Application of hydroxysafflor yellow A in preparation of medicine for treating diseases needing to be repaired by hUC-MSCs

Abstract

The invention relates to the technical field of regenerative medicine, in particular to application of hydroxysafflor yellow A in preparation of medicines for treating diseases needing to be repaired by hUC-MSCs. The hUC-MSCs are selected as a research object, the HSYA is taken as an intervention factor, and the function of the HSYA on the hUC-MSCs function is comprehensively evaluated by detecting multiple experiments such as multiplication capacity, migration capacity and paracrine level of the hUC-MSCs. The application preliminarily proves that the HSYA can promote the multiplication capacity and homing capacity of the hUC-MSCs, meanwhile, by promoting paracrine of the hUC-MSCs, the hUC-MSCs can secrete more bioactive factors such as BDNF and VEGF, the factors can possibly improve the angiogenesis promoting capacity and nerve repair promoting capacity of the hUC-MSCs, and a reference basis is provided for improving the treatment effect of clinical research of the hUC-MSCs.

Description

Technical Field

[0001] This application relates to the technical field of regenerative medicine, and particularly to the application of hydroxysafflor yellow A in the preparation of medicaments for treating diseases that require repair using hUC-MSCs. Background Art

[0002] Tissue repair and regenerative medicine are current hotspots in medical research. Due to their characteristics of self-renewal, multi-directional differentiation, and immunomodulation, mesenchymal stem cells have shown great potential in regenerative medicine. Human umbilical cord mesenchymal stem cells (hUC-MSCs), as a type of stem cell with rich sources, convenient acquisition, and low immunogenicity, have attracted much attention and play a key role in the process of tissue repair and regeneration. hUC-MSCs can differentiate into various cell types, such as osteoblasts, chondrocytes, adipocytes, etc., and participate in the repair and regeneration of tissues such as bone, cartilage, and fat. In addition, hUC-MSCs also have immunomodulatory functions, can inhibit the over-activation of immune cells, reduce the inflammatory response, and create a good microenvironment for tissue repair.

[0003] In clinical applications, improving the function of hUC-MSCs is of great significance for the treatment of various diseases. In recent years, researchers have been committed to finding substances that can effectively promote the function of mesenchymal stem cells to improve their effects in clinical treatment.

[0004] Hydroxysafflor yellow A (HSYA), as the main natural active ingredient of safflower, has gradually attracted the interest of researchers. Safflower is a traditional Chinese medicinal material with effects such as promoting blood circulation to remove blood stasis and dredging meridians to relieve pain. Research shows that safflower has effects such as anti-myocardial ischemia, anticoagulation, and blood pressure lowering. HSYA is one of the components with relatively high content and important biological activities in safflower. It has been found that HSYA can promote the proliferation and osteogenic differentiation of bone marrow mesenchymal stem cells by affecting vitamin D receptors. At the same time, HSYA can reduce the apoptosis rate of bone marrow mesenchymal stem cells and delay their aging process.

[0005] However, there has been no report on the effect of HSYA on hUC-MSCs. Summary of the Invention

[0006] This application provides an application of hydroxysafflor yellow A in the preparation of medicaments for treating diseases that require repair using hUC-MSCs.

[0007] This application aims to explore the effect of HSYA on the repair function of hUC-MSCs and provide new ideas and methods for its application in the field of tissue repair and regenerative medicine.

[0008] hUC-MSCs are extracted from the umbilical cords of newborns. Umbilical cords are usually regarded as medical waste after the birth of newborns. Therefore, compared with stem cells from other sources, such as bone marrow mesenchymal stem cells which require bone marrow aspiration for acquisition, hUC-MSCs have the advantages of rich sources, easy access, and no harm to newborns and mothers.

[0009] HUC-MSCs also have low immunogenicity. When hUC-MSCs are transplanted into patients, it is difficult for the patient's immune system to recognize hUC-MSCs, and the possibility of causing immune rejection reactions is small. This enables hUC-MSCs to be used for treatment between different individuals and allows allogeneic transplantation without strict matching, expanding its scope of application. Especially in emergency situations, such as acute tissue injury or disease deterioration, its advantages are more obvious.

[0010] The multi-lineage differentiation potential and strong self-renewal ability of hUC-MSCs enable them to be amplified in large quantities in vitro, providing sufficient cell guarantee for clinical regenerative applications. They can differentiate into various cells for tissue repair such as bone, cartilage, and nerves.

[0011] In addition, there is another very important aspect. hUC-MSCs also have paracrine effects and can secrete a variety of bioactive molecules. These molecules can regulate cell growth, differentiation, and immune responses, and promote tissue repair and regeneration. Since hUC-MSCs are derived from the umbilical cords of newborns, they have high safety and are relatively stable during in vitro culture and amplification, and are not prone to mutation and carcinogenesis.

[0012] Due to these advantages, the application potential of hUC-MSCs in the fields of tissue engineering, immune disease treatment, etc. is becoming increasingly prominent. Therefore, more and more clinical trials now tend to use hUC-MSCs as a stem cell treatment method.

[0013] Although hUC-MSCs have many therapeutic advantages, due to problems such as the slow proliferation of hUC-MSCs after transplantation, the abnormal microenvironment at the disease injury site, and the small number of transplanted stem cells reaching the lesion area, the clinical treatment application effect of hUC-MSCs is limited. Therefore, how to improve the biological performance of hUC-MSCs is the key to solving the problem.

[0014] This application selects hUC-MSCs as the research object and HSYA as the intervention factor, and comprehensively evaluates the effect of HSYA on the functions of hUC-MSCs through multiple experiments such as detecting the proliferation ability, migration ability, and paracrine level of hUC-MSCs.

[0015] In a first aspect, the present application provides an application of hydroxysafflor yellow A in the preparation of a medicament for treating diseases that require repair by utilizing the proliferation ability, migration ability, and / or paracrine ability of hUC-MSCs.

[0016] Optionally, the hydroxysafflor yellow A can improve the viability of hUC-MSCs.

[0017] Optionally, the hydroxysafflor yellow A can promote the proliferation ability of hUC-MSCs.

[0018] Optionally, the hydroxysafflor yellow A can promote the migration ability of hUC-MSCs.

[0019] Optionally, the concentration of the hydroxysafflor yellow A is 100 - 400 μmol·L -1 .

[0020] Optionally, the concentration of the hydroxysafflor yellow A is 200 μmol·L -1 .

[0021] After stem cells are transplanted into the body through various channels, whether the stem cells can play an effective role, including the number of surviving stem cells after transplantation and whether these cells can successfully reach the lesion area (homing ability), is an important aspect.

[0022] Stem cells need to migrate to the damaged tissue site through blood circulation and other channels in the body. However, during this process, they are affected by various factors, such as hemodynamic factors and tissue barriers, resulting in a limited number of stem cells that can truly reach the lesion area and play a role. Even if a small number of stem cells reach the lesion area, their survival and function may be restricted under the influence of an adverse microenvironment. Therefore, enhancing the proliferation ability and migration ability of stem cells is one of the effective ways to solve the problem.

[0023] The present application finds that HSYA at three concentrations can significantly improve the proliferation ability of hUC-MSCs, and the 200 μmol·L -1 group has the best effect. In addition, the results of the scratch experiment show that HSYA has a promoting effect on the migration ability of hUC-MSCs, and the 200 μmol·L -1 group also has the best effect. Therefore, HSYA may play a promoting role in the treatment of stem cell transplantation-related diseases by improving the proliferation and homing ability of hUC-MSCs.

[0024] Optionally, the hydroxysafflor yellow A can promote hUC-MSCs to secrete VEGF and BDNF.

[0025] Optionally, the hydroxysafflor yellow A can promote the expression of FGF2, BDNF, and VEGF in hUC-MSCs.

[0026] As is well known, a key role of stem cell therapy for diseases is the paracrine mechanism, and the paracrine effect plays an important role in angiogenesis and nerve restoration. Stem cells exert their therapeutic effects by secreting a variety of bioactive factors, such as VEGF, BDNF, FGF2, etc. FGF2 is a polypeptide growth factor that has the functions of promoting cell proliferation, angiogenesis, and nerve repair, and plays an important role in the field of tissue repair. FGF2 can induce the differentiation of hUC-MSCs, help them transform into various cell types, and enhance the anti-apoptotic ability of cells; VEGF also has an angiogenic effect and is highly specific. hUC-MSCs improve local blood circulation and promote angiogenesis by secreting VEGF. At the same time, VEGF acts on hUC-MSCs in an opposite way to regulate their functions and survival status; BDNF is crucial for the survival, growth, and differentiation of neurons. It can enhance synaptic plasticity and play a neuroprotective role. hUC-MSCs play a nerve repair role by secreting BDNF.

[0027] The Western blot results of this application show that HSYA can increase the protein contents of BDNF and VEGF in hUC-MSCs, and the ELISA detection results show that HSYA can promote hUC-MSCs to secrete more BDNF and VEGF. In summary, HSYA exerts its effects by promoting hUC-MSCs to synthesize BDNF and VEGF, thereby secreting more BDNF and VEGF.

[0028] In a second aspect, this application provides a composition. The composition includes the above-mentioned hydroxysafflor yellow A.

[0029] In a third aspect, this application provides the use of the above composition in the preparation of a medicament for treating diseases that require repair by utilizing the proliferation ability, migration ability, and / or paracrine ability of hUC-MSCs.

[0030] In summary, this application includes at least one of the following beneficial technical effects:

[0031] This application preliminarily proves that HSYA can promote the proliferation ability and homing ability of hUC-MSCs, and at the same time, by promoting its paracrine effect, hUC-MSCs secrete more bioactive factors such as BDNF and VEGF. These factors may enhance the angiogenesis-promoting ability and nerve repair-promoting ability of hUC-MSCs, providing a reference basis for improving the therapeutic effect of hUC-MSCs in clinical research. Description of the Drawings

[0032] Figure 1 Observation results of hUC-MSCs under an inverted microscope (A. 40×; B. 100×).

[0033] Figure 2 Results of detecting the expression of surface markers on hUC-MSCs by flow cytometry (A. Images of surface marker expression; B. Positive expression rates of surface markers).

[0034] Figure 3 Results of identifying the osteogenic and adipogenic differentiation abilities of hUC-MSCs (A. Control; B. Osteogenic differentiation; C. Adipogenic differentiation).

[0035] Figure 4 Results of detecting the effect of HSYA on hUC-MSCs at different concentrations and different times.

[0036] Figure 5 Results of detecting the effect of HSYA on the proliferation ability of hUC-MSCs (A. Staining results; B. Ratios of proliferating cells at different concentrations of HSYA).

[0037] Figure 6 Results of detecting the effect of HSYA on the paracrine ability of hUC-MSCs (ELISA method, A. Contents of VEGF in the supernatant at different concentrations of HSYA; B. Contents of BDNF in the supernatant at different concentrations of HSYA).

[0038] Figure 7 Results of detecting the effect of HSYA on the paracrine ability of hUC-MSCs (Western Blot method, A. Electrophoresis results of various proteins in hUC-MSCs at different concentrations of HSYA; B. Relative expression levels of FGF2 at different concentrations of HSYA; C. Relative expression levels of BDNF at different concentrations of HSYA; D. Relative expression levels of VEGF at different concentrations of HSYA).

[0039] Figure 8 Results of detecting the effect of HSYA on the migration ability of hUC-MSCs (A. Observation results of hUC-MSCs at each time point under an inverted microscope at different concentrations of HSYA; B. Cell migration rates of hUC-MSCs at each time point at different concentrations of HSYA). Detailed implementation manners

[0040] Before describing the embodiments of the present application in detail, it should be understood that the terms used herein are for the purpose of describing specific embodiments only. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which this term belongs.

[0041] It should be noted that the terms "first" and "second" are only used for descriptive purposes and should not be construed as indicating or implying relative importance or implicitly specifying the quantity of the indicated technical features. Thus, features defined with "first" and "second" may explicitly or implicitly include one or more of such features. Further, in the description of the present application, unless otherwise specified, the meaning of "a plurality" is two or more.

[0042] In the ranges disclosed in the present application, the endpoints and any values of the ranges are not limited to the exact ranges or values. These ranges or values should be understood to include values close to these ranges or values. For numerical ranges, between the endpoint values of each range, between the endpoint values of each range and individual point values, and between individual point values, they can be combined with each other to obtain one or more new numerical ranges, and these numerical ranges should be regarded as specifically disclosed herein.

[0043] In the present application, the term "comprising" or "including" is an open expression, that is, it includes the content specified in the present application, but does not exclude other aspects.

[0044] To make the purpose, technical solutions and advantages of the present application clearer, the technical solutions in the embodiments of the present application will be described clearly and completely below. Based on the embodiments of the present application, all other embodiments obtained by those skilled in the art without creative efforts fall within the scope of protection of the present application. The following described embodiments are exemplary and are only used to explain the present application and should not be construed as a limitation of the present application.

[0045] For those not specifying specific techniques or conditions in the embodiments, follow the techniques or conditions described in the literature in the field or according to the product specifications. For reagents or instruments not indicating the manufacturer, they are all conventional products that can be obtained through commercial purchase.

[0046] In the following embodiments, HSYA was purchased from Shanghai Ronghe Pharmaceutical Technology Development Co., Ltd., batch number: 230109, product number: THT317 - 20mg.

[0047] The present application will be further described in detail below in conjunction with the embodiments and the test results.

[0048] Embodiment

[0049] Embodiment 1

[0050] This embodiment provides a process for the extraction and identification of hUC - MSCs.

[0051] Specifically, it includes the following steps:

[0052] (I) Extraction of hUC - MSCs

[0053] Soak the umbilical cord tissue in sterile normal saline; remove blood stains, mucus and coagulation; put it into 75% alcohol for disinfection for 2 minutes, and then wash it with sterile normal saline; cut the umbilical cord tissue into 2-3 cm segments; remove blood vessels.

[0054] Wash Wharton's jelly (a special gel-like connective tissue in umbilical cord tissue) 2-3 times; cut Wharton's jelly into pieces of 1-4 mm 3 Paste; take 2 g of Wharton's jelly and inoculate it into a T75 cell culture flask with 15 ml of high-glucose DMEM / F12 complete medium containing 10% fetal bovine serum (FBS); culture it in a constant temperature incubator at 37 °C and 5% CO2; change the medium after 5-6 days, and passage the cells at a ratio of 1:3 when 80-90% of the cells are confluent. The third-generation hUC-MSCs are used for subsequent experiments.

[0055] The extraction results are as follows:

[0056] Place the third-generation hUC-MSCs extracted and cultured under an inverted microscope to observe their morphology. The observation results are as Figure 1 shown.

[0057] It can be seen from Figure 1 that the cell morphology is consistent, most of them are spindle-shaped and fibrous, growing adherently, and showing a radial or vortex shape after cell fusion, which conforms to the morphological characteristics of mesenchymal stem cells.

[0058] (2) Identification of hUC-MSCs

[0059] (1) Identification by flow cytometry

[0060] Prepare the cells to be tested into a single-cell suspension, and add CD45, CD44, CD105, and CD34 antibodies respectively; incubate at room temperature in the dark for 15 minutes, wash the cells with PBS buffer; detect the expression of the labeled proteins on the cell surface by flow cytometry, record the data, and analyze and process it.

[0061] The identification results are as follows:

[0062] The results of flow cytometry detection are as Figure 2 shown.

[0063] Figure 2 It shows that the positive expression rates of CD105 and CD44 in hUC-MSCs are 99.70% and 96.86% respectively, and the positive expression rates of CD45 and CD34 are 0.41% and 0.40% respectively, which conform to the characteristics of mesenchymal stem cell surface markers.

[0064] (2) Identification of osteogenic and adipogenic differentiation

[0065] Inoculate the cells to be tested into a 24-well plate at an inoculation density of 5×104 Cells / well were placed in a constant-temperature and humidified incubator for culture. When the confluence reached 60 - 70%, they were respectively replaced with osteogenic induction differentiation culture medium (manufacturer: Cyagen Biosciences Inc.; model: HUXXC - 90021) or adipogenic induction differentiation culture medium (manufacturer: Haixing Bio; model: UBHX - D102R). Meanwhile, a group without replacing the culture medium was set as the control group.

[0066] After culturing for 21 days according to the operation instructions of the differentiation culture medium products respectively, they were washed 3 times with PBS, fixed with 4% paraformaldehyde for 30 min, stained with alizarin red staining solution (for osteogenesis detection, manufacturer: Cyagen Biosciences Inc.; model: ALIR - 10001) / oil red "O" staining solution (for adipogenesis detection, manufacturer: Cyagen Biosciences Inc.; model: OILR - 10001) for 10 min, washed 1 - 2 times with PBS, and observed and photographed under an inverted microscope.

[0067] The identification results were as follows:

[0068] The staining results of hUC - MSCs after osteogenic and adipogenic induction differentiation were as Figure 3 shown.

[0069] It can be Figure 3 seen that a large number of cells in hUC - MSCs could be stained red by alizarin red and oil red "O", indicating that hUC - MSCs could differentiate into calcium nodules and lipid droplets after induced differentiation. Thus, it can be known that the extracted hUC - MSCs have the potential of osteogenic and adipogenic differentiation.

[0070] Example 2

[0071] In this example, HSYA was used to act on hUC - MSCs, and the effect of HSYA on the viability of hUC - MSCs was explored.

[0072] The specific process was as follows:

[0073] The cells to be tested were respectively inoculated into 96 - well culture plates with a cell density of 0.8×10 4 cells / well, 100 μL per well, placed in an incubator at 37°C with 5% CO2 for overnight adhesion. After washing the cells with PBS, 0, 100, 200, 400, 600 μmol·L -1HSYA at the concentration was added into 96-well culture plates, with 3 replicate wells in each group and 3 replicate wells set at each time point. At 1d, 2d, 3d, and 4d of culture respectively, the drugs were aspirated, and 100 μL of basal medium and 10 μL of CCK-8 reagent (manufacturer: Dalian Meilun Biotechnology Co., Ltd.; model: MA0218-2) were added to each well. After incubation in a constant temperature incubator at 37 °C and 5% CO2 for 1 h, the absorbance at 450 nm was measured using an enzyme-linked immunosorbent assay (ELISA) reader, with a reference wavelength of 600 nm. The group without adding HSYA was the control group.

[0074] The detection results are as follows:

[0075] The CCK-8 method was used to determine the optimal drug concentration and action time of HSYA on hUC-MSCs. The detection results are as Figure 4 shown.

[0076] As Figure 4 can be seen, HSYA can improve the viability of hUC-MSCs. Compared with the control group, at 1d, only the cell viability of the 200 μmol·L -1 group was significantly increased (P < 0.01), and there were no significant differences in the other groups; at 2d, HSYA at each concentration group could increase the viability of hUC-MSCs (P < 0.01); at 3d, the cell viability of the 100, 200, and 400 μmol·L -1 cell groups was significantly increased (P < 0.05 or P < 0.01); at 4d, the cell viability of the 100 and 200 μmol·L -1 groups was significantly increased (P < 0.05 or P < 0.01), and the cell viability of the 600 μmol·L -1 group was lower than that of the control group (P < 0.01). At the same time, as Figure 4 can be seen, only the cell viability of the 200 μmol·L -1 group was significantly increased at different time periods, and the cell viability was the highest at 2d. At 2d, compared with the 100, 400, and 600 μmol·L -1 groups, the cell viability of the 200 μmol·L -1 group was the highest (P < 0.01).

[0077] Therefore, it is considered that 200 μmol·L -1 of HSYA is the optimal drug concentration, and 2d is the optimal action time of HSYA. In subsequent experiments, the concentrations of HSYA were 100, 200, and 400 μmol·L -1 respectively, and the action time was 2d.

[0078] Example 3

[0079] This example detected the effect of HSYA on the proliferation ability of hUC-MSCs.

[0080] The specific process is as follows:

[0081] The hUC-MSCs were divided into 4 groups, namely the control group, the 100 μmol·L -1 group, the 200 μmol·L -1 group, and the 400 μmol·L -1 group. The cells to be tested were inoculated into a 24-well culture plate containing cell slides at a density of 5×10 4 . After overnight adherence, the cells were washed. Then, HSYA at concentrations of 0, 100, 200, and 400 μmol·L -1 was added to the culture plate respectively. After 2 days of culture, half of the medium was replaced with pre-warmed EdU working solution (manufacturer: Shanghai Beyotime Biotechnology Co., Ltd.; model: C0071S) at 37 °C, and the culture continued for another 2 days. The cells were fixed with 4% paraformaldehyde for 30 min, permeabilized with 0.2% Triton X-100 for 10 min, washed 3 times with the washing solution, and then incubated with Click reaction solution (manufacturer: Shanghai Beyotime Biotechnology Co., Ltd.; model: C0071S, containing the fluorescent labeling component Azide 488) in the dark for 30 min, followed by washing 3 times with the washing solution. The nuclei were stained with Hoechst 33342 (a nuclear fluorescent dye mainly used for DNA staining of live or fixed cells, CAS: 23491-52-3) for 10 min, and then mounted with a fluorescence quenching mounting solution (manufacturer: Shanghai Beyotime Biotechnology Co., Ltd.; model: P0137). Five fields of view were randomly selected at 100× and photographed under an upright fluorescence microscope. The number of EdU-positive cells and the total number of nuclei were counted respectively using Image J software, and the average values were calculated.

[0082] The calculation formula for the EdU-positive cell rate is as follows: EdU-positive cell rate = number of EdU-positive cells / total number of cells × 100%.

[0083] The detection results are as follows:

[0084] The results of EdU staining detection are as Figure 5 shown.

[0085] It can be seen from Figure 5 that compared with the control group, the number of EdU-positive cells in the 3 HSYA concentration groups was significantly increased (P < 0.01); compared with the 100 μmol·L -1 group and the 400 μmol·L -1 group, the 200 μmol·L -1The number of EdU-positive cells in the [group name] was the highest (P < 0.01 or P < 0.01). It can be seen that HSYA in the three concentration groups can significantly promote the proliferation ability of hUC-MSCs, and compared with the 100 μmol·L -1 group and the 400 μmol·L -1 group, the 200 μmol·L -1 group had the best proliferation effect. The results show that HSYA has a promoting effect on the proliferation of hUC-MSCs.

[0086] Example 4

[0087] In this example, the effect of HSYA on the paracrine ability of hUC-MSCs was detected.

[0088] The specific process is as follows:

[0089] (1) Detection by enzyme-linked immunosorbent assay

[0090] The specific process is as follows:

[0091] hUC-MSCs were divided into 4 groups, namely the control group, the 100 μmol·L -1 group, the 200 μmol·L -1 group, and the 400 μmol·L -1 group. The cells to be tested were inoculated into a 24-well culture plate at a density of 5×10 4 cells / well, and HSYA at concentrations of 0, 100, 200, and 400 μmol·L -1 was added to the culture plate respectively, and then cultured in a constant temperature and humidified incubator for 2 days. The culture supernatants of the 4 groups of hUC-MSCs were collected respectively, and the contents of VEGF and BDNF in the supernatants were detected with reference to the instructions of an enzyme-linked immunosorbent assay (ELISA) kit (manufacturer: andygene company; model: AD10657Hu9 (VEGF), AD11145Hu (BDNF)).

[0092] The detection results are as follows:

[0093] The ELISA detection results are as Figure 6 shown.

[0094] It can be Figure 6 seen that compared with the control group, the VEGF and BDNF in the supernatants of hUC-MSCs in the three HSYA concentration groups were significantly increased (P < 0.05 or P < 0.01). It shows that HSYA can promote the secretion of VEGF and BDNF by hUC-MSCs. Compared with the 100 μmol·L -1 group and the 400 μmol·L-1 Compared with the [group name], 200 μmol·L -1 The levels of VEGF and BDNF in the supernatant of the [group name] were the highest (P < 0.05), indicating that 200 μmol·L -1 The concentration of HSYA had the best promoting effect on the secretion of VEGF and BDNF by hUC-MSCs.

[0095] (2) Detection by Western Blot

[0096] The specific process is as follows:

[0097] hUC-MSCs were divided into 4 groups, namely the control group, 100 μmol·L -1 group, 200 μmol·L -1 group, 400 μmol·L -1 group. RIPA protein lysate (manufacturer: Shanghai Beyotime Biotechnology Co., Ltd.; model: P0013B) was used to extract the cell proteins of the 4 groups of hUC-MSCs. After taking 30 μg of protein and performing 10% SDS-PAGE gel electrophoresis, it was transferred to a PVDF membrane. It was blocked with 5% skim milk powder at room temperature for 2 h. The primary antibodies (the concentrations of BDNF, VEGF, FGF2, and Tubulin were 1:500, 1:500, 1:1000, and 1:2000 respectively) were incubated overnight at 4°C, and the secondary antibody (the concentration of goat anti-rabbit antibody was 1:8000) was incubated for 1.5 h. The membrane was washed with TBST. After adding the developing solution, a protein chemiluminescence imaging system was used to take pictures. Using Image J software, with Tubulin as the internal reference, the relative gray value was calculated. The protein expression levels of FGF2, BDNF, and VEGF in hUC-MSCs were detected.

[0098] The calculation method of the relative protein expression level is as follows: Relative protein expression level = gray value of the target protein / gray value of Tubulin.

[0099] The detection results are as follows:

[0100] The detection results of Western Blot are as Figure 7 shown.

[0101] As Figure 7 can be seen, compared with the control group, the 3 HSYA concentration groups could all promote the increase in the protein expression of FGF2, BDNF, and VEGF (P < 0.05, P < 0.01). It can be seen from the statistical chart that the 200 μmol·L -1 group had the best promoting effect on the expression levels of the 3 proteins. The results showed that HSYA in each concentration group had a promoting effect on the protein expression of FGF2, BDNF, and VEGF in hUC-MSCs.

[0102] Example 5

[0103] This example detected the effect of HSYA on the migration ability of hUC-MSCs.

[0104] The specific process is as follows:

[0105] Scratch assay: hUC-MSCs were seeded in 6-well culture plates at a density of 5×10 5 cells / well. After overnight attachment, the cells were washed with PBS. HSYA at concentrations of 0, 100, 200, and 400 μmol·L -1 was added to the culture plates. After 2 days of culture, a "cross" scratch was made in the center of each well with a 200-μL sterile pipette tip. The wells were gently washed 1-2 times with PBS and then replaced with basal medium. The plates were placed in an incubator at 37°C with 5% CO2 and observed and photographed under an inverted microscope at 0, 12, 24, 48, and 72 h, respectively.

[0106] The detection results are as follows:

[0107] The detection results of the scratch assay are as Figure 8 shown.

[0108] It can be Figure 8 seen that the migration speeds of the three HSYA concentration groups at each time period were better than those of the control group. Compared with the control group, the 200 μmol·L -1 group had the fastest migration speed at 12 h (P < 0.05); after 1 day, the three HSYA concentration groups all showed a faster migration speed (P < 0.01). Compared with the 100 μmol·L -1 group and the 400 μmol·L -1 group, the 200 μmol·L -1 group had the fastest migration speed at each time period, and the migration speed was more significant after 2 days (P < 0.01). The results indicate that HSYA can promote the migration ability of hUC-MSCs, and the promoting effect is best at a concentration of 200 μmol·L -1 concentration.

[0109] In the description of this specification, the descriptions referring to terms such as "one embodiment", "some embodiments", "examples", "specific examples", or "some examples" mean that the specific features, structures, materials, or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of this 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, without contradiction, those skilled in the art can combine and combine the different embodiments or examples described in this specification and the features of different embodiments or examples.

[0110] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of this application, rather than to limit them; although this application has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions recorded in the foregoing embodiments, or perform equivalent replacements on some of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of this application.

Claims

1. Application of hydroxysafflor yellow A in the preparation of a drug for treating a disease requiring repair of the proliferation ability, migration ability and / or paracrine ability of hUC-MSCs.

2. The use according to claim 1, characterized in that: The hydroxysafflor yellow A can improve the activity of hUC-MSCs.

3. The use according to claim 1, characterized in that: The hydroxysafflor yellow A can promote the proliferation ability of hUC-MSCs.

4. The use according to claim 1, characterized in that: The hydroxysafflor yellow A can promote the migration ability of hUC-MSCs.

5. The use according to claim 1, characterized in that: The hydroxysafflor yellow A can promote hUC-MSCs to secrete VEGF and BDNF.

6. The use according to claim 1, characterized in that: The hydroxysafflor yellow A can promote the expression of FGF2, BDNF and VEGF in hUC-MSCs.

7. The use according to claim 1, characterized in that: The concentration of hydroxysafflor yellow A is 100-400 μmol·L -1 .

8. The use according to claim 1, characterized in that: The concentration of hydroxysafflor yellow A is 200 μmol·L -1 .

9. A composition, characterized in that The composition comprises the hydroxysafflor yellow A according to any one of claims 1 to 8.

10. Use of the composition according to claim 9 in the preparation of a composition for treating a disease requiring restoration of the proliferation ability, migration ability and / or paracrine ability of hUC-MSCs.

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