Use of DNJ or derivative thereof in preparing Anti-senescence drug

Abstract

Use of DNJ or a derivative thereof in preparing an anti-senescence drug. The DNJ or the derivative thereof has a significant anti-senescence effect in vitro and in vivo. The in-vitro anti-senescence effect relates to various tissue cells, such as endothelial cells, cardiomyocytes, and mesenchymal stem cells. Various cell models such as continuous passage replicative senescence or stress senescence induced by H2O2, D-galactose, and the like, and various administration modes such as pre-administration, simultaneous administration, or administration after modeling all show that the DNJ and the derivative thereof have good anti-senescence activity. In-vivo experiments show that DNJ can significantly delay the senescence of mice.

Description

Application of DNJ or its derivatives in the preparation of anti-aging drugs Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to the application of DNJ or its derivatives in the preparation of anti-aging drugs. Background Art

[0002] Aging is a process of degenerative changes in bodily functions. As we age, the functions of our cells, tissues, and organs gradually decline, significantly increasing the risk of various age-related diseases, including cardiovascular disease, neurodegenerative disease, metabolic disease, and immune system disorders.

[0003] Cellular senescence is a driving factor in aging. With the exception of germline stem cells, normal cells can only undergo a limited number of divisions, after which they enter a state of proliferation stagnation, also known as cellular senescence. Cellular senescence is divided into replicative senescence and stress-induced senescence. Replicative senescence refers to the phenomenon that occurs after cells have undergone a limited number of divisions and the gradual shortening of chromosome telomeres, resulting in the loss of cell differentiation ability and proliferation stagnation. Stress-induced senescence refers to the phenomenon that occurs when cells respond to pathological stimuli (such as DNA damage and oxidative stress). The balance of the autophagy-lysosome system in senescent cells is disrupted, leading to an increase in lysosome content and an increase in the expression of lysosomal senescence-associated β-galactosidase (SA-β-Gal). Therefore, the expression of SA-β-Gal is considered one of the typical signs of cellular senescence (López-Otín C, et al. Hallmarks of aging: An expanding universe. Cell. 2023; 19; 186(2): 243-278). In addition, although senescent cells are growth-stalled, they still have metabolic activity and secrete numerous bioactive factors, including inflammatory cytokines, chemokines, growth factors, matrix metalloproteinases, etc., collectively referred to as the senescence-associated secretory phenotype (SASP), which is another important feature of senescent cells. Existing studies have shown that SASP includes dozens or even hundreds of bioactive factors. There are some differences in the composition and content of SASP in different tissues and aging models, but in all types of in vitro generated senescent cells, the core program of SASP mainly includes pro-inflammatory interleukin-6 (IL-6), CXC chemokine ligand 8 (CXCL8, also known as IL-8) and monocyte chemoattractant protein 1 (MCP1, also known as CCL2) (Di Micco R, et al., Cellular senescence in ageing: from mechanisms to therapeutic opportunities. Nat Rev Mol Cell Biol. 2021, 22(2): 75-95).

[0004] Cellular senescence involves multiple mechanisms, including telomere dysfunction, DNA damage, oncogene activation, mitochondrial dysfunction, and chromatin modifications. Mitochondrial dysfunction is closely associated with the induction and maintenance of cellular senescence phenotypes. Mitochondria are not only the powerhouses of the cell but also crucial sites for regulating apoptosis, calcium storage, and signaling. Normal mitochondrial function is essential for maintaining health, and their gradual degeneration leads to the aging phenotype. Mitochondrial dysfunction in senescent cells is often characterized by various mitochondrial dysfunctions, including bioenergetics imbalance, decreased ATP production, reduced mitochondrial membrane potential, disrupted mitochondrial dynamics, and impaired autophagy. As highly dynamic organelles, the balance between mitochondrial fusion and fission is crucial for maintaining mitochondrial function and quality control. A prominent feature of cellular senescence is the dramatic changes in mitochondrial dynamics and organization, manifested by mitochondrial elongation and enlargement, and damage to mitochondrial cristae. The loss of cristae in senescent cells impairs the supply of ATP necessary for essential cellular function, while also reducing the activity of respiratory complexes, increasing electron leakage, and increasing ROS generation.

[0005] Given the key role of senescent cells in a variety of related diseases, a lot of investment has been made in the development of drugs targeting senescent cells in recent years. Improving the mitochondrial dysfunction of senescent cells has been considered as a way to develop anti-aging drugs, and some related clinical trials are underway. For example, β-nicotinamide mononucleotide (NMN) is a naturally occurring biologically active nucleotide that is a coenzyme I (NAD + ) is a key intermediate in the synthesis of β-actin and is considered a supplement for preventing aging; ABT-263 (navitoclax) and others exert their anti-aging effects by targeting the anti-apoptotic B-cell lymphoma 2 (BCL-2) family proteins located in mitochondria; Elamipretide has a positive effect on various aging-related phenotypes in mice by targeting cardiolipin in the inner mitochondrial membrane; some other plant-derived polyphenols, triterpenes and sterol compounds such as quercetin and rosmarinic acid have also been found to have anti-aging activity.

[0006] Optic atrophy protein-1 (OPA1) is a type of motor protein localized to the inner mitochondrial membrane. It plays an essential role in mitochondrial cristae remodeling and regulates mitochondrial inner membrane fusion, thereby increasing oxidative phosphorylation. Current research indicates that OPA1 is a key regulator of skeletal muscle and cardiac pathology and physiology. 1-Deoxynojirimycin (DNJ) is a polyhydroxy alkaloid abundant in mulberry trees that exhibits multiple activities, including antidiabetic, antioxidant, anti-inflammatory, and anti-obesity. Our previous work showed that DNJ promotes oligomerization by targeting OPA1, thereby rebuilding mitochondrial cristae and rescuing mitochondrial function, and is a potential drug for the treatment of mitochondrial hypertrophic cardiomyopathy (Zhuang Q, et al. 1-Deoxynojirimycin promotes cardiac function and rescues mitochondrial cristae in mitochondrial hypertrophic cardiomyopathy. J Clin Invest. 2023; 17; 133(14): e164660.) (Patent No.: 202111036977.5). To improve the bioavailability of DNJ and expand its applications, we have also optimized its structure and synthesized a series of DNJ derivatives. Some of these compounds, compared to the lead compound DNJ, can better bind to the target protein OPA1, significantly rescue mitochondrial function, effectively improve cellular physiology, and exhibit lower cytotoxicity. They are intended for the treatment of diseases associated with imbalanced OPA1 multimer formation, including hypertrophic cardiomyopathy, deafness, optic atrophy, progressive external ophthalmoplegia and ataxia, progressive myoclonic epilepsy, spastic paresis, intestinal dysmotility, and retinal degeneration. (Patent No.: 202211631240.2) (International Application No.: PCT / CN2023 / 127606). Summary of the Invention

[0007] The present invention further provides the use of DNJ or its derivatives (DNJ-1, DNJ-3a, DNJ-3b and DNJ-5a) in the preparation of anti-aging drugs.

[0008] Use of a 1-deoxynojirimycin compound in the preparation of an anti-aging drug, wherein the 1-deoxynojirimycin compound is at least one of the following:

[0009] (1) 1-deoxynojirimycin or a pharmaceutically acceptable salt thereof; (2) a 1-deoxynojirimycin derivative or a pharmaceutically acceptable salt thereof.

[0010] Preferably, the 1-deoxynojirimycin derivative is any one of the following:

[0011] (1) DNJ-1; the structure is shown in Formula II:

[0012] (2) The structure is shown in the general formula:

[0013] wherein R is selected from: -H, -NO2, -X, wherein X is F, Cl, Br or I;

[0014] (3) DNJ-5a; the structure is shown in Formula V:

[0015] More preferably, when R is -H, the 1-deoxynojirimycin derivative is DNJ-3a, the structure of which is shown in Formula III:

[0016] More preferably, when R is -X, X is F, and the 1-deoxynojirimycin derivative is DNJ-3b; the structure is shown in Formula IV:

[0017] (i) When the 1-deoxynojirimycin derivative is (1), compound a and b1 are subjected to a condensation reaction in a solvent to obtain the corresponding 1-deoxynojirimycin derivative, wherein compound a is 1-deoxynojirimycin and compound b1 is:

[0018] (ii) When the 1-deoxynojirimycin derivative is (4), compound a and compound b4 are subjected to a condensation reaction in a solvent to obtain the corresponding 1-deoxynojirimycin derivative, wherein compound a is 1-deoxynojirimycin and compound b4 is:

[0019] (iii) When the 1-deoxynojirimycin derivative is (2), 1-deoxynojirimycin is first reacted with benzyl chloroformate to form intermediate 1:

[0020] Intermediate 1 is then reacted with PivCl to give intermediate 2:

[0021] Finally, the intermediate product 2 is reduced to obtain the 1-deoxynojirimycin derivative.

[0022] Preferably, the anti-aging effect of the anti-aging drug is prevention before aging occurs, alleviation during aging, or repair after aging occurs.

[0023] Preferably, the anti-aging drug further comprises a pharmaceutically acceptable excipient.

[0024] Preferably, the dosage form of the anti-aging drug is at least one of a suspension, granules, capsules, powders, tablets, emulsions, pills, injections, suppositories, enemas, aerosols, patches or drops.

[0025] The present study found that DNJ or its derivatives have anti-aging effects: cells from various tissue sources, such as human umbilical vein endothelial cell line (Huvec cell line), rat cardiomyocyte cell line (H9c2 cell line) and human iPSC-derived mesenchymal stem cells (iPSC-MSC), were treated with H2O2 and / or galactose (D-gal) to construct a stress-induced cell senescence model and a replicative cell senescence model was constructed by continuous cell passage. Three different administration methods, including pre-administration, simultaneous administration and post-modeling, were adopted. NMN treatment was used as the positive control group, and the expression of senescence-associated β-galactosidase (SA-β-Gal) and senescence-associated secretory phenotype (SASP) in the cells was detected to evaluate the anti-aging activity of DNJ or its derivatives in vitro; the doxorubicin (Dox)-induced aging mouse model was used to evaluate the anti-aging effect of DNJ in vivo. Results showed that in vitro, whether administered pre-administered, concurrently administered, or administered post-modeling, DNJ or its derivatives exhibited robust anti-aging activity against a variety of tissue cells, including endothelial cells, cardiomyocytes, and mesenchymal stem cells, and across different aging induction pathways. In vivo, DNJ significantly delayed aging in mice. This invention represents a novel application of DNJ or its derivatives. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 is the reaction equation of structural formula II.

[0027] Figure 2 is a mass spectrometry analysis of DNJ-1.

[0028] Figure 3 is the reaction equation of structural formula III.

[0029] FIG4 is the NMR resonance spectrum of DNJ-3a.

[0030] FIG5 is a reaction equation of structural formula IV.

[0031] FIG6 is the NMR resonance spectrum of DNJ-3b.

[0032] FIG7 is a reaction equation of structural formula VII.

[0033] FIG8 is the NMR resonance spectrum of DNJ-5a.

[0034] Figure 9 shows the protective effect of pretreatment with DNJ or its derivatives against H2O2-induced senescence in human umbilical vein endothelial cells. A: SA-β-Gal activity staining of H2O2 (Sigma)-induced senescence in Huvec cells 24 hours after pretreatment with DNJ or its derivatives (DNJ-1, DNJ-3a, DNJ-3b, and DNJ-5a) or a positive drug (NMN); B: The percentage of SA-β-Gal activity-positive cells in Huvec cells after pretreatment. n = 3; ***P < 0.001.

[0035] Figure 10 shows the anti-senescence effects of simultaneous treatment with DNJ or its derivatives and H2O2 on human umbilical vein endothelial cells. A: Representative SA-β-Gal activity staining of Huvec cells 24 hours after simultaneous treatment with DNJ or its derivatives (DNJ-1, DNJ-3a, DNJ-3b, and DNJ-5a) and H2O2; B: The percentage of SA-β-Gal-positive cells in Huvec cells after simultaneous treatment. n = 3; ***P < 0.001.

[0036] Figure 11 shows the protective effect of DNJ or its derivatives on H2O2-induced senescence in human umbilical vein endothelial cells. A: Representative SA-β-Gal activity staining of Huvec cells treated with DNJ or its derivatives (DNJ-1, DNJ-3a, DNJ-3b, and DNJ-5a) 24 hours after H2O2 induction; B: The percentage of SA-β-Gal-positive cells in Huvec cells after simultaneous drug treatment. n = 3, ***P < 0.001.

[0037] Figure 12 shows the protective effect of pretreatment with DNJ or its derivatives against D-galactose-induced senescence in human umbilical vein endothelial cells. A: Representative SA-β-Gal activity staining of D-galactose (Sigma)-induced Huvec cell senescence 24 hours after pretreatment with DNJ or its derivatives (DNJ-1, DNJ-3a, DNJ-3b, and DNJ-5a); B: The percentage of SA-β-Gal-positive Huvec cells after pretreatment. n = 3, ***P < 0.001.

[0038] Figure 13 shows the protective effect of DNJ against serial passage-induced senescence in human umbilical vein endothelial cells. A: Representative SA-β-Gal activity staining of serial passage-induced senescence Huvec cells 24 hours after DNJ treatment; B: The percentage of SA-β-Gal-positive cells in Huvec cells after DNJ treatment. n = 12.

[0039] Figure 14 shows the protective effect of pretreatment with DNJ or its derivatives against H2O2-induced senescence in rat cardiomyocytes (H9c2 cells). A: Representative SA-β-Gal activity staining of H9c2 cells induced by H2O2 24 hours after pretreatment with DNJ or its derivatives (DNJ-1, DNJ-3a, DNJ-3b, and DNJ-5a); B: The percentage of SA-β-Gal-positive cells in H9c2 cells after pretreatment. n = 3, ***P < 0.001.

[0040] Figure 15 shows the protective effect of pre-treatment with DNJ or its derivatives on H2O2-induced senescence of human iPSC-differentiated mesenchymal stem cells (iPSC-MSCs). A: Representative SA-β-Gal activity staining of H2O2-induced iPSC-MSC senescence 24 hours after pre-treatment with DNJ or its derivatives (DNJ-1, DNJ-3a, DNJ-3b, and DNJ-5a); B: The percentage of SA-β-Gal-positive cells in iPSC-MSCs after pre-treatment. n = 3, ***P < 0.001.

[0041] Figure 16 shows the improvement of DNJ pretreatment on the senescent inflammatory phenotype of human umbilical vein endothelial cells induced by H2O2. n=4.

[0042] Figure 17 shows the improvement effect of DNJ treatment on the senescent inflammatory phenotype of human umbilical vein endothelial cells induced by serial passage. n=4.

[0043] Figure 18 shows that DNJ treatment significantly delayed aging in mice. Control group n=6, aging group n=6, and drug-treated group n=6. DETAILED DESCRIPTION

[0044] To investigate the anti-aging activity of DNJ or its derivatives, three different cell senescence models (H2O2 stimulation, D-gal stimulation, and continuous cell passage) and three different administration methods (pre-administration, simultaneous administration, and post-modeling) were selected. DNJ or its derivatives were tested in cells derived from various tissues (human umbilical vein endothelial cells, rat cardiomyocytes, and hiPSC-derived mesenchymal stem cells) to determine whether they could reduce the number of SA-β-Gal-positive cells and inhibit the expression of inflammatory factors associated with the SASP phenotype. C. elegans were fed DNJ continuously for 30 days to determine whether DNJ could prolong their survival.

[0045] The structural formulas of DNJ or its derivatives DNJ-1, DNJ-3a, DNJ-3b, and DNJ-5a are shown in Formulas I to V, respectively.

[0046] NMN, used as a positive control, is β-nicotinamide mononucleotide.

[0047] Each DNJ derivative was prepared as follows:

[0048] (1) Compound name: DNJ-1; Molecular formula: C 14 H 25 NO5S2, structural name: 5-((R)-1,2-dithiolan-3-yl)-1-((2R,3R,4R,5S)-3,4,5-trihydroxy-2-(hydroxymethyl)piperidin-1-yl)pentan-1-one; the structure is shown in Formula II:

[0049] The reaction equation is shown in Figure 1.

[0050] Compounds a (193.97 mg, 1.19 mmol) and b (245 mg, 1.19 mmol) were placed in a round-bottom flask and added with DCM (10 ml). The temperature was lowered to 0°C, and HOBT (0.177 g, 1.31 mmol) and EDCl (0.251 g, 1.31 mmol) were added. The mixture was allowed to react at room temperature for 10 hours. After completion of the reaction, the solvent was evaporated, and the product was separated by column chromatography to obtain the product.

[0051] Figure 2 is a mass spectrometry analysis of DNJ-1.

[0052] (2) Compound name: DNJ-3a; Molecular formula: C 14 H 21 NO4, structural name: (2R,3R,4R,5S)-2-(hydroxymethyl)-1-phenethylpiperidine-3,4,5-triol ((2R,3R,4R,5S)-2-(hydroxymethyl)-1-phenethylpiperidine-3,4,5-triol); the structure is shown in Formula III:

[0053] The reaction equation is shown in Figure 3.

[0054] Compound a (163 mg, 1 mmol), compound b (220.79 mg, 1.2 mmol), and potassium carbonate (414 mg, 3 mmol) were placed in a sealed tube. DMF (5 mL) was added as solvent. The mixture was heated at 80°C for 4 h. Completion of the reaction was monitored by TLC. The solvent was evaporated and column chromatography was performed to obtain approximately 80 mg of the product.

[0055] FIG4 is the NMR resonance spectrum of DNJ-3a.

[0056] (3) Compound name: DNJ-3b; Molecular formula: C 14 H 20 FNO4, structural name: (2R,3R,4R,5S)-1-(4-fluorophenethyl)-2-(hydroxymethyl)piperidine-3,4,5-triol; the structure is shown in Formula IV:

[0057] The reaction equation is shown in Figure 5.

[0058] Compound a (163 mg, 1 mmol), compound b (242.38 mg, 1.2 mmol) and potassium carbonate (414 mg, 3 mmol) were measured and placed in a sealed tube. DMF was used as solvent and the reaction was heated at 80°C for 4 h. The reaction was monitored on a plate until completion. The solvent was evaporated and column chromatography was performed to obtain about 70 mg of the product.

[0059] FIG6 is the NMR resonance spectrum of DNJ-3b.

[0060] (4) Compound name: DNJ-5a; Molecular formula: C 11 H 21 NO5, structural name: ((2R,3R,4R,5S)-3,4,5-trihydroxypiperidin-2-yl)methylpivalate; the structure is shown in Formula VII:

[0061] The reaction equation is shown in Figure 7.

[0062] Compound a (115 mg, 0.7 mmol) was dissolved in a mixture of dioxane / water (1:1, 12 mL). Sodium chloride (1.75 equiv) and benzyl chloroformate (1.54 equiv) were then added and stirred at room temperature for 18 hours. The dioxane was then removed by rotary evaporation, and the mixture was extracted with dichloromethane. The organic layer was retained. Column chromatography afforded product b. Compound b (149 mg, 0.5 mmol) was dissolved in pyridine (3 mL), and PivCl (2 equiv) was slowly added at 0°C. After 1 hour, further PivCl (2 equiv) was added, and after 3 hours, the reaction mixture was diluted with ethyl acetate and methanol. The solvent was removed under reduced pressure, and column chromatography afforded compound c.

[0063] Compound c (150 mg, 0.39 mmol) was dissolved in MeOH (5 mL), and then 10% Pd / C (30 mg) was added. The mixture was stirred at room temperature and protected with H2 overnight. Compound d was obtained by column chromatography.

[0064] FIG8 is the NMR resonance spectrum of DNJ-5a.

[0065] Example 1

[0066] The culture conditions of human umbilical vein endothelial cells (Huvec cells) are as follows:

[0067] Huvec cells were cultured in ECM medium (SclenCell) and passaged and seeded when the cells reached a density of 70%-80% in a culture dish. After washing once with PBS, the cells were digested with 0.25% trypsin (Gibco) for 2-3 minutes. ECM medium was then added to terminate digestion. The cells were then blown off and collected in a 15ml centrifuge tube. The tube was centrifuged at 1000 rpm for 5 minutes, the supernatant discarded, and fresh ECM medium was added and evenly distributed. The tubes were then seeded in 12- or 24-well plates (at a density of 70%-80%) and cultured in a 37°C, 5% CO2 incubator. Once the cells adhered, the next step could be performed.

[0068] The experimental operation and grouping are as follows:

[0069] Experimental groups: control group (Control), model group (H2O2), DNJ drug group (H2O2+DNJ, H2O2+DNJ-1, H2O2+DNJ-3a, H2O2+DNJ-3b, H2O2+DNJ-5a), positive drug group (H2O2+NMN).

[0070] The working solution concentrations of each drug and modeling agent: DNJ, DNJ-1, DNJ-3a, DNJ-3b, DNJ-5a, and NMN are all 30 μM; modeling agent: H2O2, 200 μM.

[0071] Experimental operation: After discarding the supernatant, the cells were washed three times with PBS and then added with the drug solution pre-prepared in the culture medium. The blank group and the model group were replaced with blank culture medium without drugs. After culturing for 24 hours, the supernatant was discarded and the cells were washed three times with PBS. The blank group was added with blank culture medium, and the model group and the drug-treated group were added with culture medium containing 200μM H2O2. After treatment for 24 hours, the cell senescence β-galactosidase staining kit (Beyotime, C0602) was used for cell SA-β-Gal staining.

[0072] The workflow for cell SA-β-Gal staining is as follows:

[0073] For cells cultured in 6-well plates, aspirate the cell culture medium, wash once with PBS, add 1 mL of SA-β-Gal staining fixative, and fix at room temperature for 15 minutes. For other culture plate types, refer to the following ratios for the amounts of fixative and subsequent solutions. Aspirate the cell fixative and wash the cells three times with PBS for 3 minutes each. Aspirate the PBS and add 1 mL of working staining solution to each well. Prepare the working staining solution as follows: β-Galactosidase Staining Solution A (10 μL) + β-Galactosidase Staining Solution B (10 μL) + β-Galactosidase Staining Solution C (930 μL) + X-Gal solution (50 μL). Incubate overnight at 37°C. Cover the 6-well plate with parafilm or plastic wrap to prevent evaporation. The cells were counted using ImageJ software (National Institutes of Health, v 1.8.0), and the ANOVA data were analyzed among multiple groups (mean ± SD) using GraphPad Prism software (GraphPad Software, v 9.0.0).

[0074] Compared with normal cells, senescent cells have the characteristic of high expression of SA-β-Gal. The Cell Senescence β-Galactosidase Staining Kit uses X-Gal as a substrate. Under the catalysis of senescence-specific β-galactosidase, a dark blue product is generated, which can be used to determine whether the cells are senescent.

[0075] The results are shown in Figure 9. Compared with the control group, the number of positively stained (blue) cells increased significantly after 24 hours of stimulation with 200 μM H2O2, indicating that the senescent cell model was successfully constructed. Pre-administration of DNJ or its derivatives DNJ-1, DNJ-3a, DNJ-3b, and DNJ-5a for 24 hours significantly inhibited the increase in the number of senescent cells compared with the H2O2 group, and the aging protective effects of DNJ-1, DNJ-3a, and DNJ-5a were significantly better than those of NMN (with significant differences), indicating that DNJ or its derivatives have good aging prevention effects.

[0076] Example 2

[0077] The basic method is similar to that of Example 1, except for the mode of administration: while inducing H2O2 and establishing a human umbilical vein endothelial cell aging model, DNJ or its derivatives were administered to detect the anti-aging activity of the drug.

[0078] The results are shown in Figure 10. The staining results showed that DNJ or its derivatives DNJ-1, DNJ-3a, DNJ-3b and DNJ-5a could significantly reduce the production of H2O2-induced senescent cells. Statistical analysis showed that the number of SA-β-Gal staining-positive cells was significantly reduced, indicating that DNJ or its derivatives have good anti-aging activity.

[0079] Example 3

[0080] The basic method is similar to that of Example 1, except for the mode of administration: after H2O2 induction and construction of a human umbilical vein endothelial cell aging model, the cells were treated with DNJ or its derivatives to detect the anti-aging activity of the drugs.

[0081] The results are shown in Figure 11. After 24 hours of H2O2 stimulation and induction of senescence, the addition of DNJ or its derivatives DNJ-1, DNJ-3a, DNJ-3b and DNJ-5a, respectively, can significantly reduce SA-β-Gal expression, thereby reducing cell senescence, indicating that DNJ or its derivatives have a good delaying and therapeutic effect on cell senescence.

[0082] Example 4

[0083] The anti-aging activity of the drug was tested in a D-galactose-induced stress-induced senescence model of human umbilical vein endothelial cells. The basic method was similar to that in Example 1, except that the modeling method was to stimulate the cells with 40 g / L D-gal for 24 hours to establish the senescence model.

[0084] The results are shown in Figure 12. After 24 hours of D-gal stimulation, the number of cells positive for β-galactosidase staining in the D-gal group was significantly increased compared to the control group, demonstrating the successful establishment of a senescent cell model. Pre-treatment with DNJ or its derivatives DNJ-1, DNJ-3a, DNJ-3b, and DNJ-5a for 24 hours significantly reduced the number of cells positive for SA-β-Gal staining, demonstrating that DNJ or its derivatives have a potent anti-aging effect in different senescent cell models and the senescence process.

[0085] Example 5

[0086] The anti-aging activity of the drug was tested in a replicative senescence model of human umbilical vein endothelial cells induced by serial passage. The basic method was similar to that of Example 1, with the following differences: cell passages were performed when the cells reached approximately 80% confluence, and experiments were performed using cells from passages 4, 6, 9, and 12. Compared to passage 4 cells, cells from passages 6, 9, and 12 showed progressive senescence.

[0087] Experimental operation and grouping: After the cells grew to 70-80% density, they were passaged and divided into control group and treatment group at the 4th, 6th, 9th and 12th passages, respectively. The control group was cultured in normal culture medium, and the treatment group was cultured in culture medium containing 30μM DNJ. After 24 hours, the cells were stained with SA-β-Gal.

[0088] The results, as shown in Figure 13, show that after serial passages, the number of β-galactosidase-positive cells in passages 6, 9, and 12 significantly increased compared to young cells at passage 4, confirming the successful establishment of a senescent cell model. DNJ treatment significantly reduced the number of SA-β-Gal-positive cells and the degree of senescence in passages 6, 9, and 12, compared to the untreated group, demonstrating that DNJ has a potent anti-aging effect across various senescent cell models and the senescence process.

[0089] Example 6

[0090] Rat cardiomyocytes (H9c2 cells) culture conditions are as follows:

[0091] Cells were cultured in high-glucose DMEM medium containing 10% FBS and passaged and seeded when the cells reached a density of 70%-80% in a culture dish. The cells were washed once with PBS and digested with 0.25% trypsin for 2-3 minutes. Digestion was terminated by adding high-glucose DMEM medium containing 10% FBS. The cells were collected by blowing off and transferred to a 15ml centrifuge tube. The tube was centrifuged at 1000 rpm for 5 minutes, and the supernatant was discarded. Fresh ECM medium was added and evenly mixed. The tube was then seeded into a 12-well or 24-well plate (density of 70%-80%) and cultured in a 37°C, 5% CO2 incubator. The cells could be attached before the next step.

[0092] The anti-aging activity of the drug was tested in H9c2 cells. In H9c2 cells, the model was established by stimulating them with 400 μM H2O2 for 48 hours. The results are shown in Figure 14. Compared with the control group, the number of SA-β-Gal-positive cells in the H2O2 group increased significantly, indicating that the H9c2 cell aging model has been successfully established. The drug activity of DNJ or its derivatives DNJ-1, DNJ-3a, DNJ-3b, and DNJ-5a was tested by pre-treatment for 24 hours. DNJ or its derivatives were also able to reduce the number of SA-β-Gal-positive H9c2 cells, indicating that DNJ or its derivatives also have an anti-aging effect on cardiomyocytes.

[0093] Example 7

[0094] Human iPSC-differentiated mesenchymal stem cells (iPSC-MSC) culture conditions:

[0095] Cells were cultured in MesenCult-ACF Plus Medium (STEMCELL, Cat#05446) and passaged and seeded when the cells reached a density of 70%-80% in culture dishes. After washing once with DPBS (Gibco), cells were digested with ACF Enzymatic Dissociation Solution (STEMCELL, Cat#05426) for 2-3 minutes. Digestion was terminated by adding ACF Enzyme Inhibition Solution (STEMCELL, Cat#05426). Cells were collected by dislodging into a 15 ml centrifuge tube and centrifuged at 300 g for 8 minutes. The supernatant was discarded and fresh MesenCult-ACF Plus Medium was added and then homogenized. The cells were seeded into 12- or 24-well plates (at a density of 70%-80%) and cultured in a 37°C, 5% CO2 incubator. Once the cells adhered, the next step was performed.

[0096] The anti-aging activity of the drug was detected in iPSC-MSC. In iPSC-MSC, the culture medium was replaced with 40μM H2O2 for 2h and then cultured for 24h to construct a cell aging model. The results are shown in Figure 15. Compared with the control group, the significant increase in the number of SA-β-Gal positive cells in the H2O2 group proved the successful establishment of the iPSC-MSC aging model. The drug activity of DNJ or its derivatives was detected by pre-dose treatment for 24h. DNJ or its derivatives DNJ-1, DNJ-3a, DNJ-3b and DNJ-5a can also reduce the number of SA-β-Gal positive iPSC-MSC cells, indicating that DNJ or its derivatives treatment also has anti-aging effects on mesenchymal stem cells.

[0097] Example 8

[0098] The activity of drugs in inhibiting aging-related inflammatory phenotypes was detected in the H2O2-induced stress aging model of human umbilical vein endothelial cells. The basic method refers to Example 1, except that the experimental operation is as follows: after discarding the supernatant of the cells, wash them three times with PBS, and then add a culture medium containing 30μM DNJ pre-prepared with the culture medium. The blank group and the modeling group were replaced with a blank culture medium without drugs. After culturing for 24 hours, the supernatant was discarded, and the cells were washed three times with PBS. The blank group was added with blank culture medium, and the modeling group and the drug-treated group were added with culture medium containing 200μM H2O2. After 24 hours of treatment, the RNA sample was extracted using Steady Pure Quick RNA Extraction Kit (Aikerui), and the RNA was reverse transcribed into cDNA using Evo M-MLV Reverse Transcription Premix Kit (Aikerui), and then the RNA was reverse transcribed into cDNA using Green Pro Taq HS premixed qPCR kit (Acryl) was used for qRT-PCR reaction to detect the mRNA expression level of SASP factors.

[0099] The cell RNA extraction process is as follows:

[0100] Aspirate the culture medium from the culture dish and wash the cells once with 1× PBS buffer. Aspirate the PBS buffer and add 500 μL of Buffer QLS Lysis Buffer. Gently shake the culture dish to ensure that the Buffer QLS solution is evenly distributed on the cell surface. Use a pipette to repeatedly pipette to dislodge the cells. Then transfer the entire volume of the cell-containing homogenate to a centrifuge tube. Mix by high-speed vortexing or pipetting until the homogenate is clear and non-viscous. Let the homogenate stand at room temperature for 2 minutes. Carefully transfer the supernatant to a new RNase-free EP tube and proceed with the following steps:

[0101] (1) Add an equal volume of 100% ethanol to the homogenate and mix thoroughly by pipetting. If there is obvious sticky matter or precipitation, pipette several times until the precipitation is broken up.

[0102] (2) Immediately transfer the entire mixture to the Quick RNA Mini Column, centrifuge at 12,000 rpm for 2 minutes at room temperature, and discard the filtrate.

[0103] (3) Add 700 μL of Buffer QWB (Buffer QWB and anhydrous ethanol in a volume ratio of 3:7) to the Quick RNA Mini Column, centrifuge at 12,000 rpm for 1 min at room temperature, and discard the filtrate.

[0104] (4) Remove the Quick RNA Mini Column vertically, preventing the column head from touching the wall of the collection tube, and place it on a new 2.0 ml Collection Tube. Centrifuge at 12,000 rpm for 2 minutes at room temperature.

[0105] (5) Place the Quick RNA Mini Column on a new RNase-Free Tube. Add 50 μl to 200 ml of RNase-Free Water to the center of the column membrane. Allow to stand at room temperature for 3 minutes to dissolve the RNA. Centrifuge at 12,000 rpm for 2 minutes at room temperature to elute the RNA. Measure the RNA concentration of the sample using a Nano-100 microspectrophotometer and the RNA purity by absorbance at 260 / 280 nm. Store at -80°C.

[0106] The reverse transcription and qRT-PCR reaction procedures for cell RNA samples are as follows:

[0107] (1) Remove genomic DNA. Calculate the required volume of RNase-free water and RNA based on the corresponding RNA concentration. Add 2 μL of 5×g DNA Clean Reaction Mix and the corresponding volume of RNase-free water and RNA to a 200 μL EP tube, ensuring that the total liquid system is 10 μL. Place the EP tube in the reverse transcription instrument and perform the genomic DNA removal reaction according to the following procedure: 42°C for 2 minutes, then cool to 4°C.

[0108] (2) Reverse transcription reaction. Remove the reaction solution from step (1) and add 4 μL of 5×Evo M-MLV RT Reaction Mix and 6 μL of RNase-free water, respectively, to ensure that the total liquid system is 20 μL. Place the EP tube in the reverse transcription instrument and perform the denaturation and annealing reaction according to the following procedure: 37°C for 15 minutes, 85°C for 5 seconds, and then cool to 4°C. It can be used for qRT-PCR experiments.

[0109] use Green Pro Taq HS premixed qPCR kit (Acryl) was used for qRT-PCR reaction. 2 μL of cDNA solution of the corresponding sample, 0.08 μL of forward and reverse primers, 5 μL of 2× Add Green Pro Taq HS Premix and 2.92 μL of RNase-free water to a new EP tube, ensuring a total volume of 10 μL per replicate. (Forward and reverse primer sequences for target genes, see Table 1.) Set the assay program to: pre-denaturation (95°C, 30 sec; 95°C, 5 sec), annealing (60°C, 30 sec), cycle 40 times, and then analyze.

[0110] Data Analysis:

[0111] (1) The Ct value of the sample group minus the control group (H1), that is, ΔCt,

[0112] (2) The Ct value of the target genome minus the reference genome (B-actin), i.e., ΔΔCt,

[0113] (3) Calculate the relative expression of the target gene, i.e., mRNA expression (fold change, FC) = 2^-(ΔΔCt).

[0114] Table 1 Primer sequences

[0115] Compared with normal cells, senescent cells are characterized by high expression of SASP factors. The core program of SASP mainly includes pro-inflammatory interleukin-6 (IL-6), CXC chemokine ligand 8 (CXCL8, also known as IL-8) and monocyte chemoattractant protein 1 (MCP1, also known as CCL2). The results are shown in Figure 16. Compared with the control group, the mRNA expression level of SASP factors was significantly increased after 24 hours of stimulation with 200 μM H2O2. After 24 hours of pre-treatment with DNJ, the increase in mRNA expression of SASP factors was significantly inhibited compared with the H2O2 group, indicating that DNJ has the effect of improving the SASP phenotype of senescent cells.

[0116] Example 9

[0117] The drug's ability to inhibit the aging-associated inflammatory phenotype was tested in a replicative senescence model of human umbilical vein endothelial cells induced by serial passage. The modeling method and experimental grouping were similar to those in Example 8, and the basic procedures were similar to those in Example 9. As shown in Figure 17, the mRNA expression levels of SASP factors in senescent cells from passages 6, 9, and 12 were significantly increased compared to those in young cells from passage 4. DNJ treatment for 24 hours significantly inhibited the increase in SASP factor mRNA expression compared to the control group, demonstrating that DNJ has the effect of ameliorating the SASP phenotype of senescent cells.

[0118] Example 10

[0119] A mouse model was used to test the drug's anti-aging effect in vivo. C57BL / 6J male mice were randomly divided into a normal group (Control), an aging group (Aging), and a drug-treated group (Aging+DNJ). Dox was injected intraperitoneally into the mice for one week to establish an aging mouse model; the drug-treated group was treated with DNJ while establishing the aging mouse model. The status of the mice was recorded, and the mice were killed one week later. Organs such as the liver and kidney were immediately collected for subsequent testing. Using the Cell Senescence β-Galactosidase Staining Kit (Beyotime), frozen sections were fixed with fixative for 15 minutes, rinsed three times with PBS, and incubated in SA-β-gal solution at 37°C in the dark for 16-18 hours. ImageJ (NIH) software was used to quantitatively determine the stained area ratio.

[0120] The results, as shown in Figure 18, show that compared with the normal control group, Dox-induced senescent mice exhibited hunched bodies, sluggishness, and significantly reduced motor ability. Cryosection staining of organs such as the kidneys revealed a significant increase in the number of SA-β-Gal-positive cells, demonstrating an aging phenotype. In the DNJ-treated senescent mice, also known as the drug-treated group, significant improvements in physical condition and motor ability were observed, with a significant decrease in the number of SA-β-Gal-positive cells in organs such as the kidneys, suggesting that DNJ or its derivatives have a potent anti-aging effect in vivo.

Claims

1. Use of 1-deoxynojirimycin compounds in the preparation of anti-aging drugs, wherein the 1-deoxynojirimycin compounds are at least one of the following: 1-deoxynojirimycin or its pharmaceutically acceptable salt; 1-deoxynojirimycin derivative or its pharmaceutically acceptable salt.

2. The application according to claim 1, wherein, The 1-deoxynojirimycin derivative is any one of the following: (1) DNJ-1; The structure is as shown in Formula II: (2) The structure is as shown in the general formula: wherein R is selected from: -H, -NO2, -X, where X is F, Cl, Br or I; (3) DNJ-5a; The structure is as shown in Formula V:

3. The application according to claim 2, characterized in that when R is selected as -H, the 1-deoxynojirimycin derivative is DNJ-3a; The structure is as shown in Formula III:

4. The application according to claim 2, wherein When R selects -X, X is F, and the 1-deoxynojirimycin derivative is DNJ-3b; the structure is shown in Formula IV:

5. The use according to claim 2, wherein (i) When the 1-deoxynojirimycin derivative is (1), the corresponding 1-deoxynojirimycin derivative is obtained by condensing compound a and b1 in a solvent, where compound a is 1-deoxynojirimycin and compound b1 is: (ii) When the 1-deoxynojirimycin derivative is (4), the corresponding 1-deoxynojirimycin derivative is obtained by condensation reaction of compound a and b4 in a solvent, where compound a is 1-deoxynojirimycin and compound b4 is: (iii) When the 1-deoxynojirimycin derivative is (2), first react 1-deoxynojirimycin with benzyl chloroformate to form intermediate 1: Then react the intermediate 1 with PivCl to obtain intermediate 2: finally, the intermediate product 2 is reduced to obtain the 1-deoxynojirimycin derivative.

6. The application according to claim 1, characterized in that The anti-aging effect of the anti-aging drug is prevention before the occurrence of aging, alleviation during the occurrence of aging, or repair after the occurrence of aging.

7. The application according to claim 1, characterized in that, The anti-aging drug further comprises a pharmaceutically acceptable excipient.

8. The application according to claim 1, characterized in that The dosage form of the anti-aging drug is at least one of suspension, granule, capsule, powder, tablet, emulsion, dropping pill, injection, suppository, enema, aerosol, patch or drop.

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