Application of substances targeting the CYTB-187AA gene and / or protein in the preparation of drugs for delaying aging and / or prolonging life.

Abstract

This invention provides the application of substances targeting the CYTB-187AA gene and / or protein in the preparation of drugs for delaying aging and / or prolonging lifespan. The research results of this invention indicate that by reducing the expression of CYTB-187AA protein and / or inhibiting its activity, the aging process can be effectively delayed, thereby achieving the goal of extending healthy lifespan or lifespan. The research results of this invention are of great significance for scientific research on aging mechanisms and the development of anti-aging intervention strategies.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to the application of substances targeting the CYTB-187AA gene and / or protein in the preparation of drugs for delaying aging and / or prolonging life. Background Technology

[0002] Aging is a complex biological process of functional decline that occurs over time in all multicellular organisms, and it is the most significant risk factor for many chronic diseases, such as neurodegenerative diseases, cardiovascular diseases, and metabolic diseases. Currently, the global population is aging rapidly, making the development of strategies to slow the aging process and extend healthy lifespan of great social and economic importance.

[0003] CN116676379A discloses ERV and its downstream signaling pathway proteins as biomarkers of neuronal aging and their application in interfering with neuronal aging. Specifically, it includes using LMNB1 and LMNB2 as intervention targets to inhibit the activation of the ERV or cGAS pathway to intervene in human neuronal aging, and also involves the application of the reverse transcriptase inhibitor abacavir in interfering with neuronal aging in primates.

[0004] CN116041477A discloses the application of the TDGF1 gene in the preparation of drugs for treating age-related diseases or reversing cellular senescence. This invention discovers that overexpression of the TDGF1 gene can reverse cellular senescence and treat age-related diseases. The expression vector overexpressing the TDGF1 gene can effectively reverse the senescence of key cells in the body, thus achieving effective treatment for various diseases caused by aging, with good results. Cell experiments have demonstrated that it effectively increases the proportion of non-senescent cells, and mouse experiments have shown that overexpression of the TDGF1 gene through AAV2 effectively alleviates the symptoms of age-related cataracts.

[0005] Despite numerous research findings on aging, specific intervention targets for the core processes of aging remain very limited, and effective anti-aging drugs and methods are still to be developed. Summary of the Invention

[0006] To address the shortcomings of existing technologies, the present invention aims to provide the application of substances targeting the CYTB-187AA gene and / or protein in the preparation of drugs for delaying aging and / or prolonging lifespan. This invention offers a novel strategy for delaying aging and / or prolonging lifespan, and is of great significance for scientific research on aging mechanisms and the development of anti-aging intervention strategies.

[0007] To achieve this objective, the present invention adopts the following technical solution:

[0008] In a first aspect, the present invention provides the use of substances targeting the CYTB-187AA gene and / or the CYTB-187AA protein in the preparation of medicaments for delaying aging and / or prolonging lifespan.

[0009] The results of this invention show that by reducing the expression of CYTB-187AA protein and / or inhibiting its activity, the aging process can be effectively delayed, thereby achieving the purpose of extending the healthy period or lifespan.

[0010] Preferably, the nucleotide sequence of the CYTB-187AA gene includes that shown in SEQ ID NO:1.

[0011] Preferably, the amino acid sequence of the CYTB-187AA protein includes that shown in SEQ ID NO:2.

[0012] Preferably, the substance targeting the CYTB-187AA gene is an inhibitor that reduces the activity and / or expression level of the CYTB-187AA gene.

[0013] Preferably, the inhibitors that reduce the activity and / or expression of the CYTB-187AA gene include: gene editing reagents that specifically knock out / knock down the CYTB-187AA gene or interference molecules that specifically interfere with the expression of the CYTB-187AA gene.

[0014] Preferably, the interfering molecule is a small interfering RNA, antisense nucleic acid, microRNA, or dsRNA that targets the CYTB-187AA gene or its transcript for suppression or silencing, or a construct that can express or form the small interfering RNA, antisense nucleic acid, microRNA, or dsRNA.

[0015] Preferably, the substance targeting CYTB-187AA protein is an inhibitor that reduces the expression and function of CYTB-187AA protein.

[0016] Preferably, the inhibitor that reduces the expression and function of CYTB-187AA protein is a CYTB-187AA protein inhibitor.

[0017] Preferably, the CYTB-187AA protein inhibitor comprises: a small molecule compound, an antibody that specifically recognizes the CYTB-187AA protein, a functional antagonist of the CYTB-187AA protein, a competitive inhibitor of the CYTB-187AA protein, a reagent that downregulates the expression of the CYTB-187AA protein, an interfering RNA that targets the CYTB-187AA gene, or a knockdown or knockout vector that targets the CYTB-187AA gene.

[0018] Secondly, the present invention provides the application of CYTB-187AA protein activator in the preparation of a kit for the study of accelerating the aging process; the CYTB-187AA protein activator enhances the activity of CYTB-187AA protein.

[0019] The results of this invention demonstrate that by promoting the expression of CYTB-187AA protein and / or enhancing its activity, aging-related phenotypes can be constructed or accelerated for use in the study of aging mechanisms.

[0020] In this invention, the phenotypes related to "accelerated aging process" include, but are not limited to:

[0021] (1) Overall physiological function decline: manifested as decreased exercise endurance and coordination, decreased basal metabolic rate, disordered diurnal rhythm, and weakened stress tolerance.

[0022] (2) Decreased function and pathological changes of tissues and organs: including skin wrinkling and thinning, sparse and gray hair, delayed wound healing; reduced muscle mass and strength (sarcomustosis); decreased bone density; decreased functional indicators, atrophy or fibrosis of important organs (such as liver, kidney, heart and lungs); cognitive and behavioral deficits related to neurodegenerative diseases.

[0023] (3) Accumulation of cell senescence markers: decreased cell proliferation capacity, significantly increased activity of senescence-related β-galactosidase (SA-β-gal), and increased expression and secretion of senescence-related secretory phenotype (SASP) factors (such as IL-6 and TNF-α).

[0024] (4) Changes at the molecular level: Increased genomic instability (such as accumulation of DNA damage focal γ-H2AX), telomere loss or dysfunction, mitochondrial dysfunction, weakened autophagic flux, and accelerated epigenetic age.

[0025] Thirdly, the present invention provides a method for screening drugs to delay aging and / or prolong lifespan, the method comprising: administering a candidate drug to a test animal; measuring the expression level and / or activity of CYTB-187AA protein in the blood or tissue of the test animal before and after administration; and indicating that the expression level and / or activity of CYTB-187AA protein in the blood or tissue of the test animal is reduced after administration compared to before administration, indicating that the candidate drug is the target drug.

[0026] Fourthly, the present invention provides a method for constructing an animal model of accelerated aging-related phenotypes, the method comprising: increasing the expression level and / or activity of CYTB-187AA protein in the blood or tissues of test animals to obtain an animal model of accelerated aging-related phenotypes.

[0027] Compared with the prior art, the present invention has the following beneficial effects:

[0028] (1) This invention reveals for the first time the direct link between the expression level or activity of CYTB-187AA protein and the aging process, providing new molecular targets and regulatory pathways for the study of aging mechanisms and filling the technological gap in this field.

[0029] (2) The drug screening system and animal model construction method established by the present invention based on the above findings have clear detection indicators and operability. They can not only be used for high-throughput screening of anti-aging drugs, but also provide stable and reliable animal models for the study of aging-related diseases. They have important scientific research and translational application value. Attached Figure Description

[0030] Figure 1 The results are from Western blotting analysis of young and old mice.

[0031] Figure 2 The results of Western blot analysis of mice with systemic knockdown of CYTB-187AA.

[0032] Figure 3 Survival curves for mice with systemic knockdown of CYTB-187AA and wild-type WT mice. Detailed Implementation

[0033] The embodiments of the present invention are described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention.

[0034] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of indicated technical features. Therefore, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. Furthermore, in the description of this invention, unless otherwise stated, "a plurality of" means two or more.

[0035] In this document, the terms “comprising” or “including” are open-ended expressions, meaning that they include the contents specified in this invention, but do not exclude other aspects.

[0036] In this paper, the term "CYTB-187AA protein" refers to the mitochondrial DNA-encoded protein arising from cytosolic translation (mPACT) encoded by the mitochondrial gene cytochrome b (CYTB), and its amino acid sequence is shown below (SEQ ID NO:1).

[0037] MMKLWVPSRSLPNSPNHYRSFLSHTLHIRYNNSLFISNTHLSRRKLRVTNPIYTRKRSLNIFYLLIPSCRTRLILWIIYIYRNLKHWSTSTVRSHSHSIYRLRPSMRTNIILRCHSYYKPPISHPIYWNNPSRMNLRGLLSRQSHLDPILRFPLHLTIYYRGPSNRSPPLPPRNRIKQPNRIKLRCR。

[0038] The nucleotide sequence encoding the CYTB-187AA protein is shown in SEQ ID NO:2.

[0039] atgatgaaactttgggtcccttctaggagtctgcctaatagtccaaatcattacaggtcttttcttagccatacactacacatcagatacaataacagccttttcatcagtaacacacatttgtcgagacgtaaattacgggtgactaatccgatatatacacgcaaacggagcctcaatattttttatttgcttattccttcatgtcggacgaggcttatattatggatcatatacatttatagaaacctgaaacattggagtacttctactgttcgcagtcatagccacagcatttataggctacgtccttccatgaggacaaatatcattctgaggtgccacagttattacaaacctcctatcagccatcccatatattggaacaaccctagtcgaatgaatttgagggggcttctcagtagacaaagccaccttgacccgattcttcgctttccacttcatcttaccatttattatcgcggccctagcaatcgttcacctcctcttcctccacgaaacaggatcaaacaacccaacaggattaaactcagatgcagataa。

[0040] In this document, the term "CYTB-187AA protein inhibitor" refers to an agent capable of specifically reducing the activity of the CYTB-187AA protein. This agent can be a protein, a small molecule compound, a macromolecule, a nucleic acid reagent, or an agent used to induce specific mutations (e.g., gene editing). Reducing the activity of the CYTB-187AA protein can mean reducing the intracellular activity of the CYTB-187AA protein by at least 10%, such as at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%, compared to the absence of the inhibitor. For example, it can reduce the intracellular protein expression level of CYTB-187AA by at least 10%, such as at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%.

[0041] According to embodiments of the present invention, the CYTB-187AA protein inhibitor includes an antibody that specifically recognizes the CYTB-187AA protein, a functional antagonist of the CYTB-187AA protein, a competitive inhibitor, and / or a reagent that downregulates the expression of the CYTB-187AA protein.

[0042] In some embodiments, the agents for downregulating CYTB-187AA protein expression include at least one of an antisense oligonucleotide ASO specifically targeting the CYTB-187AA gene, a small hairpin RNA (shRNA), and interfering RNA (RNAi).

[0043] In this document, the term "CYTB-187AA protein activator" refers to a protein whose activity is enhanced in the presence of an activator compared to the absence of an activator. For example, the activity of CYTB-187AA protein in the presence of a CYTB-187AA protein activator is approximately 1.2 times, approximately 1.5 times, approximately 2 times, approximately 3 times, approximately 4 times, approximately 5 times, approximately 6 times, approximately 7 times, approximately 8 times, approximately 9 times, approximately 10 times, approximately 15 times, approximately 20 times, approximately 25 times, approximately 50 times, or higher than that in the absence of the activator. In some embodiments, the CYTB-187AA protein activator comprises an expression vector containing CYTB-187AA.

[0044] In this article, the term "animal" can refer to humans, rats, pigs, cattle, sheep, rabbits, horses, fruit flies, etc.

[0045] The technical solution of the present invention will be further illustrated below through specific embodiments. Those skilled in the art should understand that the embodiments described are merely illustrative of the present invention and should not be construed as limiting the invention in any way.

[0046] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.

[0047] Example 1

[0048] The expression level of CYTB-187AA protein increases with natural aging in mice.

[0049] 1. Laboratory animals and sample collection.

[0050] Animals and grouping: Wild-type mice with a C57BL / 6J background were selected.

[0051] Young adult group: 3-month-old mice (n=6), representing the young adult stage after reaching maturity.

[0052] Older group: 20-month-old mice (n=6), representing late natural aging.

[0053] Tissue sample collection: The mice in both groups were euthanized, and various tissue samples were quickly dissected and collected. The samples were immediately flash-frozen in liquid nitrogen and then transferred to a -80°C freezer for storage for protein extraction.

[0054] 2. Western blot detection of CYTB-187AA protein expression.

[0055] The results are as follows Figure 1 As shown, compared with the 3-month-old young mice, the expression level of CYTB-187AA protein was significantly increased in various tissues of the heart, liver, spleen, lungs, and small intestine of the 20-month-old aged mice, indicating that CYTB-187AA is associated with aging in mice.

[0056] Example 2

[0057] Mice with systemic knockdown of CYTB-187AA were constructed.

[0058] The H11-shCYTB-187A sequence was synthesized by adding the upstream U6 promoter and downstream poly T to the validation sequence of the silenced mouse CYTB-187AA (sh187#2: CGCAAACGGAGCCATCATATT, SEQ ID NO:3), and then cloned into the donor plasmid. The sgRNA was designed to target the H11 locus. Then, Cas9 mRNA, sgRNA (5'-GGGGCCACTAGGGACAGGAT-3', SEQ ID NO:4), and the H11-shCYTB-187AA donor plasmid were co-microinjected into C57BL / 6J zygotes, which were then transferred to pseudopregnant females. The obtained live pups were subsequently screened by PCR analysis and sequencing verification. At 4 weeks of age, the genotypes of male and female mice were identified by genomic analysis of the mouse tail. Finally, proteins were extracted from various mouse tissues, and the expression level of CYTB-187AA protein was detected by Western blot to verify successful knockdown. The results are as follows Figure 2 As shown, compared with normal mice, the expression of CYTB-187AA in all tissues of CYTB-187AA knockdown mice was downregulated, indicating that the CYTB-187AA knockdown mice were successfully constructed.

[0059] Example 3

[0060] Survival study of mice with systemic knockdown of CYTB-187AA.

[0061] 1. Animal strain and genotype: Mice with a C57BL / 6J background were used in the experiment.

[0062] Experimental group: Mice with systemic knockdown of CYTB-187AA (n=7, male; n=9, female).

[0063] Control group: wild-type WT mice from the same litter (n=7, male; n=8, female).

[0064] 2. Husbandry conditions: All mice were kept in a specific pathogen-free (SPF) barrier environment, with constant temperature (22±2℃), constant humidity (50±10%) and 12-hour light-dark cycle, and free access to standard irradiated feed and sterile water.

[0065] 3. Survival Rate Observation Experiment: Survival status was formally recorded starting at 8 weeks of age (juvenile stage) and continued until natural death. Data Recording: The unique identifier, date of birth, group, date of death, and number of days of survival for each mouse were recorded. A complete survival database was established.

[0066] 4. Survival curve plotting: Use the Kaplan-Meier method to plot the survival curves for the WT group and the KD group.

[0067] 5. Statistical comparison: The log-rank test was used to compare the overall differences between the survival curves of the two groups.

[0068] The results are as follows Figure 3 As shown, knocking down CYTB-187AA prolongs the lifespan of female mice compared to normal mice, indicating that CYTB-187AA is associated with aging in female mice.

[0069] In summary, this invention, based on a study of the survival of mice with systemic CYTB-187AA knockdown and wild-type mice, found that knocking down the CYTB-187AA gene can prolong the lifespan of female mice. The findings of this invention are of great significance for scientific research on aging mechanisms and the development of anti-aging intervention strategies.

[0070] The applicant declares that the above description is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention fall within the protection and disclosure scope of the present invention.

Claims

1. The use of substances that target the CYTB-187AA gene and / or the CYTB-187AA protein in the preparation of drugs for delaying aging and / or prolonging life.

2. The application according to claim 1, characterized in that, The nucleotide sequence of the CYTB-187AA gene includes that shown in SEQ ID NO:

1.

3. The application according to claim 1, characterized in that, The amino acid sequence of the CYTB-187AA protein includes that shown in SEQ ID NO:

2.

4. The application according to any one of claims 1-3, characterized in that, The substance targeting the CYTB-187AA gene is an inhibitor that reduces the activity and / or expression level of the CYTB-187AA gene.

5. The application according to claim 4, characterized in that, The inhibitors that reduce the activity and / or expression of the CYTB-187AA gene include: gene editing reagents that specifically knock out / knock down the CYTB-187AA gene or interference molecules that specifically interfere with the expression of the CYTB-187AA gene. Preferably, the interfering molecule is a small interfering RNA, antisense nucleic acid, microRNA, or dsRNA that targets the CYTB-187AA gene or its transcript for suppression or silencing, or a construct that can express or form the small interfering RNA, antisense nucleic acid, microRNA, or dsRNA.

6. The application according to any one of claims 1-3, characterized in that, The substance that targets CYTB-187AA protein is an inhibitor that reduces the expression and function of CYTB-187AA protein.

7. The application according to claim 6, characterized in that, The inhibitor that reduces the expression and function of CYTB-187AA protein is a CYTB-187AA protein inhibitor; Preferably, the CYTB-187AA protein inhibitor comprises: a small molecule compound, an antibody that specifically recognizes the CYTB-187AA protein, a functional antagonist of the CYTB-187AA protein, a competitive inhibitor of the CYTB-187AA protein, a reagent that downregulates the expression of the CYTB-187AA protein, an interfering RNA that targets the CYTB-187AA gene, or a knockdown or knockout vector that targets the CYTB-187AA gene.

8. The application of CYTB-187AA protein activator in the preparation of the kit, characterized in that, The kit is used for research on accelerating the aging process; the CYTB-187AA protein activator enhances the activity of the CYTB-187AA protein.

9. A method for screening drugs to delay aging and / or prolong lifespan, characterized in that, The method includes: administering a candidate drug to a test animal; measuring the expression level and / or activity of CYTB-187AA protein in the blood or tissue of the test animal before and after administration; if the expression level and / or activity of CYTB-187AA protein in the blood or tissue of the test animal decreases after administration compared to before administration, the candidate drug is considered the target drug.

10. A method for constructing an animal model of accelerated aging-related phenotypes, characterized in that, The method includes: increasing the expression level and / or activity of CYTB-187AA protein in the blood or tissues of test animals to obtain an animal model of accelerated aging-related phenotypes.

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