Use of intracellular endogenous metabolites for the preparation of a medicament for the treatment and / or prevention of aging

Abstract

The application discloses application of cell endogenous metabolites in preparation of medicines for treating and / or preventing aging. The cell endogenous metabolites are any one or combination of phosphocreatine, hypotaurine and / or phosphoethanolamine. The application finds that phosphocreatine, hypotaurine and phosphoethanolamine can effectively restore cell growth, reduce cell SA-beta-Gal staining intensity, improve cell mitochondrial function and increase ATP yield, restore cell mitochondrial membrane potential, alleviate damage caused by cell oxidative stress and premature aging, and also prolong the life of nematodes and restore exercise capacity in the middle of aging. Meanwhile, the three metabolites have a synergistic effect, and by adjusting the matching concentration of the three metabolites, the same cell protection effect can be achieved by mixed administration. Since the endogenous metabolites are accurate, efficient and safe, the three cell endogenous metabolites provided by the application can be used for preparing anti-aging medicines.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of medicine, in particular to the application of intracellular endogenous metabolites in the preparation of drugs for treating and / or preventing aging. BACKGROUND

[0002] With the continuous intensification of social aging, anti-aging, life extension and healthy aging have become one of the popular research directions. Cell aging is an important reason for individual aging and tissue degeneration, and the regulation of cell aging has become an important link for anti-aging and intervention of aging-related diseases. However, cell aging involves the regulation of multiple levels and factors such as gene expression, epigenetic modification, signal pathway cascade reaction, and is related to multiple levels of mechanisms such as metabolic small molecules, biological macromolecules and organelles, and is a very complex process, and its detailed mechanism has not been fully elucidated.

[0003] Cell aging is the result of continuous accumulation of cell damage, and oxidative stress is a common cell damage and can induce premature aging of cells (premature senescence), which is a special form of cell aging. Current research and technology development are mainly exogenous intervention, including small molecule inhibitors, signal pathway modulators and other drugs, and artificially synthesized drugs, etc. These exogenous intervention means improve cell aging and have certain efficacy.

[0004] For example, patent CN202080042529.3 discloses extracellular vesicles from human liver stem cells (HLSC-EV) for reducing cell senescence, which uses human liver stem cell-derived extracellular vesicles to prepare a preparation that can improve cell senescence and related diseases in in vitro and ex vivo experiments. For example, patent CN202111376415.5 discloses the application of mannose glucuronan oligosaccharides and polysaccharides and derivatives in the preparation of drugs for treating and / or preventing aging, which has prepared a series of mannose glucuronan oligosaccharides and polysaccharides and derivatives, which have anti-aging effects. For example, patent CN202111499257.2 discloses an anti-aging composition and its application, which has prepared 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 ratio, 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 subject's central nervous system cognitive impairment or neuroinflammation related to aging. For example, patent CN202010819194.3 discloses a molecular marker related to aging and health and its application in improving healthy aging, which discloses that knocking down FABP4 gene expression or inhibiting FABP4 activity in old mice can significantly improve cognitive function, inhibit aging and related inflammation indicators, suggesting that FABP4 gene has the effect of promoting aging, and that the inhibitor of FABP4 gene can improve the cognitive function of animals and has anti-aging effect.

[0005] The above patents have achieved certain effects by exogenous intervention methods to intervene and regulate aging. However, these methods also have some problems. For example, the safety of small molecule drugs, such as inhibitors, which regulate genes and signaling pathways. However, due to the interaction between genes, the network regulation characteristics of signaling pathways, and the targeting of drug molecules, the range of their action may be wider than what is known, and their effects may be more complex and extensive than what is known, making it difficult to rule out 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 body itself, and it is necessary to find endogenous metabolites that can restore the vitality of senescent cells, which can provide a new direction for aging research.

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

[0007] Although there are a large number of documents in recent years that report the use of small molecule inhibitors and other drugs to effectively extend the lifespan of nematodes, the mechanism is mainly related to the regulation of cell signaling pathways. However, such inhibitors have a wide range of effects, and their effects are complex and extensive. As exogenous drugs, their safety needs to be studied, and there are few studies on the regulation of nematode lifespan by endogenous metabolites. At the same time, nematode motor ability is an important indicator of aging. Previous studies have mainly extended the lifespan of nematodes, but few have reported improvements in the activity of aging nematodes. Improving and protecting body functions during the aging process is an important aspect of aging research, but there are few studies in this area. Therefore, discovering endogenous metabolites that can extend the lifespan of nematodes and improve the activity of aging nematodes can also provide a new direction for aging research. SUMMARY

[0008] In view of the deficiencies in the prior art, the present application provides a technology for intervening in cell aging by using endogenous metabolites. The technology directly uses existing metabolites in cells to resist oxidative stress damage and slow down cell aging. It can also extend the lifespan of nematodes while delaying the decline of physiological functions, and has the characteristics of safety and efficiency.

[0009] To solve the above technical problems, the present application provides the following technical solutions:

[0010] The present application uses the method of supplementing key metabolites to the culture medium for metabolic intervention. When the induction of cells is completed, the culture medium containing metabolites is replaced. The level of oxidative stress and premature aging of cells is evaluated by 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 in cells, phosphocreatine, hypotaurine and O-PE, can effectively restore cell growth, reduce cell SA-β-Gal staining intensity, improve cell mitochondrial function and increase ATP yield, restore cell mitochondrial membrane potential, and alleviate the damage caused by cell oxidative stress and premature aging.

[0011] In addition, the present application finds that the three metabolites have a synergistic effect through the method of mixed metabolic intervention, and the same cell protection effect can be achieved by adjusting the concentration ratio of the three metabolites, i.e. using 1 / 5 of the optimal concentration of each of the three metabolites (it is detected that the effect is not as good as the mixed effect of 1 / 5 of the optimal concentration under other mixing ratios), and then performing mixed administration.

[0012] In addition, the present application finds that the three metabolites have a synergistic effect through the method of mixed metabolic intervention, and the same cell protection effect can be achieved by adjusting the concentration ratio of the three metabolites, i.e. using 1 / 5 of the optimal concentration of each of the three metabolites (it is detected that the effect is not as good as the mixed effect of 1 / 5 of the optimal concentration under other mixing ratios), and then performing mixed administration.

[0013] Therefore, In one aspect, The present application provides an application of a cell endogenous metabolite in preparing a medicine for treating and / or preventing aging.

[0014] Preferably, the cell endogenous metabolite is any one or a combination of phosphocreatine, hypotaurine and / or O-PE.

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

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

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

[0018] More preferably, in another specific embodiment, the endogenous metabolite of the cell 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 more further preferably 0.04 mmol / L.

[0019] More preferably, in another specific embodiment, the endogenous metabolite of the cell is a combination of phosphocreatine, hypotaurine and phosphoethanolamine, and the concentration ratio of phosphocreatine, hypotaurine and phosphoethanolamine in the drug for treating and / or preventing aging is 12.5:25:1. Further preferably, the concentration of phosphocreatine 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] More preferably, the drug is a drug for prolonging the life of nematodes or improving the movement ability of aging nematodes.

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

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

[0024] More preferably, in another specific embodiment, the endogenous metabolite of the cell 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 more further preferably 0.1 mmol / L.

[0025] In another aspect, The present application also provides a drug for treating and / or preventing aging, which contains a combination of any one or more of phosphocreatine, hypotaurine and / or phosphoethanolamine.

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

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

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

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

[0030] (1) The three endogenous metabolites of cells, creatine phosphate, hypotaurine and phosphoethanolamine, provided by the present application can effectively restore cell growth, reduce cell SA-β-Gal staining intensity, improve cell mitochondrial function and increase ATP yield, restore cell mitochondrial membrane potential, alleviate damage caused by cell oxidative stress and premature aging at specific concentrations. Meanwhile, the three metabolites have a synergistic effect, and the same cell protection effect can be achieved by adjusting the ratio of the concentrations of the three metabolites and mixing administration. Therefore, the three endogenous metabolites of cells provided by the present application have a significant anti-aging effect, have small toxic and side effects, are safe and effective, and can be used for preparing anti-aging drugs.

[0031] (2) By using the endogenous metabolites of creatine phosphate, hypotaurine and phosphoethanolamine for intervention, it is found that the three metabolites have the effects of prolonging the lifespan of nematodes and restoring the exercise capacity of nematodes in the middle stage of aging at specific concentrations, and promoting the healthy aging of nematodes. Since the endogenous metabolites have the characteristics of accuracy, high efficiency and safety, the three endogenous metabolites of cells provided by the present application can be used for preparing nematode anti-aging drugs, can prolong the lifespan of nematodes or improve the exercise capacity of aging nematodes, and provide a new direction for aging research. BRIEF DESCRIPTION OF DRAWINGS

[0032] Figure 1 Results of using different concentrations of H2O2 to induce premature aging of cells.

[0033] Figure 2 Results of creatine phosphate intervention alleviating premature aging of cells induced by oxidative stress. Wherein, a is cell density, and b is the staining intensity of senile galactosidase.

[0034] Figure 3 Results of hypotaurine intervention alleviating premature aging of cells induced by oxidative stress. Wherein, a is cell density, and b is the staining intensity of senile galactosidase.

[0035] Figure 4 Results of phosphoethanolamine intervention alleviating premature aging of cells induced by oxidative stress. Wherein, a is cell density, and b is the staining intensity of senile galactosidase.

[0036] Figure 5 The results of intervention to alleviate aging by administering three metabolites mixedly. Wherein, a is cell density, and b is aging galactosidase staining intensity.

[0037] Figure 6 The results of intervention to improve cell mitochondrial function by administering three metabolites respectively at the optimal concentration.

[0038] Figure 7 The results of the test of alleviating aging of nematodes by different doses of subsulfocine.

[0039] Figure 8 The results of the test of alleviating aging of nematodes by different doses of phosphocreatine.

[0040] Figure 9 The results of the test of alleviating aging of nematodes by different doses of phosphoethanolamine.

[0041] Figure 10 The results of the test of protecting the movement ability of nematodes by three metabolites. DETAILED DESCRIPTION

[0042] The present application will be further described below in conjunction with specific examples. The following examples are not used to limit the present application, but only to illustrate the present application. Unless otherwise specified, the experimental methods used in the following examples are generally performed according to conventional conditions. Unless otherwise specified, the materials, reagents, etc. used in the following examples can be obtained from commercial channels.

[0043] Terms and explanations:

[0044] In the present application, the term "premature senescence" refers to a special aging form of normal dividing cells that enters aging prematurely under the stimulation of external damage factors, showing growth inhibition, increased expression of senescence-associated galactosidase, etc.

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

[0046] In the present application, the term "senescence-associated galactosidase (SA-β-Gal)" is a specific enzyme of senescent cells, which is used as a marker of cell senescence.

[0047] In the present application, the term "Caenorhabditis elegans (C. elegans)" is a kind of worm isolated from soil, which has a short life cycle and is commonly used as a model for aging research.

[0048] In the present application, the term "Thrashing" refers to the movement of the nematode in the buffer solution, and each movement of about 180 degrees is counted as one thrashing, which is an evaluation index of the movement ability of the nematode.

[0049] In the present application, the term "metabolic intervention" refers to the intervention method of adding specific metabolites to the culture plate when the nematode enters the adult stage.

[0050] In the present application, the term "NGM plate" refers to a plate prepared with nematode growth medium as the nematode feeding environment.

[0051] Example 1 Cell Anti-aging Related Test - Single Metabolite Administration and Mixed Administration

[0052] The test method steps used in this example are as follows:

[0053] 1. Cell culture

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

[0055] 2. Cell aging induction

[0056] Cells were treated with 80 micromolar hydrogen peroxide for 1 hour, fresh medium was replaced, and 48 hours were restored, which completed the modeling of aging induction.

[0057] 3. Cell oxidative stress detection

[0058] DCFDA fluorescent probe was used for detection, the probe was used at a concentration of 10 micromolar in the detection buffer, incubated for 25 minutes, and then washed with buffer to remove the probe. Observed and photographed under a fluorescence microscope.

[0059] 4. Cell aging staining

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

[0061] 5. Cell metabolic intervention

[0062] The three intracellular endogenous metabolite standards described in the present application were added to the culture medium for metabolic intervention.

[0063] 6. Single cell mass spectrometry detection

[0064] The detection was performed using single cell mass spectrometry technology, and the mass spectrometry detection related parameters were as follows: ion source voltage about 4 kV, 500 Hz, capillary temperature 275℃, radio frequency intensity 50%, mass spectrometry detection resolution 70000, ion injection time 10 milliseconds, and scanning mode negative example mode.

[0065] 7、Mitochondrial function detection

[0066] The detection was performed using seahorse cell metabolism detector (XF96), the change of cell oxygen consumption rate after adding oligomycin (1 μM), FCCP (1.5 μM), rotenone / antimycin (1 μM) was monitored, seahorse software was used to calculate ATP yield and respiratory efficiency, and then the mitochondrial function of cells was evaluated.

[0067] 8、Mitochondrial membrane potential detection

[0068] The detection was performed using TMRE mitochondrial membrane potential fluorescent probe to evaluate the damage of mitochondrial membrane potential and the recovery thereof by metabolic intervention. According to the kit instruction, 1x TMRE staining working solution was prepared using serum-free DMEM medium, the cells were incubated for 15 minutes, the cells were washed once with PBS, the serum-free DMEM medium was replaced, and the photographing was performed under a fluorescence microscope.

[0069] 9、Image processing and analysis

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

[0071] 10、Cell metabolome data analysis

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

[0073] The specific test conditions were as follows:

[0074] I. Test method

[0075] 1. HEK293T cells were planted in 3.5 mm culture dishes;

[0076] 2. After the cells were grown overnight, the cell density was confirmed, about 50%;

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

[0078] 4. Replace the culture medium with one containing a specific metabolite, and administer three different endogenous cellular metabolites separately or in combination.

[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 then perform aging-related galactosidase assays.

[0081] II. Test Results

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

[0083] like Figure 1 As shown, treatment of HEK293T cells with H2O2 concentrations ranging from 50 μmol / L to 120 μmol / L induced premature senescence. Furthermore, the intensity of senescence-associated galactosidase (SA-β-Gal) was positively correlated with the H2O2 treatment concentration, indicating that H2O2 treatment within this concentration range successfully induced premature senescence. Therefore, the model of premature senescence induced by oxidative stress is stable and reliable.

[0084] 2. Phosphocreatine metabolic intervention trial

[0085] The phosphocreatine dosage sequence design is as follows (e.g.) Figure 2 (As shown): 0.1 mmol / L, 0.25 mmol / L, 0.5 mmol / L, 1 mmol / L.

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

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

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

[0089] 3. Hypotaurine metabolic intervention trial

[0090] The dosage sequence of hypotaurine is as follows (as shown in the table): 0.25 mmol / L, 0.5 mmol / L, 1 mmol / L, and 2 mmol / L. Figure 3

[0091] Through the above test method, under the treatment of hypotaurine at each dose, the growth inhibition caused by cell aging is relieved. Among them, 1 mmol / L hypotaurine has the best effect, and the cell density after 48 hours is closest to the control group.

[0092] Cell aging galactosidase staining shows that, except for 2 mmol / L hypotaurine which fails to improve cell staining intensity, the rest of the doses are effective in improving the staining intensity of aging galactosidase. Among them, 1 mmol / L phosphocreatine has the best effect, and the staining intensity of aging galactosidase is the lowest.

[0093] Therefore, when using hypotaurine alone to intervene in cell aging, the best dose is 1 mmol / L.

[0094] 4. Phosphoethanolamine (O-PE) metabolism intervention test

[0095] The dosage sequence of phosphoethanolamine is as follows (as shown in the table): 10 μmol / L, 20 μmol / L, 40 μmol / L, and 80 μmol / L. Figure 4

[0096] Through the above test method, except for 20 μmol / L phosphoethanolamine which fails to effectively relieve the growth inhibition caused by cell aging, the rest of the doses are effective in relieving the growth inhibition caused by cell aging. Among them, 40 μmol / L and 80 μmol / L have better effects, and the cell density after 48 hours is close to the control group.

[0097] Cell aging galactosidase staining shows that 40 μmol / L phosphoethanolamine effectively reduces the staining intensity of aging galactosidase, which is closest to the control group.

[0098] Therefore, when using phosphoethanolamine alone to intervene in cell aging, the best dose is 40 μmol / L.

[0099] 5. Mixed administration metabolism intervention test

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

[0101] ​​Specifically, the optimal concentration of each of the three metabolites is 1 / 5, i.e., 0.1 mmol / L of creatine phosphate, 0.2 mmol / L of hypotaurine, and 8 μmol / L of phosphoethanolamine, and the ratio of the three is 12.5:25:1. When mixed and administered, the same cell protection effect as when the metabolites are administered alone can be achieved. Growth analysis and cell senescence-related galactosidase staining results show that mixed administration at 1 / 5 of the optimal concentration greatly restores cell growth and effectively reduces the intensity of cell senescence-related galactosidase staining. At a lower total dose, the same effect as that of single metabolite administration is achieved, and the safety is higher.

[0102] Due to the synergistic effect, the alleviation of aging is achieved at other mixing ratios (as shown in Figure 5 ). Mixed administration at 1 / 4 of the optimal concentration can alleviate aging. Mixed administration at 1 / 8 of the optimal concentration cannot alleviate cell aging due to a too low dose. In order to reduce the total dose as much as possible, mixed administration at 1 / 5 of the optimal concentration is selected as the optimal ratio.

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

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

[0105] The optimal concentrations of the three metabolites are used for intervention, and the oxygen consumption rate of the cells is analyzed using a cell metabolism detector, as shown in Figure 6 . The baseline respiration level is measured in the initial stage, and after the addition of oligomycin, the ATP synthesis is inhibited, the oxygen consumption rate decreases, and the degree of decrease in the oxygen consumption rate reflects the ability of ATP synthesis. It can be seen that phosphoethanolamine and hypotaurine improve the baseline respiration level and greatly improve the ability of ATP synthesis of the cells, and therefore it is presumed that this is a related mechanism of the metabolic intervention in alleviating cell aging.

[0106] Example 2. Intervention of endogenous metabolites in aging and improvement of exercise capacity of nematodes

[0107] The test method steps used in this example are as follows:

[0108] 1. Nematode culture

[0109] The nematode strain is N2, i.e., the wild type. All experimental nematodes are cultured at 20°C, and OP50 E. coli is used as the nematode food. For the metabolic intervention life span analysis experiment, inactivated OP50 E. coli is used as the food to exclude the interference caused by the metabolism thereof.

[0110] 2. Synchronization of nematodes

[0111] L4 and young adults were selected for egg collection using bleach method: NGM plates were rinsed with sterile water, and worms were captured and placed in a centrifuge tube. The tube was filled with 3.5 mL of sterile water, and a bleach solution (0.5 mL of 5 M NaOH + 1 mL of 5% NaClO) was prepared and added to the tube. The tube was shaken and allowed to stand for 2 minutes, and the shaking and standing were repeated for a total of 10 minutes. The tube was centrifuged at 1300 g for 30 seconds, and the supernatant was discarded. The tube was filled with 5 mL of sterile water, shaken, and centrifuged at 1300 g for 5 seconds to obtain sterile eggs, which were inoculated into NGM medium. Synchronized larvae were obtained the next day.

[0112] 3. Preparation of metabolic intervention plates

[0113] 300 μL of a standard solution of the metabolite was applied to the NGM plate, which was prepared by diluting a stock solution of a specific concentration with sterile water to the administration concentration, and incubated overnight. Inactivated OP50 E. coli was also uniformly applied to the plate. All metabolite-coated NGM plates were prepared at least 12 hours before the start of the experiment.

[0114] 4. Analysis of nematode lifespan

[0115] NGM plates were pre-incubated with 0.1 mg / mL 5'-FUDR before the start of the experiment to inhibit the hatching of eggs and prevent interference with the lifespan analysis experiment. When nematodes stopped laying eggs after reaching adulthood and began to age, 5'-FUDR was stopped on the NGM plate. Nematodes that developed to the L4 stage were transferred to the prepared metabolic intervention plate, and the plate was changed every 2-3 days to ensure the effectiveness of the metabolic intervention. Nematodes were observed under a microscope every 1-2 days, and the number of surviving and dead nematodes was recorded. The survival rate was calculated based on the number of surviving and dead nematodes, and survival analysis was performed. Nematodes that crawled out of the plate, accidentally burst, or hatched larvae inside the body were excluded from the experiment.

[0116] 5. Nematode plate transfer

[0117] Multiple sedimentation was used for plate transfer in the lifespan analysis experiment. The plate was washed with M9 buffer, and the nematodes were captured and transferred to a centrifuge tube. The nematodes were allowed to naturally sediment using gravity, which removed impurities and accidentally hatched larvae, ensuring the stability of the synchronized system.

[0118] 6. Measurement of nematode motility

[0119] Nematodes were transferred to M9 buffer in a 24-well plate and allowed to adapt for 30 seconds. The number of times the nematodes moved left and right in the buffer was observed under a stereomicroscope, and the number of movements per minute was calculated to compare the motility of the nematodes.

[0120] The specific test conditions were as follows:

[0121] I. Main experimental steps

[0122] 1. Life span analysis

[0123] (1) Obtain synchronized eggs: select several adults and place them on empty NGM plates to lay eggs;

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

[0125] (3) Obtain synchronized adults: cultivate at 22°C, and the nematodes develop completely in 2-3 days to obtain synchronized adults;

[0126] (4) Metabolite-coated NGM plates: coat 300 μL of metabolite standard solution on NGM plates, and the specific metabolite doses are as follows: hypotaurine 2 mmol / L; creatine phosphate 1 mmol / L; phosphoethanolamine 0.1 mmol / L. These metabolites are components already present in nematodes and are endogenous intervention means;

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

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

[0129] (7) Replace the NGM plates coated with metabolites every 2-3 days during the experiment to ensure the effect of metabolite intervention;

[0130] (8) Record the life span of all nematodes and compare them with the control group.

[0131] 2. Evaluation of movement ability during nematode aging process

[0132] (1) Obtain synchronized eggs: select several adults and place them on empty NGM plates to lay eggs;

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

[0134] (3) Obtain synchronized adults: cultivate at 22°C, and the nematodes develop completely in 2-3 days to obtain synchronized adults;

[0135] (4) Metabolite-coated NGM plates: coat 300 μL of metabolite standard solution on NGM plates, and the specific metabolite doses are as follows: hypotaurine 2 mmol / L; creatine phosphate 1 mmol / L; phosphoethanolamine 0.1 mmol / L. These metabolites are components already present in nematodes and are endogenous intervention means;

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

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

[0138] (7) On the 9th day after adulthood, the nematodes were transferred into buffer solution, and after 1 minute of adaptation, the number of body bends was observed under a stereomicroscope and recorded within 1 minute. The more the number of body bends, the better the movement ability.

[0139] II. Experimental results

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

[0141] Metabolic intervention of nematodes with 5 mM and 2 mM hypotaurine can effectively prolong the lifespan of nematodes, but 2 mM hypotaurine is better. There is a specific dosage requirement. 2 mM hypotaurine treatment increased the median lifespan of nematodes by 46.7% and the maximum lifespan by 20.8% compared with the control group; 5 mM hypotaurine treatment increased the median lifespan of nematodes by 13.3% and the maximum lifespan by 16.7% compared with the control group, which was significantly lower than that of 2 mM treatment. Therefore, specific concentration of hypotaurine treatment is required to obtain the optimal anti-aging effect.

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

[0143] Phosphocreatine can also prolong the lifespan of nematodes, but there is a specific dosage effect. Metabolic intervention with 5 mM phosphocreatine only increased the median lifespan of nematodes, which was 33.3% higher than the control group, and the maximum lifespan did not increase. Metabolic intervention with 1 mM phosphocreatine increased the median lifespan of nematodes by 46.7% and the maximum lifespan by 25%, which was significantly better than the 5 mM phosphocreatine intervention group. This indicates that phosphocreatine has a good lifespan prolonging effect at a specific concentration.

[0144] 3. O-phosphoethanolamine (O-PE) has the effect of prolonging the lifespan of nematodes and has specific dosage requirements (as shown in Figure 9 ).

[0145] Metabolic intervention using different doses of ethanolamine phosphate also showed anti-aging effect at specific doses. Metabolic intervention using 0.5 mM and 0.1 mM ethanolamine phosphate respectively, only 0.1 mM ethanolamine phosphate had anti-aging effect, and its effect was better than that of various metabolite interventions described above, with an increase of 53.3% in median lifespan and an increase of 50% in maximum lifespan. But 0.5 mM ethanolamine phosphate had almost no anti-aging effect. It is shown that ethanolamine phosphate only has anti-aging effect at specific concentration dose.

[0146] 4. All three metabolites can improve the movement ability of nematodes in the middle of aging (as shown in Figure 10

[0147] The control group of nematodes showed a decrease in movement ability during aging, and the movement ability of the nematodes was detected at the 9th day after the nematodes became adults (middle of aging), and it was found that the movement ability of the nematodes was significantly decreased compared with that of young nematodes. The movement ability of the nematodes intervened by ethanolamine phosphate at the 9th day after becoming adults was at the same level as that of the control group of nematodes when they were young, indicating that the intervention of ethanolamine phosphate greatly improved the movement ability of the nematodes during aging, and the physical function of the nematodes was protected. After metabolic intervention of hypotaurine and creatine phosphate, although the effect was not as obvious as that of ethanolamine phosphate, the movement ability of the nematodes during aging was also improved to a certain extent.

[0148] Finally, it should be noted that the above content is only used to illustrate the technical solutions of the present application, and is not a limitation on the protection scope of the present application. Simple modifications or equivalent replacements of the technical solutions of the present application made by those skilled in the art do not deviate from the essence and scope of the technical solutions of the present application.​

Claims

1. The application of endogenous cellular metabolites in the preparation of anti-aging drugs, characterized in that, The cellular endogenous metabolites are a combination of creatine phosphate, taurine, and ethanolamine phosphate, and the concentration ratio of creatine phosphate, taurine, and ethanolamine phosphate in the anti-aging drug is 12.5:25:

1.

2. The application according to claim 1, characterized in that, The anti-aging drug contains 0.1 mmol / L creatine phosphate, 0.2 mmol / L taurine, and 8 μmol / L ethanolamine phosphate.

3. A drug for preventing aging, characterized in that, The concentration ratio of creatine phosphate, taurine, and ethanolamine phosphate in the drug is 12.5:25:

1.

4. The drug according to claim 3, characterized in that, The drug also includes a pharmaceutically acceptable carrier, which is any one or a combination of several of the following: binders, disintegrants, lubricants, and glidants.

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