A small molecule composition for restoring the immune regulation function of senescent mesenchymal stem cells and application thereof

Abstract

This invention discloses a small molecule composition for restoring the immunomodulatory function of senescent mesenchymal stem cells (MSCs) and its applications. It belongs to the field of biomedical technology. The small molecule composition comprises resveratrol, quercetin, ginsenoside Rg1, and N-(4-phenylbutyl)acetamide in a weight ratio of 1:2:2:1. Experiments show that this composition significantly reduces the SA-β-gal positivity rate of senescent MSCs (from 76.3% to 11.2%), inhibits T cell proliferation by 89.7%, and reduces TNF-α and IFN-γ by 76.8% and 77.5%, respectively, with significantly better effects than the individual use of each component or any combination of the three drugs. Pathway inhibitor recovery experiments confirm that its synergistic effect depends on the simultaneous activation of multiple pathways. This composition can be used to prepare drugs or cell culture additives for restoring the immunomodulatory function of senescent MSCs, providing a new approach for stem cell therapy of age-related diseases.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and more specifically to a small molecule composition for restoring the immunomodulatory function of aging mesenchymal stem cells and its application. Background Technology

[0002] Mesenchymal stem cells (MSCs) are a type of pluripotent adult stem cells derived from the mesoderm in early development, possessing self-renewal, multi-lineage differentiation potential, and unique immunomodulatory properties. MSCs show broad application prospects in autoimmune diseases, inflammatory diseases, and tissue damage repair. However, during long-term in vitro expansion, MSCs gradually undergo replicative senescence, characterized by decreased proliferation capacity, morphological changes, increased positivity of senescence-associated β-galactosidase (SA-β-gal), upregulation of senescence-related proteins such as p16 / p21 / p53, and a significant decline in immunomodulatory function. Studies have shown that senescent MSCs exhibit significant functional defects in inhibiting T cell proliferation and regulating cytokine secretion; this immune dysfunction has become a major technical bottleneck limiting the clinical application of MSCs.

[0003] In recent years, natural products have attracted attention due to their wide availability and relatively high safety. Literature reports the protective effects of various natural compounds on mesenchymal stem cells (MSCs). For example, resveratrol, quercetin, and ginsenoside Rg1 have been studied for their potential to delay MSC senescence. Furthermore, some scholars have proposed the concept of "caloric restriction mimetics" (CRMs), grouping quercetin, resveratrol, and others into a category, suggesting that these compounds have the potential to inhibit cellular senescence.

[0004] N-(4-Phenylbutyl)acetamide (CAS No.: 34059-11-5) is an N-substituted acetamide small molecule compound. A search revealed no publicly available information about this compound in the field of mesenchymal stem cell anti-aging, and existing technologies do not provide any technical guidance or inspiration for its use in restoring the immune regulatory function of aging MSCs.

[0005] Although existing technologies disclose that the aforementioned natural products each have certain effects on MSCs, the following technical limitations still exist in restoring the immunomodulatory function of senescent MSCs: First, there is a lack of research on synergistic combinations of natural products. Existing studies mainly focus on the effects of single natural products on MSCs, and there is a lack of systematic research on whether there are synergistic effects between different natural products. Simply stacking compounds belonging to the same functional class may pharmacologically produce saturation or even antagonism, rather than the expected synergistic effect.

[0006] Second, the technical objectives of existing compositions differ from those of this application. Existing research on natural product compositions has mainly focused on promoting osteogenic differentiation and chondrogenic differentiation of MSCs or obtaining exosomes, and there are no reports of compositions specifically designed for the specific objective of "restoring the immune regulatory function of aging MSCs".

[0007] Third, the application of N-(4-phenylbutyl)acetamide in the field of MSC anti-aging is still in its infancy. The role of this compound in restoring the immunomodulatory function of aging MSCs has not been disclosed in any literature or patents, and its technical effects and synergistic potential in this field remain to be explored.

[0008] In summary, there is an urgent need to develop a small molecule composition that can effectively restore the immune-regulating function of aging MSCs, has clearly defined components, and exhibits synergistic effects. This is a technical problem that needs to be solved by those skilled in the art. Summary of the Invention

[0009] In view of this, the present invention provides a small molecule composition for restoring the immunomodulatory function of senescent mesenchymal stem cells and its application. Through systematic screening, the present invention has found that when resveratrol, quercetin, ginsenoside Rg1 and N-(4-phenylbutyl)acetamide are used in combination in a specific ratio, a significant synergistic effect can be produced. This not only effectively delays the replicative senescence of MSCs, but also significantly restores the immunomodulatory function of senescent MSCs, achieving unexpected technical results.

[0010] To solve the above-mentioned technical problems, this application adopts the following technical solution: The primary objective of this application is to provide a small molecule composition for restoring the immunomodulatory function of aging mesenchymal stem cells, comprising a first component resveratrol, a second component quercetin, a third component ginsenoside Rg1, and a fourth component N-(4-phenylbutyl)acetamide, wherein the weight ratio of resveratrol, quercetin, ginsenoside Rg1, and N-(4-phenylbutyl)acetamide is 1:2:2:1.

[0011] As a preferred technical solution, the final concentrations of resveratrol, quercetin, ginsenoside Rg1 and N-(4-phenylbutyl)acetamide in the culture medium are 5 μM, 10 μM, 10 μM and 5 μM, respectively.

[0012] As a preferred technical solution, the small molecule composition further comprises a pharmaceutically acceptable carrier and / or excipients.

[0013] As a preferred technical solution, the small molecule composition is prepared into at least one dosage form selected from injections, lyophilized powder injections, solutions, or culture medium additives.

[0014] One object of this application is to provide: the use of the small molecule composition in the preparation of a pharmaceutical composition for restoring the immunomodulatory function of aging mesenchymal stem cells.

[0015] As a preferred technical solution, the restoration of the immunomodulatory function of senescent mesenchymal stem cells includes one or more of the following: reducing the positivity rate of senescence-related β-galactosidase in senescent mesenchymal stem cells, downregulating the expression of senescence-related proteins p16, p21 and p53, restoring the proliferative capacity of senescent mesenchymal stem cells, restoring the inhibitory capacity of senescent mesenchymal stem cells on T cell proliferation, and restoring the ability of senescent mesenchymal stem cells to inhibit the secretion of inflammatory factors TNF-α and IFN-γ by T cells.

[0016] One object of this application is to provide a pharmaceutical composition comprising the small molecule composition and a pharmaceutically acceptable carrier.

[0017] One object of this application is to provide: the use of the small molecule composition in the preparation of a cell culture additive for delaying the aging of mesenchymal stem cells.

[0018] One object of this application is to provide: a cell culture additive that enhances the immunomodulatory function of aging mesenchymal stem cells, comprising the small molecule composition and a pharmaceutically acceptable carrier.

[0019] As can be seen from the above technical solution, compared with the prior art, the present invention has the following beneficial effects: (1) Innovative Combination Selection: This invention is the first to propose and verify the application of a composition containing four naturally derived or naturally structured small molecules—resveratrol, quercetin, ginsenoside Rg1, and N-(4-phenylbutyl)acetamide—in restoring the immunomodulatory function of aging mesenchymal stem cells (MSCs). N-(4-phenylbutyl)acetamide has never been disclosed in the field of MSC anti-aging before, and its anti-aging and immunomodulatory function restoration effects in MSCs are revealed for the first time in this invention.

[0020] (2) Breakthrough synergistic effect: The components in the small molecule composition of this invention have a significant synergistic effect. The positive rate of SA-β-gal decreased from 76.3% to 11.2%, the synergistic inhibition rate reached 85.3%, the T cell proliferation inhibition rate reached 89.7%, TNF-α and IFN-γ decreased by 76.8% and 77.5% respectively, and the synergistic index CI value reached 0.216~0.358. The effect is significantly better than the sum of the effects of each component used alone, and also significantly better than the effect of any combination of three components.

[0021] (3) Safety and accessibility advantages: All components of the small molecule composition of the present invention are small molecule compounds of natural origin or natural structure origin, which are widely available, highly safe, cost controllable, and easy to produce and promote on a large scale.

[0022] (4) Irreplaceable technical solution: Comparative experiments show that using other natural compounds (such as vitamin C, EGCG, curcumin, metformin, and rhodioloside) to replace N-(4-phenylbutyl)acetamide cannot achieve the effect of the four-component combination of the present invention, proving that the specific component selection and ratio of the present invention are irreplaceable. Detailed Implementation

[0023] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0024] Example 1, Preparation of small molecule composition: Resveratrol (CAS No.: 501-36-0, purity ≥98%, purchased from Sigma-Aldrich), quercetin (anhydrous, CAS No.: 117-39-5, purity ≥98%, purchased from Sigma-Aldrich), ginsenoside Rg1 (CAS No.: 22427-39-0, purity ≥98%, purchased from the National Institutes for Food and Drug Control (NIFDC)), and N-(4-phenylbutyl)acetamide (CAS No.: 34059-11-5, molecular formula: C12H17NO, molecular weight: 191.27, purchased from MACKLIN, catalog number N763062, purity 95%, hereinafter referred to as PBA) were weighed separately and mixed evenly in a weight ratio of 1:2:2:1 to obtain the small molecule composition of the present invention.

[0025] The above-mentioned small molecule composition was dissolved in dimethyl sulfoxide to prepare a 100 mM stock solution (in which the concentrations of resveratrol, quercetin, ginsenoside Rg1, and N-(4-phenylbutyl)acetamide were 100 mM, 200 mM, 200 mM, and 100 mM, respectively). Before use, the solution was diluted with cell culture medium to the required concentration. The final concentration of DMSO in the culture medium should not exceed 0.1%.

[0026] Example 2: Isolation and culture of human umbilical cord mesenchymal stem cells and establishment of an aging model: Umbilical cord tissue was collected from healthy mothers after cesarean section, with informed consent from the mothers and approval from the ethics committee. The umbilical cord tissue was washed in PBS containing 1% penicillin-streptomycin to remove blood stains, and cut into tissue pieces approximately 1 mm³. 0.1% type II collagenase and 0.1% hyaluronidase were added, and the tissue was digested at 37°C for 1 hour. The digestion solution was filtered through a 100-mesh sieve, and the cell suspension was collected. The cells were centrifuged at 1500 rpm for 5 minutes, the supernatant was discarded, and the cells were resuspended in α-MEM medium containing 10% fetal bovine serum. The cells were then seeded in T75 culture flasks and cultured in a 37°C, 5% CO2 incubator. The medium was changed every 3 days. When the cells reached 80% confluence, they were passaged using 0.25% trypsin-EDTA digestion.

[0027] Human umbilical cord MSCs obtained from the above isolation and culture were continuously passaged, with the third generation (P3) cells used as a young MSC control; simultaneously, homologous MSCs were passaged to the 15th generation (P15) to establish a senescent MSC model, and the senescence positivity rate was detected by SA-β-gal staining. The results showed that the SA-β-gal positivity rate in P15 generation MSCs reached 78.6±4.2%, significantly higher than the 4.2±0.8% in P3 generation MSCs (P<0.001), indicating the successful establishment of the senescent MSC model. The expression levels of p16, p21, and p53 proteins in P15 generation MSCs were also detected, and the results showed that all were significantly upregulated, further confirming the successful establishment of the senescent MSC model.

[0028] Example 3: The effect of small molecule composition on improving the aging phenotype and immunomodulatory function of senescent MSCs: (I) Experimental Grouping and Processing The P15 generation senescent MSCs were divided into the following 11 groups: (1) Blank control group: only an equal volume of DMSO solvent was added; (2) Resveratrol monotherapy group (Res): Resveratrol was added to a final concentration of 5 μM; (3) Quercetin monotherapy group (Que): Quercetin was added to a final concentration of 10 μM; (4) Ginsenoside Rg1 alone group (Rg1): Ginsenoside Rg1 was added to a final concentration of 10 μM. (5) N-(4-phenylbutyl)acetamide monotherapy (PBA): N-(4-phenylbutyl)acetamide was added to a final concentration of 5 μM; (6) Resveratrol + Quercetin combination therapy (Res+Que): Resveratrol and quercetin were added to a final concentration of 5 μM and 10 μM respectively; (7) Resveratrol + Quercetin + Ginsenoside Rg1 three-drug combination group (Res+Que+Rg1): Resveratrol, Quercetin and Ginsenoside Rg1 were added to a final concentration of 5 μM; (8) Resveratrol + Quercetin + N-(4-phenylbutyl)acetamide triple drug combination group (Res+Que+PBA): 5 μM resveratrol, 10 μM quercetin and 5 μM N-(4-phenylbutyl)acetamide were added to the final concentration; (9) Resveratrol + Ginsenoside Rg1 + N-(4-phenylbutyl)acetamide triple drug combination group (Res+Rg1+PBA): 5 μM resveratrol, 10 μM ginsenoside Rg1 and 5 μM N-(4-phenylbutyl)acetamide were added to the final concentration; (10) Quercetin + Ginsenoside Rg1 + N-(4-phenylbutyl)acetamide triple drug combination group (Que+Rg1+PBA): 10 μM quercetin, 10 μM ginsenoside Rg1 and 5 μM N-(4-phenylbutyl)acetamide were added to a final concentration; (11) Resveratrol + Quercetin + Ginsenoside Rg1 + N-(4-phenylbutyl)acetamide four-drug combination group (Res+Que+Rg1+PBA): 5 μM resveratrol, 10 μM quercetin, 10 μM ginsenoside Rg1 and 5 μM N-(4-phenylbutyl)acetamide were added.

[0029] After culturing the cells in each group at 37°C and 5% CO2 for 72 hours, various tests were performed.

[0030] (ii) SA-β-gal staining detection After treatment, the culture medium was discarded, the cells were washed twice with PBS, and fixed with SA-β-gal staining fixative at room temperature for 15 min. After washing with PBS, SA-β-gal staining working solution (pH=6.0) was added and incubated overnight at 37°C. The proportion of positive cells (blue cells) was observed and counted under an optical microscope. At least 200 cells were randomly counted in each group, and the experiment was repeated 3 times. The experimental results are shown in Table 1.

[0031] Table 1. Results of SA-β-gal staining in different groups Group SA-β-gal positivity rate (%) Reduction rate relative to Control (%) Control 76.3±3.8 - Res 52.7±3.1 30.9 Que 49.4±2.9 35.3 Rg1 47.6±3.2 37.6 PBA 51.2±3.0 32.9 Res+Que 34.1±2.5 55.3 Res+Que+Rg1 18.6±1.9 75.6 Res+Que+PBA 22.3±2.0 70.8 Res+Rg1+PBA 24.8±2.2 67.5 Que+Rg1+PBA 23.5±2.1 69.2 Res+Que+Rg1+PBA 11.2±1.2 85.3 Results Analysis: Compared with the blank control group, all treatment groups significantly reduced the SA-β-gal positivity rate (P<0.01). The SA-β-gal positivity rate in the N-(4-phenylbutyl)acetamide monotherapy group was 51.2%, indicating that N-(4-phenylbutyl)acetamide alone has a moderate anti-aging effect on senescent MSCs. The effects of any three-component combination were significantly better than the two-drug combination groups (P<0.01), with the Res+Que+Rg1 combination group showing the best effect (18.6%). However, the four-drug combination group (11.2%) was significantly better than all three-drug combination groups (P<0.01), and the SA-β-gal positivity rate was further reduced by 39.8% compared to the Res+Que+Rg1 combination group. The addition of N-(4-phenylbutyl)acetamide produced a breakthrough synergistic effect beyond the synergistic boundary of the three components.

[0032] (III) Detection of cell proliferation capacity using the CCK-8 assay After 72 hours of treatment, cells from each group were seeded in 96-well plates at a density of 5 × 10³ / well. After culturing for 24 hours, 48 ​​hours, and 72 hours, 10 μL of CCK-8 solution was added to each well, and the plates were incubated at 37°C for 2 hours. The absorbance at 450 nm was measured using a microplate reader. The experimental results are shown in Table 2.

[0033] Table 2 Results of cell proliferation assay using CCK-8 assay in different groups Group OD450 value (72h) Multiplication factor relative to Control Control 0.52±0.05 1.00 Res 0.73±0.06 1.40 Que 0.78±0.07 1.50 Rg1 0.81±0.06 1.56 PBA 0.69±0.05 1.33 Res+Que 1.04±0.08 2.00 Res+Que+Rg1 1.36±0.09 2.62 Res+Que+PBA 1.28±0.08 2.46 Res+Rg1+PBA 1.22±0.08 2.35 Que+Rg1+PBA 1.26±0.08 2.42 Res+Que+Rg1+PBA 1.58±0.10 3.04 Results analysis: The four-drug combination group showed the most significant proliferation-promoting effect, with a proliferation rate of 3.04, which was significantly higher than that of the Res+Que+Rg1 three-drug combination group (2.62, P<0.01), and also significantly higher than other three-drug combination groups.

[0034] (iv) Western blot detection of aging-related protein expression After 72 hours of treatment, cells from each group were collected, and total protein was extracted using RIPA lysis buffer. Protein concentration was determined by the BCA method. Equal amounts of protein were subjected to SDS-PAGE electrophoresis, transferred to PVDF membranes, and blocked with 5% skim milk at room temperature for 1 hour. Anti-p16 antibody (1:1000), anti-p21 antibody (1:1000), anti-p53 antibody (1:1000), and anti-GAPDH antibody (1:5000) were added, and incubated overnight at 4°C. After washing with TBST, HRP-labeled secondary antibody was added, and the membrane was incubated at room temperature for 1 hour. ECL staining was performed, followed by exposure and photography. Grayscale analysis was performed using ImageJ software, and the relative expression level of the target protein was calculated using GAPDH as an internal control. The experimental results are shown in Table 3.

[0035] Table 3. Expression results of aging-related proteins in different groups Group p16 relative expression level p21 relative expression level p53 relative expression level Control 1.00±0.08 1.00±0.07 1.00±0.08 Res 0.62±0.05 0.58±0.04 0.60±0.05 Que 0.59±0.04 0.55±0.05 0.57±0.04 Rg1 0.56±0.04 0.53±0.04 0.55±0.05 PBA 0.68±0.05 0.65±0.05 0.67±0.05 Res+Que 0.41±0.03 0.38±0.03 0.40±0.03 Res+Que+Rg1 0.28±0.03 0.22±0.02 0.25±0.03 Res+Que+PBA 0.32±0.03 0.28±0.02 0.31±0.03 Res+Rg1+PBA 0.35±0.03 0.31±0.03 0.33±0.03 Que+Rg1+PBA 0.33±0.03 0.29±0.03 0.32±0.03 Res+Que+Rg1+PBA 0.16±0.02 0.13±0.01 0.15±0.02 Results analysis: The four-drug combination group showed the most significant downregulation of p16, p21 and p53 proteins, with relative expression levels decreasing to 0.16, 0.13 and 0.15 times that of the blank control group, respectively, which was significantly better than the Res+Que+Rg1 three-drug combination group (0.28, 0.22 and 0.25 times, P<0.01).

[0036] (v) Detection of T cell proliferation inhibition Senescent MSCs from each group treated in Example 3 were treated with 25 μg / mL mitomycin C for 30 min, washed three times with PBS, and then... 5 The cells were seeded at a density of / wells in 24-well plates and allowed to adhere to the walls overnight. PBMC (purchased from Lonza, catalog number 4W-270) was then seeded at a density of 1 × 10⁻⁶ wells. 6 / The cells were added to MSC culture wells at a density of 1:10 (MSC:PBMC = 1:10), and anti-CD3 antibody (5 μg / mL) and anti-CD28 antibody (2 μg / mL) were added simultaneously to activate T cells. After co-culturing for 48 h, suspension cells were collected, and the attenuation of CFSE fluorescence intensity was detected by flow cytometry. The T cell proliferation inhibition rate was calculated, and the experimental results are shown in Table 4.

[0037] Inhibition rate (%) = (1 - T cell proliferation rate in experimental group / T cell proliferation rate in control group) × 100%.

[0038] Table 4 Results of T cell proliferation inhibition detection in different groups Group T cell proliferation inhibition rate (%) Control 22.4±2.1 Res 38.7±3.0 Que 41.2±3.2 Rg1 43.5±3.1 PBA 32.8±2.8 Res+Que 61.3±4.2 Res+Que+Rg1 82.6±5.1 Res+Que+PBA 73.5±4.8 Res+Rg1+PBA 68.4±4.5 Que+Rg1+PBA 71.2±4.6 Res+Que+Rg1+PBA 89.7±5.5 Results analysis: The inhibition rate of T cell proliferation by MSCs in the four-drug combination group reached 89.7%, which was significantly higher than that in the three-drug combination group of Res+Que+Rg1 (82.6%, P<0.01).

[0039] (vi) Detection of inflammatory factor secretion (ELISA) Collect the co-culture supernatant and, following the instructions of the ELISA kit, detect the concentrations of TNF-α and IFN-γ in the supernatant. The experimental results are shown in Table 5.

[0040] Table 5. Results of inflammatory factor detection in different groups Group TNF-α concentration (pg / mL) IFN-γ concentration (pg / mL) Control 356.4±24.6 428.9±30.2 Res 248.7±18.3 301.5±21.4 Que 235.2±17.8 289.7±20.6 Rg1 228.5±17.2 281.3±20.1 PBA 268.3±19.2 322.6±22.5 Res+Que 167.3±14.1 198.4±16.3 Res+Que+Rg1 98.6±9.3 115.7±10.2 Res+Que+PBA 118.5±10.5 138.6±11.8 Res+Rg1+PBA 135.2±11.8 152.3±12.5 Que+Rg1+PBA 125.8±11.0 145.7±12.0 Res+Que+Rg1+PBA 82.8±7.5 96.4±8.8 Results analysis: The four-drug combination treatment most effectively inhibited the secretion of pro-inflammatory factors TNF-α and IFN-γ by T cells, reducing their concentrations to 82.8 pg / mL and 96.4 pg / mL, respectively, with inhibition rates of 76.8% and 77.5%. Compared with the Res+Que+Rg1 three-drug combination group (TNF-α: 98.6 pg / mL, IFN-γ: 115.7 pg / mL), the four-drug combination group further reduced TNF-α by 16.0% and IFN-γ by 16.7%, which was significantly better than all three-drug combinations (P<0.01).

[0041] Example 4, Pathway Inhibitor Reversal Experiment: To further verify the signaling pathways upon which the synergistic effect of the composition depends, a pathway-specific inhibitor reversion experiment was designed. The following pathway-specific inhibitors were used: EX527: SIRT1 specific inhibitor (5μM); Compound C: AMPK-specific inhibitor (10 μM); Bay 11-7082: NF-κB specific inhibitor (2μM); LY294002: PI3K-AKT specific inhibitor (20μM).

[0042] Experimental Groups: Four-drug combination therapy (Res+Que+Rg1+PBA); Four drugs in combination + EX527; Four-drug combination therapy + Compound C; Four-drug combination therapy + Bay 11-7082; Four drugs in combination + LY294002; Four drugs in combination plus a mixture of four inhibitors; After 72 hours of treatment, the positive rate of SA-β-gal and the T cell proliferation inhibition rate of each group were detected. The experimental results are shown in Table 6.

[0043] Table 6 Results of pathway inhibitor response experiments in different groups Group SA-β-gal positivity rate (%) T cell proliferation inhibition rate (%) Four-drug combination (without inhibitors) 11.2±1.2 89.7±5.5 Four-drug combination + EX527 28.6±2.2 64.8±4.2 Four-drug combination therapy + Compound C 32.4±2.5 60.3±4.0 Four-drug combination + Bay 11-7082 25.8±2.0 68.5±4.5 Four-drug combination + LY294002 35.2±2.6 57.2±3.8 Four-drug combination + four inhibitors 58.6±4.0 38.5±3.2 Results analysis: The above results indicate that blocking any single pathway can only partially weaken the effect of the four-drug combination, but cannot completely eliminate it. The SA-β-gal positivity rate rebounded from 11.2% to 25.8-35.2%, while the inhibition rate decreased to 57.2-68.5%. Even when all four pathways were blocked simultaneously, the effect was still significantly better than the blank control group (SA-β-gal positivity rate 58.6% vs 76.3%, P<0.01), indicating that the synergistic effect of the four-drug combination depends on the simultaneous participation of multiple signaling pathways.

[0044] Example 5, Quantitative analysis of synergistic effects: The Chou-Talalay combination index method was used to quantitatively evaluate the synergistic effect of each component. The SA-β-gal positivity rate was used as the effect index to calculate the combination index (CI) value of each drug combination. CI < 1 indicated a synergistic effect, CI = 1 indicated an additive effect, and CI > 1 indicated an antagonistic effect.

[0045] Single-drug dose-response relationship determination: Multiple concentration gradients (1-50 μM) of resveratrol, quercetin, ginsenoside Rg1, and N-(4-phenylbutyl)acetamide were set up, and the positive rate of SA-β-gal at each concentration was measured to plot dose-response curves. The curves were fitted using CompuSyn software (a dedicated analysis software based on the Chou-Talalay method), and the theoretical concentrations required for each single drug to achieve different effect levels were calculated.

[0046] The results showed that resveratrol alone required approximately 18 μM to achieve a 20% positive rate, quercetin required approximately 20 μM, ginsenoside Rg1 required approximately 18 μM, and N-(4-phenylbutyl)acetamide required approximately 22 μM.

[0047] The target effect was calculated with a reduction in the SA-β-gal positivity rate to approximately 20%. Resveratrol alone required approximately 18 μM to achieve this effect, quercetin alone required approximately 20 μM, ginsenoside Rg1 alone required approximately 18 μM, and N-(4-phenylbutyl)acetamide alone required approximately 22 μM. When achieving this effect in combination, the amounts of resveratrol were 5 μM, quercetin 10 μM, ginsenoside Rg1 10 μM, and N-(4-phenylbutyl)acetamide 5 μM. The experimental results are shown in Table 7.

[0048] Table 7. Quantitative analysis results of synergistic effects among different groups Target effect (SA-β-gal positivity rate) CI value of four drugs in combination Synergy determination 30% 0.358 Collaboration 20% 0.287 Collaboration 15% 0.216 Collaboration Results analysis: Under all three target effects, the CI values ​​were much less than 1, which fully demonstrates the significant synergistic effect of the four-drug combination.

[0049] Example 6: To verify the necessity of the specific component selection and the specificity of the synergistic effect of the present invention, the following comparative examples were set up. In each comparative example, a representative compound known to have anti-MSC aging activity was selected to replace N-(4-phenylbutyl)acetamide, including: Vitamin C represents the classic antioxidant category; EGCG represents the polyphenol / plant-derived antioxidant category; curcumin represents the flavonoid natural product category; metformin represents a representative compound in the CRMs framework; and rhodioloside represents a traditional Chinese medicine active ingredient known to delay MSC aging.

[0050] The above-mentioned components cover known anti-MSC aging compounds with different chemical structures and mechanisms of action, aiming to systematically verify the irreplaceable role of N-(4-phenylbutyl)acetamide in the compositions of this invention, as detailed below: Comparative Example 1: Resveratrol 5 μM + quercetin 10 μM + ginsenoside Rg1 10 μM + vitamin C 50 μM were used to replace N-(4-phenylbutyl)acetamide. Comparative Example 2: Resveratrol 5 μM + quercetin 10 μM + ginsenoside Rg1 10 μM + epigallocatechin gallate (EGCG) 10 μM were used to replace N-(4-phenylbutyl)acetamide. Comparative Example 3: Resveratrol 5 μM + quercetin 10 μM + ginsenoside Rg1 10 μM + curcumin 5 μM were used to replace N-(4-phenylbutyl)acetamide. Comparative Example 4: Resveratrol 5 μM + quercetin 10 μM + ginsenoside Rg1 10 μM + metformin 50 μM replaced N-(4-phenylbutyl)acetamide. Comparative Example 5: Resveratrol 5 μM + quercetin 10 μM + ginsenoside Rg1 10 μM + rhodioloside 10 μM replaced N-(4-phenylbutyl)acetamide. The positive rate of SA-β-gal and the inhibition rate of T cell proliferation after treatment of senescent MSCs with each comparative combination were detected according to the method in Example 3. The experimental results are shown in Table 8.

[0051] Table 8. Results of SA-β-gal positivity rate and T cell proliferation inhibition rate in different groups. Group SA-β-gal positivity rate (%) T cell proliferation inhibition rate (%) This invention Res+Que+Rg1+PBA 11.2±1.2 89.7±5.5 Comparative Example 1 (+Vitamin C) 29.8±2.3 65.4±4.3 Comparative Example 2 (+EGCG) 27.5±2.2 68.2±4.5 Comparative Example 3 (+curcumin) 26.8±2.1 69.5±4.6 Comparative Example 4 (+Metformin) 24.5±2.0 72.8±4.8 Comparative Example 5 (+Rhodioloside) 23.2±1.9 74.6±4.9 Results Analysis: The effects of all the above comparative examples were significantly lower than those of the four-drug combination group of this invention (P<0.01). The additional benefit brought by N-(4-phenylbutyl)acetamide as the fourth component (SA-β-gal positive rate decreased from 18.6% to 11.2%, a reduction of 39.8%; inhibition rate increased from 82.6% to 89.7%, an increase of 8.6%) was significantly greater than the benefit of other natural compounds replacing N-(4-phenylbutyl)acetamide, proving that N-(4-phenylbutyl)acetamide has an irreplaceable special role in the composition of this invention. The application of N-(4-phenylbutyl)acetamide in the field of MSC anti-aging has never been disclosed before, and its role in restoring the immune regulatory function of aging MSCs is revealed for the first time in this invention.

[0052] Example 7, In vivo animal experiments: To further verify the in vivo efficacy of the small molecule composition of this invention, a D-galactose-induced aging mouse model was established. Seventy 8-week-old BALB / c mice (purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd., BALB / cAnNCrl inbred strain, strain code 211, SPF grade) were randomly divided into 7 groups: Control group: injected with an equal volume of PBS; Model group: D-galactose (500 mg / kg / day, subcutaneous injection, for 8 weeks); Res group: MSCs pretreated with resveratrol intravenously (1×10⁻⁶) in addition to the model. 6 / Only); Que group: MSCs pretreated with quercetin via intravenous injection based on the model; Rg1 group: MSCs pretreated with ginsenoside Rg1 were intravenously injected on the basis of the model. Three-drug combination group: MSCs pretreated with Res+Que+Rg1 were intravenously injected on the basis of the model; The four-drug combination group (the group of this invention): MSCs pretreated with Res+Que+Rg1+PBA were injected intravenously on the basis of the model; MSCs in each pretreatment group were treated according to the protocol in Example 3 for 72 hours before injection. Various indicators were measured 4 weeks after injection, and the experimental results are shown in Table 9.

[0053] Table 9 Results of in vivo animal experiments detection indicators Control Model Res Que Rg1 Triple drug combination Four-drug combination therapy (this invention) Spleen index (mg / g) 3.12±0.25 5.86±0.42 4.85±0.35 4.72±0.33 4.68±0.32 3.58±0.28 3.24±0.22 Thymus index (mg / g) 2.45±0.18 1.23±0.11 1.56±0.12 1.62±0.13 1.65±0.12 2.18±0.15 2.36±0.16 Serum TNF-α (pg / mL) 85.6±7.2 425.3±32.5 312.4±24.8 298.7±23.5 285.6±22.4 142.3±12.6 108.5±10.2 Serum IL-10 (pg / mL) 156.8±12.4 68.5±5.8 89.2±7.5 92.5±7.8 95.6±8.0 142.5±11.8 151.2±12.0 Positive rate of SA-β-gal in liver tissue (%) 5.2±0.6 38.6±3.2 26.8±2.4 25.4±2.2 24.5±2.1 12.3±1.2 9.5±0.9 Results Analysis: In vivo experimental results showed that the four-drug combination pretreatment of MSCs in this invention achieved the best therapeutic effect in an aging mouse model. All indicators were significantly better than the three-drug combination group (P<0.05), demonstrating that the addition of N-(4-phenylbutyl)acetamide also produced a significant synergistic effect at the in vivo level.

[0054] Example 8, Validation experiment with different MSC sources: To verify the universality of the small molecule composition of the present invention for MSCs from different sources, the experiments of Examples 2 to 4 were repeated using human bone marrow-derived MSCs and human adipose-derived MSCs, respectively.

[0055] The results showed that for senescent MSCs derived from human bone marrow (Poietics® Normal Human Bone Marrow Derived Mesenchymal Stem Cells (hMSCs), catalog number PT-2501, manufactured by Lonza) (passage P15, SA-β-gal positivity rate 79.2±3.9%), after treatment with the small molecule composition of this invention for 72 h, the SA-β-gal positivity rate decreased to 12.5±1.3%, and the T cell proliferation inhibition rate increased to 88.4±5.2%. For senescent MSCs derived from human adipose tissue (Poietics® Normal Human Adipose Derived Stem Cells (hADSCs), catalog number PT-5006, manufactured by Lonza) (passage P15, SA-β-gal positivity rate 77.5±3.6%), after the same treatment, the SA-β-gal positivity rate decreased to 10.8±1.1%, and the T cell proliferation inhibition rate increased to 90.2±5.4%. The above results are basically consistent with the experimental results of MSCs derived from umbilical cord, indicating that the small molecule composition of the present invention has significant anti-aging and immune regulation function restoration effects on MSCs from different tissue sources, and has good universality.

[0056] This invention provides a small molecule composition for restoring the immunomodulatory function of senescent mesenchymal stem cells (MSCs), comprising four naturally derived or structurally derived small molecule compounds: resveratrol, quercetin, ginsenoside Rg1, and N-(4-phenylbutyl)acetamide. Experimental evidence sufficiently demonstrates that this small molecule composition can significantly reduce the SA-β-gal positivity rate in senescent MSCs, downregulate the expression of senescence-related proteins p16, p21, and p53, restore the proliferative capacity and immunomodulatory function of MSCs, and significantly inhibit T cell proliferation and the secretion of inflammatory factors TNF-α and IFN-γ.

[0057] More importantly, this invention is the first to apply N-(4-phenylbutyl)acetamide to restore the immunomodulatory function of aging MSCs, a technology never before disclosed in the field of MSC anti-aging. The combined use of the four components produced a significant synergistic effect, significantly superior to the use of any single component or any combination of the three components, exhibiting unexpected technical benefits and representing a non-obvious invention. This invention not only provides a novel technical solution for restoring the immunomodulatory function of aging MSCs but also provides crucial technical support for MSC-based regenerative medicine and immunotherapy.

[0058] The above description is merely a preferred embodiment of the present invention and does not limit the patent scope of the present invention. Any equivalent structural or procedural transformations made based on the content of the present invention specification, or direct or indirect applications in other related technical fields, are similarly included within the patent protection scope of the present invention.

[0059] The various embodiments in this specification are described in a progressive manner, with each embodiment focusing on the differences from other embodiments. The same or similar parts between the various embodiments can be referred to each other.

[0060] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A small molecule composition for restoring the immunomodulatory function of senescent mesenchymal stem cells, characterized in that, It comprises a first component resveratrol, a second component quercetin, a third component ginsenoside Rg1, and a fourth component N-(4-phenylbutyl)acetamide, wherein the weight ratio of resveratrol, quercetin, ginsenoside Rg1, and N-(4-phenylbutyl)acetamide is 1:2:2:

1.

2. The small molecule composition of claim 1, wherein, The final concentrations of resveratrol, quercetin, ginsenoside Rg1, and N-(4-phenylbutyl)acetamide in the culture medium were 5 μM, 10 μM, 10 μM, and 5 μM, respectively.

3. The small molecule composition of claim 1, wherein, The small molecule composition also contains a pharmaceutically acceptable carrier and / or excipients.

4. The small molecule composition of any one of claims 1-3, wherein, The small molecule composition is prepared into at least one dosage form selected from injections, lyophilized powder injections, solutions, or culture medium additives.

5. The use of the small molecule composition according to any one of claims 1-4 in the preparation of a pharmaceutical composition for restoring the immunomodulatory function of aging mesenchymal stem cells.

6. Use according to claim 5, characterized in that, The restoration of the immunomodulatory function of senescent mesenchymal stem cells includes one or more of the following: reducing the positivity rate of senescence-related β-galactosidase in senescent mesenchymal stem cells, downregulating the expression of senescence-related proteins p16, p21 and p53, restoring the proliferative capacity of senescent mesenchymal stem cells, restoring the inhibitory capacity of senescent mesenchymal stem cells on T cell proliferation, and restoring the ability of senescent mesenchymal stem cells to inhibit the secretion of inflammatory factors TNF-α and IFN-γ by T cells.

7. A pharmaceutical composition, characterized by, It comprises the small molecule composition according to any one of claims 1-4 and a pharmaceutically acceptable carrier.

8. The use of the small molecule composition according to any one of claims 1-4 in the preparation of a cell culture additive for delaying the aging of mesenchymal stem cells.

9. A cell culture additive that enhances the immunomodulatory function of senescent mesenchymal stem cells, characterized in that, It comprises the small molecule composition according to any one of claims 1-4 and a pharmaceutically acceptable carrier.