Application of cell endogenous metabolite in preparation of medicine for treating and / or preventing senescence

Abstract

The invention discloses an application of a cell endogenous metabolite in preparation of a medicine for treating and / or preventing senescence. The cell endogenous metabolite is any one or a combination of more of phosphocreatine, hypotaurine and / or ethanolamine phosphate. The invention finds that phosphocreatine, hypotaurine and phosphoethanolamine can effectively recover cell growth, reduce SA-beta-Gal staining intensity of cells, improve mitochondrial functions of the cells, increase ATP yield, recover mitochondrial membrane potential of the cells and relieve damage and premature aging caused by oxidative stress of the cells under a specific concentration. The life of the nematodes can be prolonged; the exercise ability in the aging middle stage is recovered. Meanwhile, the three metabolites have a synergistic effect, and the same cell protection effect can be achieved by adjusting the proportioning concentration of the three metabolites and performing mixed administration. The endogenous metabolites have the characteristics of accuracy, high efficiency and safety at the same time, so that the three cell endogenous metabolites provided by the invention can be used for preparing anti-aging drugs.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to the use of endogenous metabolites in cells in preparing drugs for treating and / or preventing aging. Background Art

[0002] As the aging of society continues to intensify, anti-aging, life extension and healthy aging have become one of the hot research directions. Cellular senescence is an important cause of individual aging and tissue degeneration, and regulating cell senescence has become an important part of anti-aging and intervention in aging-related diseases. However, cell senescence involves multi-level and multi-factor regulation such as gene expression, epigenetic modification, and signal pathway cascade reactions. It is related to multi-level mechanisms such as metabolic small molecules, biological macromolecules, and organelles. It is a very complex process, and its detailed mechanism has not yet been fully elucidated.

[0003] Cell senescence is the result of the continuous accumulation of cell damage. Oxidative stress is a common cell damage and can induce premature senescence of cells. It is a special form of cell senescence. At present, related research and technology development are mainly based on exogenous intervention, including small molecule inhibitors, signal pathway regulators and other drugs, as well as synthetic drugs. These exogenous intervention methods improve cell senescence and have certain efficacy.

[0004] For example, patent CN202080042529.3 discloses extracellular vesicles (HLSC-EV) from human liver stem cells for reducing cell aging. It uses extracellular vesicles derived from human liver stem cells to prepare preparations, which can improve cell aging and related diseases in in vitro and in vitro experiments. For another example, patent CN202111376415.5 discloses the use of mannose glucuronic acid oligosaccharides and polysaccharides and derivatives in the preparation of drugs for treating and / or preventing aging. It prepares a series of mannose glucuronic acid oligosaccharides and polysaccharides and derivatives with anti-aging effects. For another example, patent CN202111499257.2 discloses an anti-aging composition and its application, which prepares a composition with mesenchymal stem cell exosomes, basic fibroblast growth factor, squalane, grape seed oil, sorbitol and hyaluronic acid as special components, and has a special proportion, which can effectively resist aging. For example, patent CN201680079142.9 discloses a method for preventing and / or treating cognitive impairment and neuroinflammation related to aging, which uses a drug Ppargc 1a activator to improve the central nervous system cognitive impairment or neuroinflammation related to aging in subjects. For another example, patent CN202010819194.3 discloses a molecular marker related to aging and healthy aging and its application in improving healthy aging. Its specification discloses that knocking down FABP4 gene expression or inhibiting FABP4 activity in old mice can significantly improve cognitive function, inhibit aging and related inflammatory indicators, indicating that FABP4 gene has the effect of promoting aging, and the inhibitor of FABP4 gene can improve the cognitive function of animals and has anti-aging effect.

[0005] The above patents have achieved certain results by intervening and regulating aging through in vitro exogenous intervention methods. However, these methods also have some problems. For example, the safety of small molecule drugs. Small molecule drugs such as inhibitors regulate genes and signal pathways, but due to gene interactions, the network regulation characteristics of signal pathways, and the targeting of drug molecules, their scope of action is likely to be wider than what is known, and the effects they produce may also be more complex and extensive than what is already known, making it difficult to eliminate potential safety risks. Therefore, the intervention of exogenous drugs or natural extracts has the disadvantage of being quite different from the components of the living organism itself. It is necessary to find endogenous metabolites that can rejuvenate aging cells, which can provide a new direction for aging research.

[0006] Caenorhabditis elegans (C. elegans) is a commonly used model for aging-related research. Its lifespan is about 3-4 weeks. It is an important and simple model for detecting aging and its regulation. Research on the natural aging of nematodes is an important part of aging research. Nematode aging is manifested in its morphological changes (such as increased body size, increased particulate matter in the body, etc.), reproductive aging, decreased motor ability, etc., and its indicators are easy to detect and observe. The extension of nematode lifespan is related to its enhanced ability to resist various environmental stresses, but there are few reports on the regulation of nematode lifespan by specific small molecule metabolites.

[0007] Although in recent years, a large number of literature reports have shown that the use of small molecule inhibitors and other drugs to intervene in nematodes can effectively extend the lifespan of nematodes, and their mechanisms are mostly related to the regulation of cell signaling pathways. However, this type of inhibitor has a wide range of action, and its effects are relatively complex and extensive. In addition, as an exogenous drug, its safety also needs to be studied. There are few studies in academia on the regulation of nematode lifespan by endogenous metabolites. At the same time, nematode movement ability is an important indicator of aging. Previous studies have mostly extended the lifespan of nematodes, but there are few reports on improving the mobility of aged nematodes. The improvement and protection of body functions during aging is an important aspect of aging research, but there are currently few studies in this area. Therefore, the discovery of endogenous metabolites that can extend the lifespan of nematodes and improve the mobility of aged nematodes can also provide new directions for aging research. Summary of the invention

[0008] In response to the shortcomings of the existing technology, the present invention provides a technology for intervening in cell aging with endogenous metabolites. It directly uses the existing metabolites in the cells to combat cell oxidative stress damage and slow down cell aging. It can also extend the lifespan of nematodes while delaying the decline of physiological functions. It is safe and efficient.

[0009] In order to solve the above technical problems, the present invention provides the following technical solutions:

[0010] The present invention performs metabolic intervention by supplementing key metabolites into the culture medium, and when the cells are induced, the culture medium containing metabolites is replaced. The levels of cellular oxidative stress and premature aging are evaluated by indicators such as cell growth, cell SA-β-Gal staining, cell mitochondrial function and ATP yield, and cell mitochondrial membrane potential. The results show that three endogenous metabolites, phosphocreatine, hypotaurine, and phosphoethanolamine (O-PE), can effectively restore cell growth, reduce cell SA-β-Gal staining intensity, improve cell mitochondrial function, increase ATP yield, and restore cell mitochondrial membrane potential at specific concentrations, which can alleviate the damage caused by cell oxidative stress and premature aging.

[0011] In addition, the present invention uses a mixed metabolite intervention method to find that the above three metabolites have a synergistic effect. By adjusting the ratio concentration of the three metabolites, that is, using 1 / 5 of the optimal concentration of each of the above three metabolites (after testing, at other mixing ratios, the effect is not as good as the 1 / 5 optimal concentration mixing effect), the same cell protection effect can be achieved by mixed administration. The results of growth analysis and cell SA-β-Gal staining showed that the mixed intervention of 1 / 5 optimal concentration greatly restored cell growth and effectively reduced the cell SA-β-Gal staining intensity, achieving the same effect of single metabolite intervention at a lower total dose.

[0012] In addition, the present invention uses the key metabolites of stress damage and aging discovered in the cell model to perform metabolic intervention on nematodes, and discovers the effect of endogenous small molecule metabolism in controlling nematode aging, and effectively improves the motor ability of nematodes during aging, indicating that metabolic intervention effectively prolongs life and can improve the body function of nematodes during aging, and play a protective role. Specifically, the present invention found that endogenous metabolites, phosphocreatine, hypotaurine and phosphoethanolamine (O-PE) can prolong the life of nematodes at specific doses, and also have the effect of improving the activity ability of nematodes in the middle stage of aging. The above three metabolites are all endogenous metabolites, which can play an anti-aging effect through the regulation of existing metabolic pathways, and have the advantages of being milder and safer than traditional drug interventions.

[0013] therefore, on the one hand, The present invention provides the use of endogenous metabolites in cells in preparing drugs for treating and / or preventing aging.

[0014] Wherein, the endogenous metabolite is any one or a combination of creatine phosphate, hypotaurine and / or ethanolamine phosphate.

[0015] Preferably, the drug for treating and / or preventing aging is a drug for treating and / or preventing cellular aging.

[0016] More preferably, in a specific embodiment, the endogenous metabolite is creatine phosphate, and the concentration of creatine phosphate in the drug for treating and / or preventing aging is 0.2-5 mmol / L, more preferably 0.25-1 mmol / L, and even more preferably 0.5 mmol / L.

[0017] More preferably, in another specific embodiment, the endogenous metabolite is hypotaurine, and the concentration of hypotaurine in the drug for treating and / or preventing aging is 0.25-10 mmol / L, further preferably 0.25-1 mmol / L, and further preferably 1 mmol / L.

[0018] More preferably, in another specific embodiment, the endogenous metabolite is phosphoethanolamine, and the concentration of phosphoethanolamine in the drug for treating and / or preventing aging is 0.01-0.5 mmol / L, further preferably 0.01-0.08 mmol / L, and further preferably 0.04 mmol / L.

[0019] More preferably, in another specific embodiment, the endogenous metabolite is a combination of creatine phosphate, hypotaurine and phosphoethanolamine, and the concentration ratio of creatine phosphate, hypotaurine and phosphoethanolamine in the drug for treating and / or preventing aging is 12.5:25:1. Further preferably, the concentration of creatine phosphate, the concentration of hypotaurine, and the concentration of phosphoethanolamine in the drug for treating and / or preventing aging is 0.1 mmol / L, the concentration of hypotaurine is 0.2 mmol / L, and the concentration of phosphoethanolamine is 0.008 mmol / L.

[0020] Preferably, the drug for treating and / or preventing aging is a drug for treating and / or preventing aging of nematodes.

[0021] Furthermore, the drug is a drug that prolongs the life span of nematodes or improves the movement ability of aged nematodes.

[0022] More preferably, in a specific embodiment, the endogenous metabolite is creatine phosphate, and the concentration of creatine phosphate in the drug is 0.2-5 mmol / L, and further preferably 1 mmol / L.

[0023] More preferably, in another specific embodiment, the endogenous metabolite is hypotaurine, and the concentration of hypotaurine in the drug is 0.25-10 mmol / L, more preferably 0.4-10 mmol / L, and even more preferably 2 mmol / L.

[0024] More preferably, in another specific embodiment, the endogenous metabolite is phosphoethanolamine, and the concentration of phosphoethanolamine in the drug is 0.01-0.5 mmol / L, further preferably 0.02-0.5 mmol / L, and further preferably 0.1 mmol / L.

[0025] on the other hand, The present invention also provides a drug for treating and / or preventing aging, wherein the drug contains any one or a combination of creatine phosphate, hypotaurine and / or ethanolamine phosphate.

[0026] Preferably, the concentration ratio of creatine phosphate, hypotaurine and phosphoethanolamine in the drug is 12.5:25:1.

[0027] More preferably, the concentration of creatine phosphate in the drug is 0.1 mmol / L, the concentration of hypotaurine is 0.2 mmol / L, and the concentration of ethanolamine phosphate is 8 μmol / L.

[0028] Preferably, the drug further comprises a pharmaceutically acceptable carrier, and the pharmaceutically acceptable carrier is any one or a combination of a binder, a disintegrant, a lubricant, and a glidant.

[0029] Compared with the prior art, the present invention has the following beneficial effects:

[0030] (1) The three endogenous metabolites provided by the present invention, creatine phosphate, hypotaurine and ethanolamine phosphate, can effectively restore cell growth, reduce cell SA-β-Gal staining intensity, improve cell mitochondrial function and increase ATP production, restore cell mitochondrial membrane potential, and alleviate cell damage and premature aging caused by oxidative stress. At the same time, the three metabolites have a synergistic effect. By adjusting the ratio and concentration of the three metabolites, the same cell protection effect can be achieved by mixed administration. Therefore, the three endogenous metabolites provided by the present invention have significant anti-aging effects, small toxic and side effects, safe and effective, and can be used to prepare anti-aging drugs.

[0031] (2) The present invention uses endogenous metabolites creatine phosphate, hypotaurine and phosphoethanolamine for intervention respectively, and finds that the three metabolites have the effect of extending the lifespan of nematodes and restoring the motility of mid-aging nematodes at specific concentrations, thereby promoting the healthy aging of nematodes. Since endogenous metabolites are accurate, efficient and safe at the same time, the three cell endogenous metabolites provided by the present invention can be used to prepare nematode anti-aging drugs, which can extend the lifespan of nematodes or improve the motility of aging nematodes, providing a new direction for aging research. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 The results are shown in Figure 2. ...

[0033] Figure 2 The results show that creatine phosphate intervention can alleviate premature cell senescence induced by oxidative stress. Wherein, a is the cell density, and b is the staining intensity of senescence β-galactosidase.

[0034] Figure 3 The results show that hypotaurine intervention can alleviate premature cell senescence induced by oxidative stress. Wherein, a is the cell density, and b is the staining intensity of senescence β-galactosidase.

[0035] Figure 4 The results show that phosphoethanolamine intervention can alleviate premature cell senescence induced by oxidative stress. Wherein, a is the cell density, and b is the staining intensity of senescence β-galactosidase.

[0036] Figure 5 The results of the mixed administration of three metabolites to alleviate aging. Among them, a is the cell density and b is the staining intensity of senescence β-galactosidase.

[0037] Figure 6 The results are as follows: intervention with the three metabolites at the optimal concentrations improved mitochondrial function.

[0038] Figure 7 These are the test results of the alleviating effect of different doses of hypotaurine on nematode aging.

[0039] Figure 8 These are the experimental results on the alleviating effect of different doses of creatine phosphate on nematode aging.

[0040] Fig. 9 These are the test results of the alleviating effect of different doses of phosphoethanolamine on nematode aging.

[0041] Fig.10 These are the test results of the protective effects of three metabolites on the motility of nematodes. DETAILED DESCRIPTION

[0042] The present invention will be further described in detail below in conjunction with specific examples. The following examples are not intended to limit the present invention, but are only intended to illustrate the present invention. The experimental methods used in the following examples are generally conventional, unless otherwise specified, and the materials, reagents, etc. used in the following examples are commercially available, unless otherwise specified.

[0043] Terms and explanations:

[0044] In the present invention, the term "premature senescence" refers to a special form of senescence in which normally dividing cells enter senescence prematurely under the stimulation of external damaging factors, which is manifested by growth inhibition, increased expression of senescence-related β-galactosidase, etc.

[0045] In the present invention, the term "metabolic intervention" refers to the process of adding a specified metabolite into a cell culture medium and culturing the cell culture medium.

[0046] In the present invention, the term "cellular senescence-associated β-galactosidase (SA-β-Gal)" is an enzyme specific to senescent cells and serves as a marker of cell senescence.

[0047] In the present invention, the term "Caenorhabditis elegans (C. elegans)" refers to a worm isolated from soil, which has become a common model for aging research due to its short life cycle.

[0048] In the present invention, the term "thrashing" refers to the movement of nematodes in a buffer solution, and each left-right movement is counted as one thrashing, which is used as an evaluation index of the nematode's motility.

[0049] In the present invention, the term "metabolic intervention" refers to an intervention method by adding specific metabolites to the culture plate when the nematodes enter the adult stage.

[0050] In the present invention, the term "NGM plate" refers to a plate prepared with a nematode growth medium, which serves as a nematode rearing environment.

[0051] Example 1 Cellular anti-aging related experiments - single metabolite administration and mixed administration

[0052] The test method steps adopted in this embodiment are as follows:

[0053] 1. Cell culture

[0054] HEK293 or HEK293T cells were cultured in an incubator at 37°C and 5% carbon dioxide, with water added to ensure humidity. Cells were passaged every 2-3 days, with a density greater than 70% during passage. All experimental cells were cultured using high-glucose DMEM medium containing 10% fetal bovine serum and 1% double antibiotics.

[0055] 2. Induction of cell senescence

[0056] Cells were treated with 80 μM hydrogen peroxide for 1 h, replaced with fresh medium, and allowed to recover for 48 h to complete the modeling of induced senescence.

[0057] 3. Cellular oxidative stress detection

[0058] The DCFDA fluorescent probe was used for detection. The probe concentration in the detection buffer was 10 micromolar. The cells were incubated for 25 minutes, and then the probe was washed away with the buffer. The cells were observed and photographed under a fluorescence microscope.

[0059] 4. Cell Senescence Staining

[0060] The SA-β-Gal (senescence-associated galactosidase) staining method was used for detection. The cells were fixed according to the instructions in the kit, stained with a staining agent in a 37°C incubator overnight, and photographed under a bright field microscope.

[0061] 5. Cellular metabolism intervention

[0062] The three cell endogenous metabolite standards of the present invention are added to the culture medium to perform metabolic intervention.

[0063] 6. Single cell mass spectrometry detection

[0064] Single-cell mass spectrometry was used for detection. The relevant parameters of mass spectrometry detection were as follows: ion source voltage was about 4 kV, 500 Hz, capillary temperature was 275 °C, radio frequency intensity was 50%, mass spectrometry detection resolution was 70000, ion injection time was 10 milliseconds, and scanning mode was negative sampling mode.

[0065] 7. Mitochondrial function testing

[0066] The seahorse cell metabolism detector (XF96) was used to monitor the changes in cellular oxygen consumption rate after the addition of oligomycin (1μM), FCCP (1.5μM), and rotenone / antimycin (1μM). The seahorse software was used to calculate ATP production and respiratory efficiency, thereby evaluating cellular mitochondrial function.

[0067] 8. Mitochondrial membrane potential detection

[0068] TMRE mitochondrial membrane potential fluorescent probe was used for detection to evaluate the damage of mitochondrial membrane potential and its recovery after metabolic intervention. According to the instructions of the kit, serum-free DMEM medium was used to prepare 1x TMRE staining working solution, incubate cells for 15 minutes, wash cells once with PBS, replace serum-free DMEM medium, and take pictures under a fluorescence microscope.

[0069] 9. Image processing and analysis

[0070] All staining and probe images were analyzed using imageJ to calculate cellular oxidative stress, mitochondrial membrane potential, and senescence-associated β-galactosidase staining intensity.

[0071] 10. Cellular metabolomics data analysis

[0072] Matlab was used for k-medios clustering and UMAP was used for visualization. Metabolic model construction, validation, and prediction were completed using Matlab classification learner and regression learner. Metabolic pathway enrichment analysis was performed using the metaboanalyst (www.metaboanalyst.ca) online tool.

[0073] The specific test results are as follows:

[0074] 1. Test methods

[0075] 1. Plant HEK293T cells in a 3.5 mm culture dish;

[0076] 2. After the cells have grown overnight, confirm the cell density, which is about 50%;

[0077] 3. Treat cells with 80 μmol / L H2O2 for 1 hour;

[0078] 4. Replace the culture medium containing specific metabolites, and administer or mix three endogenous metabolites of cells;

[0079] 5. Take photos at 0, 8, 24, and 48 hours after treatment to observe cell density and evaluate cell growth;

[0080] 6. Culture the cells for 48 hours and perform senescence-associated β-galactosidase assay.

[0081] 2. Test results

[0082] 1. Oxidative stress can induce premature aging of cells

[0083] like Figure 1 As shown in the figure, HEK293T cells were treated with H2O2 at a concentration of 50μmol / L-120μmol / L, which induced the cells to enter a state of premature senescence. The intensity of senescence-associated β-galactosidase (SA-β-Gal) was positively correlated with the H2O2 treatment concentration, indicating that H2O2-treated cells within this concentration range can successfully induce premature senescence. Therefore, the model of using oxidative stress to induce premature cellular senescence is stable and reliable.

[0084] 2. Phosphocreatine metabolism intervention trial

[0085] The creatine phosphate dosage sequence is designed as follows (eg Figure 2 As shown): 0.1mmol / L, 0.25mmol / L, 0.5mmol / L, 1mmol / L.

[0086] The above test method showed that except for 0.1mmol / L creatine phosphate, which failed to improve the growth inhibition caused by cell senescence, the growth inhibition caused by cell senescence was alleviated at other doses. Among them, 0.5mmol / L creatine phosphate had the best effect, and the cell density at 48 hours was closest to the control group.

[0087] The results of cell senescence galactosidase staining showed that except for 0.1mmol / L creatine phosphate, which failed to improve the cell staining intensity, the other doses effectively improved the senescence galactosidase staining intensity. Similarly, 0.5mmol / L creatine phosphate had the best effect and the lowest senescence galactosidase staining intensity.

[0088] Therefore, when using creatine phosphate alone to intervene in cell aging, the optimal dose is 0.5mmol / L.

[0089] 3. Hypotaurine metabolic intervention trial

[0090] The hypotaurine dosage sequence was designed as follows (eg Figure 3 As shown): 0.25mmol / L, 0.5mmol / L, 1mmol / L, 2mmol / L.

[0091] The above test method showed that the growth inhibition caused by cell senescence was alleviated under the treatment of various doses of hypotaurine. Among them, 1mmol / L hypotaurine had the best effect, and the cell density at 48 hours was closest to the control group.

[0092] Cell senescence galactosidase staining showed that except for 2mmol / L hypotaurine, which failed to improve the cell staining intensity, the other doses effectively improved the senescence galactosidase staining intensity. Among them, 1mmol / L creatine phosphate had the best effect and the lowest senescence galactosidase staining intensity.

[0093] Therefore, when hypotaurine is used alone to intervene in cell senescence, the optimal dose is 1mmol / L.

[0094] 4. Phosphoethanolamine (O-PE) metabolic intervention trial

[0095] The dosage sequence of phosphoethanolamine was designed as follows (eg Figure 4 As shown): 10 μmol / L, 20 μmol / L, 40 μmol / L, 80 μmol / L.

[0096] The above test method showed that except for 20 μmol / L phosphoethanolamine, which failed to effectively alleviate the growth inhibition caused by cell senescence, the other doses all effectively alleviated the growth inhibition caused by cell senescence. The effects were better at 40 μmol / L and 80 μmol / L doses, and the cell density at 48 hours was close to that of the control group.

[0097] Cell senescence β-galactosidase staining showed that 40 μmol / L phosphoethanolamine effectively reduced the senescence β-galactosidase staining intensity, which was closest to the control group.

[0098] Therefore, when phosphoethanolamine is used alone to intervene in cell senescence, the optimal dose is 40 μmol / L.

[0099] 5. Mixed drug administration metabolic intervention trial

[0100] Through the method of mixed metabolite intervention, the present invention discovered that the above three key metabolites have a synergistic effect.

[0101] Specifically, 1 / 5 of the optimal concentration of the above three metabolites when used alone, namely 0.1mmol / L of creatine phosphate, 0.2mmol / L of hypotaurine, and 8μmol / L of phosphoethanolamine, with a ratio of 12.5:25:1, was taken for mixed intervention to achieve the same cell protection effect as when a single metabolite was intervened. The results of growth analysis and cell senescence-related β-galactosidase staining showed that the mixed intervention of 1 / 5 of the optimal concentration significantly restored cell growth and effectively reduced the intensity of cell senescence-related β-galactosidase staining. At a lower total dose, it achieved the same effect as single metabolite intervention and was safer.

[0102] Since the synergistic effect has a specific ratio, it has been tested that it also has the effect of alleviating aging at other mixing ratios (such as Figure 5 1 / 4 optimal concentration mixed intervention can alleviate the effects of aging. 1 / 8 optimal concentration mixed intervention cannot alleviate cell aging because the dose is too low. In order to reduce the total dose as much as possible, 1 / 5 optimal concentration mixed intervention is selected as the optimal ratio.

[0103] Therefore, when mixed administration is used to intervene in cell aging, the concentrations of the three endogenous cellular metabolites are selected as creatine phosphate 0.1mmol / L, hypotaurine 0.2mmol / L, and phosphoethanolamine 8μmol / L.

[0104] 6. Metabolic intervention alleviates aging by improving mitochondrial respiratory function

[0105] The optimal concentrations of the three metabolites were used for intervention, e.g. Figure 6 As shown, a cell metabolism detector was used to analyze the oxygen consumption rate of cells and determine the basal respiration and ATP synthesis capacity of cells. The baseline respiration level was measured in the initial stage. After the addition of oligomycin, ATP synthesis was inhibited and the oxygen consumption rate decreased. The degree of decrease in the oxygen consumption rate reflects the ability to synthesize ATP. It can be seen that phosphoethanolamine and hypotaurine improved the baseline respiration level and greatly improved the ability of cells to synthesize ATP. Therefore, it is speculated that this is a related mechanism for metabolite intervention to alleviate cell aging.

[0106] Example 2 Experiment on the intervention of endogenous metabolites in nematode aging and improvement of motor ability decline

[0107] The test method steps adopted in this embodiment are as follows:

[0108] 1. Nematode culture

[0109] The nematode strain was N2, i.e., wild type. All experimental nematodes were cultured at 20°C using solid NGM medium and OP50 Escherichia coli as nematode food. In the metabolic intervention lifespan analysis experiment, inactivated OP50 Escherichia coli was used as food to eliminate the interference caused by its metabolism.

[0110] 2. Synchronization of nematodes

[0111] Select L4 and young adults, and use the bleach method to obtain eggs: rinse the NGM plate with sterile water, capture the nematodes and place them in a centrifuge tube, add sterile water to 3.5mL, prepare bleach solution (0.5mL 5mol / L NaOH + 1mL 5% NaClO) and add it to the centrifuge tube, shake, let it stand for 2 minutes, repeat shaking and standing until it lasts for 10 minutes. Centrifuge at 1300g for 30s, discard the supernatant, add 5mL of sterile water, shake, centrifuge at 1300g for 5s, obtain sterile eggs, inoculate them in NGM culture medium, and obtain synchronized larvae the next day.

[0112] 3. Metabolic intervention plate preparation

[0113] 300 μL of metabolite standard solution was applied to NGM plates. The solution was diluted from the stock solution with sterile water to the dosing concentration and incubated overnight. Inactivated OP50 Escherichia coli was evenly applied. All metabolite-coated NGM plates were prepared at least 12 hours before the start of the experiment.

[0114] 4. Analysis of nematode lifespan

[0115] Before the experiment, pre-incubate the NGM plates with 0.1 mg / mL 5'-FUDR to inhibit the hatching of eggs and avoid interfering with the lifespan analysis experiment. When the nematodes age and no longer lay eggs after adulthood, stop incubating 5'-FUDR on the NGM plates. Nematodes that have developed to the L4 stage are transferred to the prepared metabolic intervention plates, and the plates are transferred every 2-3 days to ensure the effectiveness of the metabolite intervention. Nematodes are examined under a microscope every 1-2 days, and the number of surviving and dead nematodes is recorded. The survival rate is calculated and survival analysis is performed later. Nematodes that crawl out of the plate, burst accidentally, hatch larvae in the body, and other unnatural deaths will be excluded from the experiment.

[0116] 5. Nematode transfer plate

[0117] In the life span analysis experiment, the plate was transferred by multiple sedimentation method. The plate was washed with M9 buffer, and the nematodes were captured and transferred to a centrifuge tube. The nematodes were allowed to settle naturally by gravity, which could wash away impurities and accidentally hatched larvae and ensure the stability of the synchronization system.

[0118] 6. Determination of nematode activity

[0119] The nematodes were transferred to the M9 buffer in a 24-well plate and adapted for 30 seconds. The number of times the nematodes swam in the buffer was observed under a stereoscope. Movement to the left and right was recorded as one time. The number of movements per minute was calculated to compare the nematode movement ability.

[0120] The specific test results are as follows:

[0121] 1. Main test steps

[0122] 1. Lifespan analysis

[0123] (1) Obtaining synchronized eggs: Select several adult worms, place them on an empty NGM plate, and wait for them to lay eggs;

[0124] (2) Obtaining synchronized larvae: The synchronized eggs are hatched overnight, and synchronized nematodes can be obtained the next day;

[0125] (3) Obtaining synchronized adults: Cultivating at 22°C, the synchronized adults are obtained after 2-3 days of antennae development.

[0126] (4) Metabolite coating NGM plates: 300 μL of metabolite standard solution was coated on the NGM plates. The specific metabolite dosages were as follows: 2 mmol / L hypotaurine; 1 mmol / L creatine phosphate; 0.1 mmol / L phosphoethanolamine. These metabolites are components already present in the nematode body and are endogenous intervention measures.

[0127] (5) Transfer synchronized adults to NGM plates pre-coated with metabolites;

[0128] (6) Record the number of nematodes alive and dead every 1-2 days;

[0129] (7) During the experiment, the NGM plates coated with metabolites were replaced every 2–3 days to ensure the effectiveness of the metabolite intervention;

[0130] (8) The lifespan of all nematodes was recorded and compared with that of the control group.

[0131] 2. Evaluation of motor ability during aging of nematodes

[0132] (1) Obtaining synchronized eggs: Select several adult worms, place them on an empty NGM plate, and wait for them to lay eggs;

[0133] (2) Obtaining synchronized larvae: The synchronized eggs are hatched overnight, and synchronized nematodes can be obtained the next day;

[0134] (3) Obtaining synchronized adults: Cultivating at 22°C, the synchronized adults are obtained after 2-3 days of antennae development.

[0135] (4) Metabolite coating NGM plates: 300 μL of metabolite standard solution was coated on the NGM plates. The specific metabolite dosages were as follows: 2 mmol / L hypotaurine; 1 mmol / L creatine phosphate; 0.1 mmol / L phosphoethanolamine. These metabolites are components already present in the nematode body and are endogenous intervention measures.

[0136] (5) Transfer synchronized adults to NGM plates pre-coated with metabolites;

[0137] (6) During the experiment, the NGM plates coated with metabolites were replaced every 2–3 days to ensure the effectiveness of the metabolite intervention;

[0138] (7) On the 9th day after adulthood, transfer the nematodes to the buffer solution. After acclimation for 1 minute, observe the nematodes' swinging motion under a stereoscope and record the number of swings in 1 minute. The more swings, the better the motility.

[0139] 2. Test results

[0140] 1. Hypotaurine (Hypt) has the effect of extending the lifespan of nematodes and has specific dosage requirements (such as Figure 7 (shown)

[0141] Metabolic intervention with 5mM and 2mM hypotaurine can effectively extend the lifespan of nematodes, but 2mM hypotaurine is more effective and has specific dosage requirements. 2mM hypotaurine treatment increased the median lifespan of nematodes by 46.7% and the maximum lifespan by 20.8% relative to the control group; 5mM hypotaurine treatment increased the median lifespan of nematodes by 13.3% and the maximum lifespan by 16.7% relative to the control group, which is significantly lower than the 2mM treatment. Therefore, hypotaurine treatment requires a specific concentration to achieve the optimal anti-aging effect.

[0142] 2. Phosphocreatine (PCr) has the effect of extending the lifespan of nematodes and has specific dosage requirements (such as Figure 8 (shown)

[0143] Creatine phosphate can also extend the lifespan of nematodes, but there is also a specific dose effect. Metabolic intervention with 5mM creatine phosphate only increased the median lifespan of nematodes by 33.3% relative to the control group, but the maximum lifespan did not increase. Metabolic intervention with 1mM creatine phosphate increased the median lifespan of nematodes by 46.7% and the maximum lifespan by 25%, and the anti-aging effect was significantly better than that of the 5mM creatine phosphate intervention group. This shows that creatine phosphate has a good lifespan extension effect at a specific concentration.

[0144] 3. Phosphoethanolamine (O-PE) has the effect of extending the life span of nematodes and has specific dosage requirements (such as Fig. 9 (shown)

[0145] Metabolic intervention using different doses of phosphoethanolamine also showed anti-aging effects at specific doses. Metabolic intervention using 0.5mM and 0.1mM phosphoethanolamine respectively showed that only 0.1mM phosphoethanolamine had anti-aging effects, which was better than the above-mentioned various metabolite interventions, with the median lifespan increased by up to 53.3% and the maximum lifespan increased by up to 50%. However, 0.5mM phosphoethanolamine had almost no anti-aging effect. This shows that phosphoethanolamine only exerts anti-aging effects at specific concentrations.

[0146] 4. All three metabolites have the ability to improve the motility of nematodes in the middle stage of aging (e.g. Fig.10 (shown)

[0147] The nematodes in the control group showed a decline in their motor ability during the aging process. On the 9th day after adulthood (mid-aging), the nematodes were tested for their motor ability and found that their motor ability was significantly reduced compared to that of young nematodes. However, the motor ability of the nematodes that had undergone phosphoethanolamine metabolic intervention on the 9th day after adulthood was at the same level as that of the control nematodes when they were young, indicating that phosphoethanolamine intervention significantly improved the motor ability of nematodes during aging and protected their body functions. Although the effects of hypotaurine and creatine phosphate metabolic intervention were not as obvious as those of phosphoethanolamine, they could also improve the motor ability of nematodes during aging to a certain extent.

[0148] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, rather than to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions of the technical solution of the present invention by ordinary technicians in this field do not deviate from the essence and scope of the technical solution of the present invention.

Claims

1. Application of endogenous metabolites in the preparation of drugs for treating and / or preventing aging.

2. The use according to claim 1, characterized in that: The cell endogenous metabolite is any one or a combination of creatine phosphate, hypotaurine and / or ethanolamine phosphate.

3. The use according to claim 2, characterized in that: The endogenous metabolite of the cell is creatine phosphate, and the concentration of creatine phosphate in the drug for treating and / or preventing aging is 0.2-5mmol / L.

4. The use according to claim 2, characterized in that: The endogenous metabolite of the cell is hypotaurine, and the concentration of hypotaurine in the drug for treating and / or preventing aging is 0.25-10 mmol / L.

5. The use according to claim 2, characterized in that: The cell endogenous metabolite is phosphoethanolamine, and the concentration of phosphoethanolamine in the drug for treating and / or preventing aging is 0.01-0.5 mmol / L.

6. The use according to claim 2, characterized in that: The endogenous cell metabolites are a combination of creatine phosphate, hypotaurine and ethanolamine phosphate, and the concentration ratio of creatine phosphate, hypotaurine and ethanolamine phosphate in the drug for treating and / or preventing aging is 12.5:25:

1.

7. The use according to claim 6, characterized in that: The concentration of creatine phosphate, the concentration of hypotaurine, and the concentration of ethanolamine phosphate in the drug for treating and / or preventing aging are 0.1 mmol / L, 0.2 mmol / L, and 8 μmol / L, respectively.

8. A drug for treating and / or preventing aging, characterized in that: The medicine contains any one or a combination of creatine phosphate, hypotaurine and / or ethanolamine phosphate.

9. The use according to claim 8, characterized in that: The concentration ratio of creatine phosphate, hypotaurine and phosphoethanolamine in the medicine is 12.5:25:

1.

10. The use according to claim 9, characterized in that: The medicine also includes a pharmaceutically acceptable carrier, and the pharmaceutically acceptable carrier is any one or a combination of a binder, a disintegrant, a lubricant, and a glidant.

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