Application of arbutin in delaying replicative senescence of mesenchymal stem cells

Abstract

The invention discloses an application of arbutin in delaying replicative senescence of mesenchymal stem cells. Experimental results show that the arbutin has an obvious improvement effect on MSC aging caused by in-vitro replication and amplification and is related to the dose, and the highest dose designed by the research still does not generate any toxicity to cells. Therefore, the addition of the arbutin in the culture medium is helpful for the in-vitro batch culture of the MSC, and the development of clinical application can be accelerated and promoted.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology and relates to the application of bearberry extract in delaying the replicative aging of mesenchymal stem cells. Background Technology

[0002] Cellular senescence is a complex, multi-step process involving various signal transduction pathways and molecular mechanisms. It manifests as a decline and eventual loss of proliferative capacity, ultimately leading to permanent cell cycle arrest. Based on its causes, it can be mainly divided into two types: replicative senescence and physiological senescence. Theoretically, stem cells possess "unlimited" proliferative capacity, but current research shows that one characteristic of aging is in vitro senescence of stem cells, and in vitro culture has failed to achieve "immortality" of cells. Stem cells are susceptible to senescence due to various factors, subsequently losing their repair and regeneration capabilities. These factors primarily manifest as DNA damage, telomerase shortening caused by continuous DNA replication stress, mitochondrial dysfunction, oxidative stress, and dysregulation of stem cell autophagy.

[0003] Mesenchymal stem cells (MSCs) are a type of adult stem cell derived from the mesoderm. They possess unique immunomodulatory functions and can promote the repair of damaged tissues through paracrine effects. Currently, they have shown promising applications in preclinical studies of various intractable diseases. However, significant individual differences exist in the efficacy of MSCs in clinical applications, while cell viability is positively correlated with therapeutic efficacy. In vitro culture and expansion of MSCs show a slowing of proliferation and decreased activity with increasing passage number, leading to replicative senescence, which greatly limits their clinical application. Delaying MSC replicative senescence is a crucial issue that urgently needs to be addressed.

[0004] Senescent MSCs undergo morphological changes. Senescent MSCs exhibit increased cell surface area, flattened cell bodies, loss of three-dimensionality, decreased length-to-width ratio, and the appearance of vacuoles in the cytoplasm. During in vitro passage culture, MSCs divide a limited number of times (Hayflick limit), after which cell proliferation slows down, differentiation potential decreases, while SA-β-gal activity and ROS levels significantly increase.

[0005] Argus root extract, also known as galangal root extract (PubChem CID: 5318869), is widely found in Chinese medicinal herbs such as astragalus, licorice, cardamom, clove, and patchouli, and is present in high amounts. Its extraction process is mature and simple.

[0006] Previous studies have confirmed that bearberry extract possesses various pharmacological activities, including antioxidant activity, antitumor activity, inhibition of α-glucosidase, and antibacterial activity, demonstrating broad potential for drug development. Several studies have confirmed that monomers extracted from traditional Chinese medicines, such as ligustrazine and astragalus extract, have good inhibitory effects on mesenchymal stem cell senescence, but there are no reports of bearberry extract's anti-cellular senescence effects. Summary of the Invention

[0007] To address the above problems, this invention provides the application of arbutin in delaying the replicative senescence of mesenchymal stem cells. Specifically, it includes the following steps: Application of arbutin in enhancing SIRT3 expression and / or delaying the replicative senescence of mesenchymal stem cells.

[0008] Optionally, the mesenchymal stem cells include one or more of the following: umbilical cord mesenchymal stem cells, adipose mesenchymal stem cells, bone marrow mesenchymal stem cells, placental mesenchymal stem cells, dental pulp mesenchymal stem cells, or menstrual blood mesenchymal stem cells.

[0009] Optionally, the mesenchymal stem cells are human umbilical cord mesenchymal stem cells.

[0010] Optionally, the delay in the replicative senescence of mesenchymal stem cells includes inhibiting the senescence phenotype of cells; inhibiting the senescence phenotype of cells includes: inhibiting senescence-related morphological changes, significantly reducing the activity of the senescence marker β-galactosidase (SA-β-gal), reducing the proportion of staining positive cells, and maintaining the proliferative potential of high-generation mesenchymal stem cells.

[0011] A method for delaying the replicative senescence of mesenchymal stem cells includes adding arbutin to the culture medium of the mesenchymal stem cells during the mesenchymal stem cell culture process.

[0012] Optionally, the culture medium contains DMEM-F12 and fetal bovine serum; Preferably, the culture medium further contains an antibiotic; the antibiotic is penicillin and / or streptomycin.

[0013] Optionally, the final concentration of the arbutin in the culture medium is 1~100µM.

[0014] Optionally, the final concentration of the arbutin in the culture medium is 50~100µM.

[0015] Optionally, the mass ratio of the DMEM / F12 culture medium to fetal bovine serum is 85–95:5–15.

[0016] A culture medium for delaying the replicative senescence of mesenchymal stem cells, comprising DMEM / F12 medium, fetal bovine serum, and arbutin.

[0017] The technical solution of this invention has the following advantages: This invention provides arbutin, which delays or prevents the replicative aging of MSCs during in vitro expansion by reducing oxidative stress, thus providing a solid foundation for the application of MSCs in tissue engineering.

[0018] The experimental results of this invention show that arbutin significantly improves MSC aging caused by in vitro replication and expansion, and this effect is dose-dependent. Furthermore, even at the highest dose (100 μM) designed for this study, no toxicity was observed in the cells. Therefore, we believe that adding arbutin to the culture medium facilitates the large-scale in vitro culture of MSCs and can accelerate the development of clinical applications. Attached Figure Description

[0019] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0020] Figure 1 Image showing umbilical cord mesenchymal stem cells and their identification.

[0021] Figure 2 The image shows the results of MSC proliferation promoted by bearberry extract.

[0022] Figure 3 Figures showing the morphology and corresponding cell diameter of MSCs under different conditions (*P<0.05, **P<0.005).

[0023] Figure 4 Figure 1 shows the proliferation activity of MSCs in different groups (*P<0.05, **P<0.005).

[0024] Figure 5 Figure showing the results of SA-β-gal staining and quantitative analysis (***P<0.001, ****P<0.0001).

[0025] Figure 6 Figure 1 shows the results of MDA detection in MSC cells (*P<0.05,**P<0.005,****P<0.0001).

[0026] Figure 7 Figure showing the expression results of the antioxidant enzyme SOD2 in MSCs (***P<0.001,****P<0.0001). Detailed Implementation

[0027] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention. It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the present invention.

[0028] Furthermore, regarding the numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, are also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0029] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar to or equivalent to those described herein may be used in the implementation or testing of this invention.

[0030] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0031] Statistical methods used in this embodiment: The obtained data were statistically analyzed using R language (version 3.6.1) and its statistical computing packages. Quantitative data are expressed as mean ± standard deviation (SD). Pairwise t-tests were used for pairwise comparisons between groups, and the Bonferroni method was applied for multiple comparison correction. A Bonferroni-corrected p-value < 0.05 was used to determine whether the difference was statistically significant.

[0032] Arbutin, also known as galangaloid (Jaranol or Kumatakenin, PubChem CID: 5318869), is widely found in high concentrations in traditional Chinese medicinal herbs such as Astragalus membranaceus, licorice, cardamom, clove, and patchouli. Its extraction process is mature and simple. Its chemical formula is as follows:

[0033] This invention proposes the application of arbutin in enhancing SIRT3 expression and / or delaying the replicative senescence of mesenchymal stem cells.

[0034] SIRT3 is an NAD+-dependent histone deacetylase that plays a crucial role in regulating mitochondrial function, energy metabolism, antioxidant defense, and cell survival. Through experiments, we found that arbutin can reduce MSC senescence by increasing SIRT3 expression, reducing intracellular MDA, and increasing SOD2 activity. This was manifested in significantly reduced SA-β-gal activity and increased proliferation efficiency in high-passage cells. Therefore, our study demonstrates that arbutin exerts its anti-MSC senescence effect by increasing SIRT3 expression, reducing oxidative stress, and restoring mitochondrial activity.

[0035] The aforementioned delay in the replicative senescence of mesenchymal stem cells is specifically manifested in effectively inhibiting the senescence phenotype of cells, including: inhibiting senescence-related morphological changes, such as preventing cell volume increase and flattening; significantly reducing the activity of the senescence marker β-galactosidase (SA-β-gal) and reducing the proportion of its staining positive cells; and maintaining the proliferative potential of high-generation mesenchymal stem cells.

[0036] A method for delaying the replicative senescence of mesenchymal stem cells based on arbutin includes: adding arbutin to the culture medium of the mesenchymal stem cells during the culture of the mesenchymal stem cells.

[0037] Furthermore, the culture medium contains DMEM-F12 and fetal bovine serum; Furthermore, the culture medium also contains antibiotics; the antibiotics are penicillin and / or streptomycin.

[0038] Furthermore, the final concentration of the arbutin in the culture medium is 1~100µM, preferably 50~100µM.

[0039] Furthermore, the mass ratio of the DMEM / F12 culture medium to fetal bovine serum is 85–95:5–15.

[0040] The use of the aforementioned culture medium to culture mesenchymal stem cells can not only effectively enhance cell activity, promote cell proliferation and migration, but also maintain stable cell metabolism and reduce the proportion of senescent cells, thereby effectively delaying cell senescence (i.e., replicative senescence) of mesenchymal stem cells during continuous in vitro passage culture. This significantly improves the expansion efficiency and cell activity of in vitro cultured mesenchymal stem cells. Therefore, a culture medium containing DMEM / F12 medium, fetal bovine serum and arbutin that delays replicative senescence of mesenchymal stem cells should also be within the scope of protection of this invention.

[0041] Experimental Example 1: Cell Isolation and Identification Cell preparation: Fetal umbilical cord tissue was used in this study. The umbilical cord was thoroughly washed with PBS. The umbilical cord was split longitudinally, and the two arteries and one vein within it were removed. The Walton gel was cut into 1cm x 1cm fragments and placed in 10cm culture dishes. The culture medium was DMEM-F12 (Corning) containing 10% fetal bovine serum (GiBco) and 1% penicillin-dextrin antibiotics and incubated in a 5% CO2 incubator. Medium was added the following day, and the culture medium was changed every 72 hours. When cells were observed extending from the edge of the tissue block and the local cell confluence reached over 85%, the cells were passaged and then cryopreserved for later use. Identification of HUCMSCs: The expression of CD45, CD29, CD105, CD34, CD90, and CD73 (BioLegend) was detected by flow cytometry. The expression results are as follows: Figure 1 As shown.

[0042] Experimental Example 2: Arbutin Intervention for Anti-Cellular Replication and Senescence: MSC P3 generation cells at 5*10 3 Cells were seeded at a density of 1 µM in 96-well plates, with 6 replicates per group. After 24 h of seeding, the corresponding concentration of arbutin was added and the cells were cultured for 48 h. Cell proliferation was then assessed using a CCK-8 assay. Four concentrations of arbutin were selected: 1 µM, 25 µM, 50 µM, and 100 µM.

[0043] Experimental groups: P3, P5, and P10 MSC cells were selected as representatives of the three stages of cell replication and senescence. Groups: 1. Control group: P3, P5, P10; 2. Bearberry extract anti-aging group: P10 cells were co-cultured with bearberry extract for 48 hours before various tests were performed.

[0044] Observe cell morphology and detect cell size: The morphology of different groups of cells was observed under a microscope and photographed. The cells were then digested with 0.25% trypsin, and the number and size of cells were measured using a Countstar cell counter.

[0045] 1. Effects of different doses of arbutin on MSC proliferation activity: Four concentrations (1µM, 25µM, 50µM, and 100µM) of arbutin were added to P3 generation MSCs and co-cultured for 48 h. The proliferation activity was then detected by CCK-8 assay. The results showed that arbutin promoted MSC proliferation in a positively dose-dependent manner and did not exhibit toxicity. Therefore, in subsequent experiments, a high concentration of 100µM was selected.

[0046] 2. Morphological changes in senescent MSC cells: P3 generation cells, P6 generation cells, and P10 generation cells were respectively fed at 1*10 5After seeding in 6-well plates and culturing for 48 hours, cells were observed and photographed under a phase-contrast microscope. With increasing passage number, cell volume increased, cell bodies became irregular and more spread out, and cell boundaries became indistinct. After intervention with arbutin, the cell morphology became spindle-shaped.

[0047] Experimental Example 3: Cell Proliferation Cells from each group were digested with pancreatic enzyme digestion solution and centrifuged to prepare cell suspensions. Cells were then counted at 5 x 10⁻⁶ cells / mL. 3 Cells were seeded at a density in 96-well plates, with 6 replicates per group (control group cultured continuously for 72 h; intervention group, 100 μM arbutin added 24 h after seeding and cultured for 48 h). Cell proliferation rate (Uelandy) was detected using a CCK-8 assay kit 72 h after seeding.

[0048] Cell proliferation was detected in both groups of cells, such as... Figure 2 , Figure 3 , Figure 4 As shown, the results indicated that the higher the passage number of MSCs, the lower their proliferation activity. However, after 48 hours of intervention with 100 μM arbutin, the proliferation activity of P10 cells was significantly increased (p < 0.05), which was statistically significant.

[0049] Experiment 4: Detection of β-galactosidase (SA-β-gal) activity Assay for aging-related β-galactosidase (SA-β-gal) activity (Beyotime) (1) Cells in good growth condition were divided into groups of 5×10 4 10 cells / well were seeded in a 24-well plate. When the cells reached 60-70% confluence, the cell culture medium was removed and the cells were washed twice with PBS. (2) Add 500 μl of staining fixative to the well plate and fix for 20-25 min at room temperature. Then, wash thoroughly with PBS 3 times, 5 min each time. (4) Prepare the staining working solution, each milliliter containing 10 μl of SA-β-gal staining solution A, 10 μl of staining solution B, 930 μl of staining solution C, and 50 μl of X-Gal solution, and mix well; (5) Add 500 μl of staining working solution to each well and incubate overnight in a CO2-free incubator at 37°C. Seal with plastic wrap or sealing film to prevent the staining solution from evaporating. (6) The next day, observe the staining under a phase contrast microscope, select multiple fields of view in different areas of the culture dish, collect images, and calculate the positive cell rate in the field of view (number of blue-stained cells / total number of cells × 100%).

[0050] Experimental results: SA-β-gal staining results showed that, Figure 5As shown, the number of blue-stained cells in high-passage MSCs increased significantly, while the number of blue-stained cells in P10 cells decreased significantly after 48 h of arbutin intervention (P<0.001).

[0051] Experimental Example 5 Antioxidant Stress Response Oxidative stress index detection: Select cells from each group with good growth status, digest them with trypsin to detach the adherent cells, centrifuge at 1500 rpm for 5 min at room temperature to collect the cell pellet, and detect SOD2 and MDA according to the kit (Nanjing Jiancheng) instructions.

[0052] (1) Detection of malondialdehyde (MDA) content MDA content reflects the severity of intracellular lipid peroxidation and is one of the indirect indicators of the degree of free radical attack on cells. Cell passaging leads to an increase in intracellular MDA with each passaging cycle. Figure 6 As shown, when MSCs of P10 were treated with 100 μM arbutin for 48 h, the activity of MDA was reduced, and the result was statistically significant (P < 0.05).

[0053] (2) Detection of expression and activity of antioxidant enzyme SOD2 SOD2 is an antioxidant metalloenzyme in living organisms, playing a crucial role in maintaining the balance between intracellular oxidation and antioxidation. For example... Figure 7 As shown in the figure, the SOD2 activity assay showed that the SOD2 activity decreased with increasing passage number, while the SOD2 activity of the P10+ arbutin group increased significantly after intervention with arbutin (P<0.0001).

[0054] Experimental Example 6: Sirt3 Expression Level Detection SIRT3 is a mitochondrial deacetylase, abundant in tissues with high metabolic levels, and plays a crucial role in regulating mitochondrial homeostasis, oxidative stress, and cellular senescence through its deacetylation action. We detected the expression level of SIRT3 in different groups of MSCs using RT-qPCR(A) and Western blot. The results showed that compared with low-passage cells, the expression levels of SIRT3 mRNA and protein in high-passage MSCs were significantly downregulated (P<0.001), while the expression level of SIRT3 increased after intervention with arbutin (P<0.001, P<0.0001).

[0055] mRNA level detection: (1) Total RNA extraction: Collect logarithmic growth phase cells from each experimental group and add Trizol reagent. (Invitrogen) Lyse cells and extract total RNA according to the kit instructions. Determine RNA concentration and purity using a UV spectrophotometer (A260 / A280 ratio should be between 1.8 and 2.0). (2) Reverse transcription to synthesize cDNA: Take 2 μg of total RNA and use a reverse transcription kit (Takara) to synthesize first-strand cDNA. The reverse transcription system is 20 μL, and the reaction conditions are: 37℃ for 15 minutes and 85℃ for 5 seconds. (3) Real-time quantitative PCR detection: Using cDNA as a template, Real-time PCR amplification was performed using the SYBR Green fluorescent dye method. The reaction system was 20 μL, containing 10 μL of SYBR Green premix, 0.4 μL each of upstream and downstream primers (final concentration 0.2 μM), 2 μL of cDNA template, and 7.2 μL of ddH2O. The amplification conditions were: 95℃ pre-denaturation for 10 minutes, 95℃ denaturation for 15 seconds, and 60℃ annealing / extension for 60 seconds, for 40 cycles. β-actin was used as an internal reference gene, and the relative expression level of Sirt3 gene was calculated using the 2^(-ΔΔCt) method.

[0056] Protein level testing (1) Protein extraction: Collect logarithmic growth phase cells from each experimental group, discard the culture medium, and wash twice with pre-cooled PBS (phosphate buffer, pH 7.4); add an appropriate amount of RIPA lysis buffer (containing protease inhibitor, Solarbio), and incubate on ice for 5 minutes; scrape adherent cells thoroughly with a cell scraper, transfer the cell suspension to a 1.5 mL centrifuge tube, and continue lysis on ice for 15 minutes; centrifuge at 4℃ and 12,000 rpm for 15 minutes, and collect the supernatant as total protein.

[0057] (2) Protein quantification and denaturation: The protein concentration of each sample was determined using the BCA protein concentration assay kit (Beyotime). The protein concentration of each sample was adjusted to be consistent according to the assay results. 5×SDS-PAGE loading buffer (Solepro) was added at a ratio of 4:1 and heated in a 100℃ metal bath or boiling water bath for 5 minutes to fully denature the protein.

[0058] (3) SDS-PAGE electrophoresis and transfer: Prepare 10-12% SDS-PAGE separating gel, load an equal amount of protein (20μg) per lane, concentrate at 80 V for 30 minutes, and separate at 120 V for about 1.5 hours; after electrophoresis, transfer the protein to a PVDF membrane (pre-activated with methanol) using wet transfer, with transfer conditions of 300 mA constant current for 60 minutes.

[0059] (4) Immunoblotting detection: PVDF membranes were blocked with 5% skim milk-TBST solution at room temperature for 1 hour; incubated overnight at 4°C with Sirt3 primary antibody (Santa Cruz) and internal control GAPDH primary antibody (Proteintech); washed the membranes 3 times with TBST for 10 minutes each time; added the corresponding horseradish peroxidase (HRP) labeled secondary antibody (Proteintech) and incubated at room temperature for 1-2 hours; washed the membranes 3 times with TBST for 10 minutes each time.

[0060] (5) Color development and data analysis: Enhanced chemiluminescence (ECL, Millipore) was used for signal development, and the Bio-Rad chemiluminescence imaging system captured protein band images. Subsequently, the integrated optical density of the target protein Sirt3 and the internal reference protein GAPDH bands was measured using ImageJ software. The ratio of the two was taken as the relative expression level of Sirt3 protein and normalized with the control group as the benchmark.

[0061] Experimental results: After 48 hours of intervention with 100 μM arbutin, high-generation MSCs (P10) showed reduced diameter, increased proliferation capacity, and decreased β-galactosidase activity. Cellular senescence and oxidative stress are closely related. Further studies revealed that arbutin intervention increased SIRT3 expression and SOD2 activity, while reducing intracellular MDA activity. This indicates that arbutin reduces intracellular oxidative stress through the SIRT3 / SOD2 pathway, reversing replicative senescence during MSC expansion.

[0062] The above experimental results indicate that arbutin significantly improves MSC aging caused by in vitro replication and expansion, and this effect is dose-dependent. Furthermore, the highest dose (100 μM) designed in this study did not produce any toxicity to the cells. Therefore, we believe that adding arbutin to the culture medium facilitates the large-scale in vitro culture of MSCs and can accelerate the development of clinical applications.

[0063] In summary, this invention utilizes bearberry extract to delay the replicative senescence of mesenchymal stem cells, specifically by effectively inhibiting cellular senescence phenotypes, including: suppressing senescence-related morphological changes, such as preventing cell enlargement and flattening; significantly reducing the proportion of senescence marker β-galactosidase (SA-β-gal)-positive cells; and maintaining the proliferative potential of high-generation mesenchymal stem cells. Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. Use of ursolic acid in improving expression of SIRT3 and / or delaying replicative senescence of mesenchymal stem cells.

2. Use according to claim 1, characterized in that, The mesenchymal stem cells comprise one or more of umbilical cord mesenchymal stem cells, adipose tissue-derived mesenchymal stem cells, bone marrow mesenchymal stem cells, placental mesenchymal stem cells, dental pulp mesenchymal stem cells or menstrual blood-derived mesenchymal stem cells.

3. Use according to claim 2, characterized in that, The mesenchymal stem cells are human umbilical cord mesenchymal stem cells.

4. Use according to claim 1, characterized in that, The delaying of replicative senescence of mesenchymal stem cells comprises inhibiting the senescence phenotype of cells, which comprises inhibiting senescence-associated morphological changes, significantly reducing the activity of senescence-associated β-galactosidase (SA-β-gal), reducing the proportion of cells staining positive, and maintaining the proliferative potential of mesenchymal stem cells of high passage number.

5. A method of delaying replicative senescence of mesenchymal stem cells, characterized in that, The method comprises: adding ursolic acid to the culture solution of mesenchymal stem cells during the culture of mesenchymal stem cells.

6. The method of claim 5, wherein the method is for delaying replicative senescence of mesenchymal stem cells. The culture solution comprises DMEM-F12 and fetal bovine serum. Preferably, the culture solution further comprises antibiotics; the antibiotics are penicillin and / or streptomycin.

7. The method of claim 5, wherein the method is for delaying replicative senescence of mesenchymal stem cells. The final concentration of ursolic acid in the culture solution is 1-100 µM.

8. The method of claim 5, wherein the method is for delaying replicative senescence of mesenchymal stem cells. The final concentration of ursolic acid in the culture solution is 50-100 µM.

9. The method for delaying replicative senescence of mesenchymal stem cells according to claim 5, characterized in that, The mass ratio of DMEM / F12 medium to fetal bovine serum is 85-95:5-15.

10. A culture solution for delaying replicative senescence of mesenchymal stem cells, characterized by, The culture solution comprises DMEM / F12 medium, fetal bovine serum and ursolic acid.

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