Anti-aging two-factor composition based on epigenetic reprogramming and application thereof

Abstract

The invention discloses an anti-aging two-factor composition based on epigenetic reprogramming and application thereof, and belongs to the technical field of biological medicine. The composition comprises a Wnt pathway small molecule activator Laduvilliub and an ERK pathway activator bFGF, and through synergistic activation of Wnt and ERK signal pathways, epigenetic reprogramming of senescent cells is induced, and senescence-related chromatin open change, histone modification imbalance and DNA methylation clock drift are significantly reversed. At the cellular level, the composition can reverse various senescence phenotypes, namely restoring multiplication capacity, down-regulating expression of senescence-related secretory phenotype factors, relieving DNA damage, improving telomerase activity and telomere length and increasing nucleofibrin. At an individual level (an old mouse model), the composition is injected in the intraperitoneal cavity, so that the cognitive ability and the exercise ability are remarkably improved, the aging pathological characteristics of multiple organs are reduced, and the aging-related gene expression profiles of the organs such as the liver and muscles are reversed. The double-factor composition can be applied to anti-aging drugs, health care products or cosmetics.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological medicine, and particularly relates to an anti-aging factor composition based on epigenetic reprogramming and application thereof. BACKGROUND

[0002] With the aggravation of social aging, the research on aging mechanism has become a hot topic in life science. Aging is manifested as a multi-level biological decline: at the cellular level, aging cells enter an irreversible proliferative arrest state, accompanied by telomere shortening, DNA damage accumulation and activation of senescence-associated secretory phenotype (SASP), which continuously releases pro-inflammatory factors to destroy the tissue microenvironment; at the individual level, aging leads to the synchronous degradation of multiple organ functions, such as muscle loss, cognitive decline, metabolic disorder, and significantly increases the risk of senile diseases such as Alzheimer's disease and cardiovascular disease. This complexity is due to the multi-pathway driving characteristics of aging (such as nine major markers such as genomic instability, epigenetic disorder and mitochondrial dysfunction), making it difficult for single-target intervention strategies to comprehensively reverse the aging process.

[0003] Current aging assessment mainly relies on three types of technologies: cell aging marker detection (such as beta-galactosidase staining, p16 / p21 protein expression analysis), which can only reflect the local cell state and cannot assess the degree of aging at the organ or system level; epigenetic clock, which can quantify biological age but cannot reveal the mechanism of aging pathways; functional indicators (such as animal movement ability, memory test), which can reflect the overall aging phenotype but lack precise molecular analysis. These technologies are limited in their respective fields, making it difficult for anti-aging drug development to establish a precise correlation between "mechanism-phenotype", and most drugs can only delay a single aging phenotype (such as reducing the number of senescent cells), and cannot achieve systemic reversal.

[0004] Traditional anti-aging drugs (such as rapamycin, metformin) can partially improve the aging phenotype, but are limited by single-pathway mechanisms and cannot coordinately regulate the core network of aging. SUMMARY

[0005] The purpose of the present application is to provide a composition for delaying cell aging and individual aging. The present application first discovers the combination of the small molecule Wnt pathway small molecule activator Laduviglusib and the ERK activator bFGF, which breaks through the limitation of single target. The combination of the two factors in the composition produces a synergistic effect, enhancing its anti-aging effect, and can significantly delay the aging of multiple cell types and multiple indicators of mice.

[0006] To achieve the above technical purposes, the present application provides the following technical solutions:

[0007] The present invention first provides a composition for delaying cellular aging and individual aging, the composition containing two components: bFGF (basic fibroblast growth factor) and Laduviglusib.

[0008] This invention uses the recombinant protein bFGF produced by Escherichia coli (E. coli), which is a single-chain polypeptide composed of 154 amino acids, and its sequence is as follows:

[0009] AAGSITTLPALPEDGGSGAFPPGHFKDPKRLYCKNGGFFLRIHPDGRVDGVREKSDPHI KLQLQAEERGVVSIKGVCANRYLAMKEDGRLLASKCVTDECFFFERLESNNYNTYRSRKY TSWYVALKRTGQYKLGSKTGPGQKAILFLPMSAKS(SEQ ID NO: 1)

[0010] bFGF is a member of the fibroblast growth factor family and has a high affinity for heparin. It is an ERK signaling pathway activator. bFGF activates downstream ERK signaling pathways by binding to the FGFR receptor and heparin, thereby regulating cell proliferation and differentiation. Furthermore, bFGF negatively regulates the NF-κB pathway, reducing the release of SASP factors such as TNF-α and IL-6.

[0011] Laduviglusib is an aminopyrimidine derivative with the following structural formula:

[0012]

[0013] Laduviglusib, a highly selective GSK-3β inhibitor, significantly enhances telomere maintenance and promotes tissue stem cell regeneration by stabilizing β-catenin and activating the Wnt / β-catenin pathway.

[0014] Preferably, in the anti-aging composition provided by the present invention, the mass ratio of bFGF to Laduviglusib is 1:20 to 10:1. In some specific embodiments of the present invention, the mass ratio of bFGF to Laduviglusib is 1:14. Through extensive research, the inventors have discovered that the combined use of bFGF and Laduviglusib exhibits a significant synergistic effect in anti-aging and functional maintenance in various cells and mouse individuals, significantly delaying cellular and individual aging.

[0015] The study of this invention shows that combined treatment with bFGF and Laduviglusib can significantly improve the anti-aging ability of cells through the synergistic effect of multiple mechanisms, specifically as follows: the combination of bFGF and Laduviglusib reverses aging by inducing epigenetic reprogramming, wherein the epigenetic reprogramming includes at least one of the following: reversing aging-related changes in chromatin openness; reversing histone modification imbalance, including upregulation of at least one of H3K27ac, H3K4me3, and H3K9ac and / or downregulation of at least one of H3K9me3, H3K27me3, and H4K20me3; and reversing the aging drift of the DNA methylation clock.

[0016] The aging phenotypes reversed at the cellular level by the bFGF and Laduviglusib dual-factor combination include at least one of the following: restoration of cell proliferation capacity (manifested as increased Ki67 positivity and decreased p16 and p21 protein expression levels); downregulation of aging-associated secretory phenotype (SASP) factor expression and secretion; reduction of DNA damage (manifested as decreased γH2AX levels); improvement of telomerase activity and telomere length; and upregulation of lamin protein expression levels (including Lamin A / C and Lamin B1).

[0017] The bFGF and Laduviglusib dual-factor combination improves at the individual level aging phenotypes including at least one of the following: improved cognitive abilities (manifested as increased Y-maze exploration attempts in mice); improved motor abilities (manifested as enhanced grip strength and prolonged rotarod exercise duration in mice); reduced serum SASP factor levels; alleviated pathological changes in multiple organs of aging, including the heart, liver, kidneys, spleen, lungs, pancreas, cerebral cortex, hippocampus, and muscles, with pathological changes including reduced fibrosis, decreased glycogen deposition, reduced fat accumulation, and reduced amyloid deposition; improved liver function (manifested as decreased serum ALT and AST levels) and / or improved kidney function (manifested as decreased serum UREA and UA levels); and reversed the expression profile of aging-related genes in multiple organs.

[0018] The anti-aging composition provided by this invention can be used in products for delaying aging, reversing aging phenotypes, or treating age-related diseases. These products can be pharmaceuticals, health products, or cosmetics, and in addition to containing bFGF and Laduviglusib, may also contain pharmaceutically or cosmetically acceptable excipients. The excipients can be one or more pharmaceutically or cosmetically acceptable salts, carriers, or excipients, including surfactants, thickeners, conditioning agents, humectants, preservatives, fragrances, deionized water, etc.

[0019] The aforementioned drug or health product, as an anti-aging agent, can be in oral or injectable dosage forms. The working solution can be prepared by separately preparing bFGF and Laduviglusib powders, or by simultaneously fusing the powders together; the preparation process is the same. Taking 1 mL of working solution as an example, 100 μL of 20.8 mg / mL clarified DMSO stock solution is added to 400 μL of PEG300 and mixed thoroughly. Then, 50 μL of Tween-80 is added to the above system and mixed thoroughly. Finally, 450 μL of physiological saline is added to bring the volume to 1 mL.

[0020] The cosmetics mentioned are mainly skin care products, which can be made into various dosage forms including toners, lotions, nourishing creams, massage creams, serums, eye creams, cleansing creams, body lotions, facial cleansers, face masks, pressed powders, etc.

[0021] The beneficial effects of this invention are:

[0022] Current mainstream aging interventions can be broadly categorized into lifestyle health initiatives (such as dietary restrictions, exercise, and circadian rhythm regulation), allogeneic blood exchange, partial reprogramming, and pharmacological intervention. While dietary restrictions, exercise, circadian rhythm regulation, and allogeneic blood exchange have been proven to extend the lifespan and improve the health of experimental animals, their clinical application is limited, with allogeneic blood exchange facing ethical constraints. Although current mainstream anti-aging drugs are more readily applicable clinically, they often target only a single pathway, only delaying aging phenotypes at the cellular senescence level, and failing to reverse cellular senescence. Partial reprogramming, while reversing cellular senescence fate through epigenetic reprogramming at the cellular senescence level and improving individual aging characteristics at the individual aging level, requires gene editing and is limited by the potential increased risk of cancer. This invention, through large-scale screening of small molecules and cytokines with anti-aging potential, and guided by the theory of multi-molecule combined targeting of multiple aging pathways, ultimately yielded a dual-factor combination capable of reversing cellular senescence fate. The bFGF and Laduviglusib dual-factor combination of the present invention induces epigenetic reprogramming in senescent cells by synergistically activating the Wnt and ERK signaling pathways, significantly reversing age-related changes in chromatin openness, histone modification imbalance, and DNA methylation clock drift. It can not only reverse multiple aging phenotypes at the cellular level, but also significantly improve cognitive and motor abilities at the individual level, reduce age-related pathological features in multiple organs, and reverse the expression profile of age-related genes in multiple organs. Attached Figure Description

[0023] Figure 1The results of Example 1 show that the combined treatment of senescent MEF cells with bFGF and Laduviglusib was significantly more effective than single-drug treatment. The left panel shows Ki67-positive cells, the top right panel shows β-gal staining results (scale bar: 150 μm), and the bottom right panel shows statistical results. In the figures, bFGF represents the bFGF treatment group, CHIR99021 represents the highly selective GSK-3β inhibitor Laduviglusib treatment group, FC represents the combined bFGF and Laduviglusib treatment group, and Ctrl represents the blank control group.

[0024] Figure 2 This figure shows the decrease in β-gal staining and the recovery of proliferation capacity after combined treatment of senescent MEF cells with the bFGF and Laduviglusib dual-factor combination in Example 2. Figure A shows the β-gal staining results of PD12 senescent MEF cells after treatment with the bFGF and Laduviglusib dual-factor combination for 0, 48, 96, and 144 hours, and after treatment for 0, 3, 6, and 9 days (scale bar: 400 μm); Figure B shows the statistical results of positive cells in Figure A.

[0025] Figure 3 This figure shows the changes in the proportion of Ki67-positive cells and the expression levels of p16 and p21 after combined treatment of senescent MEF cells with bFGF and Laduviglusib in Example 3. Figure A shows the expression of Ki67 in senescent PD12 MEF cells after 6 days of combined treatment with bFGF and Laduviglusib, as detected by immunofluorescence (scale bar: 400 μm); Figure B shows the statistical results of the proportion of positive cells in Figure A; Figure C shows the expression levels of p16 and p21 in PD12 MEF cells after Western blotting for PD4, PD8, PD12, and PD12 cells after 48, 96, and 144 hours of combined treatment with bFGF and Laduviglusib, and the statistical results. Error bars in the statistical figures represent the standard deviation obtained from three independent experiments, and a two-tailed unpaired t-test was performed.

[0026] Figure 4 This illustrates the cell cycle reversal observed in senescent MEF cells after combined treatment with bFGF and Laduviglusib in Example 4. A shows the cell growth curves of mouse embryonic fibroblasts during senescence, after combined treatment with the two factors, and after drug withdrawal; B shows the changes in G1, S, and G2 phases of senescent MEF cells treated with PD12 for 6 days, as detected by flow cytometry; C shows the statistical proportions of MEF cells at each stage of the cell cycle. The statistical graphs were obtained from three independent experiments and subjected to a two-tailed unpaired t-test.

[0027] Figure 5This figure shows the reversal of SASP expression and secretion in senescent MEF cells after combined treatment with bFGF and Laduviglusib in Example 5. A and B represent the changes in SASP levels in the cell culture medium of senescent MEF cells treated with the combined bFGF and Laduviglusib for 6 days, as detected by a SASP microarray, and the statistical results. C represents the statistical analysis of RNA expression levels of SASP-related genes in senescent MEF cells treated with the combined bFGF and Laduviglusib for 6 days. Error bars in the figure represent the standard deviation obtained from three independent experiments, analyzed using a two-tailed unpaired t-test.

[0028] Figure 6 This illustrates the reversal of posterior granular wear in senescent MEF cells treated with a combination of bFGF and Laduviglusib in Example 6. In this diagram, Young represents solvent-treated young PD2 MEF cells, Old represents solvent-treated senescent PD12 MEF cells, and Cocktail FC represents bFGF and CHIR99021-treated senescent PD12 MEF cells. Figure A shows the changes in posterior granular length and fluorescence intensity of senescent PD12 MEF cells treated with the combination of bFGF and CHIR99021 after 6 days, detected by in situ hybridization (scale bar: 20 μm). Figure B shows the quantitative detection of changes in posterior granular length and posterior granzyme activity of senescent PD12 MEF cells treated with the combination of bFGF and CHIR99021 after 6 days using the TRAP method (top figure) and (bottom figure). Error bars in the statistical graphs represent the standard deviation obtained from three independent experiments, analyzed using a two-tailed unpaired t-test.

[0029] Figure 7 This illustrates the reversal of DNA damage in senescent MEF cells after combined treatment with bFGF and Laduviglusib in Example 7. In this diagram, Young represents solvent-treated PD2 young MEF cells, Old represents solvent-treated PD12 senescent MEF cells, and Cocktail FC or FC represents bFGF and CHIR99021 combined treatment of PD12 senescent MEF cells. Figure A shows the change in γH2AX expression level in PD12 senescent MEF cells after 6 days of combined treatment with the combined factor, detected by immunofluorescence (scale bar: 100 μm). Figure B shows the percentage of positive cells in Figure A. Figure C shows the changes in γH2AX and p53 protein levels in PD12 senescent MEF cells after 6 days of combined treatment with the combined factor, detected by Western blotting. Error bars in the statistical graphs represent the standard deviation obtained from three independent experiments, analyzed using a two-tailed unpaired t-test.

[0030] Figure 8This figure shows the changes in cellular senescence phenotypes after treatment with a combination of bFGF and Laduviglusib in Example 8. Cocktail FC or FC represents bFGF and CHIR99021 dual-factor treatment; A shows the changes in β-gal staining in mouse lung fibroblasts, mouse mesenchymal stem cells, and human mesenchymal stem cells 3 days after dual-factor treatment (scale bar: 400 μm); B shows the percentage of positive cells in Figure A; C shows the changes in cell proliferation capacity in mouse lung fibroblasts, mouse mesenchymal stem cells, and human mesenchymal stem cells 3 days after dual-factor treatment. Error bars in the statistical graphs represent the standard deviations obtained from three independent experiments, analyzed using a two-tailed unpaired t-test.

[0031] Figure 9 This demonstrates the reversal of the DNA methylation clock after combined treatment of senescent MEF cells with the bFGF and Laduviglusib dual-factor combination in Example 9. Here, YC represents solvent treatment of PD2 young MEF cells, OC represents solvent treatment of PD12 senescent MEF cells, and Cocktail FC represents bFGF and CHIR99021 dual-factor treatment of PD12 senescent MEF cells.

[0032] Figure 10 This demonstrates how the combination of bFGF and Laduviglusib in Example 10 enhanced muscle strength and endurance in aged mice. Figures A and B show the changes in movement in 18-month-old mice four weeks after treatment with the combination of bFGF and Laduviglusib, as detected by the mouse grip test and mouse rotator bar test, respectively. Error bars in the statistical graphs represent standard deviations obtained from more than three independent experiments, analyzed using a two-tailed unpaired t-test.

[0033] Figure 11 This illustrates how the combined treatment of bFGF and Laduviglusib in Example 11 enhanced short-term memory in aged mice. In Figure A, the route map of 18-month-old mice exploring a Y-maze 4 weeks after treatment with the Cocktail FC is shown, with color intensity representing the time spent in different areas of the Y-maze. Figure B shows the number of entry arms and alternations in the Y-maze for mice in the Cocktail FC treatment group and the control group. Error bars in the statistical graph represent the standard deviation obtained from more than three independent experiments, analyzed using a two-tailed unpaired t-test.

[0034] Figure 12This diagram illustrates how the combined treatment of bFGF and Laduviglusib in Example 12 enhanced anxiety and exploratory behaviors in aged mice. Figure A shows the route map of 18-month-old mice exploring an open field 4 weeks after treatment with the two-factor combination (Cocktail FC). Figure B shows the movement time, average speed, number of times entering the central area, movement distance, number of times standing, standing time, and rest time of mice in the two-factor combination treatment group and the control group during exploration in the open field. Error bars in the statistical graphs represent the standard deviation obtained from more than three independent experiments, analyzed using a two-tailed unpaired t-test.

[0035] Figure 13 This example illustrates how the combined treatment of bFGF and Laduviglusib in Example 13 reduced serum liver and kidney function indicators in aged mice. Specifically, it shows changes in serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), albumin (ALB), alkaline phosphatase (ALP), urea (UREA), uric acid (UA), and low-density lipoprotein (LDL) levels in 18-month-old mice four weeks after treatment with the combined factor combination (Cocktail FC). Error bars in the statistical graph represent standard deviations obtained from more than three independent experiments, and a two-tailed unpaired t-test was performed.

[0036] Figure 14 This illustrates the pathological staining characteristics of the cortex, hippocampus, heart, and islets of aged mice improved by the combined treatment of bFGF and Laduviglusib in Example 14. In Figure A, changes in Congo red, PAS, H&E, and Sirius red staining of the cerebral cortex, hippocampus, heart, and islets of 18-month-old mice four weeks after treatment with the two-factor combination (Cocktail FC) are shown (scale bar 500 μm). Figure B shows the statistical results of the proportion of positive regions; error bars in the statistical graph represent the standard deviation obtained from more than three independent experiments, and a two-tailed unpaired t-test was performed.

[0037] Figure 15 This diagram illustrates the improvement in pathological staining characteristics of the kidneys of aged mice by the combined treatment of bFGF and Laduviglusib in Example 14. In Figure A, changes in PAS, H&E, Sirius red, and Oil Red O staining of the kidneys of 18-month-old mice four weeks after treatment with the two-factor combination (Cocktail FC) are shown, with a scale bar of 500 μm. Figure B shows the statistical results of the proportion of positive regions. Error bars in the statistical graph represent the standard deviation obtained from more than three independent experiments, and a two-tailed unpaired t-test was performed.

[0038] Figure 16This illustrates the pathological staining characteristics of the spleen, lung, and liver in aged mice treated with a combination of bFGF and Laduviglusib in Example 14. A shows the changes in PAS, H&E, and Sirius red staining of the spleen, lung, and liver in 18-month-old mice 4 weeks after treatment with the two-factor combination (Cocktail FC), with a scale bar of 500 μm. B shows the statistical results of the proportion of positive areas; error bars in the statistical graph represent the standard deviation obtained from more than three independent experiments, and a two-tailed unpaired t-test was performed.

[0039] Figure 17 This illustrates the reversal of SASP levels in the blood of aged mice after treatment with the bFGF and Laduviglusib dual-factor combination in Example 15. In the figure, A represents the change in serum SASP levels in 18-month-old mice after 4 weeks of treatment with the dual-factor combination (Cocktail FC) as detected by a SASP chip, and B represents the statistical results. Error bars in the statistical graph represent the standard deviation obtained from three independent experiments, and a two-tailed unpaired t-test was performed. Detailed Implementation

[0040] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments. The embodiments given are only for illustrating the present invention and are not intended to limit the scope of the present invention.

[0041] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the materials and reagents used in the following examples are conventional biochemical reagents and are commercially available.

[0042] Example 1. The combination of bFGF and Laduviglusib showed significantly better anti-aging effects than single-drug treatments.

[0043] I. Methods

[0044] β-gal staining is the most commonly used method for identifying senescent cells, applicable to both in vitro cultured cells and tissue sections. This method utilizes the difference in optimal pH between senescent and non-senescent cells for β-galactosidase activity. In a buffer solution at pH 6, X-gal can be catalyzed by β-galactosidase in cells to produce a blue product, thus distinguishing senescent cells from non-senescent cells. In this study, the β-gal staining experiment was performed using a senescent histochemical staining kit (Sigma-Aldrich, CAS0030). Cells were first prepared at 2.4 × 10⁶ cells per well. 5 Seeds were placed in six-well cell culture plates and allowed to grow to 1×10⁶ cells per well. 6Density was used for staining experiments. First, adherent cells were washed twice with 1 mL PBS, then incubated at room temperature for 7 minutes with 1.5 mL of cell fixative. After cell fixation, the fixative was discarded, and the cells were rinsed three times with 1 mL PBS. Then, 1 mL of freshly prepared staining solution was added, and the cells were incubated overnight at 37°C. After staining, the cells were observed and photographed under a microscope, and the images were analyzed using ImageJ software. The percentage of senescent cells was calculated based on the β-gal signal to assess the degree of cell senescence. Ki67 is a marker related to proliferating cells, and its function is closely related to mitosis, making it essential for cell proliferation. The experiment was divided into a blank group (Ctrl, solvent treatment), a CHIR99021 treatment group (CHIR99021 single-factor treatment), a bFGF treatment group (bFGF single-factor treatment), and an FC treatment group (bFGF and CHIR99021 dual-factor combined treatment). The cell treatment concentration of bFGF was 100 ng / mL, the cell treatment concentration of CHIR99021 was 3 μM, and the concentration of the dual-factor combined treatment was the same as above. The cell treatment time was 6 days, and the medium was changed every two days to add single-factor or dual-factor combination.

[0045] II. Results

[0046] The results are as follows Figure 1 The proportion of Ki67-positive cells in senescent MEF cells (mouse embryonic fibroblasts) treated with the combination of bFGF and Laduviglusib was significantly higher than that in senescent MEF cells treated with a single factor. In addition, the proportion of β-gal-positive cells in senescent MEF cells treated with the combination of the two factors was significantly lower than that in senescent MEF cells treated with a single factor.

[0047] Example 2. β-gal staining of MEF cells reversing senescence using a combination of bFGF and Laduviglusib.

[0048] I. Method:

[0049] The method was the same as in Example 1, with single-factor or dual-factor combined treatment added to PD12 senescent MEF cells. The population doubling (PD or pd) number is an approximation of the number of times the cell population doubles. This is more meaningful for cell lines with finite lifespans. For example, when the cell division ratio in passages is 1:2, the passage number equals the PD number; when the cell division ratio is 1:4, the passage number equals 2 PDs, and so on. In cell culture, the PD number can reflect the degree of proliferation. Primary cells have a finite lifespan; after a certain degree of cell division, cells will age (stop dividing). The cell treatment concentration of bFGF was 100 ng / mL, the cell treatment concentration of CHIR99021 was 3 μM, and the dual-factor combined treatment concentration was the same as above. The cell treatment time was 6 days, with the medium changed every two days and single-factor or dual-factor combinations added again. The β-gal staining status of MEF cells was detected on days 0, 2, 4, and 6. Six days after treatment, the addition of the two factors was stopped, and the β-gal staining status of MEF cells was re-examined on days 0, 3, 6, and 9.

[0050] II. Results:

[0051] MEF cells that have undergone multiple passages have entered a state of cell arrest, and most cells (approximately 50%) are β-gal positive. After treating MEF cells that have entered a state of replicative senescence with a combination of two factors, the proportion of β-gal positive cells significantly decreased, and within 144 hours of treatment, it gradually decreased to a low level (<10%) similar to that of MEF cells that have not undergone senescence. Figure 2 As shown.

[0052] Example 3. The combination of bFGF and Laduviglusib upregulated the proportion of Ki67-positive cells and downregulated the expression of p16 and p21.

[0053] I. Method:

[0054] Ki67 is a marker associated with cell proliferation, and its function is closely related to mitosis, making it essential for cell proliferation. p16 and p21 are both negative regulators of the cell cycle; upregulation of their expression leads to cell cycle arrest in the G1 phase. Elevated levels of p16 and p21 are also widely considered a broad-spectrum characteristic of cellular senescence. Experimental groups included a blank group (Ctrl, solvent-treated) and an FC-treated group (bFGF and CHIR99021 dual-factor treatment), with treatment concentrations identical to those in Example 1. Immunofluorescence staining and proteoblotting were used to detect Ki67 and the negative regulators of cell proliferation (p16 and p21), respectively.

[0055] II. Results:

[0056] After 6 days of treatment with a combination of bFGF and Laduviglusib, the proportion of Ki67-positive cells in senescent MEF cells was significantly higher than before treatment (see...). Figure 3 (See A and B). Furthermore, after treatment with a combination of bFGF and Laduviglusib, p16 and p21 in senescent MEF cells were significantly downregulated at the protein level compared to PD12-treated senescent MEF cells (see [reference]). Figure 3 (C)

[0057] Example 4. Reversal of cell cycle arrest using a combination of bFGF and Laduviglusib

[0058] I. Method:

[0059] To demonstrate that the reversal of the classic senescent phenotype after combined treatment with bFGF and Laduviglusib was not due to immortalization of MEF cells, this study stopped the combined treatment 144 hours later and continued multiple passages (approximately every 3 days). The proportion of β-gal positive cells and proliferation rate were continuously monitored, with the treatment concentration and cell state remaining the same as in Example 1. Furthermore, to further demonstrate that the bFGF and Laduviglusib combination can affect the cell cycle of senescent cells, this study stained the DNA in the cells with PI and then used flow cytometry to detect the proportion of MEF cells at various stages of the cell cycle before and after combined treatment with bFGF and Laduviglusib.

[0060] II. Results:

[0061] The results showed that after stopping the dual-factor combination treatment and passaged three times consecutively (for a total of 9 days), MEFs gradually re-entered cell cycle arrest with repeated passages (see...). Figure 4 (See Figure A). This indicates that the process by which the dual-factor combination reverses the classic aging phenotype of senescent MEFs is reversible, and that the combined treatment of the dual-factor combination does not lead to the immortalization transformation of senescent MEFs. Before the combined treatment of bFGF and Laduviglusib, the proportion of cells in G1 phase in MEFs was 65.6%, while the proportions in G2 / M and S phases were 27.5% and 6.9%, respectively. After the combined treatment, the proportion of cells in G1 phase decreased to 56.3%, while the proportions in G2 / M and S phases increased to 32.4% and 11.3%, respectively (see Figure A). Figure 4 (B)

[0062] Example 5. The combination of bFGF and Laduviglusib reversed the upregulation of SASP expression.

[0063] I. Method:

[0064] SASP expression and secretion are also important characteristics of cellular senescence. Therefore, this study used RNA-seq and SASP protein microarray technology to analyze the expression differences of senescence-related secretory genes and the expression of SASP-related proteins in the microarray of young, aged, and dual-factor-treated MEF cells. The experimental groups were divided into a blank group (Ctrl, solvent-treated) and an FC-treated group (bFGF and CHIR99021 dual-factor treatment), with the same treatment concentrations and cell states as in Example 1.

[0065] II. Results:

[0066] After combined treatment with bFGF and Laduviglusib, the downregulated pathways in senescent MEF cells were closely related to immune and inflammatory factors. Many classic chemokines, such as Ccl, Cxcl, and insulin-like growth factor IgF, were significantly downregulated after combined treatment with the two factors (see [link to treatment]). Figure 5 These genes are classic members of the aging-associated secretory phenotype SASP. Furthermore, combined treatment with bFGF and Laduviglusib significantly reduced the secretion of SASP factors in senescent MEF cells, including angiotensin-Ang-2, chemokines CCL2, CCL11, CXCL1, CXCL16, CXCL5, insulin-like growth factor IGFBP-2, IGFBP-3, IGFBP-5, tumor necrosis factor TNFRSF11b, TSG14, and metalloproteinase MMP-3 (see [link to article]). Figure 5 (A, B)

[0067] Example 6. Reversal of telomere wear using a two-factor combination of bFGF and Laduviglusib.

[0068] I. Method:

[0069] Telomere wear is a key characteristic of cellular senescence and even individual aging. Telomeres gradually shorten as cells age, and when the length of telomeres shortens to a critical point, it leads to genomic instability and subsequently cell senescence or death. Telomere shortening in senescent cells is generally considered to be due to the gradual decrease in telomerase activity during cellular senescence. TRAP method: Culture an appropriate amount of cells and collect the cell pellet. Lyse the cells with cell lysis buffer and collect the cell extract. The extract can be used directly for telomerase activity detection after lysis. Add reaction components to a PCR tube and perform the reaction using a PCR instrument, depending on the sample and primers. Detect the telomerase reaction products using gel electrophoresis. Mix the PCR products with DNA loading dye and add it to the gel wells. Run electrophoresis at 100–120V for 60 minutes. Use DNA markers for molecular weight reference. Observe the PCR product bands using an ultraviolet imaging system. The key to telomerase activity detection is to see if there are products of a specific size. The band size is usually 6–10 TTAGGG repeat units. The relative intensity of telomerase activity was assessed by comparing the bands in the experimental and control groups using ImageJ analysis software. Telomerase activity could be quantitatively analyzed by calculating the band intensity. FISH assay: A suitable amount of mouse cells were cultured, ensuring they were in the logarithmic growth phase. Cells were fixed with 4% paraformaldehyde (PFA) at room temperature for 10–20 minutes. After fixation, the cells were washed twice with PBS for 5 minutes each time. Cells were permeabilized with 0.5% Triton X-100 (a solution in PBS) and incubated at room temperature for 5–10 minutes. Cells were washed twice with PBS for 5 minutes each time to remove the permeabilization solution. DNA probes labeled with fluorescent dye targeting telomere repeat sequences were prepared. The telomere probes were added to hybridization buffer at a ratio of 1:10. The mixed probe buffer was added dropwise to the cells, ensuring complete coverage. The slides were placed in a humidified chamber and incubated at 37°C for 16–18 hours (overnight hybridization). Remove the slides and wash them with 2×SSC buffer at 60°C for 30 minutes to remove unbound probes. Continue washing the slides with 2×SSC at room temperature 2–3 times, 5 minutes each time. Stain the cell or tissue slides with DAPI dye. The DAPI concentration is usually 1 μg / mL, and the staining time is 5–10 minutes. Wash with PBS to remove excess DAPI. Mount the slides with an anti-fluorescence quencher to prevent fluorescence signal attenuation. Observe using a fluorescence microscope. Telomere location can be identified by fluorescence signal, while cell nuclei are revealed by DAPI staining. Observe the cells under a microscope and record the fluorescence signal of telomeres. Analyze the length, number, and distribution of telomeres using image analysis software (ImageJ).In this experiment, the experimental groups were PD2 young MEF cells (solvent-treated), PD12 senescent MEF cells (solvent-treated), and the Cocktail FC treatment group (PD12 senescent MEF cells treated with bFGF and CHIR99021 dual factors). The concentration of the dual factor combined treatment was the same as in Example 1, and the cell treatment time was 6 days. The medium was changed every two days to re-add either a single factor or a combination of dual factors.

[0070] II. Results:

[0071] The results showed that combined treatment with bFGF and Laduviglusib not only significantly reversed the decrease in telomerase activity caused by senescence in MEF cells, but also that FISH staining of telomeres and TRAP quantitative detection of telomere length showed that the combined treatment reversed the shortening of telomere length in senescent MEF cells (see...). Figure 6 ).

[0072] Example 7. The combination of bFGF and Laduviglusib reduces DNA damage.

[0073] I. Method:

[0074] DNA damage is one of the main causes of cellular senescence. With increasing age, damage to nuclear DNA increases significantly and is associated with declining bodily functions. γH2AX is a DNA damage marker, and its dynamic changes during senescence reflect the level of cellular DNA damage. With increasing age, the level of γH2AX in cells gradually increases, indicating the accumulation of DNA damage during senescence. p53 is an important tumor suppressor gene that is activated when cells suffer DNA damage, thereby inducing cell cycle arrest, DNA repair, cellular senescence, or apoptosis. Furthermore, when the telomere length of cells shortens to a certain extent, it also leads to DNA double-strand breaks, thereby activating DNA damage signaling pathways, ultimately causing p53 activation and the cells entering a senescent state. In this experiment, the cells were divided into three groups: PD2 young MEF cells (solvent-treated), PD12 senescent MEF cells (solvent-treated), and a Cocktail FC treatment group (PD12 senescent MEF cells treated with bFGF and CHIR99021 dual-factor therapy). The concentration of the dual-factor combination treatment was the same as in Example 1, and the cell treatment time was 6 days, with the medium changed every two days to re-add either a single factor or a combination of factors. γH2AX was detected by immunofluorescence staining, and the expression levels of γH2AX and p53 were detected by Western blotting.

[0075] II. Results:

[0076] Experimental results showed that combined treatment of senescent MEF cells with bFGF and Laduviglusib significantly reversed the upregulation of γH2AX and p53 protein expression levels caused by cellular senescence (see [link to study]). Figure 7 (C). Furthermore, immunofluorescence results for γH2AX also showed that the combined treatment with the two factors significantly reversed the upregulation of the proportion of γH2AX-positive cells with senescence (see [link to article]). Figure 7 (A, B). The results showed that the combined treatment of the two factors had the ability to reduce DNA damage in senescent cells.

[0077] Example 8. The combination of bFGF and Laduviglusib has the ability to reverse aging phenotypes in multiple cell types.

[0078] I. Method:

[0079] Although different cell types share similar aging-related phenotypes, their intrinsic pathways of cellular senescence and the number of cell replications required for replicative senescence differ significantly. For example, most mouse cell types reach the Hayflick threshold and enter a state of cellular senescence with proliferative arrest after 10-14 replications, while human cells often require more than 50 replications to reach the Hayflick threshold and enter a state of cellular senescence with proliferative arrest. Therefore, to verify the aging effects and broad-spectrum efficacy of a novel anti-aging molecule, it is often necessary to conduct validation in different species and cell types. Therefore, this study further selected mouse lung fibroblasts, mouse mesenchymal stem cells, and human mesenchymal stem cells. In this experiment, the cells were divided into a blank group (Ctrl, solvent-treated) and an FC-treated group (bFGF and CHIR99021 dual-factor treatment), with treatment concentrations and cell states as in Example 1. Cell β-gal staining status was detected using the method described in Example 1. In addition, other cell types selected included mouse lung fibroblasts (PD12, MPF), mouse mesenchymal stem cells (PD12, mMSC), and human mesenchymal stem cells (PD50, hMSC). Cells were divided into a blank control group (Ctrl, solvent-treated) and an FC-treated group (bFGF and CHIR99021 dual-factor treatment). The treatment concentrations were 100 ng / mL for bFGF and 3 μM for CHIR99021, with a treatment time of 3 days. Cell density and cell proliferation were measured.

[0080] II. Results:

[0081] Mouse lung fibroblasts, mouse mesenchymal stem cells, and human mesenchymal stem cells were selected and cultured to PD12 and PD50, respectively, to induce typical cellular replicative senescence. The cellular senescence status was verified by β-gal staining, and the cell cycle arrest was verified by cell proliferation assay (see [link to study]). Figure 8 The results showed that combined treatment with the two factors significantly reduced the proportion of β-gal-positive cells in senescent mouse lung fibroblasts, mouse mesenchymal stem cells, and human mesenchymal stem cells (see [link to study]). Figure 8 This allows these three different types of cells to break through the Hayflick limit and escape the state of cell cycle arrest.

[0082] Example 9. Reversal of the DNA methylation clock in MEF by a combination of bFGF and Laduviglusib

[0083] I. Method:

[0084] During cellular senescence, in addition to significant changes in histone modifications, DNA methylation modifications also undergo a series of programmed changes at various modification sites. In particular, some methylation site modifications change gradually with the aging process, and conserved DNA methylation modification sites with consistent changes exist across different cell types and organ tissues during aging. Currently, various methods exist to assess the DNA methylation clock of an individual's physiological age by detecting the modification levels of specific DNA methylation sites. This study utilized whole-genome bisulfite sequencing (WGBS) technology to detect changes in DNA methylation in MEF (metacellular fluid fragments) after 6 days of treatment with a combination of young PD4, senescent PD12, and a dual-factor combination. Furthermore, a mature DNA methylation clock was used to further determine the ability of the dual-factor combination to reverse cellular senescence. The four methylation databases selected in this experiment were derived from public data: blood (Blood, Petkovich et al., 2017), liver (Liver, Wang et al., 2018), and multiple tissues (WLMT, Stubbs et al., 2017, YOMT, Margarita V Meer et al., 2017). In this experiment, the cells were divided into three groups: PD2 young MEF cells (solvent-treated), PD12 senescent MEF cells (solvent-treated), and Cocktail FC treatment group (PD12 senescent MEF cells treated with bFGF and CHIR99021 dual factors).

[0085] II. Results:

[0086] DNA methylation is often thought to silence gene expression, especially when CpG islands in gene promoter regions are hypermethylated. Methyl groups hinder transcription factor binding to DNA and recruit methyl-binding proteins and histone deacetylases, leading to chromatin compression into heterochromatin, ultimately suppressing gene expression. The DNA methylation scores of senescent MEF cells treated with the bFGF and Laduviglusib combination were significantly lower than those of untreated senescent MEF cells in four public databases, and were closer to those of young MEF cells (see...). Figure 9 This indicates that the combination of bFGF and Laduviglusib has a reversal effect on cellular senescence that is not limited to the cellular senescence phenotype, but rather reverses the cellular senescence fate by influencing changes in the epigenetics of senescent cells.

[0087] Example 10. The combination of bFGF and Laduviglusib improves motor function in aged mice.

[0088] I. Method:

[0089] Following cellular-level studies, this study further investigated the anti-aging effects of the bFGF and Laduviglusib dual-factor combination on mice. Eighteen-month-old male C57BL / 6 mice were selected and administered the drugs via intraperitoneal injection. The control group was treated with 0.9% saline, while the experimental group was treated with 2 mg / kg Laduviglusib and 0.1 mg / kg bFGF. Mice in the experimental group received the dual-factor combination injection every three days for a total of 10 injections over 30 days. The control group received the same frequency and dosage of saline as a control. With increasing age, the skeletal muscle mass of the mice showed a significant decline, exhibiting characteristics such as decreased motor function. Therefore, after treatment, this study first assessed their motor function using a mouse rotarod test and a mouse grip strength tester. The rotundus test for mice is a commonly used behavioral testing method to assess the exercise endurance and balance ability of mice. In the experiment, mice must maintain their balance on a rotating rod, adjusting their center of gravity to avoid falling off. The duration of time the mouse can maintain its position on the rotundus test is used to evaluate its exercise endurance and balance. The mouse grip strength test is a commonly used experimental tool for assessing the muscle strength and motor ability of mice, and can be used to observe the effects of a two-factor combination therapy on muscle function in mice.

[0090] II. Results:

[0091] The combined treatment with the two factors significantly enhanced the grip strength and retention time on the rotarod in aged mice. These results indicate that combined treatment with bFGF and Laduviglusib can improve muscle strength and endurance, as well as cerebellar balance in aged mice (see [link to study]). Figure 10 ).

[0092] Example 11. The combination of bFGF and Laduviglusib improves memory in aged mice.

[0093] I. Method:

[0094] As mice age, their memory declines, which is related to changes in brain structure and function. Extensive research has reported that appropriate anti-aging interventions, such as exercise training, dietary adjustments, or transfusion of young blood, can improve cognitive function in aged mice. To investigate the effects of combined treatment with bFGF and Laduviglusib on cognitive function in aging mice, this study first used the Y-maze test to examine the improvement in short-term memory after treatment. The Y-maze test is a simple and effective method for assessing mouse learning and memory; by observing the mice's exploratory behavior in the Y-maze, it can reflect their spatial cognition, working memory, and other cognitive functions. In this experiment, the mice were treated as in Example 10, and the total number of entries and the number of maximum alternations were recorded.

[0095] II. Results:

[0096] Mice treated with the dual-factor combination bFGF and Laduviglusib showed significantly higher total number of entries and maximum number of alternations in the Y maze compared to the control group (see [link to data]). Figure 11 The experimental results showed that aged mice treated with the dual-factor combination of bFGF and Laduviglusib exhibited stronger exploratory drive and enhanced short-term memory.

[0097] Example 12. The combination of bFGF and Laduviglusib improves anxiety and exploratory behaviors in aged mice.

[0098] I. Method:

[0099] This study also utilized the mouse open field test to examine comprehensive behavioral indicators in mice after treatment. The mouse open field test is a commonly used experimental method in neurobehavioral research to assess exploratory, excitatory, and motor behaviors in mice, and can be used to evaluate the influence of various factors on mouse behavior. In this experiment, the mice were treated in the same manner as in Example 10. The ambulatory distance, ambulatory time, average speed, entries in the center, vertical counts, and resting time of the mice in the open field were recorded.

[0100] II. Results:

[0101] Aged mice treated with a two-factor combination therapy exhibited superior ambulatory distance, ambulatory time, average speed, entries in center, and vertical counts in open field activity, as well as shorter resting time (see [link to treatment]). Figure 12 The results showed that mice treated with the two-factor combination exhibited higher motor skills and a greater desire to explore.

[0102] Example 13. The combination of bFGF and Laduviglusib improves liver and kidney function in aged mice.

[0103] I. Method:

[0104] To further verify the intervention effect of the bFGF and Laduviglusib dual-factor combination on individual aging in mice, this study further examined several serum biochemical indicators in mice. Alanine aminotransferase (ALT), aspartate aminotransferase (AST), albumin (ALB), and alkaline phosphatase (ALP) are indicators of liver function, which significantly increased when liver function was impaired. Urea (UREA) and uric acid (UA) are indicators of kidney function, which significantly increased when kidney function was impaired. Elevated low-density lipoprotein (LDL) is often considered a risk factor for atherosclerosis. In this experiment, the mice were treated in the same manner as in Example 10, and the corresponding serum indicators were measured, including ALT, AST, albumin (ALB), alkaline phosphatase (ALP), urea (UREA), uric acid (UA), and serum low-density lipoprotein (LDL) levels.

[0105] II. Results:

[0106] In aged mice treated with a combination of bFGF and Laduviglusib, serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), albumin (ALB), and alkaline phosphatase (ALP) were significantly reduced. Furthermore, the combination treatment also significantly reduced serum levels of urea (UREA) and uric acid (UA) in aged mice. The treated mice also exhibited lower serum low-density lipoprotein (LDL) levels (see [link to article]). Figure 13 ).

[0107] Example 14. The combination of bFGF and Laduviglusib improves the pathological features of multi-organ aging in aged mice.

[0108] I. Method:

[0109] This study further analyzed the heart, liver, spleen, kidney, lung, cerebral cortex, hippocampus, and pancreatic islets of mice treated with a combination of bFGF and Laduviglusib, as well as aged control mice, using various staining and pathological methods. The mouse treatment methods in this experiment were the same as in Example 10. HE staining: The sections were immersed in xylene for 5 minutes, repeated twice to remove paraffin. The sections were then immersed sequentially in 100%, 95%, 80%, and 70% ethanol, 5 minutes in each ethanol solution, and finally transferred to distilled water. The sections were stained with hematoxylin for 10 minutes, depending on the tissue type. The sections were washed with running water to remove excess hematoxylin until the surface of the sections turned pale blue. The sections were stained with eosin for 3 minutes. The sections were washed with running water until the surface of the sections turned pink. The sections were rapidly incubated in 0.1% hydrochloric acid-alcohol solution for 5 seconds to observe the tissue contrast. Then, the sections were rinsed with running water. The sections were then cleared sequentially in xylene for 5 minutes, repeated twice. Add neutral resin to a glass slide and cover with a coverslip. Observe the structure of the tissue sections under a microscope and analyze the morphological changes after H&E staining. PAS staining: a commonly used histochemical staining method, can be used to detect carbohydrates, such as glycoproteins and glycolipids, and can be used to diagnose various diseases. After dewaxing and rehydrating the tissue sections, place them in a 0.5% periodic acid solution (0.5g periodic acid dissolved in 100mL distilled water) and let them stand at room temperature for 10 minutes. Rinse with distilled water to remove the periodic acid. Immerse the sections in the prepared Schiff reagent for 15 minutes. Schiff reagent contains a complex formed by the reaction of sulfite and peroxide, which can significantly bind to carbohydrate components. Wash the sections thoroughly with distilled water to remove excess Schiff reagent. Clear the sections sequentially in xylene for 5 minutes, repeating twice. Add neutral resin and gently cover with a coverslip, avoiding air bubbles. Observe the tissue sections under a microscope. After PAS staining, glycogen and other sugar-containing components appear purplish-red or red, while non-carbohydrate components appear pale purple or colorless. Sirius red staining: A simple and effective method for detecting tissue fibrosis, playing an important role in the diagnosis and treatment assessment of various fibrosis-related diseases. Organ fibrosis is usually caused by a decline in tissue regeneration capacity. After dewaxing and rehydration, tissue sections are immersed in Sirius red staining solution for 30 minutes, the specific time depending on the tissue type and the cells or structures to be stained. The sections are thoroughly rinsed with PBS buffer or distilled water to remove excess dye. The stained sections are then cleared in xylene twice, 5 minutes each time. Neutral resin is added, and a coverslip is gently placed on top. Under a microscope, the stained tissue structures will appear red or purplish-red, while the background will appear light blue. Congo red staining: An important indicator for diagnosing amyloidosis and also an indicator of Alzheimer's disease, a classic age-related disease.After dewaxing and rehydration, tissue sections are immersed in Congo Red staining solution for 30 minutes to 1 hour, with the specific time optimized according to tissue type and the amount of amyloid deposits. The stained sections are then treated with 0.02M hydrochloric acid for 3 minutes to remove the background color. The sections are rinsed with distilled water to completely remove the hydrochloric acid. The sections are then immersed in 10% ammonia solution for 3 minutes to neutralize the acidic environment. The sections are rinsed with distilled water to remove the ammonia. The sections are then cleared by immersing them in xylene twice, 5 minutes each time. Neutral resin is added, and a coverslip is gently placed on top. The sections are then observed under a microscope. Under normal conditions, amyloid deposits appear red or bright red and exhibit characteristic green birefringence (obvious birefringence can be observed using a polarizing microscope). Oil Red staining: A commonly used method for detecting fat deposits in tissues. Reports indicate that fat deposits in the kidneys increase with age, which may be related to abnormal fat metabolism during kidney aging. After dewaxing and rehydration, tissue sections are immersed in Oil Red O staining solution for 15 minutes. During staining, Oil Red O binds to the fatty portions of the tissue, turning the adipose tissue red. Gently wash the sections with 70% isopropanol to remove excess dye until it no longer sheds. Rinse the sections with distilled water to thoroughly remove any excess dye. To enhance the contrast of the adipose tissue, aquamarine can be used for background staining. Immerse the sections in aquamarine for 3 minutes, then rinse thoroughly under running water. Clear the stained sections twice in xylene, 5 minutes each time. Add neutral resin, gently cover with a coverslip, and observe the staining results under a microscope. The adipose tissue will appear red or bright red, especially the intracellular fat droplets or fat deposits. The background will appear pale blue.

[0110] II. Results:

[0111] HE staining results showed that after treatment with the dual factor combination of bFGF and Laduviglusib, mice exhibited more intact alveolar structure, more intact heart and liver tissue structure, smaller pancreatic islet cell clusters, and significant improvement in glomerular atrophy (see...). Figures 14-16 PAS staining results showed that aged mice treated with a two-factor combination therapy exhibited a significant reduction in positive staining areas in the lungs, cerebral cortex, liver, hippocampus, and kidneys (see [link to PAS staining results]). Figures 14-16 Sirius red staining results showed that the dual-factor combination therapy significantly reduced fibrosis levels in the lungs, heart, liver, pancreas, kidneys, and spleen. Congo red staining showed that the dual-factor combination therapy reduced the accumulation of amyloid protein deposits in the cerebral cortex and hippocampus of aged mice (see...). Figure 14 Oil Red staining results showed that the dual-factor combination treatment improved fat accumulation in the kidneys of aged mice (see...). Figure 15 ).

[0112] Example 15. The combination of bFGF and Laduviglusib reduced SASP levels in the blood of aged mice.

[0113] I. Method:

[0114] During aging, the level of SASP factors in the blood is significantly upregulated. These SASP factors can serve as biomarkers of aging to assess an individual's physiological age and health risks. Changes in blood SASP factors may affect vascular function, leading to reduced blood flow and increased blood coagulability. These changes accelerate the aging process of tissues such as skin. This study utilized SASP protein chip technology for aging-related secretory phenotypes to detect the level of SASP-related proteins in the serum of aged mice after treatment with a combination of bFGF and Laduviglusib. The mouse treatment in this experiment was the same as in Example 10.

[0115] II. Results:

[0116] The results showed that the combined treatment with the two factors significantly reduced the secretion of angiotensin-Ang-1 and Ang-2, chemokines CCL17, CCL11, CCL22, CXCL5, CXCL16, and CXCL13, insulin-like growth factor IGFBP-6, IGFBP-3, and IGFBP-5, interleukin IL12, and metalloproteinases MMP-3 and MMP-9 in the blood of aged mice (see [link to article]). Figure 17 The combined treatment with the two factors significantly reduced the level of SASP in the blood of aged mice, which is consistent with the results that the two factors can reduce the expression and secretion of SASP in senescent MEF cells.

[0117] The present invention has been described in detail above through embodiments. For those skilled in the art, the present invention can be implemented or further modified under equivalent parameters, concentrations, and conditions within a wide range without departing from its spirit and scope. In summary, any modifications, applications, or improvements to the present invention made using conventional techniques in the art, based on the principles of the present invention, fall within the scope of protection of the present invention.

Claims

1. An anti-aging composition comprising recombinant protein bFGF and aminopyrimidine derivative Laduviglusib, wherein, The structural formula of Laduviglusib is shown below:

2. The anti-aging composition according to claim 1, characterized in that, The amino acid sequence of the recombinant protein bFGF is shown in SEQ ID NO: 1 in the sequence listing.

3. The anti-aging composition according to claim 1, characterized in that, The mass ratio of bFGF to Laduviglusib is 1:20 to 10:

1.

4. The anti-aging composition according to claim 3, characterized in that, The mass ratio of bFGF to Laduviglusib is 1:

14.

5. The anti-aging composition according to claim 1, characterized in that, The composition comprises one or more pharmaceutically and cosmetically acceptable excipients.

6. The use of the anti-aging composition according to any one of claims 1 to 5 in the preparation of a medicament, health product or cosmetic for delaying aging, reversing aging phenotypes and / or treating aging-related diseases.

7. The application as described in claim 6, characterized in that, The drug or health product is in oral or injectable form.

8. The application as described in claim 6, characterized in that, The cosmetics in question are skincare products.

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