New application of 20 (S)-protopanoxadiol in delaying senescence of bone marrow mesenchymal stem cells

Abstract

The invention provides a new application of 20 (S)-protopanoxadiol (20 (s)-protopanoxadiol, 20 (s)-PPD) in delaying the senescence of bone marrow mesenchymal stem cells (MSCs), and provides a new application of 20 (S)-protopanoxadiol (20 (s)-PPD) in delaying the senescence of bone marrow mesenchymal stem cells. The invention successfully constructs a BM-MSCs cell aging model, and researches find that 20 (S)-protopanoxadiol has in vitro efficacy of delaying BM-MSCs aging, including improvement of cellular morphology, SA-beta-gal staining, intracellular ROS and other aging phenotypic changes, has protein and mRNA expression of senescence related molecules p53, p21 and p16 at molecular level, and has the efficacy of delaying BM-MSCs aging, and has the efficacy of delaying BM-MSCs aging, inhibiting BM-MSCs aging, inhibiting BM-MSCs aging, inhibiting BM-MSCs aging, inhibiting BM-MSCs aging, and inhibiting BM-MSCs aging. And the reduction of adipogenic differentiation and osteogenic differentiation capability of the BM-MSCs induced by the AAPH is obviously improved. The 20 (S)-protopanoxadiol has the effects of promoting proliferation of the bone marrow mesenchymal stem cells and inhibiting replicative senescence, so that the quality of the bone marrow mesenchymal stem cells is guaranteed, and the culture and production cost of the bone marrow mesenchymal stem cells is greatly reduced; the method provides important guarantee for in-vitro amplification, stemness maintenance and subsequent application of the bone marrow mesenchymal stem cells, has positive significance in promoting clinical application of the bone marrow mesenchymal stem cells to the public, and has important economic value and social significance.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and specifically provides a new use of 20(S)-protopanaxadiol in delaying the aging of bone marrow mesenchymal stem cells. Background Art

[0002] Bone marrow mesenchymal stem cells (BM-MSCs) are non-hematopoietic, multipotent stem cells derived from the bone marrow and capable of differentiation. Due to their low immunogenicity, ease of collection, and stable biological properties, they have attracted significant attention as seed cells for tissue and cell engineering. Studies have shown that BM-MSCs undergo rapid senescence during transplantation, leading to a gradual loss of their stemness and an inability of the transplanted cells to proliferate and differentiate into functional cells, ultimately failing to achieve the desired therapeutic effect. Replicative senescence has become one of the most significant bottlenecks in the clinical application of BM-MSCs. Understanding the regulatory mechanisms of stem cell senescence is crucial for identifying pathways to activate stem cell activity and holds promise for the prevention and treatment of age-related degenerative diseases. Therefore, identifying and discovering components that can accelerate the rate and extent of MSC expansion and inhibit replicative senescence is a key step in accelerating and expanding the clinical application of stem cells, with potential implications for clinical transplantation.

[0003] Ginseng is a traditional Chinese medicine remedy for replenishing qi. Ginsenosides are important medicinal components of ginseng. Ginsenosides are composed of numerous monomeric components, including protopanaxadiol (PPD), protopanaxatriol (PPT), aconitol (OCT), and oleanane (OA), each with distinct effects. Research has demonstrated that ginsenosides possess diverse pharmacological activities, including anti-tumor, antioxidant, and anti-inflammatory properties, as well as metabolic stimulation and regulation of cell proliferation and differentiation. 20(S)-protopanaxadiol (20(S)-PPD), the final metabolite of protopanaxadiol saponins metabolized by the human intestinal microbiota, possesses numerous pharmacological effects. However, the application of 20(S)-protopanaxadiol in delaying the aging of bone marrow mesenchymal stem cells has not been reported. Summary of the Invention

[0004] In order to solve the above technical problems, the present invention provides a new use of 20(S)-protopanaxadiol in delaying the aging of bone marrow mesenchymal stem cells.

[0005] Specifically, the present invention provides the use of 20(S)-protopanaxadiol in delaying the aging of bone marrow mesenchymal stem cells.

[0006] Furthermore, the present invention also provides the use of 20(S)-protopanaxadiol in the preparation of a drug for delaying the aging of bone marrow mesenchymal stem cells.

[0007] In the aforementioned use, 20(S)-protopanaxadiol is applied to bone marrow mesenchymal stem cells in vitro to delay their aging.

[0008] In the aforementioned use, 20(S)-protopanaxadiol can delay the aging of bone marrow mesenchymal stem cells by enhancing the cell viability of bone marrow mesenchymal stem cells.

[0009] In the aforementioned use, 20(S)-protopanaxadiol can delay the aging of bone marrow mesenchymal stem cells by reducing the ROS level of bone marrow mesenchymal stem cells.

[0010] In the aforementioned use, 20(S)-protopanaxadiol can delay the aging of bone marrow mesenchymal stem cells by reducing the expression levels of p53, p21 and p16 proteins and increasing the expression level of p-Rb protein.

[0011] In the use described above, 20(S)-protopanaxadiol can delay the aging of bone marrow mesenchymal stem cells by reducing the mRNA levels of p53, p21 and p16 in bone marrow mesenchymal stem cells.

[0012] In the aforementioned use, 20(S)-protopanaxadiol can delay the aging of bone marrow mesenchymal stem cells by improving their adipogenic differentiation ability.

[0013] In the aforementioned use, 20(S)-protopanaxadiol can delay the aging of bone marrow mesenchymal stem cells by improving their osteogenic differentiation ability.

[0014] The screening platform of the present invention is a self-built library of more than 500 small molecule monomer compounds from traditional Chinese medicine and natural products. By establishing a replicative aging model of BM-MSCs and an AAPH-induced premature aging model, a more comprehensive stem cell adipogenic and osteogenic differentiation ability test is used to further evaluate the differentiation ability of stem cells and conduct a preliminary exploration of its influencing mechanism.

[0015] The present invention successfully constructed a BM-MSCs cell aging model. Through research, the present invention found that 20(S)-protopanaxadiol has the in vitro efficacy of delaying BM-MSCs aging, including improving cell morphology, SA-β-gal staining, and intracellular ROS and other aging phenotypic changes. At the molecular level, it has the ability to regulate the protein and mRNA expression of aging-related molecules p53, p21, and p16, and significantly improves the decline in AAPH-induced adipogenic and osteogenic differentiation abilities of BM-MSCs.

[0016] The present invention discovered that 20(S)-protopanaxadiol can promote the proliferation of bone marrow mesenchymal stem cells and inhibit replicative senescence. While ensuring the quality of bone marrow mesenchymal stem cells, it will greatly reduce the cost of bone marrow mesenchymal stem cell culture and production, provide important guarantees for the in vitro expansion, stemness maintenance and subsequent applications of bone marrow mesenchymal stem cells, and have positive significance for promoting the clinical application of bone marrow mesenchymal stem cells for the public, and have important economic value and social significance. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 is the morphological changes of BM-MSCs treated with different concentrations of AAPH; Figure 2 The effect of different concentrations of AAPH treatment on the ROS level of BM-MSCs, where A is the fluorescence image obtained by flow cytometry; B is the quantitative analysis of ROS level; Figure 3 is the effect of AAPH treatment on the expression of senescence-related proteins in BM-MSCs; Figure 4 The effect of 20(S)-protopanaxadiol on BM-MSCs cell viability; Figure 5 The effect of 20(S)-protopanaxadiol on the ROS level induced by AAPH, where A is a fluorescence image obtained by flow cytometry; B is a quantitative analysis of the ROS level; Figure 6 The effect of different concentrations of 20(S)-protopanaxadiol pretreatment on senescence-related proteins in BM-MSCs induced by AAPH; Figure 7 is the effect of 20(S)-protopanaxadiol pretreatment on AAPH-induced BM-MSCs-related mRNA; Figure 8 is the change of senescence-related protein levels in BM-MSCs treated with 20(S)-protopanaxadiol; Figure 9 is the effect of 20(S)-protopanaxadiol treatment on senescence-related mRNA in BM-MSCs; Figure 10 is the effect of 20(S)-protopanaxadiol treatment on adipogenic differentiation; Figure 11 The effect of 20(S)-protopanaxadiol treatment on osteogenic differentiation. DETAILED DESCRIPTION

[0018] Main reagents and instruments: 20(S)-protopanaxadiol (Shanghai Shidande Standard Technology Service Co., Ltd., China); rat bone marrow mesenchymal stem cells (BM-MSCs), Basal Medium (complete medium for rat bone marrow mesenchymal stem cells), 0.25% trypsin-EDTA digestion solution (Saiye (Guangzhou) Biotechnology Co., Ltd., China); Cell Counting Kit-8, phosphatase inhibitor (PhosSTOP), protease inhibitor, protein marker, 2×SYBR Green, 10× Tris-Glycine SDS electrophoresis buffer (pH 8.3), 10× blotting membrane transfer buffer (Wuhan Abotek Biotechnology Co., Ltd., China); RNA rapid extraction kit (Shanghai Yishan Biotechnology Co., Ltd., China); Dimethyl sulfoxide (DMSO) sulfoxide, DMSO), polyethylene glycol dehydrated sorbitan monolaurate (Tween 20), 2,2'-azobis-2-methyl-propanimidamide, dihydrochlori (AAPH), tetramethylethylenediamine solution (TEMED), ammonium persulfate (Sigma-Aldrich, USA); Western primary antibody, secondary antibody diluent, Western primary and secondary antibody removal buffer (strong alkaline), 5× protein loading buffer (containing DTT), RIPA lysis buffer (strong) (Shanghai Beyotime Biotechnology Co., Ltd., China); acrylamide / methylenebisacrylamide 40% solution (29:1), 4× Tris-HCl / SDS buffer (pH 6.8), 4× Tris-HCl / SDS buffer (pH 8.8) (Shanghai Bioengineering Co., Ltd., China); senescence-associated β-galactosidase staining (Shanghai Biotechnology Co., Ltd., China); GAPDH antibody (Wuhan Abbotec Biotechnology Co., Ltd.); p21 antibody (Abcam (Shanghai) Trading Co., Ltd.); p16 antibody (Wuhan Abbotec Biotechnology Co., Ltd.); p53 antibody (Wuhan Abbotec Biotechnology Co., Ltd.); pRb antibody (Wuhan Abbotec Biotechnology Co., Ltd.); fluorescence microscope (ZEN Blue Lite, Carl Zeiss, Germany); PCR instrument (LightCyler96, Thermo Fisher Scientific, USA); chemiluminescence imaging system (ChemiDocXS+, BIO-RAD, USA).

[0019] Example 1 BM-MSCs recovery and culture and calculation of passage age (number of doublings)

[0020] After removing the cells from the liquid nitrogen tank, quickly place them in a 37°C water bath and shake them to thaw rapidly. When only a small ice crystal remains, remove them from the water bath, spray them with 75% alcohol, wipe them dry, and transfer them to a biosafety cabinet. Transfer the thawed cells to 10 volumes of preheated low-glucose medium and centrifuge at 800 rpm for 5 minutes. Discard the supernatant, resuspend the cells in BM-MSCs medium, plate them in an appropriately sized cell culture dish, shake them evenly, and incubate them in an incubator at 37°C, 5% CO2. Change the medium after 24 hours and continue culturing. When the cells reach approximately 80-90% confluence, passage them at a 1:2 or 1:4 ratio. Count cells before and after passage, and count a sample of cells 20 hours after passage to calculate the cell attachment rate (R). The cell passage age (cumulative population doublings, CPD) is calculated as ∑log²(D / D0 / R), where D and D0 are the cell densities at harvest and seeding, respectively, and R is the cell attachment rate.

[0021] Example 2 Detection of cell viability by CCK8 method

[0022] BM-MSCs in the logarithmic growth phase were collected and the cells were divided into 1×10 4 Cells were seeded into 96-well plates at 100 μL per well. After 24 hours of incubation, a gradient of test drug concentrations was added, with six replicate wells set up for each concentration. A control group (solvent without drug) and a blank group without cells were also set up. After 3 days of incubation, 10 μL of CCK-8 solution was added to each well and mixed thoroughly by vortexing. The cells were then incubated in a constant-temperature incubator for 1-2 hours. Absorbance was measured at 450 nm using a microplate reader. Relative viability was calculated, with the blank control group containing only cells as 100%. Cell viability = (OD of the treated group - OD of the blank group / OD of the control group - OD of the blank group) * 100%.

[0023] Example 3 Construction of AAPH-induced premature senescence model of BM-MSCs

[0024] The cells were treated with different concentrations of the free radical inducer AAPH (0-20 mmol / L) for 24 hours. The optimal AAPH concentration was selected based on Western Blot and reactive oxygen species (ROS) level detection experiments. BM-MSCs were treated to construct a free radical-induced premature aging cell model.

[0025] 1. Flow cytometry detection of cellular reactive oxygen free radical levels BM-MSCs in the logarithmic growth phase were collected and the cells were divided into 5×10 4Cells were seeded in 12-well plates. After 24 hours, the culture medium was discarded. Cells were pretreated with drugs containing different concentrations for 12 hours and then treated with AAPH for 24 hours. The cells were then returned to the drug-containing medium and cultured for an additional 72 hours. ROS levels were then measured. The culture medium was aspirated, and cells were washed three times with PBS. 1 mL of the reactive oxygen species (ROS) free radical (ROS) fluorescent probe DCFH-DA (prepared in DMEM basal medium) was added to each well at a molar concentration of 10 μM. The cells were then incubated in a cell culture incubator for 20 minutes. The probe was discarded, and the cells were washed three times with DMEM basal medium. Cells were trypsinized, harvested, centrifuged, and washed once with PBS. Cells were collected by centrifugation again, resuspended in 500 μL of PBS, and transferred to flow cytometers for analysis. 10,000 cells were collected from each group, and cell fluorescence intensity (FL1) was analyzed. Higher fluorescence indicates higher levels of ROS free radicals.

[0026] 2. Western blot detection of aging-related protein expression (1) Extraction of total protein BM-MSCs in the logarithmic growth phase were collected and the cells were divided into 5×10 4 Cells were seeded in 12-well plates at 100 μL / well. After 24 hours, the old culture medium was discarded and cells were pretreated with drugs containing different concentrations for 12 hours. The cells were then treated with AAPH for 24 hours. The culture medium was then returned to the drug-containing medium and cultured for another 72 hours. The cell culture medium was removed and the cells were washed with 1 mL of PBS. The PBS was then removed and 100 μL of RIPA lysis buffer was added to each well. The cells were incubated at 4°C for 30 minutes to complete cell lysis. The cells were gently scraped with a cell scraper and transferred to a 1.5 mL centrifuge tube. The supernatant was then transferred to a new 1.5 mL centrifuge tube to obtain the total protein sample.

[0027] (2) Determination of protein concentration According to the instructions of the BCA protein quantification kit, mix BCA reagent and Cu reagent in a 50:1 ratio to prepare a working solution. Take a 5 mg / mL BSA standard solution and dilute it to 0.5 mg / mL with PBS. Add different volumes of standard protein to a 96-well plate to generate a standard curve. Add 2 μL of extracted protein sample to each well to be tested, make up to a total volume of 20 μL with PBS, and add 200 μL of BCA working solution to incubate at 37°C for 30 minutes. Measure the absorbance at 562 nm and calculate the sample protein concentration based on the BSA standard curve. Standardize the samples to the same concentration for later use.

[0028] (3) Protein denaturation treatment The protein sample was mixed with 5× Loading Buffer in a volume ratio of 4:1, and then heated in a 100°C metal bath for 8 min for denaturation. After mixing, the mixture was centrifuged at low speed and stored in a -20°C refrigerator.

[0029] (4) SDS-PAGE protein electrophoresis Prepare the separating gel and stacking gel in the order described. Select a comb with the appropriate number of wells based on your experimental needs. Store the prepared gel in electrophoresis buffer at 4°C or use directly. After thawing the protein sample, gently shake to mix and centrifuge at low speed. After adding the protein sample, run the electrophoresis at 80 V for 30 minutes, then adjust to 120 V and continue electrophoresis for 60 minutes.

[0030] (5) Immunoimprinting Depending on the size of the gel, pre-cut two layers of filter paper and a PVDF membrane to match the size of the gel. Soak the PVDF membrane in methanol for 2 minutes, then soak the filter paper and gel in transfer buffer. After electrophoresis, carefully remove the gel and place it in transfer buffer. Then, perform a semi-dry or wet transfer. The semi-dry transfer procedure is as follows: Place a layer of filter paper on the anode of a semi-dry transfer apparatus. Place the PVDF membrane on top. Place the gel on top of the membrane, then top it with the filter paper. Align the layers carefully and remove any bubbles. Finally, close the cathode of the transfer apparatus and transfer at 0.65-0.80 mA / cm² for 2 hours. For wet transfer, chill the transfer buffer at 4°C. Follow the transfer instructions to create a "sandwich": Place the gray transfer box downwards, align the pre-soaked transfer buffer pad, filter paper, gel, and PVDF membrane in that order, and then cover with the filter paper and pad. Gently remove any bubbles with a glass rod. After the electroporation box is assembled, place it in the electroporation tank, install the cooling box, add about 500 ml of transfer buffer, and transfer the membrane at 320 mA for 1 hour.

[0031] After transfer, the membrane was removed and briefly soaked in methanol to fully immobilize the proteins on the PVDF membrane. The membrane was then blocked with shaking in TBST containing 5% nonfat dry milk for 1 hour. The blocked membrane was placed in a hybridization bag and reacted with the primary antibody solution at room temperature for 1 hour or overnight at 4°C. Unbound antibody was then washed three times with TBST containing 0.1% Tween 20 for 5 minutes each to remove the membrane. In a new hybridization bag, the membrane was reacted with a horseradish peroxidase-conjugated secondary antibody solution at room temperature for 1 hour and then washed three times with TBST for 5 minutes each. Proteins were detected using chemiluminescence, developed according to the ECLPLUS Western Blotting Kit instructions, and images were captured using a Chemi Imager 5500 gel imaging system.

[0032] 3. qRT-PCR detection of aging-related gene expression Total RNA was extracted from BM-MSCs in different treatment groups using an RNA rapid extraction kit and reverse transcribed to obtain cDNA. Using cDNA as a template, the PCR reaction system was prepared according to the instructions of the SYBR® Premix Ex TaqTM Fluorescence Quantitation Kit, and the transcription levels of aging pathway-related genes (p16, p21, and p53) were detected using a Roche real-time fluorescence quantitative PCR instrument.

[0033] 4. β-Galactosidase Staining Follow the kit instructions: Prepare the working staining solution by mixing the components of the staining solution in the appropriate proportions. Discard the old culture medium and wash the cells once with 500 μL of PBS per well. Discard the PBS and add 500 μL of fixative solution. Incubate at room temperature for 15 minutes. Aspirate the fixative solution and wash the cells three times with 500 μL of PBS per well for 3 minutes each. Remove the PBS after each wash. Stain the cells by adding 500 μL of the working staining solution to each well. Wrap the cell culture plate with plastic wrap to prevent evaporation of the solution and changes in the staining solution concentration. Incubate and stain at 37°C for 3-16 hours. Count and observe under a microscope (positive cells will appear blue-green after staining).

[0034] 5. Adipogenic Differentiation Ability Detection The test was performed according to the Oricell® Rat Bone Marrow Mesenchymal Stem Cell Adipogenic Differentiation Kit; the rat bone marrow mesenchymal stem cells to be induced were divided into 1*10 4 Cells were seeded at a density of 10 cells / cm in 12-well plates. After adipogenesis induction, the cells were fixed with 4% paraformaldehyde for 3 minutes, and the accumulation of intracellular lipid droplets was measured using Oil Red 0. The culture plates were placed under a microscope to observe the adipogenic staining effect.

[0035] 6. Osteogenic Differentiation Ability Detection The test was performed according to the Oricell® Rat Bone Marrow Mesenchymal Stem Cell Adipogenic Differentiation Kit; the rat bone marrow mesenchymal stem cells to be induced were divided into 1*10 4 Cells were seeded at a density of 100 cells / cm in a 12-well plate and stained with Alizarin Red after induction of osteogenesis. Osteogenic differentiation was quantified by adding an aqueous solution containing 20% methanol and 10% acetic acid to the 96-well plate after osteogenic differentiation staining was completed, incubating at room temperature for 15 minutes, and then measuring the A value on a microplate reader. 450 value.

[0036] Statistical analysis of results: The experimental results presented in this section were obtained from three independent experiments or three replicates within a single experiment. All data were analyzed and plotted using Graphpad 9.5. One-way analysis of variance was used to compare means between multiple groups, and independent sample t-tests were used to compare means between two groups. P < 0.05 was considered statistically significant.

[0037] Example 4 AAPH-induced premature senescence of BM-MSCs

[0038] The free radical generator AAPH with better stability was selected to construct a premature cell aging model.

[0039] 1. Effects of different concentrations of AAPH on BM-MSC morphology After 24 h of treatment with different concentrations of AAPH, the culture medium was returned to normal and the cell status was observed after 72 h of culture. Figure 1 ), after AAPH treatment, the cells shrank significantly and the density decreased, indicating that the cell proliferation rate decreased.

[0040] 2. Effect of AAPH treatment on the level of reactive oxygen species in BM-MSCs The accumulation of reactive oxygen species (ROS) is considered to be one of the main causes of cell senescence. To verify the effect of AAPH on BM-MSCs, we used flow cytometry to detect the level of cellular reactive oxygen species. Figure 2 After 24 hours of treatment with different concentrations of AAPH, the cells were returned to normal culture medium and cultured for another 72 hours. The ROS levels in the treated groups were significantly increased compared to those in the control group.

[0041] Effects of AAPH treatment on senescence-related proteins in BM-MSCs To further verify whether AAPH treatment causes premature senescence of BM-MSCs, we used Western Blot to detect the protein levels of senescence marker proteins p53, p21, and p16. The results showed that treatment with different concentrations of AAPH led to a significant increase in the expression levels of p53, p21, and p16 proteins. Figure 3 As shown. This indicates that AAPH can induce premature senescence of BM-MSCs. Based on the experimental results, we chose to treat the cells with 1mM AAPH for 24h.

[0042] Example 5 20(S)-protopanaxadiol delays BM-MSCs aging

[0043] 1. Effect of 20(S)-protopanaxadiol on BM-MSCs cell viability After treatment with different concentrations of 20(S)-protopanaxadiol (1-40 μM), the cell viability of BM-MSCs increased significantly with the increase of 20(S)-protopanaxadiol concentration. Figure 4 shown.

[0044] 2. Effect of 20(S)-protopanaxadiol pretreatment on premature senescence of BM-MSCs induced by AAPH (1) Effects of different concentrations of 20(S)-protopanaxadiol pretreatment on ROS levels in AAPH-induced BM-MSCs In order to verify the effect of different concentrations of 20(S)-protopanaxadiol pretreatment on AAPH-induced BM-MSCs, we used flow cytometry to detect the level of cellular reactive oxygen free radicals. Figure 5 As shown, NAC served as a positive control at a concentration of 2 mM. After 12 hours of drug pretreatment, cells were treated with 1 mM AAPH for 24 hours and then returned to the drug-containing Basal Medium for an additional 72 hours. We found that ROS levels were significantly elevated in the AAPH-treated and solvent-treated groups compared to the control group. However, ROS levels were significantly reduced in drug-pretreated cells compared to the AAPH-treated group. At 5 μM, 20(S)-protopanaxadiol levels were comparable to those of NAC, and ROS levels decreased with increasing concentrations.

[0045] (2) Changes in protein levels of AAPH-induced BM-MSCs after pretreatment with different concentrations of 20(S)-protopanaxadiol In order to further verify whether 20(S)-protopanaxadiol pretreatment can improve the premature senescence of BM-MSCs induced by AAPH, we used Western Blot to detect the protein levels of aging marker proteins p-Rb, p53, p21, and p16, respectively. NAC was used as a positive control with a concentration of 2mM. The results showed that the protein levels of p53, p21, and p16 in BM-MSCs induced by AAPH were significantly increased compared with the solvent control group (DMSO), while p-Rb decreased. After treatment with different concentrations of drugs, the expression levels of p53, p21, and p16 proteins were significantly decreased, while p-Rb was significantly increased. Among them, the protein levels of cells pretreated with 20μM 20(S)-protopanaxadiol were comparable to those of the Control group, indicating that 20(S)-protopanaxadiol pretreatment can improve the premature senescence of BM-MSCs induced by AAPH. Figure 6 shown.

[0046] (3) Effects of different concentrations of 20(S)-protopanaxadiol pretreatment on AAPH-induced changes in BM-MSCs-related mRNA qRT-PCR was used to further examine the effects of 20(S)-protopanaxadiol pretreatment on the senescence-related genes p53, p21, and p16 in BM-MSCs induced by AAPH. NAC was used as a positive control at a concentration of 2 mM. The results showed that the mRNA expression of p53, p21, and p16 in BM-MSCs induced by AAPH was significantly increased compared with the solvent control group (DMSO). However, the mRNA expression of p53, p21, and p16 was significantly decreased after treatment with different concentrations of drugs, indicating that 20(S)-protopanaxadiol pretreatment can improve the premature senescence of BM-MSCs induced by AAPH. Figure 7 shown.

[0047] 3. 20(S)-Protopanaxadiol Delays Replicative Senescence of BM-MSCs (1) Changes in protein levels of BM-MSCs treated with 20(S)-protopanaxadiol When cells were cultured to passage 20, BM-MSCs were continuously treated with 20(S)-protopanaxadiol (10μM). Proteins extracted from passage 28 showed a significant decrease in p-Rb levels, while p53, p21, and p16 protein levels were significantly increased compared to passage 10 cells. Compared to the control group at passage 28, BM-MSCs treated with 20(S)-protopanaxadiol (10μM) showed a significant increase in p-Rb levels, while p53, p21, and p16 protein levels were significantly decreased, indicating that 20(S)-protopanaxadiol has excellent in vitro anti-aging activity. Figure 8 shown.

[0048] (2) Effects of 20(S)-protopanaxadiol treatment on BM-MSCs-related mRNA qRT-PCR was used to further examine the effects of drug treatment on the expression of BM-MSC aging-related genes p53, p21, and p16. The results showed that the mRNA levels of p53, p21, and p16 were significantly decreased after treatment with 20(S)-protopanaxadiol (10 μM). This further demonstrates that 20(S)-protopanaxadiol has excellent in vitro anti-aging activity. Figure 9 shown.

[0049] Effects of 20(S)-protopanaxadiol pretreatment on the adipogenic and osteogenic differentiation of BM-MSCs induced by AAPH (1) Effect of 20(S)-protopanaxadiol pretreatment on AAPH-induced adipogenic differentiation of BM-MSCs To further verify the effect of AAPH-induced premature senescence of BM-MSCs on the adipogenic differentiation ability of cells, as well as the effect of drug pretreatment on it, we used Oricell ®The rat bone marrow mesenchymal stem cell adipogenic differentiation kit was used for testing. The results showed that premature senescence of BM-MSCs induced by AAPH weakened the adipogenic differentiation ability of BM-MSCs. However, drug treatment significantly improved the adipogenic differentiation ability of BM-MSCs induced by AAPH. Figure 10 shown.

[0050] (2) Effect of 20(S)-protopanaxadiol pretreatment on AAPH-induced osteogenic differentiation of BM-MSCs To further verify the effect of AAPH-induced premature senescence of BM-MSCs on the osteogenic differentiation ability of cells, as well as the effect of drug pretreatment on it, we used Oricell ® The rat bone marrow mesenchymal stem cell osteogenic differentiation kit was used for testing. The results showed that premature aging of BM-MSCs induced by AAPH weakened the osteogenic differentiation ability of BM-MSCs. However, drug treatment significantly improved the osteogenic differentiation ability of BM-MSCs induced by AAPH. Compared with the solvent control group (DMSO),*** P < 0.001 .like Figure 11 shown.

[0051] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention are included in the scope of protection of the present invention.

Claims

1. Use of 20(S)-protopanaxadiol in delaying the aging of bone marrow mesenchymal stem cells.

2. The use according to claim 1, characterized in that Use of 20(S)-protopanaxadiol in preparing a drug for delaying aging of bone marrow mesenchymal stem cells.

3. The method according to claim 1 or 2, wherein the 20(S)-Protopanaxadiol was applied to bone marrow mesenchymal stem cells in vitro to delay their aging.

4. The method according to claim 1 or 2, wherein the 20(S)-Protopanaxadiol can delay the aging of bone marrow mesenchymal stem cells by enhancing the cell viability of bone marrow mesenchymal stem cells.

5. The method according to claim 1 or 2, wherein the method is characterized in that 20(S)-Protopanaxadiol can delay the aging of bone marrow mesenchymal stem cells by reducing the ROS level of bone marrow mesenchymal stem cells.

6. The use according to claim 1 or 2, characterized in that 20(S)-protopanaxadiol can delay the aging of bone marrow mesenchymal stem cells by reducing the expression levels of p53, p21 and p16 proteins and increasing the expression level of p-Rb protein.

7. The method according to claim 1 or 2, wherein the 20(S)-protopanaxadiol can delay the aging of bone marrow mesenchymal stem cells by reducing the mRNA levels of p53, p21 and p16 in bone marrow mesenchymal stem cells.

8. The use according to claim 1 or 2, characterized in that 20(S)-protopanaxadiol can delay the aging of bone marrow mesenchymal stem cells by improving their adipogenic differentiation ability.

9. The method according to claim 1 or 2, wherein the method is characterized in that 20(S)-Protopanaxadiol can delay the aging of bone marrow mesenchymal stem cells by improving their osteogenic differentiation ability.

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