Use of parishin a in the preparation of a product for treating mesenchymal stem cell senescence

Abstract

The application provides application of Parishin A in preparation of a product for treating mesenchymal stem cell aging, relates to the technical field of medicines, and realizes antioxidation homeostasis reconstruction through activation of an NRF2 channel. It effectively inhibits mesenchymal stem cell aging caused by physiological aging in a body microenvironment, significantly restores bone tissue homeostasis regulation functions thereof, and confirms that it reverses the aging microenvironment in the body. Parishin A can specifically repair self-renewal disorders of aging mesenchymal stem cells. It is confirmed that the anti-aging effect of Parishin A on aging mesenchymal stem cells strictly depends on specific activation of the NRF2 channel. It is first disclosed that Parishin A can be used for treating or delaying mesenchymal stem cell aging caused by physiological aging, can be used for preparing medicines for treating diseases caused by mesenchymal stem cell aging, and provides a new method and new idea for preparing medicines for inhibiting aging-related diseases.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, and in particular to the application of Parishin A in the preparation of products for treating aging of mesenchymal stem cells. Background Technology

[0002] With the accelerating aging of the population, the incidence of age-related diseases is increasing year by year, becoming a significant issue affecting public health and socio-economic development. Cellular senescence is a core factor leading to aging, characterized by multiple stable and irreversible cell cycle arrests, accompanied by impaired cell proliferation, migration, and secretion. Senescent cells typically express a series of characteristic markers, such as senescence-associated β-galactosidase. associated β galactosidase, SA β Increased activity of Gal, upregulated expression of cell cycle inhibitors such as P21 and P16, accumulation of DNA damage and activation of related signaling pathways, and decreased cellular antioxidant capacity are also important molecular characteristics of cellular senescence.

[0003] Mesenchymal stem cells (MSCs) are a type of adult stem cell with self-renewal capacity and multi-lineage differentiation potential, playing a crucial role in maintaining tissue homeostasis and repairing damage. Furthermore, MSCs possess advantages such as low immunogenicity, strong immunomodulatory capacity, and good homing ability, leading to their widespread application in the treatment of systemic diseases. However, MSCs are prone to senescence during aging, pathological stimulation, or long-term in vitro expansion and culture, exhibiting decreased proliferative capacity and reduced oxidative stress resistance. This results in clinical problems such as slowed extraction wound healing and poor implant bone healing, limiting their clinical efficacy. Therefore, exploring safe and effective methods to delay MSC senescence and restore their biological functions has significant theoretical and practical value.

[0004] Naturally derived small-molecule bioactive substances have attracted widespread attention for their ability to delay cellular aging due to their well-defined origins, good biocompatibility, and relatively high safety. Parishin A is a representative bioactive component of the traditional Chinese medicine Gastrodia elata, and existing research has mainly focused on its biological effects, such as neuroprotection and anti-inflammation. However, current research lacks reports on the role of Parishin A in regulating mesenchymal stem cell aging.

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

[0006] One of the objectives of this invention is to provide the application of Parishin A in the preparation of products for treating mesenchymal stem cell aging, thus providing a new therapeutic direction for treating or delaying mesenchymal stem cell aging.

[0007] In order to achieve the above-mentioned objectives of the present invention, the following technical solution is adopted: In a first aspect, the present invention provides the use of Parishin A in the preparation of products for treating and / or delaying the aging of mesenchymal stem cells.

[0008] Furthermore, Parishin A treats and / or delays mesenchymal stem cell aging by activating the NRF2 signaling pathway.

[0009] Furthermore, activation of the NRF2 signaling pathway includes upregulating NRF2 protein expression levels in mesenchymal stem cells; and / or promoting mRNA and / or protein expression of NRF2 downstream target genes in mesenchymal stem cells.

[0010] Furthermore, the downstream target genes of NRF2 include at least one of GCLC, SRX1, and NQO1.

[0011] Furthermore, the mesenchymal stem cell senescence mentioned is mesenchymal stem cell senescence caused by physiological aging.

[0012] Preferably, the mesenchymal stem cells include bone marrow mesenchymal stem cells.

[0013] Furthermore, the product possesses at least one of the functions listed in A1 to A6: A1. Inhibits the activity of β-galactosidase (SA-β-gal) in damaged mesenchymal stem cells; A2. Promotes the expression of senescence factors in damaged mesenchymal stem cells; A3. Promotes the proliferation of damaged mesenchymal stem cells; A4. Promotes osteogenic differentiation of damaged mesenchymal stem cells; A5. Inhibits the adipogenic differentiation of damaged mesenchymal stem cells; A6. Reduces the level of reactive oxygen species in damaged mesenchymal stem cells.

[0014] Furthermore, the aging factors include p16 and / or p21; Preferably, the damaged mesenchymal stem cells are damaged mesenchymal stem cells caused by physiological aging.

[0015] Secondly, this invention provides the application of Parishin A in the preparation of products for treating impaired self-renewal of mesenchymal stem cells caused by physiological aging.

[0016] Furthermore, the product includes Parishin A; Preferably, the product further includes pharmaceutically acceptable excipients.

[0017] Furthermore, the pharmaceutically acceptable excipients include at least one of absorbents, diluents, disintegrants, wetting agents, lubricants, binders, colorants, solvents, coating materials, or antimicrobial agents.

[0018] This invention relates to the application of Parishin A in the preparation of products for treating mesenchymal stem cell aging. Western blot analysis confirmed a dose-dependent increase in NRF2 protein expression with Parishin A. qRT-PCR verified the upregulation of NRF2 downstream target genes GCLC, SRX1, and NQO1, revealing that Parishin A achieves antioxidant homeostasis restoration by activating the NRF2 pathway. Oral administration in aged mice demonstrated that Parishin A effectively inhibits physiological aging-induced mesenchymal stem cell aging in the body's microenvironment, significantly restoring its bone tissue homeostasis regulation function and improving the integrity of cancellous bone structure. Simultaneously, femoral bone section β-galactosidase staining showed a significant reduction in the number of SA-β-gal positive cells, confirming its ability to reverse the aging microenvironment in vivo. In vitro functional validation showed that Parishin A treatment restored the proliferative activity of senescent hBMMSCs and specifically repaired the self-renewal impairment of senescent mesenchymal stem cells. Using β-galactosidase (SA-β-gal) as a classic aging marker, Parishin A treatment reduced the SA-β-gal positivity rate in senescent hBMMSCs and significantly downregulated the mRNA expression of the core aging factors p21 and p16. Furthermore, ROS probe detection confirmed that it could reduce the reactive oxygen species level in H2O2-induced mBMMSCs to near the level of the blank control group, with an antioxidant effect comparable to that of the positive control drug DMF. In the NRF2 gene knockdown experiment (siNRF2), it was found that siNRF2 completely blocked the ameliorative effects of Parishin A on SA-β-gal, p21 / p16, and proliferative function, confirming that the anti-aging effect of Parishin A on senescent mesenchymal stem cells is strictly dependent on the specific activation of the NRF2 pathway. This study reveals for the first time that Parishin A can treat or delay the aging of mesenchymal stem cells caused by physiological aging, and can be used to prepare drugs for treating diseases related to the aging of mesenchymal stem cells, providing a new method and new ideas for the preparation of drugs to inhibit aging-related diseases. Attached Figure Description

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

[0020] Figure 1 The β of hBMMSCs after Parishin A treatment provided in Embodiment 1 of the present invention Images showing galactosidase staining results, where A is the staining observation image and B is the SAβ-gal staining image. + Cellular statistics graph; Figure 2 The images shown are the colony formation results and Ki67 immunofluorescence staining results of hBMMSCs after Parishin A treatment provided in Example 2 of this invention. In this figure, A is a comparison of colony formation, B is a statistical chart of the number of colonies formed, C is a comparison of immunofluorescence staining, and D is a statistical chart of Ki67 protein positive cells. Figure 3 The results of Alizarin Red (ARS) staining of hBMMSCs treated with Parishin A after osteogenic induction provided in Example 3 of the present invention are shown in Figure A, where A is a staining comparison diagram and B is a statistical diagram of ARS positive areas. Figure 4 The results of Oil Red O staining of Parishin A-treated hBMMSCs after adipogenic induction provided in Example 4 of the present invention are shown in Figure A, where A is a staining comparison image and B is a lipid region statistical image. Figure 5 The femoral micro-injuries of aged mice after oral administration of Parishin A, as provided in Example 5 of this invention, were observed. Results of CT three-dimensional reconstruction and quantitative analysis of bone microstructure parameters, where A represents Micro CT scan results, B represents the quantitative analysis and statistical results of bone microstructure parameters; Figure 6 According to Example 5 of this invention, after oral administration of Parishin A to aged mice, the β-cell length of the femur was reduced. Images showing β-galactosidase staining results, where A is the staining observation image and B is the SAβ-gal staining image. + Cellular statistics graph; Figure 7 The figure shows the gene set enrichment analysis (GSEA) results of the NRF2 pathway in senescent hBMMSCs after Parishin A treatment provided in Example 6 of this invention. Figure 8This is an immunoblot image showing the results of Parishin A enhancing NRF2 protein expression in senescent hBMMSCs, as provided in Example 7 of this invention. Figure 9 The image provided in Example 8 of this invention shows the results of real-time quantitative reverse transcription polymerase chain reaction (RT-PCR) of Parishin A promoting the expression of NRF2 downstream target genes GCLC, SRX1, and NQO1, where A is GCLC, B is SRX1, and C is NQO1. Figure 10 The image shows the results of Parishin A promoting the scavenging of reactive oxygen species (ROS) in bone marrow mesenchymal stem cells (mBMMSCs) of aging mice, as provided in Example 9 of this invention. In the image, A is the ROS staining observation image and B is the ROS average intensity statistical image. Figure 11 The β-blocking effect of Parishin A on aging by knocking down NRF2 expression in hBMMSCs, as provided in Example 10 of this invention, is achieved by... Images showing galactosidase staining results, where A is a staining comparison image and B is the SAβ-gal staining result. + Cellular statistics graph; Figure 12 This is an immunofluorescence staining result of NRF2 in hBMMSCs that knocks down NRF2 expression to block the anti-aging function of Parishin A, as provided in Example 11 of this invention. In this image, A is a staining comparison image, and B is an NRF2 staining image. + Cellular statistics graph; Figure 13 The image shows the results of real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) detection of aging-related gene expression in hBMMSCs, which blocks the anti-aging function of Parishin A by knocking down NRF2 expression in hBMMSCs, as provided in Example 12 of this invention. In the image, A represents P21 and B represents P16. Detailed Implementation

[0021] Unless otherwise defined herein, the scientific and technical terms used in conjunction with this invention shall have the meanings commonly understood by one of ordinary skill in the art. The meaning and scope of terms shall be clear; however, in any case of potential ambiguity, the definitions provided herein shall prevail over any dictionary or foreign definitions. In this application, unless otherwise stated, the use of "or" means "and / or". Furthermore, the use of the term "comprising" and other forms is non-limiting.

[0022] Generally, the nomenclature and techniques used in cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization, together with those described herein, are those well-known and commonly used in the art. Unless otherwise stated, the methods and techniques of the present invention are generally carried out according to conventional methods well-known in the art and described in various general and more specific references, which are cited and discussed throughout this specification. Enzymatic reactions and purification techniques are carried out according to the manufacturer's instructions, as commonly practiced in the art, or as described herein. The nomenclature, laboratory procedures, and techniques used in analytical chemistry, synthetic organic chemistry, and medical and medicinal chemistry, together with those described herein, are those well-known and commonly used in the art.

[0023] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments. 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 skilled in the art without creative effort are within the scope of protection of the present invention.

[0024] This invention used Western blot to confirm a dose-dependent increase in NRF2 protein expression in relation to Parishin A. qRT-PCR verified the upregulation of NRF2 downstream target genes GCLC, SRX1, and NQO1, revealing that Parishin A restores antioxidant homeostasis by activating the NRF2 pathway. Oral administration to aged mice demonstrated that Parishin A effectively inhibits physiological aging-induced mesenchymal stem cell senescence in the body's microenvironment, significantly restoring its bone tissue homeostasis regulation function and improving the integrity of cancellous bone structure. Simultaneously, femoral bone section β-galactosidase staining showed a significant reduction in the number of SA-β-gal positive cells, confirming its ability to reverse the aging microenvironment in vivo. In vitro functional validation showed that Parishin A treatment restored the proliferative activity of senescent hBMMSCs and specifically repaired the self-renewal impairment of senescent mesenchymal stem cells. Using β-galactosidase (SA-β-gal) as a classic aging marker, Parishin A treatment reduced the SA-β-gal positivity rate in senescent hBMMSCs and significantly downregulated the mRNA expression of the core aging factors p21 and p16. Furthermore, ROS probe detection confirmed that it could reduce the reactive oxygen species level in H2O2-induced mBMMSCs to near the level of the blank control group, with an antioxidant effect comparable to that of the positive control drug DMF. In the NRF2 gene knockdown experiment (siNRF2), it was found that siNRF2 completely blocked the ameliorative effects of Parishin A on SA-β-gal, p21 / p16, and proliferative function, confirming that the anti-aging effect of Parishin A on senescent mesenchymal stem cells is strictly dependent on the specific activation of the NRF2 pathway.

[0025] This invention provides the use of Parishin A in the preparation of products for treating and / or delaying the aging of mesenchymal stem cells.

[0026] This study reveals for the first time that Parishin A can treat or delay the aging of mesenchymal stem cells caused by physiological aging, and can be used to prepare drugs for treating diseases related to the aging of mesenchymal stem cells, providing a new method and new ideas for the preparation of drugs to inhibit aging-related diseases.

[0027] In this invention, physiological aging refers to the gradual and systematic functional decline that occurs naturally with age in the absence of clear pathological stimuli (such as diabetes, gene defects, radiation exposure or chemical toxicity), specifically including the progressive decline in the ability of cells and tissues to maintain homeostasis.

[0028] In some specific embodiments, the senescent mesenchymal stem cells are physiologically senescent mesenchymal stem cells. In some specific embodiments, the mesenchymal stem cells include bone marrow mesenchymal stem cells.

[0029] In some specific embodiments, Parishin A treats and / or delays mesenchymal stem cell senescence by activating the NRF2 signaling pathway. In some specific embodiments, activating the NRF2 signaling pathway includes upregulating NRF2 protein expression levels in mesenchymal stem cells; and / or promoting mRNA and / or protein expression of NRF2 downstream target genes in mesenchymal stem cells. In some specific embodiments, the NRF2 downstream target genes include at least one of GCLC, SRX1, and NQO1.

[0030] In some specific embodiments, the product possesses at least one of the following functions: inhibiting β-galactosidase (SA-β-gal) activity in damaged mesenchymal stem cells, promoting the expression of aging factors in damaged mesenchymal stem cells, promoting the proliferation of damaged mesenchymal stem cells, promoting osteogenic differentiation of damaged mesenchymal stem cells, inhibiting adipogenic differentiation of damaged mesenchymal stem cells, or reducing the level of reactive oxygen species in damaged mesenchymal stem cells. In some specific embodiments, the aging factors include p16 and / or p21. In some specific embodiments, the damaged mesenchymal stem cells are damaged mesenchymal stem cells caused by physiological aging.

[0031] According to another aspect of the invention, the use of Parishin A in the preparation of products for treating impaired self-renewal of mesenchymal stem cells due to physiological aging is also provided.

[0032] In some specific embodiments, the product includes Parishin A; in some specific embodiments, the product further includes a pharmaceutically acceptable excipient. In some specific embodiments, the pharmaceutically acceptable excipient includes at least one of an absorbent, diluent, disintegrant, wetting agent, lubricant, binder, colorant, solvent, coating material, or antimicrobial agent.

[0033] The present invention will be further illustrated by the following examples. Unless otherwise specified, the materials in the examples are prepared according to existing methods or purchased directly from the market.

[0034] Material: Parishin A, with a purity of 99.58%, was purchased from Targetmol, a US company.

[0035] Type I collagenase, α-modified Eagle medium, fetal bovine serum, glutamine, and penicillin / streptomycin: purchased from Thermo Fisher Scientific.

[0036] Dispersing enzyme: purchased from Roche Pharmaceuticals, Shanghai.

[0037] β-Galactosidase staining fixative and β-Galactosidase staining working solution: purchased from Beyotime Biotechnology Co., Ltd.

[0038] Human bone marrow mesenchymal stem cells (hBMMSCs): hBMMSCs were isolated from mandibular bone tissue obtained intraoperatively from subjects aged 60-70 years. All procedures complied with the protocol approved by the Human Subject Ethics Review Committee of Nanjing Medical University (Approval No.: 2020182).

[0039] The isolation and extraction methods for hBMMSCs are as follows: Collected mandibular bone tissue was minced and placed in a digestion solution containing 3 mg / mL type I collagenase and 4 mg / mL dispersant enzyme, and digested at 37°C for 0.5 h. After digestion, the cells were filtered through a 70 μm cell filter to obtain a single-cell suspension. Cells were seeded in α-modified Eagle medium supplemented with 15% (v / v) fetal bovine serum, 2 mM glutamine, and 100 U / mL penicillin / streptomycin. Cells were cultured at 37°C in a 5% CO2 incubator, with the medium replaced with fresh medium every 2-3 days. When cell confluence reached 80%-90%, cells were digested with trypsin, centrifuged, collected, resuspended in growth medium, and further cultured and passaged. The 3rd-4th generation (P3-P4) hBMMSCs were designated as senescent hBMMSCs and used in the following examples.

[0040] Example 1: β-galactosidase staining to investigate the effect of Parishin A on the senescence of hBMMSCs 1. Preparation of Parishin A Weigh 100 mg of Parishin A and dissolve it in 1 ml of dimethyl sulfoxide (DMSO). Then add 4 ml of phosphate buffer (PBS) to prepare a 20 mM stock solution.

[0041] 2. β-galactosidase staining Senescent hBMMSCs at 4 × 10 4 Cells were seeded per well in 24-well plates. After cell adhesion, the treatment group was treated with Parishin A to a final concentration of 20 μM, while the control group was treated with an equal volume of solvent (DMSO). After 2 days of culture, 1 mL of β-galactosidase staining fixative was added to each well, and the cells were fixed at room temperature for 15 min. The cell fixative was discarded, and the cells were washed three times with PBS buffer. 1 mL of β-galactosidase staining working solution was added again, and the cells were incubated overnight at 37°C. The following day, cell senescence was observed under a light microscope, and photographs were taken and analyzed.

[0042] 3. β galactosidase staining results like Figure 1 As shown in Figures A and B, compared with the control group (DMSO), after Parishin A treatment, the treatment group (PA) had significantly more β-galactosidase-positive cells (SAβ-galactosidase). + The number of cells was significantly reduced, demonstrating that Parishin A can effectively inhibit cellular senescence in hBMMSCs.

[0043] Example 2 investigated the effect of Parishin A on the proliferation capacity of senescent hBMMSCs. 1. Configure Parishin A according to the method in Example 1.

[0044] 2. Colony Formation Experiment Senescent hBMMSCs were loaded at 5 × 10 3 Cells were seeded per well in 6-well plates. After cell adhesion, the treatment group was treated with a final concentration of 20 μM Parishin A, while the control group was treated with an equal volume of solvent (DMSO). After 7 days of culture, the culture medium was discarded, the cells were washed twice with PBS, fixed with 4% paraformaldehyde for 15 minutes, stained with 0.1% crystal violet for 30 minutes, rinsed with running water, and air-dried. The number of colonies with more than 50 cells was counted under an inverted microscope.

[0045] 3. Ki67 Experiment Cell crawling slides were placed in 12-well plates, and senescent hBMMSCs were sputtered at a density of 1×10⁻⁶. 5Cells were seeded per well in 12-well plates. Once cells adhered and reached 50% confluence, Parishin A (20 μM) was added to the treatment group, while an equal volume of DMSO was added to the control group. After one day of culture, the culture medium was removed, the cells were washed with phosphate-buffered saline (PBFS), fixed with 4% paraformaldehyde for 15 minutes, permeabilized with 1% Triton X-100 for 10 minutes, blocked with 5% fetal bovine serum (FBS) for 1 hour, and then Ki67 antibody diluted 1:200 was added. The plates were incubated overnight at 4°C. The next day, the Ki67 antibody was washed away with phosphate-buffered saline, and the plates were incubated with fluorescent antibody diluted 1:200 at room temperature for 1 hour. After washing away the fluorescent antibody, the plates were mounted with mounting medium, and Ki67 positive expression was observed under an immunofluorescence microscope.

[0046] 4. Results like Figure 2 The crystal violet staining results shown in Figures A and B indicate that, compared with the control group (DMSO), the number of colonies with more than 50 cells in the treatment group (PA) was significantly increased after Parishin A treatment, demonstrating that Parishin A can effectively promote the proliferation of senescent hBMMSCs.

[0047] like Figure 2 The immunofluorescence results shown in C and D indicate that, compared with the control group (DMSO), treatment with 20 μM Parishin A increased Ki67 expression and cell number in senescent hBMMSCs, demonstrating that Parishin A can effectively promote the proliferation of senescent hBMMSCs.

[0048] Example 3: Investigation of the effect of Alizarin Red staining on the osteogenic capacity of senescent hBMMSCs 1. Configure Parishin A according to the method in Example 1.

[0049] 2. Alizarin Red Staining Experiment Senescent hBMMSCs were loaded at 4 × 10 4 Cells were seeded per well in a 24-well plate. Once the cell density reached 70%, the medium was replaced with osteogenic induction medium. The treatment group received 20 μM Parishin A, while the control group received an equal volume of DMSO. After 14 days of osteogenic induction, the 24-well plates were removed, the medium was discarded, and cells were fixed with 4% paraformaldehyde at room temperature for 15 min. After discarding the cell fixative, the cells were washed three times with PBS buffer. Alizarin Red staining solution was added, and the plates were incubated in the dark for 10 min. The plates were then washed with distilled water until the solution was clear. The plates were scanned using a scanner, and the Alizarin Red staining was recorded under a microscope.

[0050] 3. Results like Figure 3As shown in Figures A and B, the alizarin red staining (ARS) results show that, compared with the control group (DMSO), the treatment group (PA) showed a significant increase in the alizarin red staining positive area after Parishin A treatment, demonstrating that Parishin A can effectively promote osteogenic differentiation of hBMMSCs.

[0051] Example 4: Investigating the effect of Parishin A on the adipogenic capacity of senescent hBMMSCs using Oil Red O staining. 1. Configure Parishin A according to the method in Example 1.

[0052] 2. Oil Red O staining experiment Senescent hBMMSCs were loaded at 4 × 10 4 Cells were seeded per well in 24-well plates. Once the cell density reached 90%, the medium was replaced with adipogenic induction medium. The experimental group received 20 μM Parishin A, while the control group received an equal volume of DMSO. After 21 days of adipogenic induction, the 24-well plates were removed, the medium was discarded, and cells were fixed with 4% paraformaldehyde at room temperature for 15 min. After discarding the cell fixative, the cells were washed three times with PBS buffer. Oil Red O staining solution was added, and the plates were incubated in the dark for 10 min. The cells were washed twice with washing buffer, and the Oil Red O staining was recorded under a microscope.

[0053] 3. Results like Figure 4 As shown in Oil Red staining results A and B, compared with the control group (DMSO), the lipid region of the treatment group (PA) was significantly reduced after Parishin A treatment, demonstrating that Parishin A can effectively inhibit the adipogenic differentiation of hBMMSCs.

[0054] Example 5: In vivo experiments directly demonstrated that Parishin A promotes efficient bone mass recovery in aged mice with osteoporosis. 1. Prepare Parishin A as a therapeutic drug according to the method in Example 1.

[0055] 2. Treatment of osteoporosis in aged mice (1) Animals: C57BL / 6J mice, 18-month-old aged mice, were divided into treatment group and control group.

[0056] (2) Treatment plan: Treatment group (PA): Mice were administered the treatment drug by gavage daily at a dose of 20 mg / kg based on Parishin A.

[0057] Control group (Veh): Daily gavage was performed using an equal volume of PBS instead of the treatment drug.

[0058] After 4 weeks of continuous treatment, mouse femurs were harvested, fixed overnight with 4% paraformaldehyde fixative, and scanned using Micro-CT for imaging analysis.

[0059] 3. Results Analysis like Figure 5 The results of two-dimensional and three-dimensional cross-sections of the mouse femur shown in Figure A indicate that the osteoporotic mass of the femur in the control group was lower, while the bone mass of the treatment group was significantly restored after treatment with Parishin A. The results of analyzing the microstructural parameters of the mouse femur using software are as follows: Figure 5 As shown in Figure B, compared with the control group, after treatment with Parishin A, the bone volume fraction (BV / TV), bone mineral density (BMD), and number of trabecular bones (Tb.N) all increased, while the trabecular separation (Tb.Sp) decreased, indicating an increase in bone mass after treatment.

[0060] like Figure 6 As shown in Figures A and B, HE staining and β-galactosidase staining experiments were performed on mouse femoral sections. Compared with the control group, the treatment group showed improved cancellous bone mass and reduced β-galactosidase levels. + The significant reduction in cells demonstrates that Parishin A has a significant therapeutic effect on osteoporosis in aged mice.

[0061] Example 6: Gene set enrichment analysis of the Nrf2 pathway in senescent hBMMSCs treated with Parishin A 1. Cell experiments Senescent hBMMSCs were quantified at a rate of 5 × 10 5 Cells were seeded per well in 6-well plates. Once cells adhered and reached 70% confluence, the treatment group (PA) received 20 μM Parishin A, while the control group (DM) received an equal volume of DMSO. After 48 hours of culture, the culture medium was discarded, cells were washed twice with PBS, and 1 ml of Trzio solution was added to each well to collect the cells. The cells were then transferred to 1.5 ml EP tubes and stored at -80°C for subsequent whole-transcriptome sequencing.

[0062] 2. Results like Figure 7 As shown, gene set enrichment analysis revealed that, compared with the control group, the senescent hBMMSCs treated with Parishin A showed a significant enrichment of NRF2 signaling pathway-related gene sets.

[0063] Example 7: NRF2 protein expression in senescent hBMMSCs after Parishin A enhancement 1. Protein imprinting experiment Senescent hBMMSCs were quantified at 5 × 10 5Cells were seeded per well in 6-well plates. Once cells adhered and reached 70% confluence, Parishin A (PA) was added at final concentrations of 0 μM, 10 μM, and 20 μM, respectively. After 48 hours of culture, the culture medium was discarded, the cells were washed twice with PBS, and protein was extracted for proteoblotting experiments to detect NRF2 protein expression levels.

[0064] 2. Results like Figure 8 As shown, with consistent protein expression levels of the internal reference gene, compared to the control group (PA = 0 μM), the NRF2 protein expression levels after treatment with 10 μM and 20 μM Parishin A gradually increased, indicating that Parishin A can enhance NRF2 protein expression in senescent hBMMSCs.

[0065] Example 8: Real-time quantitative reverse transcription polymerase chain reaction (qPCR) of Parishin A promoting the expression of NRF2 downstream target genes GCLC, SRX1, and NQO1 1. Real-time quantitative reverse transcription polymerase chain reaction Senescent hBMMSCs were quantified at 5 × 10 5 Cells were seeded per well in 6-well plates. Once cells adhered and reached 70% confluence, the treatment groups were treated with 10 μM and 20 μM Parishin A, while the control group received an equal volume of DMSO. After 48 hours of culture, the culture medium was discarded, and the cells were washed twice with PBS. RNA was extracted after 5 days of induction. Real-time quantitative reverse transcription polymerase chain reaction (qPCR) was performed to detect the expression of NRF22 downstream target genes GCLC, SRX1, and NQO1.

[0066] 2. Results Analysis like Figure 9 As shown, compared with the control group (DMSO), the expression levels of GCLC, SPX1, and NQO1 at the RNA level were significantly increased in the treatment groups (PA) with 10 μM and 20 μM Parishin A. This verifies that Parishin A can promote the expression of NRF2, and through NRF2, promote the expression of downstream target genes GCLC, SPX1, and NQO1.

[0067] Example 9: Parishin A promotes ROS clearance in senescent mBMMSCs 1. Configure Parishin A according to the method in Example 1.

[0068] 2. Isolation and extraction of mouse bone marrow mesenchymal stem cells (mBMMSCs): Mouse femurs and tibias were collected, and mouse-derived mBMMSCs were collected using the bone marrow flushing method. The bone marrow cell suspension was centrifuged at 1200 rpm for 5 minutes, the supernatant was discarded, and primary culture was initiated using 20% ​​serum-concentrated culture medium. When the cell density reached 90%, the cells were passaged, and then cultured again using 10% serum-concentrated culture medium, reaching passages P3-P4 for subsequent experiments.

[0069] 3. ROS testing Cell seeding and treatment: mBMMSCs in the 3rd to 5th generation logarithmic growth phase were seeded at 8 × 10⁻⁶ cells / year. 4 Cells were seeded at a density of 1 cell per well in confocal microplates and incubated at 37°C with 5% CO2 for 24 h to allow for adherent growth. The experiment was divided into three groups, with three biological replicates in each group. Blank control group: only fresh complete culture medium was added, without any induction or drug treatment; H2O2 model group (H2O2+Veh): The cells were treated with maintenance medium containing 100 μM H2O2 for 2 h to induce oxidative stress. The medium was then discarded, washed twice with PBS, and replaced with maintenance medium containing 10 μM H2O2. At the same time, DMSO of the same concentration as the drug treatment group was added throughout the modeling process, and the cells were cultured for another 48 h to establish a cell senescence model. Drug treatment groups (H2O2+DMF and H2O2+PA): The same modeling protocol as the H2O2 model group was used (100 μM H2O2 treatment for 2 h, 10 μM H2O2 maintenance for 48 h), while 10 μM positive control drug (DMF) or 20 μM Parishin A (PA) was added for intervention throughout the modeling process.

[0070] ROS Staining and Observation: After culture, the culture medium in each well was aspirated, and the cells were gently washed twice with PBS to remove residual culture medium and impurities. Following the instructions of the reactive oxygen species detection kit (Wuhan Saiweier Biotechnology Co., Ltd.), an appropriate amount of staining working solution was added, and the cells were incubated at 37°C in the dark for 25 min. The cell nuclei were counterstained with DAPI, and finally washed once with PBS. Under a laser confocal fluorescence microscope, three different fields of view were selected from each well for photography, recording the distribution and intensity of intracellular DCF (green) and nuclear DAPI (blue) fluorescence.

[0071] 4. Results like Figure 10As shown in Figures A and B, the blank control group showed regular cell morphology, intact and clear nuclei, and almost no green fluorescence, with only weak background fluorescence, indicating extremely low levels of reactive oxygen species and no significant oxidative damage. The H2O2 model group showed abundant strong green fluorescence, with a wide and dense distribution, and no obvious abnormalities in cell nucleus morphology, indicating that H2O2 successfully induced oxidative stress in cells, leading to the accumulation of ROS related to cellular senescence. In the drug treatment groups, the green fluorescence intensity in the 10 μM DMF treatment group was significantly weaker than that in the model group; the green fluorescence in the 20 μM PA treatment group was also significantly weaker than that in the model group, and close to the fluorescence intensity of the positive control drug DMF group.

[0072] Data show that Parishin A can effectively scavenge ROS induced by H2O2 and significantly reduce ROS levels in mBMMSCs. Its antioxidant and anti-cellular senescence effects are no weaker than those of the positive control drug DMF, and it can effectively inhibit H2O2-induced senescence of mBMMSCs.

[0073] Example 10: Knocking down Nrf2 expression in hBMMSCs to block the anti-aging function of ParishinA - β-galactosidase staining experiment 1. β-Galactosidase Staining Experiment Senescent hBMMSCs were quantified at 4 × 10 4 Cells were seeded per well in 24-well plates. Once cells adhered and reached 50% confluence, they were divided into four groups: Si NC, Si NC + Parishin A, Si NRF2, and Si NRF2 + Parishin A. Si NC (negative control) and Si NRF2 (NRF2 knockdown) were transfected, and Parishin A or its solvent was added to a final concentration of 20 μM. After 5 days of transfection and culture, the medium was replaced with fresh medium containing 10% serum. After another 10 days of culture, 1 mL of β-galactosidase staining fixative was added, and the cells were fixed at room temperature for 15 min. After discarding the fixative, the cells were washed three times with PBS buffer, and 1 mL of β-galactosidase staining working solution was added again. The cells were incubated overnight at 37°C. The following day, cell senescence was observed under a light microscope, and photographs were taken and analyzed.

[0074] 2. Results β The results of galactosidase staining are as follows Figure 11 As shown, NRF2 knockdown negated the anti-aging effect of Parishin A on senescent hBMMSCs.

[0075] Example 11: Immunofluorescence staining results of Nrf2 immunofluorescence staining to knock down Nrf2 expression in hBMMSCs and block the anti-aging function of Parishin A; 1. Immunofluorescence staining experiment Cell crawling slides were placed in 12-well plates, and senescent hBMMSCs were sputtered at a density of 1×10⁻⁶. 5 Cells were seeded per well in 12-well plates. Once cells adhered and reached 50% confluence, they were divided into four groups: Si NC, Si NC + Parishin A, Si NRF2, and Si NRF2 + Parishin A. Si NC (negative control) and Si NRF2 (NRF2 knockdown) were transfected with Parishin A (final concentration 20 μM) or an equal volume of DMSO. After 5 days of transfection and culture, the medium was replaced with fresh medium containing 10% serum. After another 10 days of culture, the medium was removed, the cells were washed with phosphate-buffered saline (PBFS), fixed with 4% paraformaldehyde for 15 minutes, permeabilized with 1% Triton X-100 for 10 minutes, blocked with 5% fetal bovine serum (FBS) for 1 hour, and then NRF2 antibody diluted 1:200 was added. The cells were incubated overnight at 4°C. The next day, the NRF2 antibody was washed away with phosphate-buffered saline, and the cells were incubated with fluorescent antibody diluted 1:200 at room temperature for 1 hour. After washing away the fluorescent antibody, the cells were mounted with mounting medium, and Nrf2 positive expression was observed under an immunofluorescence microscope.

[0076] 2. Results Depend on Figure 12 The immunofluorescence staining results shown in Figures A and B indicate that NRF2 knockdown negates the effect of Parishin A on promoting NRF2 expression in senescent hBMMSCs.

[0077] Example 12: Real-time quantitative reverse transcription polymerase chain reaction (qPCR) to knock down NRF2 expression in hBMMSCs and block the anti-aging function of ParishinA. 1. Immunofluorescence staining experiment Cell crawling slides were placed in 12-well plates, and senescent hBMMSCs were sputtered at a density of 1×10⁻⁶. 5 Cells were seeded per well in 12-well plates. Once cells adhered and reached 50% confluence, they were divided into four groups: Si NC, Si NRF2, Si NC + Parishin A, and Si NRF2 + Parishin A. Si NC (negative control) and Si NRF2 (NRF2 knockdown) were transfected with Parishin A or its solvent at a final concentration of 20 μM. After 5 days of transfection and culture, the medium was replaced with fresh medium containing 10% serum. After another 10 days of culture, the medium was discarded, and the cells were washed twice with PBS. RNA was extracted after 5 days of induction. Real-time quantitative reverse transcription polymerase chain reaction (qPCR) was performed on the extracted RNA to detect the expression levels of aging-related genes P21 and P16.

[0078] 2. Results like Figure 13As shown, the expression levels of aging-related genes P21 and P16 were lowest in the Si NC+Parishin A group. After the addition of Si NRF2, the expression levels of aging-related genes P21 and P16 in the Si NRF2+Parishin A group were significantly increased, which offset the anti-aging effect of Parishin A on aging hBMMSCs.

[0079] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. Application of Parishin A in the preparation of products for treating and / or delaying the aging of mesenchymal stem cells.

2. The application according to claim 1, characterized in that, Parishin A treats and / or delays mesenchymal stem cell aging by activating the NRF2 signaling pathway.

3. The application according to claim 2, characterized in that, Activation of the NRF2 signaling pathway includes upregulating NRF2 protein expression levels in mesenchymal stem cells; and / or promoting mRNA and / or protein expression of NRF2 downstream target genes in mesenchymal stem cells.

4. The application according to claim 3, characterized in that, The downstream target genes of NRF2 include at least one of GCLC, SRX1 and NQO1.

5. The application according to claim 1, characterized in that, The mesenchymal stem cell senescence mentioned above is mesenchymal stem cell senescence caused by physiological aging. Preferably, the mesenchymal stem cells include bone marrow mesenchymal stem cells.

6. The application according to claim 1, characterized in that, The product has at least one of the functions listed in A1 to A6: A1. Inhibits the activity of β-galactosidase (SA-β-gal) in damaged mesenchymal stem cells; A2. Promotes the expression of senescence factors in damaged mesenchymal stem cells; A3. Promotes the proliferation of damaged mesenchymal stem cells; A4. Promotes osteogenic differentiation of damaged mesenchymal stem cells; A5. Inhibits the adipogenic differentiation of damaged mesenchymal stem cells; A6. Reduces the level of reactive oxygen species in damaged mesenchymal stem cells.

7. The application according to claim 6, characterized in that, The aging factors include p16 and / or p21; Preferably, the damaged mesenchymal stem cells are damaged mesenchymal stem cells caused by physiological aging.

8. Application of Parishin A in the preparation of products for treating impaired self-renewal of mesenchymal stem cells due to physiological aging.

9. The application according to any one of claims 1 to 8, characterized in that, The product includes Parishin A; Preferably, the product further includes pharmaceutically acceptable excipients.

10. The application according to claim 9, characterized in that, The pharmaceutically acceptable excipients include at least one of absorbents, diluents, disintegrants, wetting agents, lubricants, binders, colorants, solvents, coating materials, or antimicrobial agents.

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