Application of enhanced and optimized mesenchymal stem cells and exosomes in anti-aging or delaying aging

Abstract

The present invention provides an application of enhanced and optimized mesenchymal stem cells and exosomes in anti-aging or delaying aging, belonging to the field of biopharmaceutical manufacturing technology. The pharmaceutical composition of the present invention exhibits significant synergistic effects in anti-aging interventions by combining the use of activity-enhanced mesenchymal stem cells and the exosomes they secrete. Experiments have confirmed that the composition can systematically improve oxidative stress damage associated with aging, significantly restore the activity of antioxidant enzymes in serum and brain tissue to near normal levels, and effectively reverse cognitive function degradation, as manifested by behavioral improvements such as shortened escape latency and improved spatial memory ability. It is suitable for the preparation of drugs or functional preparations that delay aging or anti-aging, and provides an innovative technical path for the development of efficient and safe anti-aging therapies.

Description

Technical Field

[0001] The present invention relates to the technical field of biopharmaceutical manufacturing, and in particular to the use of enhanced and optimized mesenchymal stem cells and exosomes in anti-aging or anti-aging. Background Art

[0002] With the increasing aging of the global population, anti-aging research has become a key focus in the biomedical field. Aging is essentially characterized by degenerative changes in cellular function and a decline in regenerative capacity, involving multiple mechanisms such as telomere shortening, accumulation of DNA damage, increased oxidative stress, mitochondrial dysfunction, and activation of chronic inflammation. Traditional anti-aging strategies, such as antioxidants (e.g., vitamin C, coenzyme Q10) and hormone replacement therapy (e.g., growth hormone, DHEA), can partially alleviate symptoms, but they suffer from poor targeting and questionable long-term safety. In recent years, stem cell-based regenerative medicine has provided new avenues for anti-aging. Mesenchymal stem cells (MSCs) have garnered significant attention due to their multipotential differentiation, immunomodulatory properties, and ability to secrete diverse active factors (e.g., growth factors, cytokines, and exosomes). Studies have demonstrated that MSCs can improve the tissue microenvironment, promote injury repair, and inhibit inflammatory responses through paracrine mechanisms. In animal models, they have demonstrated the potential to delay aging-related pathological phenotypes (e.g., skin atrophy, decreased bone density, and cognitive decline).

[0003] Chinese patent CN 110840819A discloses the use and preparation method of stem cell exosome gel for delaying aging. By preparing exosomes into an exosome gel, it can be easily stored and used. Applying it to the skin reduces wrinkles and slows skin aging. However, this solution does not provide guidance on how to delay aging or prolong life. Summary of the Invention

[0004] The purpose of the present invention is to provide an enhanced and optimized use of mesenchymal stem cells and exosomes in anti-aging or delaying aging, which provides an effective means for synergistically delaying the aging process and has significant clinical application prospects.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] The present invention provides a use of a pharmaceutical composition in the preparation of an anti-aging or anti-aging drug, wherein the pharmaceutical composition contains activity-enhanced mesenchymal stem cells and exosomes secreted by the activity-enhanced mesenchymal stem cells;

[0007] The method for preparing the activity-enhanced mesenchymal stem cells comprises the following steps:

[0008] inoculating mesenchymal stem cells into a culture medium containing quercetin and polygonatum saponin for pre-culture, thereby obtaining pre-cultured mesenchymal stem cells and a culture supernatant, wherein the pre-cultured mesenchymal stem cells are activity-enhanced mesenchymal stem cells;

[0009] The method for preparing exosomes comprises the following steps:

[0010] Exosomes secreted by the activity-enhanced mesenchymal stem cells are extracted from the culture supernatant.

[0011] Preferably, in the pharmaceutical composition, the concentration of the activity-enhanced mesenchymal stem cells is 10 5 ~10 6 cells / 0.2mL.

[0012] Preferably, in the pharmaceutical composition, the concentration of the exosomes is 40-60 μg exosomes / 0.2 mL.

[0013] Preferably, the concentration of the quercetin is 3-8 μM, and the concentration of the polygonatum saponin is 8-12 μM.

[0014] Preferably, the pre-culture time is 3 to 8 days.

[0015] Preferably, the exosomes secreted by the activity-enhanced mesenchymal stem cells are extracted from the culture supernatant by differential centrifugation.

[0016] Preferably, the dosage form of the anti-aging or anti-aging drug is an injection.

[0017] Preferably, the drug further comprises pharmaceutically acceptable excipients.

[0018] Preferably, the excipients include lyoprotectants, stabilizers or sustained-release matrix materials.

[0019] Preferably, the object of anti-aging or delayed aging is the body or the brain of the body.

[0020] Beneficial effects of the present invention:

[0021] The pharmaceutical composition of this invention, through the combined use of activity-enhanced mesenchymal stem cells and their secreted exosomes, exhibits significant synergistic effects in anti-aging interventions. Experimental studies have demonstrated that the composition can systemically improve aging-related oxidative stress damage, significantly restore antioxidant enzyme activity in serum and brain tissue to near-normal levels, and effectively reverse cognitive decline, manifested by behavioral improvements such as shortened escape latency and enhanced spatial memory. Compared to single stem cell or exosome therapies, this approach, through a combined "cell-exosome" delivery system, combines the dual advantages of rapid microenvironmental regulation by exosomes with the long-term repair of stem cells, significantly improving treatment efficacy and duration. Furthermore, a pretreatment strategy based on specific natural active ingredients (such as quercetin and polygonatum saponin) further enhances the anti-aging efficacy of the composition without the need for genetic modification or exogenous loading, resulting in a simplified process and high safety. This approach is suitable for the preparation of drugs or functional preparations for delaying or anti-aging, providing an innovative technical approach for the development of efficient and safe anti-aging therapies. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is a graph showing the difference in superoxide dismutase (SOD) activity among the groups;

[0023] Figure 2 This is a diagram of the difference analysis of catalase (CAT) activity;

[0024] Figure 3 This is a diagram for analyzing the difference in malondialdehyde (MDA) content;

[0025] Figure 4 This is the difference analysis diagram of total antioxidant capacity (T-AOC);

[0026] Figure 5 This is a diagram showing the difference in activity of glutathione peroxidase (GSH-Px). DETAILED DESCRIPTION

[0027] The present invention provides a use of a pharmaceutical composition in the preparation of an anti-aging or anti-aging drug, wherein the pharmaceutical composition contains activity-enhanced mesenchymal stem cells and exosomes secreted by the activity-enhanced mesenchymal stem cells. The preparation method of the activity-enhanced mesenchymal stem cells comprises the following steps: inoculating the mesenchymal stem cells into a culture medium containing quercetin and polygonatum saponin for pre-culturing, thereby obtaining pre-cultured mesenchymal stem cells and a culture supernatant, wherein the pre-cultured mesenchymal stem cells are activity-enhanced mesenchymal stem cells;

[0028] The method for preparing exosomes comprises the following steps: extracting the exosomes secreted by the activity-enhanced mesenchymal stem cells from the culture supernatant.

[0029] In the present invention, preferably, in the pharmaceutical composition, the concentration of the activity-enhanced mesenchymal stem cells is 105-106 cells / 0.2 mL. Preferably, in the pharmaceutical composition, the concentration of the exosomes is 40-60 μg exosomes / 0.2 mL. Preferably, the concentration of quercetin is 3-8 μM, and the concentration of polygonatum saponin is 8-12 μM. Preferably, the pre-culture time is 3-8 days. Preferably, the extraction method used to obtain the exosomes secreted by the activity-enhanced mesenchymal stem cells from the culture supernatant is ultracentrifugation.

[0030] In the present invention, the anti-aging or anti-aging drug is preferably in the form of an injection. Preferably, the drug further comprises a pharmaceutically acceptable excipient. Preferably, the excipient comprises a lyoprotectant, a stabilizer, or a sustained-release matrix material. Preferably, the anti-aging or anti-aging drug is administered to the human body or the brain.

[0031] The technical solutions provided by the present invention are described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0032] Example

[0033] 1. Umbilical Cord Mesenchymal Stem Cell Acquisition and Activity Optimization

[0034] 1. Experimental Materials

[0035] Neonatal umbilical cords were obtained from healthy full-term newborns born vaginally.

[0036] 2. Experimental Procedure

[0037] 2.1 Isolation of neonatal umbilical cord mesenchymal stem cells

[0038] Following medical ethics guidelines and obtaining informed consent from the mother, the umbilical cord of the newborn was aseptically collected in the delivery room and quickly placed in a sterile container containing α-MEM medium containing dual-antibody (penicillin 100 U / mL, streptomycin 100 μg / mL). The cord was then brought back to the laboratory. In a cleanroom, the cord was gently rinsed multiple times with preheated PBS buffer until any residual blood was removed from the surface.

[0039] Transfer the cleaned umbilical cord to a sterile dish, remove the surface blood vessels and connective tissue, and cut the remaining part into about 1-2 mm 3 Transfer the tissue pieces into a 50 mL centrifuge tube and add an appropriate amount of 0.25% trypsin-EDTA digestion solution to ensure that the tissue pieces are completely immersed. Digest in a shaking incubator at 37°C for 35-40 minutes, gently shaking the centrifuge tube every 10 minutes.

[0040] After digestion, add an equal volume of α-MEM medium containing 10% FBS to terminate the reaction. Gently pipette to disperse the tissue fragments into a single cell suspension. Transfer the cell suspension to a centrifuge tube and centrifuge at 1000 rpm for 5 minutes. Discard the supernatant. Resuspend the cells in α-MEM medium containing 10% FBS to a concentration of 1 × 105 cells / mL. Plate the cells into a T25 culture flask and culture in a 37°C, 5% CO2 incubator.

[0041] 24 hours after inoculation, change the medium for the first time to remove non-adherent cells and tissue debris. Change the medium every 3-4 days thereafter. Observe cell growth daily under a phase contrast microscope and passage cells when they reach 80%-90% confluence.

[0042] 2.2 Pretreatment and culture of umbilical cord mesenchymal stem cells

[0043] In this study, we selected well-grown, passage 4 umbilical cord mesenchymal stem cells (MSCs). The culture medium in the culture flask was discarded and the cells were gently rinsed 2-3 times with PBS buffer. An appropriate amount of 0.25% trypsin-EDTA digestion solution was added and digested at 37°C for 2-3 minutes. Observe under a phase-contrast microscope. When the cells became rounded and began to detach from the flask wall, digestion was terminated by adding α-MEM medium supplemented with 10% FBS. Gently pipette the cells to form a uniform suspension and seed them into new culture flasks at a ratio of 1:3-1:4 for continued culture.

[0044] When the cell confluence reaches 70%-80%, the cells are divided into four groups (the medium dosage is about 1×105 cells / mL):

[0045] ①Combined pretreatment group 1: Add α-MEM culture medium (containing 10% FBS) containing 5 μM quercetin and 10 μM polygonatum saponin.

[0046] ② Quercetin pretreatment group: add α-MEM medium (containing 10% FBS) containing 15 μM quercetin.

[0047] ③ Polygonatum saponin pretreatment group: add α-MEM culture medium (containing 10% FBS) containing 15 μM Polygonatum saponin.

[0048] ④Combined pretreatment group 2: Add α-MEM culture medium (containing 10% FBS) containing 5μM resveratrol + 10μM salidroside. This treatment group is an active treatment combination obtained based on screening in previous studies.

[0049] The cells were placed in a 37°C, 5% CO2 incubator and cultured for 5 days. The original culture medium was replaced with PBS solution to obtain pretreated mesenchymal stem cells.

[0050] 2.3. Exosome extraction

[0051] After pretreatment, collect the supernatant from each cell culture flask into a centrifuge tube and centrifuge at 300 × g for 10 minutes at 4°C to remove cell debris. Transfer the supernatant to a fresh centrifuge tube and centrifuge at 2000 × g for 20 minutes at 4°C to further remove larger cell debris. Transfer the supernatant to an ultrafiltration tube and centrifuge at 10,000 × g for 40 minutes at 4°C to concentrate the supernatant to approximately 1-2 mL. Extract exosomes using an exosome extraction reagent according to the manufacturer's instructions, resuspend in an appropriate amount of PBS buffer, and store at -80°C until use.

[0052] 2.4. Exosome Verification:

[0053] An appropriate amount of the exosome resuspension was dropped onto a copper grid and allowed to stand at room temperature for 5 minutes to allow the exosomes to adsorb. Excess liquid was gently removed with filter paper, and 2% phosphotungstic acid solution was added for negative staining at room temperature for 3-5 minutes. Excess stain was again removed with filter paper. After the grid dried naturally, the exosome morphology was observed under a transmission electron microscope. The exosomes exhibited typical exosome characteristics: flat, disc-shaped, and biconcave.

[0054] The exosome resuspension was appropriately diluted with PBS buffer to a concentration within the detection range of the Nanosight particle size analyzer. The exosomes were then injected into the instrument sample cell for particle size analysis and the exosome size distribution was recorded. The results showed a particle size range of 70-120 nm, indicating that the obtained exosomes were of high purity and could be used for subsequent experiments.

[0055] 2.5 Administration

[0056] The pretreated mesenchymal stem cells of each group were mixed with the extracted exosomes, and PBS buffer was used as the solvent. Each 0.2 mL solution contained 10 6 Mesenchymal stem cells + 50 μg exosomes were co-injected into the tail vein of mice.

[0057] 2. Anti-aging effects of different treatment groups on D-galactose-induced aging mouse model

[0058] 1. Materials and Methods

[0059] 1.1 Experimental animals and groups

[0060] Animal selection: 60 SPF C57BL / 6J female mice, 8 weeks old, weighing 18 ± 2 g, were randomly divided into 6 groups (n = 10):

[0061] Negative control group (Control): Normal feeding, no modeling, and injection of equal volume of normal saline.

[0062] Model group: D-galactose-induced aging model, injected with equal volume of normal saline.

[0063] Experimental group A (Group A): UC-MSCs pre-cultured with the compound reagent quercetin 5μM + polygonatum saponin 10μM combined with exosomes (combination dose: (10 6 cells + 50 μg exosomes) / 0.2 mL).

[0064] Experimental group B (Group B): UC-MSCs pre-cultured with 15 μM quercetin combined with exosomes (same dosage as Group A).

[0065] Experimental group C (Group C): UC-MSCs pre-cultured with 15 μM polygonatum saponin combined with exosomes (same dosage as Group A).

[0066] Experimental group D (group D): UC-MSCs pre-cultured with resveratrol 5 μM + salidroside 10 μM combined with exosomes (same dosage as group A).

[0067] 1.2 Construction of aging model

[0068] D-galactose induction: The model group and the experimental group were injected subcutaneously with 5% D-galactose solution (0.25 mL / 10 g) at the back of the neck every day for 8 consecutive weeks; the control group was injected with an equal volume of normal saline.

[0069] Assessment of aging phenotype: Body weight changes, hair loss, and activity status (e.g., slow movement, unresponsiveness) were recorded weekly.

[0070] 1.3 Dosage Regimen

[0071] Intervention time: After the model was established (starting from the 9th week), the experimental group was injected with the corresponding preparation via the tail vein once a week for 4 consecutive weeks.

[0072] Injection volume: 0.2 mL per mouse per time. The control group and model group were injected with equal volume of normal saline.

[0073] 2. Index detection

[0074] 2.1 Serum oxidative stress indicators

[0075] Sample collection: 48 h after the last administration, blood was collected from the mouse eyeballs, incubated at 4°C for 1 h, centrifuged at 3000 rpm for 10 min, and the serum was separated and stored at -80°C.

[0076] Detection method:

[0077] Superoxide dismutase (SOD): xanthine oxidase method (kit operation).

[0078] Catalase (CAT): Ammonium molybdate colorimetric method was used to detect the decomposition rate of H2O2.

[0079] Malondialdehyde (MDA): thiobarbituric acid (TBA) method was used to determine lipid peroxidation products.

[0080] 2.2 Antioxidant capacity of brain tissue

[0081] Sample processing: Mice were killed by cervical dislocation, and brain tissue was obtained and homogenized in 0.1 mol / L PBS, pH 7.4, in an ice bath. The mixture was centrifuged at 12000 rpm at 4°C for 15 min, and the supernatant was collected.

[0082] Detection indicators:

[0083] Total antioxidant capacity (T-AOC) was determined by FRAP method.

[0084] Glutathione peroxidase (GSH-Px): Detected by DTNB colorimetric method.

[0085] 2.3 Morris Water Maze

[0086] Equipment and parameters:

[0087] The circular pool (120 cm in diameter, 50 cm in height) was 30 cm deep and had a water temperature of 25 ± 1 °C.

[0088] A transparent acrylic platform (10 cm in diameter) was placed in the northeast quadrant, 1 cm below the water surface.

[0089] Markers of different shapes (triangle, circle, square) are fixed around the pool wall.

[0090] The movement trajectory of mice was recorded using the SMART 3.0 video tracking system.

[0091] Experimental process:

[0092] Adaptation phase (week 9 before intervention): mice swam freely for 60 s (without platform).

[0093] Training phase (starting from the third week of intervention, 5 consecutive days): 4 training sessions per day, with the starting position randomized (east, south, west, and north quadrants), and the escape latency was recorded (upper limit 90 s).

[0094] Testing phase (24 h after the last intervention): The platform was removed, and the number of platform crossings and the proportion of time spent in the target quadrant were recorded.

[0095] 2.4 Statistical analysis

[0096] The data were expressed as mean ± standard deviation and analyzed by one-way analysis of variance (ANOVA) using SPSS 26.0. The groups were compared using the LSD method. P < 0.05 was considered a significant difference, and P < 0.01 was considered an extremely significant difference.

[0097] 3. Results

[0098] 3.1 Serum oxidative stress indicators

[0099] The test results are shown in Table 1, and the difference analysis of superoxide dismutase (SOD) activity in each group is shown in Figure 1 As shown in the figure, the difference analysis of catalase (CAT) activity is shown in Figure 2 As shown in the figure, the difference analysis of malondialdehyde (MDA) content is as follows Figure 3 As shown:

[0100] Table 1 Results of serum oxidative stress index detection

[0101] Group SOD (U / mL) CAT (U / mL) MDA (nmol / mL) Control 121.52 ± 10.35 12.74 ± 1.38 5.21 ± 0.98 Model 63.85 ± 6.74* 5.92 ± 1.21* 16.83 ± 2.15* Group A 115.27 ± 4.66** 11.05 ± 1.65** 7.39 ± 0.24** Group B 98.41 ± 3.92# 9.12 ± 0.43# 10.25 ± 1.81# Group C 95.67 ± 6.55# 8.89 ± 0.82# 11.04 ± 1.53# Group D 95.02 ± 5.63# 8.15 ± 0.07# 10.97 ± 2.01#

[0102] Note:

[0103] *Compared with the Control group, P < 0.01;

[0104] **Compared with the Model group, P < 0.01;

[0105] #Compared with the Model group, P<0.05.

[0106] 3.2 Antioxidant capacity of brain tissue

[0107] The test results are shown in Table 2, and the difference analysis of total antioxidant capacity (T-AOC) of each group is shown in Figure 4 As shown; Glutathione peroxidase (GSH-Px) activity difference analysis diagram is shown Figure 5 As shown:

[0108] Table 2 Results of the antioxidant capacity test of brain tissue

[0109] Group T-AOC (U / mg prot) GSH-Px (U / mg prot) Control 1.38 ± 0.21 22.15 ± 1.12 Model 0.85 ± 0.12* 13.04 ± 1.07* Group A 1.32 ± 0.06** 20.85 ± 0.55** Group B 1.12 ± 0.04# 17.63 ± 0.13# Group C 1.08 ± 0.10# 16.89 ± 1.07# Group D 0.88 ± 0.02 13.92 ± 0.35

[0110] 3.3 Water maze behavioral testing

[0111] The test results are shown in Table 3:

[0112] Table 3 Water maze behavioral results

[0113] Group Escape latency (s) Number of platform crossings (times) Percentage of time spent in the target quadrant (%) Control 22.57 ± 2.15 5.82 ± 0.21 43.25 ± 3.42 Model 57.89 ± 4.42* 1.05 ± 0.31* 18.73 ± 3.05* Group A 28.47 ± 2.21** 4.12 ± 0.79** 35.64 ± 2.47** Group B 34.15 ± 2.03# 2.98 ± 0.64# 28.31 ± 2.25# Group C 36.82 ± 3.52# 2.75 ± 0.57# 26.89 ± 3.06# Group D 34.63 ± 2.91# 2.17 ± 0.42# 25.85 ± 3.72#

[0114] in conclusion:

[0115] Group A: The combined use of quercetin + polygonatum saponin pre-cultured group significantly improved oxidative stress indicators (P<0.01) and cognitive function (escape latency was significantly shortened, and the time spent in the target quadrant returned to near normal levels).

[0116] Groups B, C, and D: The combined group pre-cultured with a single reagent and the combined group pre-cultured with resveratrol + salidroside partially restored oxidative capacity (P<0.05), but the behavioral improvement effect was secondary.

[0117] 4. Discussion

[0118] This study systematically evaluated the anti-aging effects of umbilical cord mesenchymal stem cell-exosome combination therapy using different pretreatment regimens, revealing the potential benefits of synergistic regulation by natural active ingredients. The combined pretreatment group (quercetin + polygonatum saponin) demonstrated the best efficacy in reversing D-galactose-induced aging phenotypes. It not only significantly restored antioxidant enzyme activities in serum and brain tissue to near-normal levels but also achieved significant improvements in cognitive behavior. This suggests that the combined use of natural antioxidants can synergistically enhance the ability of stem cells and their exosomes to repair the body's oxidative stress network.

[0119] This study innovatively combines a stem cell preconditioning strategy with a mesenchymal stem cell-exosome combination therapy, offering new insights into anti-aging interventions. Experiments have demonstrated that exosomes directly deliver bioactive substances to modulate the microenvironment, while transplanted stem cells continuously secrete trophic factors for long-term protection, collectively establishing a dynamic repair system. The combination of mesenchymal stem cells pretreated with active ingredients and their exosomes exhibits a synergistic effect in ameliorating systemic oxidative damage and neurodegeneration.

[0120] Notably, the anti-aging effects of different pretreatment combinations varied significantly. Quercetin and polygonatum saponins exhibited a synergistic effect, while the resveratrol combination was relatively weaker. This provides important clues for subsequent mechanistic research, suggesting that the selection of active ingredient combinations should consider the complementarity of their molecular pathways. Subsequent research could further analyze the differences in exosomal miRNA profiles and key protein expression, elucidating the molecular mechanisms by which specific active ingredients regulate the stem cell secretome, laying a theoretical foundation for the development of precision anti-aging therapies.

[0121] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. Use of a pharmaceutical composition in the preparation of an anti-aging or anti-aging drug, characterized in that: The pharmaceutical composition contains activity-enhanced mesenchymal stem cells and exosomes secreted by the activity-enhanced mesenchymal stem cells; The method for preparing the activity-enhanced mesenchymal stem cells comprises the following steps: inoculating umbilical cord mesenchymal stem cells into a culture medium containing quercetin and polygonatum saponin for pre-culture, thereby obtaining pre-cultured mesenchymal stem cells and a culture supernatant, wherein the pre-cultured mesenchymal stem cells are activity-enhanced mesenchymal stem cells; The method for preparing exosomes comprises the following steps: extracting exosomes secreted by the activity-enhanced mesenchymal stem cells from the culture supernatant; The concentration of the quercetin is 5 μM, and the concentration of the polygonatum saponin is 10 μM.

2. The use according to claim 1, characterized in that In the pharmaceutical composition, the concentration of the activity-enhanced mesenchymal stem cells is 10 5 ~10 6 cells / 0.2mL.

3. The use according to claim 1, characterized in that In the pharmaceutical composition, the concentration of the exosomes is 40-60 μg exosomes / 0.2 mL.

4. The use according to claim 1, characterized in that The pre-culture time is 3 to 8 days.

5. The use according to claim 1, characterized in that The exosomes secreted by the activity-enhanced mesenchymal stem cells are extracted from the culture supernatant by differential centrifugation.

6. The use according to claim 1, characterized in that The dosage form of the anti-aging or anti-aging drug is an injection.

7. The use according to claim 6, characterized in that The drug further comprises pharmaceutically acceptable excipients.

8. The use according to claim 7, characterized in that The auxiliary materials include freeze-drying protectants, stabilizers or sustained-release matrix materials.

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