Application of GYY4137 in preparation of medicine or reagent for delaying senescence of human gingival mesenchymal stem cells

Abstract

The invention discloses application of GYY4137 in preparation of a medicine for delaying senescence of human gingival mesenchymal stem cells or a reagent, and relates to the technical field of biological medicine and stem cell senescence resistance. The exogenous supplement of the hydrogen sulfide (HS) donor GYY4137 can effectively improve or recover the expression level of the U2AF1 gene, effectively inhibit the excessive activation of an NF-kappa B signal channel, reduce the activity of an aging marker beta-galactosidase, and improve the cell proliferation ability. The discovery not only verifies the key effect of the U2AF1 gene in the aging process of the hGMSCs, but also indicates that an important clue can be provided for revealing the molecular mechanism of the aging of the hGMSCs by regulating the expression of the U2AF1 gene. The GYY4137 is used for treating the human gingival mesenchymal stem cells, so that the aging of the human gingival mesenchymal stem cells can be delayed. In addition, the U2AF1 gene is a potential molecular marker related to aging.

Description

Technical Field

[0001] This invention relates to the fields of biomedicine and stem cell anti-aging technology, and more specifically, to the application of GYY4137 in the preparation of drugs or reagents for delaying the aging of human gingival mesenchymal stem cells. Background Technology

[0002] Human gingival mesenchymal stem cells (hGMSCs) are adult stem cells with multipotent differentiation potential, showing great promise for periodontal tissue regeneration and wound repair. However, cellular senescence significantly limits the clinical application of hGMSCs. With increasing cell passage number, hGMSCs gradually lose their proliferative activity, primarily manifested as cell cycle arrest and the release of senescence-associated secretory phenotypes (SASPs).

[0003] The molecular mechanisms underlying the senescence of human gingival mesenchymal stem cells (hGMSCs) are not yet fully understood. While current research recognizes that cellular senescence involves multiple complex biological processes, a deeper understanding of the specific roles of gene regulation and signaling pathways in hGMSC senescence remains lacking. Therefore, developing effective strategies to delay hGMSC senescence remains a pressing issue.

[0004] Furthermore, there is currently a lack of specific molecular markers to accurately determine the aging state of cells, which is not conducive to taking timely and effective anti-aging measures.

[0005] In view of this, the present invention is proposed. Summary of the Invention

[0006] The purpose of this invention is to provide the application of GYY4137 in the preparation of drugs or reagents for delaying the aging of human gingival mesenchymal stem cells (hGMSCs) to delay the aging of hGMSCs.

[0007] This invention is implemented as follows:

[0008] In a first aspect, the present invention provides the application of GYY4137 in the preparation of drugs or reagents for delaying the aging of human gingival mesenchymal stem cells.

[0009] Secondly, this invention provides the application of reagents for detecting the U2AF1 gene in the preparation of human gingival mesenchymal stem cell aging prediction products.

[0010] The present invention has the following beneficial effects:

[0011] Differential gene expression analysis was performed on human gingival mesenchymal stem cells (hGMSCs) of different generations. Using bioinformatics screening methods, it was found that the expression of the U2AF1 gene in senescent hGMSCs was significantly lower than that in young hGMSCs, and its expression level was negatively correlated with the degree of cellular senescence. Therefore, the U2AF1 gene is a potential molecular marker associated with aging. Exogenous supplementation with the hydrogen sulfide (HS) donor GYY4137 effectively increased or restored the expression level of the U2AF1 gene, effectively inhibited the overactivation of the NF-κB signaling pathway, reduced the activity of the aging marker β-galactosidase, and enhanced cell proliferation. This finding not only confirms the key role of the U2AF1 gene in the senescence process of hGMSCs, but also indicates that regulating the expression of the U2AF1 gene can provide important clues for revealing the molecular mechanisms of hGMSC senescence. Treatment of human gingival mesenchymal stem cells with GYY4137 can delay the senescence of human gingival mesenchymal stem cells. Attached Figure Description

[0012] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0013] Figure 1 Morphological observation of human gingival mesenchymal stem cells (hGMSCs), showing the morphological comparison of hGMSCs of different generations under an inverted microscope (a, generation P3; b, generation P10).

[0014] Figure 2 A diagram showing the clonogenic capacity of P3 generation hGMSCs;

[0015] Figure 3 Figures showing the osteogenic and adipogenic differentiation capabilities of P3 generation hGMSCs (a shows the staining results after osteogenic induction, b shows the staining results after adipogenic induction).

[0016] Figure 4 The expression of surface antigens in hGMSCs was as follows: (a) CD105 positivity rate was 99.97%; (b) CD90 positivity rate was 99.82%; (c) CD45 positivity rate was 1.47%; (d) CD34 positivity rate was 1.24%.

[0017] Figure 5 The diagram shows the morphological changes of hGMSCs from different generations (a shows the morphology of hGMSCs from generation P5, and b shows the morphology of hGMSCs from generation P10).

[0018] Figure 6 The images show the β-galactosidase staining results and positive rate statistics of three groups of hGMSCs; ac represents the β-galactosidase staining results of cells in the P5, P10, and P10GYY groups, respectively; and d represents the statistical graph of the β-galactosidase staining positive rate of the three groups.

[0019] Figure 7 The following figures show the proliferation detection results of three groups of hGMSCs (a shows the CCK-8 detection results, and b shows the RTCA detection results).

[0020] Figure 8 The results of cell cycle changes in three groups of hGMSCs are shown in the figure. The cell cycle distribution of the P5, P10 and P10GYY groups is shown by the flow cytometry results (a shows the cell cycle distribution of P5 group, b shows the cell cycle distribution of P10 group, c shows the cell cycle distribution of P10GYY group, d and e are the statistical graphs of G0 / G1 phase and G2 phase of the three groups respectively, and the distribution ratio of each group of cells at different cell cycle stages is presented in bar chart form).

[0021] Figure 9 The images show the expression of senescence-related proteins in three groups of cells. Western blot was used to detect the expression of senescence-related proteins such as P16, P21, P53, and p-P53 in the P5, P10, and P10GYY groups (a shows the protein bands in the three groups of cells, and the protein expression levels in different groups are significantly different; be shows the bar charts showing the relative expression levels of P16, P21, P53, and p-P53 proteins in the three groups).

[0022] Figure 10 The transcriptome differential gene analysis results include statistics on the number of differentially expressed genes in the P10 vs P5 and P10GYY vs P10 groups, a Venn plot of differential gene intersection, a volcano plot of differentially expressed genes, and a heatmap of differential gene clustering analysis. Specifically: a) shows the statistics on the number of differentially expressed genes, presented as a bar chart showing the number of upregulated and downregulated genes in the two groups; b) shows the Venn plot of differential gene intersection, showing the number of shared differentially expressed genes in the two groups; c) and d) show the volcano plots of differentially expressed genes in the P10 vs P5 and P10GYY vs P10 groups, respectively, presenting the relationship between gene expression fold change and statistical significance in scatter plots, with significantly different genes marked with different colors; e) shows the heatmap of differential gene clustering analysis, using color intensity to represent gene expression levels, presenting the overall differences in gene expression between different groups.

[0023] Figure 11The images show the results of GO and KEGG enrichment analyses. Figure a shows the GO enrichment analysis results, where BP is mainly enriched in processes such as cell adhesion, bioadhesion, cell motility, and homologous cell adhesion. CC is mainly enriched in cellular components such as actin cytoskeleton, proteasome core complex, and endosomes. MF is mainly enriched in molecular functions such as ATP binding, protein kinase activity, and hydrolase activity. Figure b shows the KEGG enrichment analysis results, presented in a bubble chart format, showing the signaling pathways involved in differentially expressed genes, such as cell adhesion pathways. The size of the bubbles represents the number of genes, and the intensity of the color indicates the significance of enrichment.

[0024] Figure 12 This diagram shows the screening and validation of the key differentially expressed gene U2AF1, including statistics on the number of differentially expressed genes in the P10 vs P5 and P10GYY vs P10 groups, Venn plots of differentially expressed gene intersections, volcano plots of differentially expressed genes, Western blot bands of U2AF1 protein in the three groups, relative expression levels of U2AF1 gene RT-qPCR in the three groups, and relative expression levels of U2AF1 protein in the three groups. Specifically: a shows the statistics on the number of differentially expressed genes; b shows the Venn plot of differentially expressed gene intersections; c and d show the volcano plots of differentially expressed genes in the P10 vs P5 and P10GYY vs P10 groups, respectively; e shows the Western blot bands of U2AF1 protein in the three groups; f and g show the bar charts of relative expression levels of U2AF1 gene RT-qPCR in the three groups and relative expression levels of U2AF1 protein in the three groups, respectively.

[0025] Figure 13 To illustrate the senescence characteristics of hGMSCs induced by U2AF1 knockdown, β-galactosidase staining images of the siNC, siU2AF1, and siU2AF1+GYY4137 groups are shown. Specifically: ac represents the staining results of the siNC, siU2AF1, and siU2AF1+GYY4137 groups, respectively; d represents the Western blot results of the siNC, siU2AF1, and siU2AF1+GYY4137 groups; e represents the statistical analysis of the β-galactosidase staining positivity rates in the three groups; and fh represents bar charts showing the relative expression levels of U2AF1, P16, and P21 proteins in the three groups, respectively.

[0026] Figure 14This diagram illustrates the inhibition of NF-κB pathway activation by U2AF1. It presents the relative expression levels of three groups of NF-κB pathway proteins detected by Western blot, including the relative expression levels of U2AF1, P65, IκBα, p-P65, and p-IκBα proteins. Specifically: a shows the Western blot bands of the three groups of NF-κB pathway proteins; b and f show the bar charts representing the relative expression levels of U2AF1, P65, IκBα, p-P65, and p-IκBα proteins, respectively.

[0027] Figure 15 The experimental results for screening the optimal concentration (100 μM) of GYY4137 are shown in the figure. Detailed Implementation

[0028] Reference will now be made to detailed embodiments of the present invention, one or more of which are described below. Each example is provided for explanation and not for limitation of the invention. In fact, it will be apparent to those skilled in the art that various modifications and variations can be made to the invention without departing from its scope or spirit. For example, features described or illustrated as part of one embodiment may be used in another embodiment to produce further embodiments.

[0029] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0030] Definition of noun

[0031] GYY4137 is a water-soluble, slow-releasing chemical reagent containing hydrogen sulfide (HS), CAS No.: 106740-09-4.

[0032] In a first aspect, the present invention provides the application of GYY4137 in the preparation of drugs or reagents for delaying the aging of human gingival mesenchymal stem cells (hGMSCs).

[0033] The inventors' experiments revealed that exogenous supplementation with the hydrogen sulfide (HS) donor GYY4137 can effectively enhance or restore the expression level of the U2AF1 gene, effectively inhibit the overactivation of the NF-κB signaling pathway, reduce the activity of the aging marker β-galactosidase, and enhance cell proliferation. This discovery not only confirms the crucial role of the U2AF1 gene in the aging process of hGMSCs, but also indicates that regulating the expression of the U2AF1 gene can provide important clues for revealing the molecular mechanisms of hGMSC aging. Treatment of human gingival mesenchymal stem cells with GYY4137 can delay the aging of human gingival mesenchymal stem cells.

[0034] Therefore, GYY4137 has promising applications in drugs that delay the aging of human gingival mesenchymal stem cells. It can be used as an anti-aging agent or a drug to delay aging of hGMSCs in oral tissue repair, skin wound healing, and periodontal bone defect repair.

[0035] In a preferred embodiment of the present invention, the drug or reagent has at least one of the following uses:

[0036] (1) Restore the expression level of U2AF1 in human gingival mesenchymal stem cells;

[0037] (2) Inhibit the overactivation of the NF-κB signaling pathway;

[0038] (3) Reduces the activity of β-galactosidase, a marker of cellular senescence;

[0039] (4) Enhance or restore the proliferative capacity of human gingival mesenchymal stem cells;

[0040] (5) Downregulate the expression of aging-related proteins;

[0041] (6) Reduce the proportion of human gingival mesenchymal stem cells in the G0 / G1 phase and increase the proportion of human gingival mesenchymal stem cells in the G2 phase.

[0042] In a preferred embodiment of the present invention, the aging-related protein is selected from at least one of P16, P21, P53 and p-P53 proteins.

[0043] In a preferred embodiment of the present invention, the final concentration of GYY4137 used in the drug or reagent is 50-150 μM, for example, the final concentration of GYY4137 used in the drug or reagent is 50, 60, 70, 80, 90, 100, 110, 120, 130, 140 or 150 μM.

[0044] In a preferred embodiment of the present invention, the final concentration of GYY4137 used as the drug or reagent is 100 μM. At this final concentration, it exhibits excellent effects in delaying the aging of human gingival mesenchymal stem cells.

[0045] In a preferred embodiment of the present invention, when using the drug or reagent, each 1×10n human gingival mesenchymal stem cells / mL is mixed and incubated with 50-150 μM of GYY4137.

[0046] In a preferred embodiment of the present invention, the conditions for mixed incubation are: incubation at 37°C and 5% CO for 12 hours.

[0047] In a preferred embodiment of the present invention, the drug also includes a pharmaceutically acceptable carrier.

[0048] In one embodiment, the dosage form of the drug is a tablet, pill, powder, suspension, gel, emulsion, cream, granule, nanoparticle, capsule, suppository, injection, or spray. The dosage form of the drug is an injection or powder for injection.

[0049] In one embodiment, the aforementioned drug is a liquid pharmaceutical preparation (such as an injectable formulation), such as a solution, suspension, or gel, which typically contains a liquid carrier, such as water, and / or a pharmaceutically acceptable organic solvent. Furthermore, such liquid preparations may also contain a pharmaceutically acceptable carrier, such as those selected from excipients, diluents, pH adjusters, emulsifiers or dispersants, buffers, preservatives, wetting agents, gelling agents (e.g., methylcellulose), dyes, and / or flavoring agents, as defined above. The drugs may be isotonic, i.e., they may have the same osmotic pressure as blood. The isotonicity of the drug can be adjusted by using sodium chloride and other pharmaceutically acceptable reagents, such as glucose, maltose, boric acid, sodium tartrate, propylene glycol, and other inorganic or organic soluble substances. The viscosity of the liquid composition can be adjusted by a pharmaceutically acceptable thickener, such as methylcellulose. Other suitable thickeners include, for example, xanthan gum, carboxymethyl cellulose, hydroxypropyl cellulose, carbomer, etc. The preferred concentration of the thickener depends on the reagent selected.

[0050] In one alternative embodiment, the drug is a solid pharmaceutical preparation, such as a granule preparation.

[0051] In one alternative implementation, the drug is formulated and administered directly in vitro in combination with human gingival mesenchymal stem cells.

[0052] Secondly, this invention provides the application of reagents for detecting the U2AF1 gene in the preparation of human gingival mesenchymal stem cell aging prediction products.

[0053] In a preferred embodiment of the present invention, the human gingival mesenchymal stem cell aging prediction product is selected from reagent kits, test strips, chips, or detectors.

[0054] In a preferred embodiment of the present invention, the reagent for detecting the U2AF1 gene is selected from primers, probes, ELISA reagents, or Western blotting reagents.

[0055] A chip, also known as a suspension array or liquid array, consists of a carrier and nucleic acid molecules (such as primers and / or probes) bound to the surface of the carrier.

[0056] The aforementioned carrier can be made of various materials and in various forms, such as preferably a container with a flat bottom. A more typical preferred example is multi-well plates, microplates, microfluidic-based devices (e.g., microfluidic chips), petri dish-like containers, etc., which are widely used in biochemical assays, and are not limited thereto.

[0057] The microfluidic chip is selected from T-type chip, flow focusing chip or coaxial flow chip PDMS chip or metal droplet generator or PMMA microfluidic chip.

[0058] Furthermore, the kit may also include at least one of the following: buffer solution, detection reagent, diluent, and washing solution, and is not limited thereto.

[0059] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0060] This invention analyzes gene expression differences in hGMSCs of different generations through transcriptome sequencing and, combined with bioinformatics screening, reveals that U2AF1 gene expression is significantly lower in senescent hGMSCs than in young hGMSCs, and its expression level is negatively correlated with the degree of cellular senescence. Further experiments show that exogenous supplementation with the HS donor GYY4137 significantly restores U2AF1 expression levels, effectively inhibits the overactivation of the NF-κB signaling pathway, reduces the activity of the senescence marker β-galactosidase, and enhances cell proliferation. Functional validation experiments show that knockdown of U2AF1 leads to enhanced cellular senescence phenotypes, while GYY4137 treatment effectively reverses this phenomenon, indicating that U2AF1 is a key target for the anti-aging effects of GYY4137.

[0061] In the following examples, all experimental data were statistically analyzed and plotted using GraphPadPrism 10.1.2 software. Before statistical analysis, all experimental data underwent normality and homogeneity of variance tests. The t-test was used to compare statistical differences between two groups, and one-way ANOVA was used to compare statistical differences among multiple groups. All results are expressed as mean ± standard deviation (Mean ± SD). A p-value < 0.05 indicates a statistically significant difference.

[0062] Example 1

[0063] This embodiment establishes an aging model and verifies the aging state using cell aging-related detection methods.

[0064] The experimental method is as follows:

[0065] 1. Cell lines and culture

[0066] Human gingival mesenchymal stem cells (hGMSCs) were isolated from gingival tissue of healthy donors. After obtaining the gingival tissue, hGMSCs were screened using collagenase digestion and α-MEM medium. The isolated hGMSCs were placed in α-MEM medium containing 10% fetal bovine serum and cultured in an incubator at 37°C and 5% CO2. Cells were passaged when they reached 80% confluence at a passage ratio of 1:3, with P3-P10 passages used in the experiments.

[0067] 2. Main reagents and consumables

[0068] α-MEM medium, fetal bovine serum (FBS), collagenase IV, penicillin-streptomycin mixture, 0.25% trypsin-EDTA solution, universal tissue fixative, crystal violet staining solution, alizarin red S, Oil Red O, flow cytometry fluorescently labeled antibodies (CD105, CD90, CD45, CD34), sodium β-glycerophosphate, ascorbic acid, dexamethasone, 3-isobutyl-1-methylxanthine (IBMX), indomethacin, recombinant human insulin, HBSS, PBS, cell culture dishes, sterile centrifuge tubes, cell cryopreservation tubes, and sterile pipette tips were all purchased from relevant biotechnology companies.

[0069] 3. Establish an aging model

[0070] A natural aging model was constructed by continuously passaged to the P10 generation, while a lower generation group (P5 generation) was set up as a young control. The P10 generation cells were treated with GYY4137 (MCE, 106740-09-4) (100 μM) for 12 h to evaluate the anti-aging effect.

[0071] Figure 1Morphological observation images of human gingival mesenchymal stem cells (hGMSCs) show the morphological comparison of hGMSCs of different generations under an inverted microscope. Among them: Figure 1 Figure a shows the typical morphology of P3 generation hGMSCs. The cells grow in a long spindle-shaped or polygonal monolayer, exhibiting good growth status and typical mesenchymal stem cell morphological characteristics. Figure 1 Figure b shows the morphology of P10 generation hGMSCs. Compared with P3 generation, P10 generation cells are larger in size, more irregular in shape, and exhibit senescence-related morphological changes, such as uneven cell edges with wrinkles and protrusions. These changes reflect the evolution of morphological characteristics during cell senescence.

[0072] 4. Cell colony formation experiment

[0073] Three replicates were set up for each group. Cells were diluted to an appropriate concentration and seeded into 6-well culture plates and cultured for 10–14 days. After the culture, the cells were fixed with 4% paraformaldehyde, stained with crystal violet, and the number of colonies was counted.

[0074] clonogenic capacity diagram of hGMSCs (refer to...) Figure 2 As shown, this study presents the results of colony formation experiments for P3 generation hGMSCs. The colonies exhibit well-defined circular or elliptical structures, with diameters mostly concentrated in the range of 3-5 mm. The central region shows a dark, speckled appearance due to dense cell accumulation, while the cell density gradient decreases in the peripheral transition zone, demonstrating the self-renewal and colony formation capabilities of hGMSCs.

[0075] In summary, the experimental results show that hGMSCs gradually exhibit senescence characteristics with increasing passage number. Cell morphology observation reveals significant differences between P3 and P10 generation hGMSCs under an inverted microscope, with P10 generation cells exhibiting typical senescence characteristics.

[0076] 5. An experiment to assess the osteogenic and adipogenic differentiation capacity of hGMSCs.

[0077] The experimental method is as follows:

[0078] Osteogenic differentiation induction: (1) Selected P3 generation hGMSCs with good growth status, digested with trypsin, and calibrated for density using an automated cell counter. They were seeded in 6-well plates at a density of 1×10 cells / well and cultured in an incubator at 37℃ and 5% CO2. When the cell confluence reached 70%-80%, the original culture medium was removed, and the cells were gently rinsed 3 times with PBS to remove residual culture medium. Then, osteogenic induction medium was added, and the cells were cultured at 37℃ and 5% CO2 for 28 days. The medium was completely replaced every 3 days to maintain the activity of inducing factors. (2) After osteogenic induction, the induction medium was discarded, and the cells were gently rinsed 3 times with PBS. Then, a general tissue fixative was added, and the cells were fixed at room temperature for 30 min. Next, Alizarin Red S staining solution was added, and the cells were reacted at room temperature in the dark for 30 min. The cells were then gently rinsed 3 times with PBS until the background was clear. Finally, the experimental results were analyzed and photographed using an inverted optical microscope.

[0079] Adipogenic differentiation induction of hGMSCs: (1) Select well-growing P3 generation hGMSCs and seed them in 6-well plates using the same method as osteogenic induction. When the cells reach 100% confluence, adipogenic induction begins. Remove the original culture medium, gently wash three times with PBS to remove residual culture medium, and then replace it with adipogenic induction medium. Incubate at 37℃ and 5% CO2 for 21 days, changing the medium completely every 3 days to maintain the activity of inducing factors. (2) After adipogenic induction, discard the induction medium, gently wash three times with PBS, and then add general tissue fixative. Fix at room temperature for 30 min. Next, add Oil Red O staining solution and stain at room temperature in the dark for 30 min to mark intracellular lipid droplets. Then gently wash three times with PBS until the background is clear. Finally, analyze the experimental results and take pictures using an inverted optical microscope.

[0080] Results reference Figure 3 As shown, Figure 3 This is a diagram showing the osteogenic and adipogenic differentiation capabilities of hGMSCs, where: Figure 3 Figure 'a' shows the staining results after osteogenic induction. Alizarin Red S staining revealed the formation of numerous red calcium nodules, indicating that P3 generation hGMSCs have good osteogenic differentiation potential, and the cells can differentiate into osteogenic cells and form mineralized nodules. Figure 3 Figure b shows the staining results after adipogenic induction. Oil Red O staining reveals significant accumulation of intracellular lipid droplets, forming bright red spheres, indicating that P3 generation hGMSCs have a clear adipogenic differentiation potential, capable of differentiating in the adipogenic direction and accumulating lipid droplets.

[0081] 6. hGMSCs surface antigen expression test.

[0082] (1) Select P3 generation hGMSCs in good growth condition and prepare single-cell suspensions after trypsin digestion. Centrifuge at 1000 rpm for 5 min in a benchtop centrifuge to obtain cell pellet. Discard the supernatant, gently resuspend the cells in pre-cooled PBS, calibrate the density to 1×10 cells / mL using a cell counter, and aliquot into 1.5 mL EP tubes, retaining 100 μL of cell suspension in each tube for labeling.

[0083] (2) 5 μL of PE-labeled CD105 antibody, APC-labeled CD90 antibody, PerCP / Cy5.5-labeled CD45 antibody, and FITC-labeled CD34 antibody were added to the experimental groups, respectively. The isotype control group received a mixture of IgG antibodies at matched concentrations. After mixing, the mixture was incubated at room temperature in the dark for 15 min, followed by two washes with pre-cooled PBS to remove free antibodies. Multicolor flow cytometry was used for detection. One × 10⁶ effective cellular events were collected from each sample, and the raw data were analyzed using FlowJo V10.6.2 software.

[0084] Results reference Figure 4 As shown, where: Figure 4 (a) shows that the positivity rate of CD105 on the surface antigen of hGMSCs was 99.97%. (b) The positivity rate of CD90 was 99.82%. (c) The positivity rate of CD45 was 1.47%. (d) The positivity rate of CD34 was 1.24%.

[0085] This indicates that the surface antigen expression of hGMSCs strictly meets the international cell therapy society's criteria for the identification of mesenchymal stem cells.

[0086] 7. Compare the morphological differences between P5 and P10 generation hGMSCs under an inverted microscope.

[0087] Results reference Figure 5 As shown, Figure 5 Figure a shows the morphology of P5 generation hGMSCs. The cells are typically long spindle-shaped, grow in a spiral pattern, and exhibit good growth status and typical mesenchymal stem cell morphological characteristics. Figure 5 Figure b shows the morphology of P10 generation hGMSCs. Compared with P5 generation, P10 generation cells are larger in size and more irregular in shape, showing senescence-related morphological changes, such as uneven cell edges with wrinkles and protrusions. This indicates that the morphological characteristics of hGMSCs change significantly with increasing passage number, suggesting the occurrence of senescence.

[0088] Example 2

[0089] This example demonstrates a β-galactosidase staining experiment.

[0090] To verify the anti-aging effect of hydrogen sulfide, after referring to relevant literature and conducting preliminary experiments, the experiment was divided into three groups: P5 group, P10 group, and P10GYY group. The P10 group was a natural aging model constructed by continuous passage to the P10 generation, with the lower generation P5 group serving as a young control. The P10GYY group was treated with HS donor GYY4137 at a concentration of 100 μM for 12 hours to evaluate the anti-aging effect.

[0091] Staining was performed according to the instructions of the Cell Senescence β-Galactosidase Staining Kit (Beyotime, C0602). The specific procedure is as follows:

[0092] (1) P5 and P10 generation hGMSCs were selected, digested with trypsin, and their density was calibrated using an automated cell counter. They were seeded in 6-well plates at a density of 1×10⁶ cells / well and divided into three groups according to the experimental group, with 3 replicates per group. After 6 hours, the cells adhered to the plate. The original culture medium of the P10GYY group was removed, and the cells were gently washed 3 times with PBS to remove residual culture medium. Then, the medium was replaced with intervention medium and incubated for 12 hours. The P5 and P10 groups were replaced with control medium in the same manner.

[0093] (2) After 12 hours, remove the culture medium, gently wash three times with PBS, add 1 mL of β-galactosidase staining fixative to each well, and fix at room temperature for 15 min. Remove the cell fixative, gently wash three times with PBS for 3 min each time. Remove the PBS, add 1 mL of β-galactosidase staining working solution to each well. Seal the 6-well plate with plastic wrap to prevent evaporation, and incubate overnight in a carbon dioxide-free incubator at 37°C.

[0094] (3) Remove the staining working solution, gently rinse three times with PBS, add 2 mL of PBS, and analyze the experimental results using an inverted optical microscope. Ten fields of view were randomly selected from each well for photography, and the staining positivity rate was calculated using ImageJ software. The expression of senescence markers in P5, P10, and P10GYY cells was detected by β-galactosidase staining experiment. The results showed that the β-galactosidase positivity rate of P10 cells was significantly increased, while the positivity rate of P10GYY group treated with GYY4137 was significantly decreased, indicating that HS can reduce the proportion of senescent cells.

[0095] The results of β-galactosidase staining of the three groups of hGMSCs are shown in the figure below. Figure 6 The image shows the β-galactosidase staining status and positive rate statistics of cells from passages P5, P10, and P10GYY. Among them: Figure 6 Figure a shows the staining results of P5 generation cells. The proportion of β-galactosidase positive cells is low, indicating that P5 generation cells are in a relatively young state and express fewer aging markers. Figure 6Figure b shows the staining results of P10 generation cells. The proportion of β-galactosidase positive cells is significantly increased, indicating that P10 generation cells are in a senescent state and the expression of senescence markers is increased. Figure 6 Figure c shows the staining results of P10GYY group cells. After treatment with GYY4137, the proportion of β-galactosidase positive cells decreased significantly, indicating that GYY4137 can reduce the proportion of senescent cells and inhibit the cellular senescence process. Figure 6 Figure d in the figure shows the statistical analysis of the positive rate of β-galactosidase staining in the three groups. The expression levels of aging markers in the three groups of cells are presented in the form of a bar chart, which further quantifies the differences in aging status.

[0096] Example 3

[0097] In this embodiment, the proliferation capacity of P5, P10 and P10GYY groups were tested respectively. The cell source was the same as in Example 1. The P10GYY group was P10 generation cells treated with GYY4137 (100μM) for 12h.

[0098] CCK-8 assay: Cells were seeded in 96-well plates, and 10 μL of CCK-8 reagent was added to each well daily. The cells were incubated in a cell culture incubator for 2 hours. The absorbance (OD value) at 450 nm was measured using a microplate reader at a fixed time each day for 6 consecutive days, and a proliferation curve was plotted.

[0099] RTCA method: Real-time monitoring of cell index (CI) to assess proliferation kinetics. Cell suspension was seeded into E-Plate 16 detection plates, and CI values ​​were recorded every 15 minutes after preheating for 72 hours.

[0100] The proliferation detection results of the three groups of hGMSCs are shown in the figure. Figure 7 As shown, the cell proliferation curves plotted using CCK-8 and RTCA assays reveal the differences in cell proliferation capacity among the different treatment groups. Specifically:

[0101] Figure 7 Figure a in the table shows the CCK-8 assay results, which reflect the cell proliferation activity by detecting OD values ​​at different times. The results show that the proliferation capacity of P10 generation cells is lower than that of P5 generation cells, while the proliferation capacity of the P10GYY group partially recovered to a level close to that of P5 generation cells. Figure 7 Figure b in the figure shows the results of RTCA assays, which assess proliferation kinetics by real-time monitoring of cell indices (CI).

[0102] The results showed that as the number of passages increased, the cell proliferation capacity decreased. GYY4137 treatment could significantly improve the proliferation capacity of P10 generation cells, restoring it to a level close to that of P5 generation.

[0103] Example 4

[0104] Cell cycle analysis was performed on P5, P10 and P10GYY groups respectively. The cell source was the same as in Example 1. The P10GYY group consisted of P10 generation cells treated with GYY4137 (100 μM) for 12 h.

[0105] After trypsin digestion, cells were fixed with 70% ethanol for 2 hours. Cells were stained with propidium iodide staining solution and incubated at 37°C in the dark for 30 minutes. Cell numbers at each stage of the cell cycle were detected using flow cytometry, and data were analyzed using FlowJo software.

[0106] Figure 8 The image shows the cell cycle changes of three groups of hGMSCs. Flow cytometry analysis was used to illustrate the cell cycle distribution in groups P5, P10, and P10GYY. Among them: Figure 8 Figure a shows the cell cycle distribution of P5 group cells. The cells are mainly distributed in the G0 / G1 phase, accounting for 45.16%, while the cells in the G2 phase account for 24.99%. The cell cycle distribution is relatively uniform, indicating that the cells are in an active cell cycle process. Figure 8 Figure b shows the cell cycle distribution of P10 group cells. Compared with P5 group, the proportion of cells in G0 / G1 phase in P10 group increased significantly to 86.08%, while the proportion of cells in G2 phase was only 6.50%. The cell cycle was mainly arrested in G0 / G1 phase, and proliferation was inhibited or in a quiescent state. Figure 8 Figure c shows the cell cycle distribution of P10GYY group cells. After treatment with GYY4137, the proportion of cells in G0 / G1 phase decreased to 64.71%, while the proportion of cells in G2 phase increased to 16.82%, indicating that GYY4137 promoted the progression of cells from G0 / G1 phase to S / G2 phase and promoted the cell cycle process. Figure 8 d-graph and Figure 8 Figure e in the figure represents the statistical graphs of the three groups of G0 / G1 and G2 phases, presenting the distribution ratio of cells in each group at different stages of the cell cycle in the form of bar charts, further quantifying the characteristics of cell cycle changes.

[0107] Example 5

[0108] Western blot experiments were performed on P5, P10 and P10GYY groups respectively. The cell source was the same as in Example 1. The P10GYY group was P10 generation cells treated with GYY4137 (100μM) for 12h.

[0109] Cells were collected after different treatments, lysed with RIPA lysis buffer (containing 1% PMSF), and proteins were quantified using BCA. Samples were mixed with 5× loading buffer and incubated at 100°C for 5 min. SDS-PAGE electrophoresis (12% separating gel) was performed under the following conditions: 80V until the sample entered the separating gel, then adjusted to 120V. Transfer was performed (350mA, 90 min), followed by blocking with 5% skim milk powder for 2 h. Primary antibody incubation was performed (4°C overnight), followed by washing three times with TBST. Secondary antibody dilution (1:5000) was performed, incubated for 60-90 min, and washed three times with TBST. Chemiluminescence imaging was performed, and grayscale values ​​were analyzed.

[0110] Figure 9 This image shows the expression of senescence-related proteins in three groups of cells. Western blot was used to detect the expression of senescence-related proteins such as P16, P21, P53, and p-P53 in the P5, P10, and P10GYY groups. (The image includes a diagram showing the expression of these proteins.) Figure 9 Figure a shows the protein bands in the three groups of cells, with significant differences in protein expression levels among the different groups. Figure 9 The bar charts in the figure show the relative expression levels of P16, P21, P53, and p-P53 proteins in the three groups. Compared with the P5 group, the expression levels of aging-related proteins such as P16, P21, P53, and p-P53 in the P10 group cells were significantly upregulated, indicating that the P10 group cells were in a senescent state. In contrast, the expression levels of these proteins in the P10GYY group cells were significantly decreased, indicating that GYY4137 can significantly reverse the changes in the expression of aging-related proteins in the P10 group cells, thereby inhibiting cell senescence.

[0111] In summary, Western blot experiments further confirmed that GYY4137 treatment can downregulate the expression levels of aging-related proteins such as P16, P21, and P53.

[0112] Example 6

[0113] (1) Transcriptome sequencing.

[0114] hGMSCs from passages P5 and P10 were selected, digested with trypsin, and seeded at a density of 1 × 10⁶ cells / well in 6-well plates after density calibration using an automated cell counter. Cells were divided into three groups, with three replicates per group. After 6 hours, cells adhered to the plates. The original culture medium for the P10GYY group was removed, and the cells were gently washed three times with PBS to remove residual medium. The medium was then replaced with intervention medium and incubated for 12 hours. The P5 and P10 groups were treated with the same procedure, replacing the medium with control medium. After 12 hours, the medium was aspirated, and the cells were gently washed three times with PBS. 1 mL of TRIZOL reagent was added to each well, and the mixture was thoroughly pipetted until it was fluid and no longer viscous. The mixture was then collected in 1.5 mL EP tubes, frozen at -80°C, and transported wrapped with sufficient dry ice. Total RNA extraction, quality control, sequencing library construction, and quality control of the sequencing process and raw data were all handled by a professional sequencing company using an Illumina high-throughput sequencer.

[0115] (2) Sequencing data analysis

[0116] The obtained raw sequencing data underwent quality control and filtering to remove low-quality reads and adapter sequences. The cleaned data was aligned to the human reference genome (ensembl_110_homo_sapiens_grch38_primary) using HISAT2 software. Gene expression levels were calculated using the aligned data, yielding the FPKM value for each gene. Differentially expressed genes were screened using the DESeq2 software package, with |log2(Fold Change)|≥1 and Pvalue<0.05 set as screening thresholds. GO enrichment analysis and KEGG pathway analysis were performed on the screened differentially expressed genes to understand their potential biological functions.

[0117] Figure 10 This is a graph showing the results of transcriptome differential gene analysis, including statistics on the number of differentially expressed genes in the P10 vs P5 and P10GYY vs P10 groups, Venn plots of differential gene intersections, volcano plots of differentially expressed genes, and heatmaps of differential gene clustering analysis. Among them: Figure 10 Figure a in the chart shows the statistical count of differentially regulated genes, presenting the number of upregulated and downregulated genes in the two comparisons in the form of a bar chart. Figure 10 Figure b in the diagram shows the Venn diagram of the intersection of differentially expressed genes, which presents the number of common differentially expressed genes in the two comparisons. Figure 10 Figure c in the middle and Figure 10 The d plots in the figure show the differential gene volcano plots of the P10 vs P5 group and the P10GYY vs P10 group, respectively. The relationship between gene expression fold change and statistical significance is presented in the form of scatter plots, and significantly differentially expressed genes are marked with different colors. Figure 10The figure in Figure e shows a heatmap of differential gene clustering analysis, with the intensity of color representing the level of gene expression, presenting the overall differences in gene expression among different groups.

[0118] Figure 11 The image shows the enrichment analysis results for GO and KEGG, where: Figure 11 Figure a in the table shows the results of the GO enrichment analysis, presenting the biological processes, cellular components, and molecular functions involved in the differentially expressed genes in histogram form, including cell adhesion, bioadhesion, actin cytoskeleton organization, etc. Specifically, BP was mainly enriched in processes such as cell adhesion, bioadhesion, cell motility, and homologous cell adhesion. CC was mainly enriched in cellular components such as the actin cytoskeleton, proteasome core complex, and endosomes. MF was mainly enriched in molecular functions such as ATP binding, protein kinase activity, and hydrolase activity. Figure 11 Figure b in the diagram shows the results of the KEGG enrichment analysis. The signaling pathways involved in the differentially expressed genes, such as cell adhesion molecule pathways, are presented in the form of a bubble diagram. The size of the bubble represents the number of genes, and the intensity of the color indicates the significance of enrichment.

[0119] Further screening and validation of key differentially expressed genes will be conducted. Figure 12 This is a screening and validation diagram for the key differentially expressed gene U2AF1, including statistics on the number of differentially expressed genes in the P10 vs P5 group and the P10GYY vs P10 group, Venn plot of differentially expressed gene intersection, volcano plot of differentially expressed genes, as well as Western blot bands of U2AF1 protein in the three groups, relative expression levels of U2AF1 gene RT-qPCR in the three groups, and relative expression levels of U2AF1 protein in the three groups. Among them: Figure 12 Figure a in the chart shows the statistical count of differentially regulated genes, presenting the number of upregulated and downregulated genes in the two comparisons in the form of a bar chart. Figure 12 Figure b in the diagram shows the Venn diagram of the intersection of differentially expressed genes, which presents the number of common differentially expressed genes in the two comparisons. Figure 12 Figure c in the middle and Figure 12 The d plots in the figure show the differential gene volcano plots of the P10 vs P5 group and the P10GYY vs P10 group, respectively, and present the relationship between gene expression fold change and statistical significance in the form of scatter plots. Figure 12 Figure e in the figure shows three groups of U2AF1 protein Western blot bands, which visually present the expression level of U2AF1 protein in different groups. Figure 12 f-graph and Figure 12 The g-plots in the figure show the bar charts of the relative expression levels of the U2AF1 gene RT-qPCR in the three groups (P5, P10, and P10GYY) and the relative expression levels of the U2AF1 protein in the three groups, which further quantifies the expression differences of the U2AF1 gene in different groups.

[0120] The method for detecting the relative expression level of the U2AF1 gene by RT-qPCR is as follows:

[0121] Total RNA was extracted using the Trizol method, and cDNA was synthesized by reverse transcription. U2AF1 expression was detected using the SYBR Green PCR kit. Primer sequences are as follows:

[0122]

[0123] Reaction conditions: 95℃ pre-denaturation for 30 s, 95℃ for 5 s, 60℃ for 30 s, 72℃ for 1 min, for a total of 40 cycles. The relative expression level of U2AF1 was calculated, and the experiment was repeated three times.

[0124] Example 7

[0125] To test the senescence phenotype of hGMSCs caused by U2AF1 knockdown, β-galactosidase staining, relative expression levels of U2AF1, P16, and P21 proteins were measured. The specific experimental methods were the same as those described in Example 2 for β-galactosidase staining and Example 5 for Western blot experiments.

[0126] Three treatment groups were set up: siNC, siU2AF1, and siU2AF1+GYY4137. The siNC group refers to the treatment group where hGMSCs were transfected with blank siRNA. The siU2AF1 group consisted of hGMSCs transfected with siRNA targeting U2AF1 (siU2AF1 sequence: GAGATGCAGGAACACTATGAdTdT), with the interference sequence synthesized by a specialized company. The siU2AF1+GYY4137 group consisted of P10 generation hGMSCs transfected with siRNA treated with GYY4137 (100 μM) for 12 hours. The specific procedure is as follows:

[0127] (1) Remove the siRNA transfection reagent powder from the -20℃ freezer and let it stand at room temperature for 5 minutes. Then, centrifuge it briefly at low temperature to remove the powder adhering to the tube wall and place it at the bottom of the tube. On a clean bench, using a sterile, enzyme-free pipette tip, precisely add 250 μL of ultrapure water to 5 nmol of siRNA powder to ensure that the siRNA is fully dissolved in the ultrapure water, preparing a 20 μM storage solution. Aliquot the prepared siRNA solution into sterile, enzyme-free EP tubes. After aliquoting, store the samples properly in a -20℃ freezer to prevent unnecessary repeated freezing and thawing.

[0128] (2) 24 hours before transfection, seed cells in 6-well plates. To ensure uniform cell distribution, seed 1×10⁶ cells per well and gently shake the plate during seeding to achieve uniform cell dispersion. Observe cell growth under a microscope the next day, and perform transfection when the cell density is between 30% and 50%.

[0129] (3) Prepare the transfection complex according to Table 3-6, gently mix by pipetting, and incubate at room temperature for 10 min to prepare the transfection complex. Remove the culture medium for the cells to be transfected and replace it with antibiotic-free complete culture medium. Add the transfection complex dropwise and gently mix to ensure complete coverage. Terminate transfection after 6 h, discard the old culture medium, replace the siU2AF1 and siNC groups with control culture medium, and replace the siU2AF1+GYY4137 group with intervention culture medium. After 12 h of incubation, replace all media with complete culture medium. Subsequent experiments were conducted 36 h after transfection.

[0130] Figure 13 To illustrate the senescence characteristics of hGMSCs induced by U2AF1 knockdown, β-galactosidase staining images are shown in the siNC, siU2AF1, and siU2AF1+GYY4137 groups. Among them:

[0131] Figure 13 The AC plots in the figure show the staining results of the siNC group, siU2AF1 group, and siU2AF1+GYY4137 group, respectively. The β-galactosidase positivity rate of cells in the siU2AF1 group was significantly increased, indicating that knockdown of U2AF1 leads to enhanced cellular senescence phenotype; while the β-galactosidase positivity rate of the siU2AF1+GYY4137 group was significantly decreased after treatment with GYY4137, indicating that GYY4137 can reverse cellular senescence induced by knockdown of U2AF1. Figure 13 Figure d in the figure shows the Western blot results for each treatment group.

[0132] Figure 13 Figure e in the figure shows the statistical analysis of the positive rate of β-galactosidase staining in the three groups, and the expression level of aging markers in each group of cells is presented in the form of a bar chart.

[0133] Figure 13 The fh plots in the figure show bar charts of the relative WB expression levels of the three groups of U2AF1 protein, the three groups of P16 protein, and the three groups of P21 protein, respectively, which further quantify the changes in aging-related indicators.

[0134] In summary, functional verification of the U2AF1 gene showed that knocking down U2AF1 expression led to a senescent phenotype in cells, and HS treatment could reverse this phenomenon, indicating that U2AF1 plays a key role in the senescence of hGMSCs.

[0135] Example 8

[0136] This embodiment investigates the inhibitory effect of U2AF1 on NF-κB pathway activation. The experimental method is as follows:

[0137] (1) Protein sample preparation

[0138] Cell plating and intervention procedures are the same as those for transient transfection with small interfering RNA described above. After transfection, wash three times with pre-cooled PBS, add RIPA lysis buffer, and lyse the cells using pulsed sonication (20% amplitude, 3s on / 57s off, 3 cycles). Centrifuge at 12000×g for 15 min at 4°C and collect the supernatant. Then mix with 5× protein loading buffer at a 4:1 ratio and denature at 100°C for 5 min.

[0139] (2) SDS-PAGE electrophoretic separation

[0140] Prepare the gel plate using a 12.5% ​​PAGE gel rapid preparation kit. Take equal volumes of the lower gel solution and lower gel buffer, 4.0 mL each, and mix well. Add 80 μL of a modified coagulant and mix well. Pour the mixture into the gel preparation glass plate, ensuring the distance between the liquid surface and the upper edge of the short glass plate is 0.5 cm longer than the comb teeth. Add an appropriate amount of isopropanol to cover the lower gel. After 15 minutes, observe a refracted line between the isopropanol and the gel, indicating that the lower gel has solidified. Discard the upper layer of isopropanol. Take equal volumes of the upper gel solution and colored upper gel buffer, 1.0 mL each, and mix well. Add 20 μL of a modified coagulant to the mixture and mix well. Pour the mixture into the gel preparation glass plate and insert the comb teeth. After 15 minutes, the upper gel has solidified. Remove the comb teeth for electrophoresis.

[0141] Mount the gel into the vertical electrophoresis tank and add an appropriate amount of electrophoresis buffer, ensuring the gel is completely submerged. Depending on the gel well size and sample volume, use a micropipette to slowly and steadily inject the prepared sample into the bottom of the well. Simultaneously, add pre-stained protein markers to both sides of the gel. Turn on the power and perform concentration electrophoresis at 80V. Once the sample enters the separating gel, increase the voltage to 120V for separation electrophoresis until the bromophenol blue indicator front is close to the bottom of the gel.

[0142] (3) Semi-dry transfer

[0143] Cut a PVDF membrane the same size as the gel and activate it by soaking it in methanol for 1 min; transfer it to transfer buffer to prevent the membrane from drying out. Soak filter paper and a sponge pad in transfer buffer; use a glass rod to roll and remove air bubbles, ensuring each layer adheres tightly. Assemble using a "sandwich" structure, stacking from negative to positive electrode in the following order: 2 layers of sponge pad → 2 layers of filter paper → gel → PVDF membrane → 2 layers of filter paper → 2 layers of sponge pad. Precisely align the gel and PVDF membrane, and use a moistened roller to press from the center outwards, removing air bubbles layer by layer. Immediately after assembly, place it in the transfer tank, add an appropriate amount of pre-prepared and cooled to 4°C transfer buffer, ensuring the entire transfer system is completely submerged. Connect the power supply (250mA) and transfer for 1.5 h.

[0144] (4) Blocking and antibody incubation

[0145] After the transfer is complete, carefully remove the PVDF membrane, gently hold one corner of the membrane with tweezers, and place it into a container containing blocking buffer. Gently shake to ensure the membrane is fully submerged in the blocking buffer, avoiding the formation of air bubbles. Incubate on a shaker at room temperature for 2 hours. After blocking, gently wash the membrane three times with TBST buffer for 5 minutes each time to remove excess blocking buffer components.

[0146] Based on the molecular weight of the target protein and the marker location, the PVDF membrane was cut to size. The primary antibody was diluted 1:1000 using primary antibody dilution buffer. The diluted primary antibody was added to the blocked membrane, and the membrane was gently agitated to ensure full contact with the antibody. The membrane was incubated on a shaker at room temperature for 2 hours. After incubation, the membrane was gently washed three times with TBST buffer for 5 minutes each time to remove excess primary antibody.

[0147] Dilute the secondary antibody to 1:3000 with TBST buffer. Add the diluted secondary antibody to the washed membrane, gently agitate to ensure full contact between the membrane and the antibody, and incubate on a shaker at room temperature in the dark for 1 hour. After incubation, gently wash the membrane three times with TBST buffer for 5 minutes each time to remove excess secondary antibody components.

[0148] (5) Strip development

[0149] Mix ultrasensitive ECL chemiluminescence reagent solutions A and B in a 1:1 ratio. Then, use tweezers to pick up the washed membrane, blot off excess liquid with absorbent paper, and lay it flat on plastic wrap. Evenly drop ECL working solution onto the membrane, gently shaking to ensure full contact between the membrane and the luminescent substrate, avoiding air bubbles. Place the membrane in the cassette of the chemiluminescence imaging system, select appropriate exposure time and parameters, and perform imaging detection, recording the resulting image of the luminescence signal. Analyze the image using software to determine the expression level of the target protein. Use ImageJ software for band grayscale analysis, normalizing the target protein expression level using β-actin as an internal reference.

[0150] Figure 14 This is a diagram showing the inhibition of NF-κB pathway activation by U2AF1. Western blot analysis of three groups of NF-κB pathway protein bands reveals the relative expression levels of three groups of U2AF1 proteins, three groups of P65 proteins, three groups of IκBα proteins, three groups of p-P65 proteins, and three groups of p-IκBα proteins. Among them: Figure 14 Figure a in the diagram shows the Western blot bands of three groups of NF-κB pathway proteins, visually presenting the expression levels of each protein in different groups. Figure 14 The bf plots in the figure show the relative expression levels of U2AF1, P65, IκBα, p-P65, and p-IκBα proteins in the three groups, respectively. This further quantifies the effect of U2AF1 on the NF-κB signaling pathway, indicating that knocking down U2AF1 can activate the NF-κB pathway, while GYY4137 intervention inhibits the overactivation of this pathway.

[0151] Further mechanistic studies revealed that U2AF1 affects cellular senescence by negatively regulating the NF-κB signaling pathway. Knockdown of U2AF1 activates the NF-κB pathway, while intervention with HS inhibits its overactivation.

[0152] Therefore, GYY4137 plays an important role in the aging mechanism of hGMSCs by regulating U2AF1 and its regulated NF-κB signaling pathway, providing a key theoretical basis for developing anti-aging strategies based on GYY4137.

[0153] Example 9

[0154] In this embodiment, the proliferation capacity of P10+GYY4137 (0, 25, 50, 100, 150 μM) groups was tested. The cell source was the same as in Example 1. The P10GYY group was P10 generation cells treated with GYY4137 for 12 h.

[0155] CCK-8 assay: Cells were seeded in 96-well plates. On day 5, 10 μL of CCK-8 reagent was added to each well, and the cells were incubated for another 2 hours in a cell culture incubator. The absorbance (OD value) at 450 nm was measured using a microplate reader. Figure 15 The results showed that the optimal final concentration of GYY4137 was 100 μM.

[0156] In summary, this invention delves into the molecular mechanisms of hGMSC senescence, particularly exploring the role of the U2AF1 gene in cellular senescence. Through techniques such as transcriptome sequencing, it clarifies the expression changes of the U2AF1 gene in senescent cells and its relationship with cellular senescence markers and signaling pathways. This invention addresses the problem of ineffective existing anti-aging strategies. By discovering that GYY4137 can restore U2AF1 gene expression levels, thereby inhibiting the overactivation of the NF-κB signaling pathway, reducing the activity of the cellular senescence marker β-galactosidase, and enhancing cell proliferation, a novel anti-aging strategy based on GYY4137 is developed. Addressing the toxicity risks of existing HS donors, this invention also provides a safe method for using GYY4137 as an HS donor to delay hGMSC senescence. GYY4137 possesses good biocompatibility and sustained-release properties, avoiding the toxicity of exogenous HS and achieving effective cellular anti-aging effects. This invention also identifies the U2AF1 gene as a potential molecular marker for detecting the senescence status of hGMSCs. By accurately detecting the expression level of the U2AF1 gene, the degree of cellular aging can be assessed, providing a basis for timely and precise anti-aging treatment.

[0157] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. Application of GYY4137 in the preparation of drugs or reagents for delaying the aging of human gingival mesenchymal stem cells.

2. The application according to claim 1, characterized in that, The drug or reagent has at least one of the following uses: (1) Restore the expression level of U2AF1 in human gingival mesenchymal stem cells; (2) Inhibit the overactivation of the NF-κB signaling pathway; (3) Reduces the activity of β-galactosidase, a marker of cellular senescence; (4) Enhance or restore the proliferative capacity of human gingival mesenchymal stem cells; (5) Downregulate the expression of aging-related proteins; (6) Reduce the proportion of human gingival mesenchymal stem cells in the G0 / G1 phase and increase the proportion of human gingival mesenchymal stem cells in the G2 phase.

3. The application according to claim 2, characterized in that, The aging-related protein is selected from at least one of P16, P21, P53 and p-P53 proteins.

4. The application according to claim 1, characterized in that, The final concentration of GYY4137 used in the drug or reagent is 50-150 μM.

5. The application according to claim 4, characterized in that, The final concentration of GYY4137 used in the drug or reagent is 100 μM.

6. The application according to claim 1, characterized in that, When using the drug or reagent, each 1×10⁶ cells / mL of human gingival mesenchymal stem cells is mixed and incubated with 50-150 μM of GYY4137.

7. The application according to claim 1, characterized in that, The conditions for the mixed incubation are: incubation at 37°C and 5% CO for 12 hours.

8. The application according to any one of claims 1-7, characterized in that, The drug also includes a pharmaceutically acceptable carrier.

9. Application of reagents for detecting the U2AF1 gene in the preparation of human gingival mesenchymal stem cell aging prediction products.

10. The application according to claim 5, characterized in that, The human gingival mesenchymal stem cell aging prediction product is selected from reagent kits, test strips, chips, or detectors; Preferably, the reagent for detecting the U2AF1 gene is selected from primers, probes, ELISA reagents, or Western blotting reagents.

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