Anti-aging drug screening method based on mitochondrial multilevel analysis-targeted intervention

Abstract

The invention discloses an anti-aging drug screening method based on mitochondrial multilevel analysis-targeted intervention, and particularly relates to the technical field of biological drug manufacturing. The method comprises the following steps: constructing a rotenone-induced aging human mesenchymal stem cell model; the ATP level fluctuation, the active oxygen level, the mitochondrial network fusion and division dynamics and the autophagy flux are monitored in real time by using a fluorescent probe; calculating a baseline function entropy through multi-dimensional data analysis; evaluating the influence of the candidate drugs on the dynamic response entropy of the senescent cells under the condition of mild oxidative stress; an optimal drug is screened and combined with spermidine or urolithin A in a low dosage, and the synergistic effect of the drug in the aspects of maintaining low functional entropy, promoting removal of damaged mitochondria, not causing excessive autophagy and the like is verified. According to the method, multi-dimensional and complete-cycle mitochondrial function evaluation is realized through functional entropy quantification and dynamic stress recovery testing, the method has the advantages of high screening efficiency, objective result and good safety, and a new strategy is provided for the development of anti-aging drugs.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine manufacturing, and relates to a screening method of anti-aging drugs based on mitochondrial multi-level analysis-targeted intervention. BACKGROUND

[0002] In the current research on screening of anti-aging drugs, mitochondria, as the core organelle of cell energy metabolism and aging regulation, the evaluation of its functional state is mostly dependent on single-dimensional detection indicators such as ATP content, ROS level or mitochondrial membrane potential. Although such methods are simple to operate, they are difficult to comprehensively reflect the overall functional state of mitochondria under dynamic stress conditions and its quality control mechanism. For example, only a certain static indicator cannot effectively distinguish the comprehensive influence of drugs on the mitochondrial network structure, autophagic clearance efficiency and antioxidant defense system, etc., leading to limited effect or greater side effects of the screened drugs in the actual physiological environment. In addition, the traditional screening system lacks systematic comparison of the selective action of drugs between aging cells and young cells, making it difficult to rule out the potential toxicity to normal cells, further limiting its clinical conversion potential.

[0003] In view of the above problems, the present application provides a screening method of anti-aging drugs based on mitochondrial multi-level analysis and targeted intervention, which realizes comprehensive evaluation of the whole cycle and multiple angles of mitochondrial function by integrating real-time monitoring of four dimensions of ATP fluctuation, antioxidant capacity, mitochondrial network fusion and splitting dynamics and autophagy flux, and introducing functional entropy as a system quantitative indicator. This method not only can systematically screen candidate drugs with recovery ability under dynamic stress conditions, but also can enhance its safety and synergistic effect through low-dose combination drug strategy, thereby effectively overcoming the technical bottleneck of single evaluation dimension in the traditional screening system, which cannot systematically screen high-efficiency and low-toxicity anti-aging drugs. SUMMARY

[0004] In order to overcome the above-mentioned defects of the prior art, the present application provides a screening method of anti-aging drugs based on mitochondrial multi-level analysis-targeted intervention, which solves the problem of single evaluation dimension of mitochondrial function and the inability to systematically screen high-efficiency and low-toxicity anti-aging drugs in the prior art.

[0005] To achieve the above-mentioned purpose, the present application provides the following technical scheme: A screening method of anti-aging drugs based on mitochondrial multi-level analysis-targeted intervention, specifically comprising the following steps: S1: Take a part of standard human mesenchymal stem cells, treat them with low-dose rotenone to induce them into premature aging state, and construct them into an aging cell model; at the same time, take the other half of young cells as a control group without treatment; S2: Fluorescent ATP indicators were added to senescent cell models and young cell models respectively to monitor ATP level fluctuations in real time; at the same time, the antioxidant capacity of both was assessed using specific probes. S3: Using mitochondrial-targeted photoactivated fluorescent proteins, the fusion and division dynamics of the mitochondrial network were analyzed using fluorescence bleaching recovery technology; simultaneously, mitochondrial-targeted fluorescent proteins co-localized with LC3 protein were used to directionally analyze drug-induced mitochondrial autophagy flux. S4: Real-time monitoring of aging and youth models for 24 hours without intervention, followed by analysis of data from each dimension and integration to calculate a single baseline functional entropy value; S5: The cell models were divided into four groups: senescent cell drug administration group, senescent cell drug-free group, young cell drug administration group, and young cell drug-free group; then, the candidate drug was added to the cell models of the corresponding groups in a micro-liter array, and after incubation for 48 hours, mild oxidative stress was applied to all groups; after the stress was applied, a multi-level real-time monitoring system was immediately started for continuous monitoring; S6: Analyze the data from stress application to recovery and calculate the dynamic response entropy; select the best drug from the candidate drugs accordingly, then combine the selected best drug with an autophagy inducer at a low dose, and repeat the steps S1 to S4 of claim 1 using the drug combination to conduct a dynamic intervention response test. S7: From the test results, select drug combinations that can maintain stable low functional entropy, accelerate the clearance of mitochondria with high functional entropy after stress testing, and do not cause excessive autophagy leading to cell damage.

[0006] Preferably, the fluorescent ATP indicator in S2 is a quinoline fluorescent protein complex or a luciferase-based ATP probe; the probe used to assess antioxidant capacity is MitoSOX Red.

[0007] Preferably, in S3, when analyzing mitochondrial network dynamics using fluorescence bleaching recovery technology, the mitochondrial-targeted photoactivated fluorescent protein used is mitochondrial-targeted PA-GFP.

[0008] Preferably, the dimensions involved in S4 and S6 include ATP level fluctuations, antioxidant capacity, mitochondrial network fusion and division dynamics, and mitophagy flux.

[0009] Preferably, the mild oxidative stress described in S5 is achieved by depriving serum for 2 hours, thereby reducing the serum concentration in the culture medium to 0.5%–2%.

[0010] Preferably, the mitochondrial nutrients are selected from one or more of coenzyme Q10, L-carnitine, lipoic acid, pyrroloquinoline quinone, nicotinamide ribose, and nicotinamide mononucleotide; the natural compounds are selected from one or more of resveratrol, curcumin, quercetin, epigallocatechin gallate, and ginsenosides; and the FDA-approved drugs are selected from one or more of metformin, rapamycin, acarbose, and aspirin.

[0011] Preferably, the mild oxidative stress described in S5 is achieved by depriving serum for 2 hours, thereby reducing the serum concentration in the culture medium to 0.5%–2%.

[0012] Preferably, the optimal drug in S6 refers to the candidate drug that, in the dynamic intervention response test, reduces the dynamic response entropy of senescent cells the most and has the least impact on the mitochondrial function of young cells.

[0013] Preferably, in S7, not inducing excessive autophagy leading to cell damage means that the cell viability is determined by detecting the activity of lactate dehydrogenase in the culture medium, and the decrease in cell viability does not exceed 10%.

[0014] Preferably, the autophagy inducer in S7 is spermidine or urolithin A; the optimal drug is combined with spermidine and urolithin A in low doses.

[0015] The technical effects and advantages of the anti-aging drug screening method based on mitochondrial multi-level analysis and targeted intervention of this invention are as follows: 1. The core mitochondrial functions covered by this invention include ATP level fluctuations, antioxidant capacity, mitochondrial network fusion and division dynamics, and mitochondrial autophagy flux. By introducing a functional entropy value quantification standard, using the baseline functional entropy of S4 and the dynamic response entropy of S6, multi-dimensional data are transformed into a single comparable value, avoiding subjective judgment and making the screening results more objective and repeatable.

[0016] 2. This invention constructs a precise control model, using a low-dose rotenone-induced premature aging cell model, while simultaneously setting up an untreated young cell control, ensuring that the drug only works on senescent cells; its simulated physiological stress test, the mild oxidative stress described in S5, is achieved by depriving serum for 2 hours, reducing the serum concentration in the culture medium to 0.5%–2%, simulating aging-related oxidative stress in vivo, and only screening for drugs that can help cells cope with stress and recover quickly.

[0017] 3. The invention's low-dose combination strategy refers to combining the selected optimal drug with a known mild autophagy inducer at low doses to avoid the toxicity that may result from high doses of a single drug; the strict safety criteria are that it does not induce excessive autophagy and that the cell viability decreases by no more than 10%, ensuring that the selected drug repairs mitochondria without damaging normal cell function.

[0018] 4. This invention has a candidate drug library covering three classes of high-potential drugs, including mitochondrial nutrients such as coenzyme Q10 and nicotinamide mononucleotide, natural compounds such as resveratrol and curcumin, and FDA-approved drugs such as metformin and rapamycin; and it does not require the development of new drugs from scratch, because FDA-approved drugs already have a basis in human safety, and if they are found to be effective, their indications in anti-aging can be rapidly expanded.

[0019] 5. This invention employs full-cycle monitoring, including 24-hour baseline monitoring and continuous monitoring after stress, covering the entire process from static function to dynamic repair; and it has repair capabilities, assessing the impact of drugs on the recovery speed after stress through dynamic response entropy, and only screening drugs that can quickly clear damaged mitochondria with high functional entropy, thus better meeting the repair needs of aging cells in the body. Attached Figure Description

[0020] Figure 1 This is a flowchart of a screening method for anti-aging drugs based on mitochondrial multi-level analysis and targeted intervention proposed in this invention. Detailed Implementation

[0021] The technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0022] It should be noted that, in this document, relational terms such as "first" and "second" are used only to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Moreover, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Without further limitation, an element defined by the phrase "comprising..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes said element.

[0023] Example 1 This embodiment provides a method for screening anti-aging drugs based on mitochondrial multi-level analysis and targeted intervention, and the specific implementation steps include: Experimental materials: Basic experimental reagents include rotenone, fluorescent ATP indicator, antioxidant capacity assessment probe, mitochondrial-targeted photoactivated fluorescent protein, mitochondrial-targeted fluorescent protein for LC3 co-localization, cell culture medium, and buffer.

[0024] Experimental objective: Mitochondrial multilevel analysis was performed on senescent cells.

[0025] Experimental steps: S1: A portion of standard human mesenchymal stem cells were taken and treated with a low dose of rotenone to induce them into a premature aging state, thus constructing a senescent cell model; at the same time, the other half of the young cells were used as a control group and were not treated. S2: Fluorescent ATP indicators were added to senescent cell models and young cell models respectively to monitor ATP level fluctuations in real time; at the same time, the antioxidant capacity of both was assessed using specific probes. S3: Using mitochondrial-targeted photoactivated fluorescent proteins, the fusion and division dynamics of the mitochondrial network were analyzed using fluorescence bleaching recovery technology; simultaneously, mitochondrial-targeted fluorescent proteins co-localized with LC3 protein were used to directionally analyze drug-induced mitochondrial autophagy flux. S4: Under the uninterrupted state, the above four dimensions of aging and youth models, as well as aging and youth cells, are monitored in real time for 24 hours. The data of each dimension are then analyzed and integrated to calculate a single baseline functional entropy value.

[0026] Experimental results: The baseline functional entropy of the aging cell model was 0.75, while that of the young cell model was 0.25.

[0027] Example 2 This embodiment provides a method for screening anti-aging drugs based on mitochondrial multi-level analysis and targeted intervention, which dynamically intervenes in aging models. The specific implementation steps include: Experimental materials: Senescent cell models, young cell models, candidate drug libraries, micro-volume array drug delivery systems, multi-level real-time monitoring systems, and oxidative stress inducers; Candidate drug library: Mitochondrial nutrients: Coenzyme Q10, L-carnitine, alpha-lipoic acid, pyrroloquinoline quinone, nicotinamide ribose, nicotinamide mononucleotide; Natural compounds: resveratrol, curcumin, quercetin, epigallocatechin gallate, ginsenosides; FDA-approved drugs: metformin, rapamycin, acarbose, aspirin.

[0028] Experimental objective: To evaluate the effects of drugs in the candidate drug library on improving mitochondrial function in senescent cells and their influence on young cells.

[0029] Experimental steps: S1: The cell model was divided into four groups: senescent cell drug administration group, senescent cell unadministered group, young cell drug administration group, and young cell unadministered group; S2: The drug was added to the senescent cell model and the young cell model in a micro-liter array. After incubation for 48 hours, mild oxidative stress was applied by depriving serum for 2 hours. Immediately after the stress was applied, a multi-level real-time monitoring system was started and monitored continuously for 24 hours. S3: Analyze the data in four dimensions from the time of pressure application to the recovery period, and calculate the dynamic response entropy.

[0030] Experimental results: See Table 1 for details.

[0031] Table 1: Test Results of Example 2 Based on the data in the table, compared with the untreated aging control group, the dynamic response entropy of the candidate drug treatment group was significantly reduced and recovered to baseline levels more quickly; while the two young cell groups showed no significant changes; in the aging cell model (untreated), mitochondrial function was significantly disordered after oxidative stress, and the dynamic response entropy values ​​of each dimension were high, reflecting poor recovery ability; in the aging cell model (treated), the entropy values ​​of each dimension were significantly reduced after treatment with the candidate drug, indicating that the drug effectively improved mitochondrial functional stability and recovery ability; in the young cell model, the entropy value remained at a low level regardless of whether the drug was administered, indicating that the drug had little impact on young cells, which is consistent with the selective anti-aging goal.

[0032] Example 3 This embodiment provides a method for screening anti-aging drugs based on mitochondrial multi-level analysis and targeted intervention, and the specific implementation steps include: Experimental materials: The optimal drug selected from Example 2, spermidine, urolithin A, senescent cell model and young cell model, and reagents and equipment required for dynamic intervention response testing.

[0033] Experimental objective: Screening for the safest drug combinations.

[0034] Experimental steps: S1: Select the candidate drugs screened in Example 2; S2: The selected optimal drug is combined with an autophagy inducer (spermidine or urolithin A) at a low dose, and then the steps of Example 1 and Example 2 are repeated with this combination to conduct a dynamic intervention response test; S3: Based on the test results, select drug combinations that can maintain stable low functional entropy, accelerate the clearance of mitochondria with high functional entropy after stress testing, and do not induce excessive autophagy leading to cell damage.

[0035] Experimental results: The screening results are shown in Table 2.

[0036] Table 2: Test Results This invention establishes a mitochondrial-targeted anti-aging drug screening system. By constructing a cellular senescence model, it performs multi-level real-time monitoring from four dimensions: ATP metabolism, antioxidant capacity, mitochondrial network dynamics, and autophagy flux. It innovatively employs functional entropy to quantify mitochondrial functional status. Under mild oxidative stress conditions, it evaluates the improvement effect of candidate drugs on the dynamic response entropy of senescent cells, ultimately screening out the optimal combination that can be used in combination with low-dose autophagy inducers such as spermidine or urolithin A. This method achieves full-cycle monitoring from static functional assessment to dynamic stress recovery, providing a new strategy for developing safe and effective anti-aging therapies.

[0037] Comparative Example 1 This embodiment provides a traditional anti-aging drug, and the specific implementation steps include: Experimental materials: Polygonum cuspidatum extract (resveratrol source), ethanol, silica gel chromatography column, phosphate buffer, dimethyl sulfoxide.

[0038] Experimental objective: A high-purity resveratrol formulation was prepared for in vitro antioxidant and anti-aging activity testing.

[0039] Experimental steps: S1: Take the dried rhizome powder of Polygonum cuspidatum, extract it by heating and reflux with 70% ethanol solution, combine the extracts and concentrate under reduced pressure to obtain crude extract; S2: The crude extract was separated and purified by silica gel chromatography column, and gradient elution was performed using a petroleum ether-ethyl acetate system to collect the fraction rich in resveratrol. S3: The collected fraction is concentrated and recrystallized to obtain high-purity resveratrol crystals; S4: Dissolve resveratrol crystals in a small amount of dimethyl sulfoxide, then dilute with phosphate buffer to the required working concentration to prepare a drug stock solution.

[0040] Experimental results: Resveratrol solution with a purity of over 98% was successfully prepared by this method. The preparation process is mature, but the mechanism of action of the product is singular, mainly relying on its antioxidant activity, and lacks the ability to accurately assess the multidimensional functions such as mitochondrial network dynamics and autophagy flux.

[0041] Example 1 uses a low-dose rotenone-induced premature aging cell model and calculates baseline functional entropy by real-time monitoring of ATP level fluctuations, antioxidant capacity, mitochondrial network fusion / division dynamics, and mitochondrial autophagy flux in multiple dimensions to achieve a quantitative assessment of mitochondrial function.

[0042] Example 2 employs a micro-volume array dosing system and induces mild oxidative stress through serum deprivation. The dynamic response entropy is used to assess the recovery ability of candidate drugs, including mitochondrial nutrients, natural compounds, and FDA-approved drugs, under stress, thereby screening for drugs that are specifically effective against senescent cells.

[0043] Example 3 uses optimal drugs such as nicotinamide mononucleotide or metformin in low-dose combinations with autophagy inducers spermidine or urolithin A. Through repeated dynamic intervention response tests, a safe combination that can synergistically enhance mitochondrial function, rapidly clear mitochondria with high functional entropy, and does not induce excessive autophagy is screened out.

[0044] Comparative Example 1 prepared resveratrol using traditional extraction and purification methods, relying solely on antioxidant activity assessment, lacking precise regulation and real-time monitoring of multidimensional functions such as mitochondrial network dynamics and autophagy flux.

[0045] By comprehensively comparing the embodiments and comparative examples, Example 1 demonstrates the construction of an aging model and the quantification method of baseline functional entropy; Example 2 demonstrates the dynamic evaluation of multiple candidate drugs under stress conditions, reflecting the drug's recovery ability under stress through dynamic response entropy; Example 2 not only covers multiple candidate drugs but also evaluates the drug's recovery ability under stress through dynamic response entropy, thereby enhancing the physiological relevance of drug screening; Example 3 adopts a low-dose combination strategy, which requires further verification of its long-term safety and in vivo efficacy; the comparative examples highlight the limitations of traditional methods, which rely solely on a single antioxidant mechanism and cannot comprehensively assess mitochondrial health. Therefore, the multi-level analysis-targeted intervention method of the present invention has significant advantages in anti-aging drug screening, realizing a full-cycle assessment from static function to dynamic recovery.

[0046] refer to Figure 1This is a flowchart illustrating the anti-aging drug screening method based on mitochondrial multi-level analysis and targeted intervention described in this invention. The flowchart clearly demonstrates the entire process from aging model construction to drug screening and validation, specifically including the following key steps: First, an aging model is constructed by inducing human mesenchymal stem cells with a low dose of rotenone, with young cells serving as a control. Then, fluorescent probe technology is used to monitor four core dimensions in real time: ATP fluctuations, antioxidant capacity, mitochondrial network fusion and division dynamics, and autophagy flux. Based on this, baseline functional entropy is calculated through multi-dimensional data integration to quantify the initial functional state of mitochondria. Next, under mild oxidative stress conditions, the dynamic response entropy of cells treated with candidate drugs is evaluated to screen for the optimal drug that significantly reduces the entropy value of aging cells while having the least impact on young cells. Finally, the optimal drug is combined with low-dose autophagy inducers such as spermidine or urolithin A to verify its safety in synergistically maintaining low functional entropy, promoting the clearance of damaged mitochondria, and not inducing excessive autophagy. This flowchart highlights the multi-dimensional and full-cycle characteristics of the method, achieving systematic screening from static functional assessment to dynamic stress recovery, ensuring the high efficiency, objectivity, and safety of drug screening.

[0047] The above description is merely a specific embodiment of this application, but the scope of protection of this application is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in this application should be included within the scope of protection of this application. Therefore, the scope of protection of this application should be determined by the scope of protection of the claims.

[0048] In conclusion, the above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A screening method for anti-aging drugs based on mitochondrial multi-level analysis-targeted intervention, characterized by, Specifically comprising the following steps: S1: Take a part of standard human mesenchymal stem cells, treat them with low-dose rotenone to induce them into premature aging state, and construct them into a model of aging cells; at the same time, take another half of young cells as a control group without treatment; S2: Add fluorescent ATP indicator to the model of aging cells and the model of young cells respectively to monitor the fluctuation of ATP level in real time; at the same time, use a specific probe to evaluate the antioxidant capacity of the two; S3: Use mitochondria-targeted photoactivatable fluorescent protein to analyze the fusion and fission dynamics of mitochondrial network by fluorescence bleaching recovery technique; at the same time, use mitochondria-targeted fluorescent protein co-localized with LC3 protein to analyze the mitochondrial autophagy flux induced by drugs; S4: Real-time monitor the aging model and the young model for 24 hours without intervention, then analyze the data of each dimension and integrate to calculate a single baseline functional entropy value; S5: Divide the cell models into four groups: aging cell drug administration group, aging cell non-drug administration group, young cell drug administration group, and young cell non-drug administration group; then add candidate drugs to the corresponding cell models in microliter array, incubate for 48 hours, and then apply mild oxidative stress to all groups; immediately after the stress is applied, start the multi-level real-time monitoring system for continuous monitoring; S6: Analyze the data of each dimension from the stress application to the recovery period and calculate the dynamic response entropy; according to this, select the optimal drug from the candidate drugs, then combine the selected optimal drug with an autophagy inducer at a low dose, and use the drug combination to repeat the steps S1 to S4 in claim 1 for dynamic intervention response test; S7: From the test results, select the drug combination that can maintain stable low functional entropy, accelerate the clearance of high functional entropy mitochondria after stress test, and does not cause cell damage due to excessive autophagy.

2. The screening method for anti-aging drugs based on multi-level analysis-targeted intervention of mitochondria according to claim 1, wherein, The fluorescent ATP indicator in S2 is a quinoline fluorescent protein complex or a luciferase-based ATP probe; the probe used to evaluate antioxidant capacity is MitoSOX Red.

3. The screening method for anti-aging drugs based on multi-level analysis-targeted intervention of mitochondria according to claim 1, wherein, In S3, when analyzing mitochondrial network dynamics by fluorescence bleaching recovery technique, the mitochondria-targeted photoactivatable fluorescent protein used is mitochondria-targeted PA-GFP. ​ 4. The method for screening anti-aging drugs based on multi-level analysis-targeted intervention of mitochondria according to claim 1, wherein, The dimensions involved in S4 and S6 include ATP level fluctuation, antioxidant capacity, mitochondrial network fusion and fission dynamics, and mitochondrial autophagy flux.

5. The method for screening anti-aging drugs based on multi-level analysis-targeted intervention of mitochondria according to claim 1, wherein, The candidate drug library in S5 contains one or more of the following categories: mitochondrial nutrients, natural compounds, and FDA-approved drugs.

6. The screening method for anti-aging drugs based on multi-level analysis-targeted intervention of mitochondria according to claim 5, wherein, The mitochondrial nutrients are selected from one or more of coenzyme Q10, L-carnitine, lipoic acid, pyrroloquinoline quinone, nicotinamide riboside, and nicotinamide mononucleotide; the natural compounds are selected from one or more of resveratrol, curcumin, quercetin, epigallocatechin gallate, and ginsenosides; the FDA-approved drugs are selected from one or more of metformin, rapamycin, acarbose, and aspirin.

7. The screening method for anti-aging drugs based on multi-level analysis-targeted intervention of mitochondria according to claim 1, wherein, The mild oxidative stress in S5 is to deprive serum for 2 hours to reduce the serum concentration in the culture medium to 0.5% to 2%. ​ 8. The method for screening an anti-aging drug based on multi-level analysis-targeted intervention of mitochondria according to claim 1, wherein, The optimal drug in S6 refers to a candidate drug that can make the dynamic response entropy of senescent cells decrease the most and has the least effect on the function of young cell mitochondria in the dynamic intervention response test. ​ 9. The method for screening anti-aging drugs based on multi-level analysis-targeted intervention of mitochondria according to claim 1, wherein, The phrase "not causing excessive autophagy leading to cell damage" in S7 refers to the determination by detecting cell viability or lactate dehydrogenase activity in the culture medium, and the cell viability of the administration group decreases by no more than 10% compared with the control group. ​ 10. The method for screening anti-aging drugs based on multi-level analysis-targeted intervention of mitochondria according to claim 1, wherein, The autophagy inducer in S7 is spermidine or urolithin A; the optimal drug is combined with spermidine or urolithin A at a low dose. ​

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