Application of badoxolone methyl in preparation of stem cell aging inhibitor

Abstract

The invention discloses an application of badoxolone methyl in preparation of a stem cell aging inhibitor. According to the invention, the umbilical cord mesenchymal stem cell aging model is successfully established to inspect the effect of the badoxolone methyl on delaying the aging of the umbilical cord mesenchymal stem cells. The results show that badoxolone methyl can significantly inhibit the activity of senescence-related beta-galactosidase in cells, reduce the increased p21 expression level and gamma-H2A.X focus in senescence umbilical cord mesenchymal stem cells induced by adriamycin or etoposide, and provide a new strategy for subsequent clinical application of delaying stem cell senescence and increasing stem cells.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology and relates to the application of bardoxolone methyl in the preparation of stem cell aging inhibitors. Background Art

[0002] Human umbilical cord mesenchymal stem cells (hUC-MSCs) refer to a type of multifunctional stem cells present in the umbilical cord tissue of newborns, which have the functions of self-renewal, multidirectional differentiation and immunoregulation. First of all, hUC-MSCs can continuously self-renew and differentiate into one or more cell types under certain conditions, such as osteoblasts, chondrocytes and adipocytes, which then constitute the tissues and organs of the human body. hUC-MSCs cells themselves can also play an immunoregulatory function: intravenously injected hUC-MSCs can migrate to the site of injury and respond to inflammation. In addition, hUC-MSCs can also secrete a variety of bioactive factors through autocrine or paracrine secretion or through extracellular vesicles, such as exosomes and microvesicles, to respond to local microenvironment or signals. Compared with other stem cells, hUC-MSCs have the advantages of being easier to obtain, separate, culture, amplify and purify. And hUC-MSCs have low immunogenicity, so they have more unique advantages in clinical applications. In addition, compared with traditional treatments, hUC-MSCs can not only improve the survival rate, but also significantly improve the various clinical symptoms of the disease. In summary, hUC-MSCs has become one of the important sources of stem cells in cell therapy and regenerative medicine.

[0003] There is increasing evidence that hUC-MSCs have advantages in treating a variety of diseases, but hUC-MSCs will gradually lose their proliferation ability and therapeutic properties after in vitro expansion. hUC-MSCs can be cultured multiple times and subcultured to obtain a large number of cells, but during long-term culture, abnormalities in pathways related to the cell cycle and DNA damage response can lead to hUC-MSC genome instability, and in the later stages of subculture, the cell morphology becomes longer and larger, proliferation slows, and proliferation-related genes decrease. The aging of hUC-MSCs will affect their clinical application. For example, in the treatment of acute liver failure, the 5th generation cells are more effective than the 10th generation cells in homing to the liver, enhancing liver cell proliferation, and inhibiting apoptosis. And the elimination of senescent cells can promote stem cell proliferation and delay the appearance of aging characteristics.

[0004] hUC-MSCs aging is a complex problem that requires a variety of different approaches to alleviate or prevent aging and improve the clinical application of hUC-MSCs. Currently, there are three main methods to reverse or delay hUC-MSCs cell aging and maintain their in vitro proliferation capacity: gene modification, microRNA treatment, and pretreatment modification. Gene modification includes two types of gene reprogramming: complete reprogramming by recoding intracellular genes to reset epigenetics and partial reprogramming by DNA methylation and histone modification to make epigenetics younger. MicroRNA delays hUC-MSCs aging through multidimensional targets. Pretreatment modification refers to the in vitro treatment of cells with chemical and physical factors through a specially designed environment. Pretreatment modification can improve the interaction between cells and the immune system. However, the three existing methods for delaying hUC-MSCs aging all have certain clinical application limitations. First, the key pathway factors that regulate hUC-MSCs aging are also important physiological regulators of normal biological functions. Intervention to completely inhibit or activate these pathways may have other negative effects. Second, one or two microRNAs have little effect in delaying hUC-MSCs aging treatment, and several aging-related microRNAs need to be used in combination to exert therapeutic functions. Therefore, it is necessary to find an effective method that is relatively safe for the body to delay the aging of hUC-MSCs, so as to increase and promote the clinical application of hUC-MSCs.

[0005] Bardoxolone Methyl (BM) is a triterpenoid compound derived from the natural product oleanolic acid and is an IKK inhibitor.

[0006]

[0007] Bardoxolone methyl inhibits NF-κB transcriptional activity and is also an activator of the Keap1-Nrf2 pathway. It has anti-inflammatory and anti-cancer effects and is clinically used to treat solid tumors, type II diabetes, and chronic kidney disease. So far, there have been no reports on the inhibition of cell senescence by bardoxolone methyl. Summary of the invention

[0008] The present invention successfully established an hUC-MSCs aging model to investigate the effect of bardoxolone methyl in delaying hUC-MSCs aging. Bardoxolone methyl can significantly inhibit the aging of hUC-MSCs, providing a new strategy for delaying stem cell aging and increasing the clinical application of stem cells.

[0009] The specific technical solutions of the present invention are as follows:

[0010] Application of bardoxolone methyl in the preparation of stem cell aging inhibitors.

[0011] Preferably, the stem cells are selected from one or more of umbilical cord mesenchymal stem cells, placental mesenchymal stem cells, adipose mesenchymal stem cells or umbilical cord blood stem cells.

[0012] In the application of the present invention, the concentration of methyl bardoxolone for inhibiting cell senescence is 1.5-2 μM.

[0013] In the application of the present invention, cells are incubated with bardoxolone methyl for 1.5-2.5 hours and then replaced with ordinary culture medium for culture.

[0014] The common culture medium is a drug-free DMEM / F12 culture medium containing 10% fetal bovine serum.

[0015] Furthermore, after incubating the cells with bardoxolone methyl for 1.5-2.5 hours, the cells can be incubated with the normal culture medium for another 1.5-2.5 hours 5-6 days after the culture.

[0016] In a specific embodiment of the present invention, the cells are incubated with bardoxolone methyl for 2 hours, and then the cells are incubated with bardoxolone methyl for another 2 hours after culturing with ordinary culture medium for 5-6 days.

[0017] In the application of the present invention, when bardoxolone methyl is incubated with cells, the cell density is 60% to 70%.

[0018] Advantages of the present invention:

[0019] The present invention discovers for the first time that bardoxolone methyl can significantly inhibit the aging of hUC-MSCs, has the advantages of simple operation, controllable method and safety, and provides a new strategy for delaying stem cell aging and increasing the clinical application of stem cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 Results of the detection of β-galactosidase (SA-β-gal) activity in hUC-MSCs induced by doxorubicin. (A) SA-β-gal staining results. (B) Statistical graph of SA-β-gal activity detection results.

[0021] Figure 2 Results of the detection of β-galactosidase (SA-β-gal) activity in hUC-MSCs induced by etoposide. (A) SA-β-gal staining results. (B) Statistical graph of SA-β-gal activity detection results. Figure 3 The expression changes of p21 in hUC-MSCs after senescence induced by doxorubicin or etoposide (A) Western blotting results. (B) Semi-quantitative analysis of Western blotting results.

[0022] Figure 4Immunofluorescence staining was used to detect the expression changes of γ-H2A.X foci in the nucleus of hUC-MSCs after senescence induced by doxorubicin or etoposide. (A) γ-H2A.X foci staining results (green). Scale bar: 20 μm. (B) Statistical graph of immunofluorescence staining results.

[0023] Figure 5 The inhibitory effect of methyl bardoxolone on doxorubicin-induced hUC-MSCs senescence. (A) SA-β-gal assay to detect the effect of methyl bardoxolone on delaying doxorubicin-induced hUC-MSCs senescence. (B) Statistical graph of SA-β-gal activity assay results. (C) CCK8 assay to detect the cytotoxicity of different concentrations of methyl bardoxolone combined with doxorubicin on hUC-MSCs.

[0024] Figure 6 The inhibitory effect of methyl bardoxolone on etoposide-induced hUC-MSCs senescence. (A) SA-β-gal assay to detect the effect of methyl bardoxolone on delaying etoposide-induced hUC-MSCs senescence. (B) Statistical graph of SA-β-gal activity assay results. (C) CCK8 assay to detect the cytotoxicity of different concentrations of methyl bardoxolone combined with etoposide on hUC-MSCs.

[0025] Figure 7 The expression changes of p21 and DNA damage in hUC-MSCs induced by doxorubicin or etoposide after senescence were reduced by bardoxolone methyl. (A) Western blotting was used to detect the expression changes of p21 in senescent hUC-MSCs induced by doxorubicin. (B) Statistical graph of Western blotting results. (C) Western blotting was used to detect the expression changes of p21 in senescent hUC-MSCs induced by etoposide. (D) Statistical graph of Western blotting results. (E) Immunofluorescence staining was used to detect the DNA damage in senescent hUC-MSCs induced by doxorubicin. Scale bar: 20μm. (F) Statistical graph of immunofluorescence staining results. (G) Immunofluorescence staining was used to detect the DNA damage in senescent hUC-MSCs induced by etoposide. Scale bar: 20μm. (H) Statistical graph of immunofluorescence staining results. DETAILED DESCRIPTION

[0026] The specific steps of the present invention are described below by way of examples, but are not limited to the examples.

[0027] The terms used in the present invention, unless otherwise specified, generally have the meanings commonly understood by those of ordinary skill in the art.

[0028] The present invention will be further described in detail below in conjunction with specific examples and with reference to data. It should be understood that these examples are only intended to illustrate the present invention and are not intended to limit the scope of the present invention in any way.

[0029] In the following examples, various processes and methods not exhaustively described are conventional methods well known in the art.

[0030] Example 1 Preparation of hUC-MSCs aging model

[0031] 1. Take a fresh umbilical cord, clean the surface of the umbilical cord with PBS containing double antibodies, and squeeze out the residual blood in the blood vessels;

[0032] 2. Use sterile surgical scissors to cut off both ends of the umbilical cord and cut the remaining part into small pieces of 2-3 cm in length;

[0033] 3. Continue to wash the umbilical cord with PBS and use tweezers to clean the blood in the umbilical cord;

[0034] 4. Use forceps to peel off the umbilical cord tissue, use hooked forceps to gently tear off the vein, then tear off the artery, then wash it 3 times with PBS, and cut it into pieces in a 50mL centrifuge tube;

[0035] 5. Digest with 0.1% collagenase and hyaluronidase for 3-4 hours, filter, and centrifuge 2-3 times;

[0036] 6. Transfer the cells into culture flasks, spread them evenly, and use DMEM / F12 (1:1) culture medium (Shanghai Yuanpei Biotechnology Co., Ltd.) containing 10% fetal bovine serum for adherent subculture;

[0037] 7. Inoculate hUC-MSCs into a 6-well plate and observe under a microscope. When the cell growth density reaches about 60% to 70%, senescence induction can be performed;

[0038] 8. Take out the cell culture dish, discard the culture medium, wash once with 1×PBS buffer, and add fresh culture medium. Add a certain concentration of doxorubicin or etoposide to the cells, gently shake the cell culture dish up and down and left and right to mix evenly, make the doxorubicin concentration in the culture medium 0.05μM, move to a carbon dioxide cell culture incubator for 48h and then to a carbon dioxide cell culture incubator for 6 days; or make the etoposide concentration in the culture medium 10μM, move to a carbon dioxide cell culture incubator for 24h and then to a carbon dioxide cell culture incubator for 7 days.

[0039] The senescence of hUC-MSCs was detected using the SA-β-gal staining kit. The cells were observed and photographed under a total internal reflection fluorescence microscope, and the number of stained cells in each group was counted. Figure 1 and 2 The results showed that hUC-MSCs cells showed obvious senescence.

[0040] The total protein of hUC-MSCs was extracted. The expression of p21, a senescent cell marker and cell cycle inhibitor, in senescent cells induced by adriamycin or etoposide was detected by Western blotting. Figure 3 Immunofluorescence staining was used to detect the changes of γ-H2A.X foci in senescent cells induced by doxorubicin or etoposide. Figure 4 shown.

[0041] The results showed that when hUC-MSCs were induced to undergo premature senescence by doxorubicin or etoposide, p21 and γ-H2A.X foci in senescent cells were significantly upregulated.

[0042] Example 2 Inhibitory effect of bardoxolone methyl on hUC-MSCs senescence

[0043] hUC-MSCs were taken when the cells grew to 60%-70%, and cultured with culture medium containing different concentrations of bardoxolone methyl for a period of time, the culture medium containing bardoxolone methyl was discarded, and then the operation of step 8 of Example 1 was referred to, and doxorubicin or etoposide was used to induce hUC-MSCs senescence, and then culture medium containing different concentrations of bardoxolone methyl was added for a period of time, and then the culture medium without drug was changed to culture until the 9th day for sample collection.

[0044]

[0045] The senescence of hUC-MSCs in each group was detected using the SA-β-gal staining kit. The cells were observed and photographed under a total internal reflection fluorescence microscope, and the number of stained cells in each group was counted. Figure 5 and 6 As shown, the results showed that 1.5μM methyl bardoxolone can delay the senescence of hUC-MSCs induced by doxorubicin, and the CCK8 test found that 0.5μM-2μM methyl bardoxolone had no cytotoxicity when used in combination with doxorubicin. 1.5μM and 2μM methyl bardoxolone can significantly delay the senescence of hUC-MSCs induced by etoposide, and the CCK8 test found that 0.5μM-2μM methyl bardoxolone had no cytotoxicity when used in combination with etoposide. However, 3μM and 4μM methyl bardoxolone combined with etoposide caused a large number of hUC-MSCs cell deaths, and the surviving cells were almost all senescent cells. Therefore, 3μM and 4μM methyl bardoxolone are not suitable as hUC-MSCs senescence inhibitors.

[0046] Example 3 Bardoxolone methyl reduces senescence-related phenotypes in aged hUC-MSCs

[0047] The hUC-MSCs senescent cells in each administration group in Example 2 were used as the research object, and the total protein of each group of cells was extracted. Protein immunoblotting and immunofluorescence staining experiments were performed to detect the expression of senescent cell marker cell cycle inhibitor p21 protein and γ-H2A.X foci in hUC-MSCs senescent cells induced by doxorubicin and etoposide respectively after treatment with methyl bardoxolone. The results are as follows Figure 7 As shown. The results showed that 1.5μM bardoxolone methyl could reduce the elevated p21 expression levels and γ-H2A.X foci in doxorubicin-induced senescent hUC-MSCs. Similarly, 2μM bardoxolone methyl could delay the elevated p21 expression levels and γ-H2A.X foci in etoposide-induced senescent hUC-MSCs. In conclusion, 1.5μM and 2μM bardoxolone methyl could delay the senescence of hUC-MSCs induced by doxorubicin or etoposide.

Claims

1. Application of bardoxolone methyl in the preparation of stem cell aging inhibitors.

2. The use according to claim 1, Features The stem cells are selected from one or more of umbilical cord mesenchymal stem cells, placental mesenchymal stem cells, adipose mesenchymal stem cells or umbilical cord blood stem cells.

3. The use according to claim 1 or 2, Features The concentration of bardoxolone methyl to inhibit cell senescence is 1.5-2μM.

4. The use according to claim 3, Features After incubating the cells with bardoxolone methyl for 1.5-2.5 hours, the cells were replaced with normal culture medium.

5. The use according to claim 3, Features After incubating the cells with bardoxolone methyl for 1.5-2.5 hours, the cells were replaced with normal culture medium and incubated with bardoxolone methyl for another 1.5-2.5 hours 5-6 days after culture.

6. The use according to claim 5, Features After incubating the cells with bardoxolone methyl for 2 hours, the cells were replaced with normal culture medium and incubated with bardoxolone methyl for another 2 hours 5-6 days after culture.

7. The use according to claim 3, Features The cell density is 60% to 70%.

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