of the corresponding vascular growth factor in the control group by comparing and researching the influence of AS-IV intervention on the expression of the vascular growth factors by the EPCs. As can be seen from the data in Table 5, the comparative differences among the 5 groups of data have significant statistical significance (P <0.01), indicating that astragaloside can promote the endothelial progenitor cells to secrete the vascular growth factors.
TABLE 5 ELISA method for determining the concentration of two sets of cytosines in the culture medium
| Concentration (pg/ml) | VEGFa | VEGFb | VEGFc | FGF | Ang-1 |
| Control | 744.24±31.84 | 738.53±14.93 | 669.36±26.55 | 50.75±7.02 | 117.39±14.63 |
| AS-IV | 1356.26±102.89 | 1366.29±18.86 | 1150.23±50.91 | 127.06±19.87 | 201.14±16.53 |
| t value | 9.84 | 45.24 | 14.51 | 6.27 | 6.57 |
| P value | 0.001 | 0.000 | 0.000 | 0.003 | 0.003 |
3.3 cell supernatant of Astragaloside IV interfering with endothelial progenitor cell secretion promotes human vascular endothelial cells (HUVECs) tube forming experiment
Respectively extracting cell supernatant from the endothelial progenitor cells after the intervention of astragaloside IV and PBS for 24h to obtain cell supernatant extracted after the intervention of the EPCs by AS-IV and cell supernatant extracted after the intervention of the EPCs by PBS.
Dissolving Matrigel overnight at 4 ℃, and precooling a gun head box, an EP tube and a 96 pore plate overnight at 4 ℃; spreading glue on an ice box the next day, firstly diluting Matrigel with a culture medium 1:1 of M200 serum-free and growth factor supplement, uniformly mixing, adding 50 μ l/hole of 12-hole plate (pre-cooling the 12-hole plate in advance) to avoid generating bubbles, and placing in an incubator at 37 ℃ for 30 min; when HUVECs grow to 80-90%, they are digested and resuspended in 10% FBS-containing DMEM medium, and the cell density is adjusted to 1X 105Adding 1ml of resuspension into each well,F12-DMEM complete medium containing Control EPC-Exos is added when the cells adhere to the wall, wherein cell supernatant extracted after PBS is added into half of the pore plates in the Control group to intervene the EPCs is intervened, the three pores are repeated, the incubation is carried out in an incubator at 37 ℃, the cell supernatant is observed and photographed under an inverted microscope after 24 hours, and the result is shown in figure 22; adding AS-IV into half of the pore plates in the astragaloside IV group to intervene EPCs, intervening with the cell supernatant, repeating three pores, incubating at 37 deg.C, observing under an inverted microscope after 24 hr, and taking a picture, wherein the result is shown in FIG. 23; and 4 fields are randomly selected from each hole to calculate the tubule forming length, the average value is taken, the result is shown in figure 24, a Control Group represents that cell supernatant extracted after PBS interferes with EPCs interferes with HUVECs in vitro tube forming, and an Experimental Group represents that cell supernatant extracted after AS-IV interferes with EPCs interferes with HUVECs in vitro tube forming. As can be seen from fig. 22 and 23, the cell supernatants extracted after the intervention of the astragaloside group promoted the in vitro angiogenesis ability of human vascular endothelial cells (HUVECs) higher than that of the control group; as can be seen from FIG. 24, the cell supernatants extracted after the intervention of EPCs by AS-IV increased the tube forming number and the tube forming ability was strong, indicating that the cell supernatants extracted after the intervention of EPCs by astragaloside IV could promote the tube forming of human endothelial cells (HUVECs).
In conclusion, the astragaloside IV has the effect of promoting EPCs to secrete exosomes and angiogenesis factors, and has the potential of promoting angiogenesis and repair. The endothelial progenitor cells are cultured and separated in an in-vitro culture medium containing astragaloside IV to obtain exosomes and blood vessel growth factors secreted by the endothelial progenitor cells, and the exosomes and the blood vessel growth factors can be prepared into hydrogel which is non-toxic and harmless, can be safely used, and can be directly used for the wound surface of skin ulcer of a human body, promote angiogenesis, improve the blood circulation of the wound surface and accelerate the repair of the wound surface/wound.
The method for promoting the secretion of exosomes and angiogenic growth factors by human endothelial progenitor cells by astragaloside IV comprises the following steps:
s1, selecting P3-P6 generation human endothelial progenitor cells from a cell bank for resuscitation, culturing the recovered EPCs in a culture plate coated with human fibronectin, and pre-culturing the EPCs for 24h by adopting an M199 culture medium; m199 culture medium containing 20% fetal bovine serum, Vascular Endothelial Growth Factor (VEGF)10mg/L, penicillin 100kU/L and streptomycin 100 kU/L;
s2, taking adherent cells, digesting and centrifuging by using digestive juice, then resuspending, washing the cells by using Phosphate Buffer Solution (PBS) after the EPCs grow over 80% of a cell culture bottle, culturing by using EGM-2MV culture medium without Fetal Bovine Serum (FBS) containing human serum substitutes, and adding astragaloside and the EPCs into the culture bottle for combined culture for 48 hours; the concentration of astragaloside is 100 mg/L;
s3, collecting cell culture supernatant, centrifuging for 1 min to remove residual cells, and centrifuging for 20min to remove cell debris;
s4, collecting the cell supernatant after centrifugation, and concentrating to obtain cell suspension rich in human endothelial progenitor cell exosomes and vascular growth factors.
The medicine for promoting angiogenesis is hydrogel containing human endothelial progenitor cell exosomes and vascular growth factors. The active ingredients in the hydrogel are that after the traditional Chinese medicine active ingredient astragaloside intervenes in the human endothelial progenitor cells, the active ingredients are collected to be rich in human endothelial progenitor cell exosomes and vascular growth factors, and miR-126-3p, miR-126-5p, miR-21, miR-146a, miR-210, miR-155 and miR-214 related to angiogenesis are loaded in the human endothelial progenitor cell exosomes; vascular growth factors include VEGFa, VEGFb, VEGFc, FGF and Ang-1.
The grain diameter of the human endothelial progenitor cell exosome is 88.7-123.5nm, and the concentration is 500 mg/ml.
The hydrogel also comprises a stabilizing agent, a humectant, a hydrogel matrix, a pH regulator and an isotonic agent.
Preferably, the concentration of VEGFa in the hydrogel is 700-1400pg/ml, the concentration of VEGFb is 700-1400pg/ml, the concentration of VEGFc is 500-1200pg/ml, the concentration of FGF in the hydrogel is 50-400pg/ml, and the concentration of Ang-1 in the hydrogel is 50-400 pg/ml.
For the present invention, the hydrogel for promoting angiogenesis includes, in addition to human endothelial progenitor exosomes and angiogenic growth factors: the stabilizing agent, the humectant, the hydrogel matrix, the pH regulator and the isotonic agent are required to be clear that the stabilizing agent, the humectant, the hydrogel matrix, the pH regulator and the isotonic agent do not play a medicinal role, and are prepared into materials for conventional hydrogel, and the materials are equivalent to the solvent part of the hydrogel. The invention adopts the prior art to prepare the hydrogel, and the human endothelial progenitor cell exosome and the blood vessel growth factor are effective components with medicinal effect and are equivalent to the solute part of the hydrogel.
Specifically, the pH regulator is used for regulating the pH value of the hydrogel to be 6.2-7.8. The stabilizer is sodium hyaluronate or hydroxyethyl starch. The humectant is glycerol. The hydrogel matrix is carbomer. The isotonic agent is 0.9% sodium chloride or PBS solution.
The invention is not limited to the above alternative embodiments, and any other various forms of products can be obtained by anyone in the light of the present invention, but any changes in shape or structure thereof, which fall within the scope of the present invention as defined in the claims, fall within the scope of the present invention.
Claims (10)
1. Application of astragaloside IV (AS-IV) in preparing medicine for promoting angiogenesis is provided.
2. The use of astragaloside iv according to claim 1 for the preparation of a medicament for promoting angiogenesis, wherein the promotion of angiogenesis is achieved by promoting the secretion of exosomes (Exos) and angiogenic growth factors from human Endothelial Progenitor Cells (EPCs).
3. The use of astragaloside IV according to claim 2 in the preparation of a medicament for promoting angiogenesis, wherein the method for promoting the secretion of exosomes and angiogenic growth factors from human EPCs comprises:
s1, selecting P3-P6 generation human endothelial progenitor cells from a cell bank for resuscitation, culturing the recovered EPCs in a culture plate coated with human fibronectin, and pre-culturing the EPCs for 24h by adopting an M199 culture medium;
s2, taking adherent cells, digesting and centrifuging the adherent cells by digestive enzyme, then suspending the adherent cells, washing the cells by Phosphate Buffer Solution (PBS) after the EPCs are attached to the wall again, culturing the cells by adopting EGM-2MV medium without Fetal Bovine Serum (FBS) containing human serum substitute, and adding astragaloside and the EPCs for combined culture for 48 hours;
s3, collecting cell culture supernatant, centrifuging for 1 min to remove residual cells, and centrifuging for 20min to remove cell debris;
s4, collecting cell supernatant after centrifugation, and concentrating to obtain cell suspension rich in human endothelial progenitor cell exosome (EPC-Exos) and blood vessel growth factor.
4. The use of astragaloside IV according to claim 3 in the preparation of a medicament for promoting angiogenesis, wherein the concentration of astragaloside IV in step S2 is 100-400 mg/L.
5. The use of astragaloside IV according to claim 3 in the preparation of a medicament for promoting angiogenesis, wherein the concentration of astragaloside IV in step S2 is 100 mg/L.
6. The use of astragaloside IV according to claim 3 in the preparation of a medicament for promoting angiogenesis, wherein the M199 culture medium in step S1 contains 20% fetal bovine serum, Vascular Endothelial Growth Factor (VEGF)10mg/L, penicillin 100kU/L and streptomycin 100 kU/L.
7. The use of astragaloside iv according to claim 1 or 2 for the preparation of a medicament for promoting angiogenesis, wherein the medicament for promoting angiogenesis is a hydrogel comprising human endothelial progenitor cell exosomes and angiogenic growth factors.
8. The application of astragaloside IV in the preparation of drugs for promoting angiogenesis according to claim 7, wherein miR-126-3p, miR-126-5p, miR-21, miR-146a, miR-210, miR-155 and miR-214 related to angiogenesis are loaded in the exosomes of human endothelial progenitor cells in the hydrogel, the particle size of the exosomes is 88.7-123.5nm, and the concentration is 1000 mg/ml.
9. The use of astragaloside IV according to claim 7 in the preparation of a medicament for promoting angiogenesis, wherein the vascular growth factors comprise VEGFa, VEGFb, VEGFc, FGF and Ang-1.
10. The use of astragaloside iv according to claim 7 for the preparation of a medicament for promoting angiogenesis, wherein the hydrogel further comprises a stabilizer, a humectant, a hydrogel matrix, a PH adjusting agent and an isotonic agent.
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