Application of sialic acid, hyaluronic acid and resveratrol in preparation of medicine for promoting stem cell migration

Abstract

The invention relates to the technical field of medicine, in particular to application of sialic acid, hyaluronic acid and resveratrol in preparation of a medicine for promoting stem cell migration, the administration concentration of the sialic acid is 2.5 mM, the administration concentration of the hyaluronic acid is 300 mu g / mL, and the administration concentration of the resveratrol is 5-20 mu M. Cubilose acid, hyaluronic acid and resveratrol can improve the migration capacity of the amniotic fluid mesenchymal stem cells, and proliferation of the amniotic fluid mesenchymal stem cells is better promoted.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to the use of bird's nest acid, hyaluronic acid and resveratrol in the preparation of drugs that promote stem cell migration. Background Art

[0002] Sialic acid (SA), also known as sialic acid, is a unique α-keto acid. Currently, more than 50 forms of sialic acid have been found in nature, the most important of which is N-acetylneuraminic acid. SA is naturally present in many animal foods, such as bird's nests, breast milk and eggs. Sialic acid has the effects of promoting brain development, anti-virus, anti-Alzheimer's disease and anti-hypertension. In addition, SA also has anti-aging and antioxidant effects.

[0003] Hyaluronic acid (HA), also known as hyaluronic acid, is a naturally occurring acidic mucopolysaccharide with excellent moisturizing properties, widely used in the pharmaceutical, cosmetic, and food industries. Studies have shown that HA's biocompatibility, biodegradability, and ease of chemical modification can promote wound healing by promoting hemostasis, regulating inflammation, and promoting epithelial regeneration. Studies have combined HA with glutamic acid (Glu) to explore the efficacy of this complex, and the results show that the use of HA-Glu complexes in skincare formulations can effectively alleviate skin symptoms caused by inflammation and skin aging.

[0004] Resveratrol (RSV) is a non-flavonoid polyphenolic phytoalexin with a stilbene structure. First discovered in 1963 in the root extract of Veratum grandiflorum, it is also found in the root of Polygonum cuspidatum and is widely used in Traditional Chinese Medicine. As a natural medicine, RSV has broad therapeutic potential due to its antioxidant, anti-inflammatory, and anti-cancer properties, protecting various tissues from acute and chronic damage. Studies have shown that at concentrations of 0.1, 1, and 2.5 μM, RSV increases SIRT1 levels while inhibiting the expression of p53 and p16, thereby promoting cell viability and reducing the aging of MSCs; studies have shown that preparations containing resveratrol can stimulate the proliferation of fibroblasts and help increase the concentration of type III collagen because resveratrol has an antioxidant effect. It protects cells from oxidative damage to the skin caused by free radicals and ultraviolet radiation by reducing the expression of AP-1 and NF-kB factors, thereby slowing down the photoaging process of the skin; in 2023, Raffaele Conte et al. found that hyaluronic acid hydrogels loaded with resveratrol-containing chitosan nanoparticles can be used as an adjuvant for the treatment of atopic dermatitis.

[0005] Stem cells are a core tool in regenerative medicine. Amniotic fluid mesenchymal stem cells (AFMSCs) have multipotential differentiation potential, capable of differentiating into cells such as fat, bone, and cartilage. They are also low immunogenic and have a low risk of allogeneic transplant rejection. AFMSGs are obtained from prenatal amniotic fluid and are ethically uncontroversial. Stem cell therapy has gained significant attention in recent years, but issues such as low homing efficiency and poor microenvironmental adaptability persist. Improving stem cells' ability to migrate, enhancing their adaptability to the environment, and prolonging their duration of action are key to addressing these challenges. Summary of the Invention

[0006] In order to solve the above technical problems, embodiments of the present invention provide the use of bird's nest acid, hyaluronic acid and resveratrol in the preparation of drugs that promote stem cell migration.

[0007] To achieve the above objectives, the embodiments of the present invention adopt the following technical solutions:

[0008] The present invention provides an application of guanidine, hyaluronic acid and resveratrol in the preparation of a drug for promoting stem cell migration.

[0009] In some embodiments, the dosage concentration of the sialic acid is 2.5 mM, the dosage concentration of the hyaluronic acid is 300 μg / mL, and the dosage concentration of the resveratrol is 5-20 μM.

[0010] In some embodiments, the dosage concentration of the sialic acid is 2.5 mM, the dosage concentration of the hyaluronic acid is 300 μg / mL, and the dosage concentration of the resveratrol is 20 μM.

[0011] In some embodiments, the stem cells are amniotic fluid mesenchymal stem cells.

[0012] In some embodiments, the above-mentioned drug includes a pharmaceutical preparation prepared with an effective amount of bird's nest acid and hyaluronic acid as active ingredients, and pharmaceutically acceptable carriers and / or excipients.

[0013] An effective amount refers to the amount of a drug, compound, or pharmaceutical composition necessary to obtain any one or more beneficial or desired therapeutic results. Beneficial or desired results include: clinical improvement (such as reducing morbidity, reducing mortality, improving one or more symptoms), reducing severity, delaying the onset of symptoms (including symptoms or their complications, intermediate pathological phenotypes presented during the development of the disease, biochemistry, histology, and / or behavioral symptoms). The preparation can be an injection, spray, aerosol, nasal drops, oral preparation, or a dosage form suitable for mucosal administration. In addition, the route of administration to the subject includes, but is not limited to, intramuscular, intravenous, subcutaneous, intradermal, oral, intranasal, respiratory, transmucosal, sublingual, and parenteral.

[0014] The present invention has the following beneficial effects compared to the prior art:

[0015] The combination of bird's nest acid, hyaluronic acid and resveratrol promotes the proliferation of amniotic fluid mesenchymal stem cells by enhancing their migration ability, and the cell migration ability is significantly increased. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 This is a bright field microscope image of amniotic fluid mesenchymal stem cells (AFMSCs);

[0017] Figure 2 This is a physical picture of Sialic acid powder (SA);

[0018] Figure 3 This is a physical picture of hyaluronic acid (HA);

[0019] Figure 4 This is a physical picture of resveratrol (RSV);

[0020] Figure 5 The cell status diagram of the Control group and AFMSCs+RSV group at 0h;

[0021] Figure 6 Figure 24 shows the cell status of the Control group and the AFMSCs+RSV group at 24 hours;

[0022] Figure 7 Figure 48 shows the cell status of the Control group and the AFMSCs+RSV group;

[0023] Figure 8 Statistical analysis of cell scratch test between Control group and AFMSCs+RSV group;

[0024] Figure 9 Cell status diagrams of the Control group, AFMSCs+SA group, AFMSCs+HA, AFMSCs+RSV and AFMSCs+SA+HA+RSV groups at 0 h;

[0025] Figure 10 Cell status diagrams of the Control group, AFMSCs+SA group, AFMSCs+HA, AFMSCs+RSV and AFMSCs+SA+HA+RSV groups at 24 h;

[0026] Figure 11 Cell status diagrams of the Control group, AFMSCs+SA group, AFMSCs+HA, AFMSCs+RSV and AFMSCs+SA+HA+RSV groups at 48 h;

[0027] Figure 12Statistical analysis chart of cell scratch experiment in Control group, AFMSCs+SA group, AFMSCs+HA, AFMSCs+RSV and AFMSCs+SA+HA+RSV group. DETAILED DESCRIPTION

[0028] The following will be combined with the accompanying drawings to clearly and completely describe the technical solutions in some embodiments of the present disclosure. Obviously, the embodiments described are only some embodiments of the present disclosure, not all embodiments. Based on the embodiments provided by the present disclosure, all other embodiments obtained by ordinary technicians in this field are within the scope of protection of the present disclosure.

[0029] Unless otherwise specified, all raw materials, reagents, instruments and equipment used in this application can be purchased from the market or prepared by existing methods.

[0030] Experimental Materials:

[0031] (1) Cells

[0032] Amniotic fluid mesenchymal stem cells (AFMSCs) were derived from clinical amniotic fluid samples from the Third Affiliated Hospital of Guangzhou Medical University.

[0033] (2) Materials

[0034] Swallow acid (SA): purchased from MCE, CAS No.: 131-48-6.

[0035] Hyaluronic acid (HA): purchased from MCE, CAS No.: 9004-61-9.

[0036] DMEM culture medium: 1% fetal bovine serum + 1% P / S double antibody (final concentration of 50 U / mL) + 98% low-glucose DMEM basal culture medium.

[0037] Example

[0038] The experiment was divided into two parts. The first part was to screen out the optimal concentration of resveratrol. The second part was to conduct a scratch test using the screened optimal concentration to further explore the effect of the mixture of SA, HA and RSV on the migration of AFMSCs.

[0039] The experimental groups in the first part were: Control group, AFMSCs+RSV (5, 10, 15, 20 μM) group;

[0040] The second part of the experiment was divided into: Control group, AFMSCs + SA (2.5 mM) group; AFMSCs + HA (300 μg / mL) group, AFMSCs + SA (2.5 mM) + HA (300 μg / mL) + RSV (20 μM) group. The experimental procedures of the AFMSCs + SA (2.5 mM) group and the AFMSCs + HA (300 μg / mL) group were the same as those of the AFMSCs + SA (2.5 mM) + HA (300 μg / mL) + RSV (20 μM) group.

[0041] The specific experimental steps of the first part of the experiment are as follows:

[0042] First, 10 mg of resveratrol powder was dissolved in 43.8 mL of pure water to prepare a resveratrol solution with a concentration of 1 mM. The solution was filtered with a 0.22 μm disposable microporous membrane filter to remove impurities, and the mother liquor with a concentration of 1 mM resveratrol solution was diluted with DMEM culture medium to resveratrol solutions with concentrations of 5 μM, 10 μM, 15 μM, and 20 μM.

[0043] Place 10 5 Amniotic fluid-derived mesenchymal stem cells were evenly plated in a six-well plate. When the cell density reached 90%, the wells of the six-well plate were scratched with a 200μL pipette tip, maintaining a roughly uniform scratch width in each well. The cells were then incubated in pre-prepared culture medium containing varying concentrations of resveratrol. A control group was maintained without resveratrol, i.e., at a resveratrol concentration of 0μM.

[0044] like Figure 5 The cells were in the state at 0 h. A 1 cm wide scratch was made with a 200 μL pipette tip, and different concentrations of resveratrol were added. The cells were cultured at 37° C. and 5% CO 2 .

[0045] like Figure 6 The cells have been cultured in the medium containing resveratrol for 24 hours. The cell density has changed, and the scratch width has also narrowed. Compared with the control group (resveratrol concentration of 0μM), the cell density in the medium containing resveratrol has increased and the migration ability has increased.

[0046] like Figure 7 The cells were cultured in resveratrol-containing medium for 48 hours. The cell density changed significantly, and the scratch width became narrower. Compared to the control group (resveratrol concentration of 0 μM), the cell density in the resveratrol-containing medium reached almost 100%, and the migrating cells filled the gap in the scratch.

[0047] like Figure 8Statistical analysis was performed on the changes in the scratch width. The results showed that compared with the control group (resveratrol concentration was 0 μM), as the resveratrol concentration increased, the cell proliferation rate became faster and the cell migration ability increased.

[0048] The specific experimental steps of the second part of the experiment are as follows:

[0049] First, 100 mg of sialic acid powder was dissolved in 10 mL of pure water to prepare a sialic acid solution with a concentration of 100 mM, and the solution was filtered with a 0.22 μm disposable microporous membrane filter to remove impurities. The 100mM sialic acid solution was diluted to a 2.5mM sialic acid solution with 1% fetal bovine serum + 1% P / S double antibody (final concentration of 50U / mL) + 98% low-glucose DMEM basal culture medium; 100mg hyaluronic acid powder was dissolved in 20mL of pure water until it became a transparent colloid to prepare a 4.16μM hyaluronic acid solution, and the 4.16μM hyaluronic acid solution was diluted to a 300μg / mL hyaluronic acid solution with 1% fetal bovine serum + 1% P / S double antibody (final concentration of 50U / mL) + 98% low-glucose DMEM basal culture medium; 10mg resveratrol powder was dissolved in 43.8mL of pure water to prepare a 1mM resveratrol solution, which was filtered with a 0.22μm disposable microporous membrane filter to remove impurities. The resveratrol solution with a mother solution concentration of 1 mM was diluted to a resveratrol solution with a concentration of 20 μM using 1% fetal bovine serum + 1% P / S double antibody (final concentration of 50 U / mL) + 98% low-glucose DMEM basal culture medium solution.

[0050] 10 per well 5 Amniotic fluid mesenchymal stem cells were evenly spread in a six-well plate. When the cell density reached 90%, a 200μL pipette tip was used to scratch the wells of the six-well plate to keep the width of the scratches in each well at 1cm. 50μL of 2.5mM bird's nest acid solution + 120μL of 300μg / mL hyaluronic acid solution + 40μL of 20μM resveratrol solution + 1790μL of 1% fetal bovine serum + 1% P / S double antibody (final concentration of 50U / mL) + 98% low-glucose DMEM basal medium solution were added for incubation. The control group did not add bird's nest acid, hyaluronic acid, and resveratrol, that is, the concentrations of bird's nest acid, hyaluronic acid, and resveratrol were 0.

[0051] like Figure 9The cells were in the state of 0 h. At this time, a 200 μL pipette tip was used to scratch the cells with a width of 1 cm, and 50 μL of 2.5 mM bird's nest acid solution, 120 μL of 300 μg / mL hyaluronic acid solution, 40 μL of 20 μM resveratrol solution and 1790 μL of 1% fetal bovine serum + 1% P / S double antibody (final concentration of 50 U / mL) + 98% low-glucose DMEM basal culture medium solution were added. The cells were cultured at 37 ° C and 5% CO2.

[0052] like Figure 10 The cells were cultured for 24 hours in a medium containing sialic acid, hyaluronic acid, and resveratrol. The cell density changed, and the scratch width narrowed accordingly. Compared with the AFMSCs+SA (2.5mM) and AFMSCs+HA (300μg / mL) groups, the cells containing sialic acid, hyaluronic acid, and resveratrol had increased cell density and enhanced migration ability.

[0053] like Figure 11 The cells were cultured for 48 hours in a medium containing sialic acid, hyaluronic acid, and resveratrol. The cells had undergone a greater change in cell density and the width of the scratch became narrower. Compared with the AFMSCs+SA (2.5mM) and AFMSCs+HA (300μg / mL) groups, the cell density in the medium containing sialic acid, hyaluronic acid, and resveratrol reached almost 100%, and the migrating cells filled the gaps in the scratch.

[0054] like Figure 12 Statistical analysis was performed on the changes in the scratch width. The results showed that compared with the AFMSCs+SA (2.5mM) group and the AFMSCs+HA (300μg / mL) group, the mixed addition of bird's nest acid, hyaluronic acid and resveratrol at 24h and 48h could significantly promote the migration of AFMSCs.

[0055] The results of the first part of the experiment showed that compared with the control group, the increase of RSV concentration significantly promoted the migration of AFMSCs. At 48h, the migration rate of AFMSCs was 100% when the concentration of RSV was 20μM. The results of the second part of the experiment showed that compared with the AFMSCs+SA (2.5mM) group and the AFMSCs+HA (300μg / mL) group, the mixed addition of SA, HA and RSV at 24h and 48h could significantly promote the migration of AFMSCs.

[0056] In summary, the mixture of bird's nest acid, hyaluronic acid and resveratrol can enhance the migration ability of amniotic fluid mesenchymal stem cells and better promote the proliferation of amniotic fluid mesenchymal stem cells.

[0057] In the description of this specification, specific features, structures, materials or characteristics may be combined in an appropriate manner in any one or more embodiments or examples.

[0058] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any modifications or substitutions that can be easily conceived by a person skilled in the art within the technical scope disclosed in the present invention should be included in the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be based on the scope of protection of the claims.

Claims

1. Application of bird's nest acid, hyaluronic acid and resveratrol in the preparation of drugs that promote stem cell migration.

2. The use according to claim 1, characterized in that The administration concentration of the guanidine hydrochloride is 2.5 mM, the administration concentration of the hyaluronic acid is 300 μg / mL, and the administration concentration of the resveratrol is 5-20 μM.

3. The use according to claim 2, characterized in that The administration concentration of the sialic acid is 2.5 mM, the administration concentration of the hyaluronic acid is 300 μg / mL, and the administration concentration of the resveratrol is 20 μM.

4. The use according to claim 1, characterized in that The stem cells are amniotic fluid mesenchymal stem cells.

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