Application of baicalein in preparation of medicine for inhibiting stem cell oxidation and / or ferroptosis

Abstract

The invention relates to the technical field of medicine, in particular to application of baicalein in preparation of a medicine for inhibiting stem cell oxidation and / or ferroptosis, the administration concentration of the baicalein is 0.5-5 mu M. The baicalein can inhibit ferroptosis of mesenchymal stem cells and antagonize oxidative stress, can improve the survival rate of the stem cells and enhance the immune regulation effect, and can be used for preparing a medicine for inhibiting stem cell oxidation and / or ferroptosis. Therefore, the curative effect of stem cell treatment is promoted.

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical technology, and particularly to the application of baicalein in the preparation of drugs for inhibiting stem cell oxidation and / or ferroptosis. Background Art

[0002] Stem cells are a type of cells with unlimited or immortal self-renewal ability, capable of producing at least one type of highly differentiated progeny cells. According to different differentiation potentials, stem cells can be classified into totipotent stem cells, pluripotent stem cells, and unipotent stem cells.

[0003] Mesenchymal stem cells (pluripotent stem cells) have an immunomodulatory effect. Due to their characteristics, mesenchymal stem cell transplantation technology can be used to treat inflammatory and immune diseases. Many completed clinical trials have demonstrated the therapeutic effect of mesenchymal stem cells in acute myocardial ischemia, stroke, liver cirrhosis, amyotrophic lateral sclerosis, and graft-versus-host disease. In traditional stem cell transplantation therapy, due to the combined effects of mechanical, cellular, and host factors, cells will die within a few hours after transplantation, thus reducing the efficacy of stem cell therapy. Improving the survival rate of transplanted cells is crucial for stem cell therapy technology. Summary of the Invention

[0004] In order to solve the above technical problems, an embodiment of the present invention provides the application of baicalein in the preparation of drugs for inhibiting stem cell oxidation and / or ferroptosis.

[0005] To achieve the above object, the embodiments of the present invention adopt the following technical solutions:

[0006] On the one hand, the present invention provides the application of baicalein in the preparation of drugs for inhibiting stem cell oxidation and / or ferroptosis.

[0007] In some embodiments, the administration concentration of the baicalein is 0.5 - 5 μM.

[0008] In some embodiments, the structural formula of the baicalein is as follows:

[0009]

[0010] In some embodiments, the extraction method of the baicalein includes the following steps:

[0011] Steam the baicalein root through flowing steam for 1 h, dry it in the sun, soak it in water, boil it twice, each time for 1 h, combine the filtrates, adjust the pH to 3.5 with dilute hydrochloric acid, let it stand for 12 h, discard the gum-like mass, adjust the pH of the upper layer liquid to 1.5 - 2.0 with dilute hydrochloric acid, heat it to 80 °C, keep it warm for 0.5 h, cool it to 40 °C, pour off the mother liquor to obtain baicalein.

[0012] In some embodiments, the stem cells are mesenchymal stem cells.

[0013] In some embodiments, the above-mentioned drug comprises a pharmaceutical preparation prepared from baicalein as an active ingredient in an effective amount, plus a pharmaceutically acceptable carrier and / or excipient.

[0014] Effective amount refers to the amount of a drug, compound, pharmaceutical composition necessary to obtain any one or more beneficial or desired therapeutic outcomes. Beneficial or desired outcomes include: improving clinically (such as reducing morbidity, lowering mortality, improving one or more symptoms), alleviating severity, delaying the onset of a disease (including the disease or its complications, intermediate pathological phenotypes presented during the development of the disease, biochemical, histological, and / or behavioral symptoms). The preparation can be an injection, spray, aerosol, nasal drops, oral preparation, a dosage form suitable for mucosal administration. In addition, the administration routes to a subject include but are not limited to: intramuscular, intravenous, subcutaneous, intradermal, oral, intranasal, respiratory, transmucosal, sublingual, parenteral.

[0015] The present invention has the following beneficial effects compared with the prior art:

[0016] Baicalein can inhibit ferroptosis of mesenchymal stem cells and antagonize oxidative stress, can improve the survival rate of stem cells and enhance the immunomodulatory effect, thereby promoting the efficacy of stem cell therapy. Description of the Drawings

[0017] Figure 1 Cell survival rate after treatment with different concentrations of baicalein;

[0018] Figure 2 Toxicity of different concentrations of baicalein to cells;

[0019] Figure 3 Total glutathione level of AFSCs after treatment with baicalein and RSL3;

[0020] Figure 4 Malondialdehyde level of AFSCs after treatment with baicalein and RSL3;

[0021] Figure 5 Level of cytoplasmic divalent iron ions of AFSCs after treatment with baicalein and RSL3;

[0022] Figure 6 Ratio of lymphocyte CFSE detected after co-culture of AFSCs with ConA-stimulated PBMCs;

[0023] Figure 7 Ratio of lymphocyte CD4+, IFN-γ+ detected after co-culture of AFSCs with ConA-stimulated PBMCs. Detailed Description of the Invention

[0024] Next, in combination with the accompanying drawings, the technical solutions in some embodiments of the present disclosure will be clearly and completely described. Obviously, the described embodiments are only a part of the embodiments of the present disclosure, rather than all of the embodiments. Based on the embodiments provided by the present disclosure, all other embodiments obtained by those of ordinary skill in the art belong to the scope of protection of the present disclosure.

[0025] Unless otherwise specifically stated, various raw materials, reagents, instruments, and equipment used in this application can be obtained through market purchases or can be prepared by existing methods.

[0026] Baicalein, purchased from Selleck Chemicals (S2268), was dissolved in DMSO to obtain a stock solution containing Baicalein.

[0027] Ferroptosis inducer (RSL3), purchased from Selleck Chemicals (S8155), was dissolved in DMSO to obtain a stock solution containing RSL3.

[0028] Concanavalin A (Con-A), purchased from TargetMol (T4004), was dissolved in double-distilled water to obtain an aqueous solution containing Con-A.

[0029] DMSO, purchased from Sigma-Aldrich (C6164).

[0030] CCK-8 kit, purchased from Seventeen Innovation Biotechnology Co., Ltd. (SC119-03).

[0031] Kit for detecting the total glutathione (T-GSH) level in cells, purchased from Beyotime Biotechnology Co., Ltd. (S0053).

[0032] Kit for detecting the malondialdehyde (MDA) level, purchased from Beyotime Biotechnology Co., Ltd. (S0131).

[0033] Probe FerroOrange, purchased from DOJINDO (F374).

[0034] Experimental materials:

[0035] (1) Cells

[0036] Human amniotic fluid-derived mesenchymal stem cells (AFSCs) were derived from clinical amniotic fluid samples of the Third Affiliated Hospital of Guangzhou Medical University.

[0037] Human peripheral blood mononuclear cells (PBMCs) were derived from clinical blood samples of the Third Affiliated Hospital of Guangzhou Medical University.

[0038] (2) Materials

[0039] AFSCs medium: 10% fetal bovine serum + 1% P / S double antibody (final concentration is 50 U / mL) + 89% low-glucose DMEM basal medium.

[0040] PBMCs medium: 30% fetal bovine serum + 1% P / S double antibody (final concentration is 50 U / mL) + 69% RPMI 1640 basal medium.

[0041] Dissolve Baicalein in DMSO, and the stock solution concentration is 10 mM; when in use, dilute the Baicalein stock solution in hAFSCs medium, and the final concentration is 1 μM, which acts on AFSCs.

[0042] Ferroptosis inducer RSL3 is dissolved in DMSO, and the stock solution concentration is 5 mM; when in use, dilute the RSL3 stock solution in AFSCs medium, and the final concentration is 1 μM, which acts on AFSCs.

[0043] Concanavalin A (ConA; a phytohemagglutinin that can promote lymphocyte transformation reaction) is dissolved in ddH2O, and the stock solution concentration is 5 mg / mL; when in use, dilute ConA in PBMCs medium, and the final concentration is 5 μg / mL, which acts on PBMCs.

[0044] Example 1

[0045] This example is to evaluate the concentration and toxicity of Baicalein protection

[0046] When AFSCs are passaged to the 3rd generation, seed the cells into a 96-well plate and let them adhere overnight. The next day, treat the cells with different concentrations of Baicalein and the same concentration of RSL3 for 3 hours, and use the CCK-8 kit to detect the cell viability. Or treat AFSCs with different concentrations of Baicalein for different times, which are 12 h, 24 h, 72 h, 120 h, and 168 h respectively, and use the CCK-8 kit to detect the cell viability to evaluate the toxicity of Baicalein to AFSCs.

[0047] The results are as Figure 1 shown. Baicalein shows a protective effect on AFSCs starting from a concentration of 0.5 μM. When the concentration is 1 μM, the cell viability is close to 100%, and there is still a strong protective effect up to 5 μM.

[0048] The results are as Figure 2 shown. In the short term, Baicalein at a concentration ≤ 2 μM is almost non-toxic to AFSCs. When the concentration of Baicalein reaches 5 μM, time-dependent toxicity will occur. Therefore, this application selects a Baicalein concentration of 1 μM for subsequent research.

[0049] Example 2

[0050] This example is to evaluate the role of baicalein in RSL3-mediated ferroptosis

[0051] When AFSCs were passaged to the 3rd generation, they were treated with baicalein and RSL3. The groups were: Control group (untreated), Baicalein group, RSL3 group, Baicalein + RSL3 group, and the treatment time was 3 hours for all. After the treatment, the cells were collected, and the levels of total glutathione (T-GSH) and malondialdehyde (MDA) in the cells were detected using a commercial kit; the cytoplasmic free ferrous ions were detected by labeling the cytoplasmic divalent ferrous ions with the probe FerroOrange.

[0052] The results are as Figure 3 、 4 shown. Baicalein alone had no effect on AFSCs; when using RSL3 alone, the level of T-GSH decreased significantly and the level of MDA increased significantly; after combining with baicalein, the level of T-GSH increased significantly and the level of MDA decreased significantly. This indicates that baicalein can inhibit RSL3-mediated oxidative stress.

[0053] The results are as Figure 5 shown. After using RSL3, the level of divalent ferrous ions increased significantly, and it decreased significantly after combining with baicalein. This indicates that baicalein can inhibit RSL3-mediated iron overload.

[0054] Example 3

[0055] This example is to evaluate the role of baicalein in the treatment of mesenchymal stem cells

[0056] Using ConA to stimulate the proliferation and differentiation of PBMCs as an in vitro inflammation model. Co-culture with AFSCs treated in different ways, and use flow cytometry to detect the effects of AFSCs on inhibiting the proliferation and differentiation of PBMCs by CFSE and CD4, IFN-γ double staining respectively. The groups are negative control, positive control, normal AFSCs group, AFSCs adherent after RSL3 treatment, AFSCs suspended after RSL3 treatment, and the group of combined use of baicalein and RSL3.

[0057] The results are as Figure 6 、 7As shown. After adding ConA, the CFSE and the proportions of CD4+ and IFN-γ+ in PBMCs increased significantly, indicating the successful establishment of the in vitro model; after adding normal AFSCs, the CFSE and the proportions of CD4+ and IFN-γ+ decreased significantly, indicating that AFSCs can exert immunomodulatory ability and inhibit the proliferation and differentiation of lymphocytes; after adding ferroptotic cells, it was found that the CFSE and the proportions of CD4+ and IFN-γ+ increased significantly, indicating that ferroptosis affects the immunomodulatory ability of AFSCs; after adding AFSCs combined with baicalein, it was found that the CFSE and the proportions of CD4+ and IFN-γ+ were significantly restored, indicating that baicalein enhances its immunomodulatory ability by inhibiting the ferroptosis of AFSCs.

[0058] In the description of this specification, specific features, structures, materials or characteristics may be combined in a suitable manner in any one or more embodiments or examples.

[0059] As described above, the above are only specific embodiments of the present invention, but the protection scope of the present invention is not limited thereto. Any person skilled in the art within the technical scope disclosed by the present invention can easily think of changes or substitutions, which should all be covered by the protection scope of the present invention. Therefore, the protection scope of the present invention should be subject to the protection scope of the claims.

Claims

1. Application of baicalin in the preparation of drugs for inhibiting oxidation and / or ferroptosis of stem cells.

2. The use according to claim 1, characterized in that: The administration concentration of baicalein is 0.5-5 μM.

3. The use according to claim 1, characterized in that: The structural formula of baicalin is shown below:

4. The use according to claim 1, characterized in that: The method for extracting baicalein comprises the following steps: The root of Scutellaria baicalensis was fumigated with circulating steam for 1 hour, dried in the sun, soaked in water, boiled twice, each time for 1 hour, the filtrates were combined, diluted hydrochloric acid was added to adjust the pH to 3.5, the mixture was allowed to stand for 12 hours, the gum blocks were discarded, the pH of the supernatant was adjusted to 1.5-2.0 with dilute hydrochloric acid, heated to 80°C, kept warm for 0.5 hour, cooled to 40°C, the mother liquor was decanted, and baicalin was obtained.

5. The use according to claim 1, characterized in that: The stem cells are mesenchymal stem cells.

Images