Methods for identifying substances that affect aging

Abstract

A method for identifying a substance that can affect aging is provided, the method comprising determining the effect of a candidate substance on Zip11 activity and / or expression, and identifying a candidate substance that alters Zip11 activity and / or expression as a substance that can affect aging. Further provided is use of a substance that can increase Zip11 activity and / or expression in the preparation of a pharmaceutical product, wherein the pharmaceutical product is used for delaying or ameliorating aging.

Description

[Technical Field]

[0001] The present application relates to the field of biomedicine, in particular to a method for identifying substances capable of affecting aging, characterized in that said substances are capable of affecting the activity and / or expression of Zip11. [Background technology]

[0002] Aging is an inevitable biological process characterized by a decline in physiological function that typically leads to morbidity and mortality. This ongoing decline manifests as an overall decline in physical and mental capacity. Functional, tissue, and cellular aging are common to all multicellular organisms, but the rate at which aging occurs can vary considerably between individuals due to a combination of environmental and genetic factors. Whole-genome sequencing of the human genome has identified genetic variants in specific genes, such as ApoE, Sirtl, Foxo3, and Nrf3, that are significantly associated with longevity and health. However, regional restrictions and safety and efficacy issues have significantly limited the use of these products. Therefore, there is a need to develop novel longevity-related genes with significant potential. (See: Zeng Y, et al. Sex Differences in Genetic Associations With Longevity, JAMA Netw Open. 2018 Aug;l(4). Pii: el81670). DISCLOSURE OF THE INVENTION [Problem to be solved by the invention]

[0003] This application unexpectedly discovered the Zip11 gene, which encodes a member of the solute carrier family 39 (SLC39) that plays important roles in multiple areas, including inflammation, tumors, and metabolism, and is highly expressed in multiple organs, including the small intestine, stomach, reproductive organs, and liver. By controlling Zip11 protein expression, it is possible to regulate related reactions that affect the degree of aging and lifespan of an organism, and the Zip11 gene is expected to be an excellent longevity-related gene. [Means for solving the problem]

[0004] The present application provides a method for identifying a substance capable of affecting aging, the method comprising determining the effect of a candidate substance on Zip11 activity and / or expression, and identifying a candidate substance that causes said change in Zip11 activity and / or expression as a substance capable of affecting aging. The present invention also provides a use of a substance capable of increasing Zip11 activity and / or expression for the preparation of a medicament for use in delaying or ameliorating aging.

[0005] On the other hand, the present application provides a method for identifying a substance that can affect aging, which is characterized by including determining the effect of a candidate substance on the activity and / or expression of Zip11, and identifying a candidate substance that causes the change in the activity and / or expression of Zip11 as a substance that can affect aging.

[0006] In some embodiments, determining the effect of the candidate substance on Zip11 activity and / or expression comprises determining the effect of the candidate substance on the expression level and / or activity of the Zip11 nucleic acid.

[0007] In some embodiments, the Zip11 nucleic acid expression level and / or activity is determined by measuring the level of Zip11 Nucleic acids and a primer capable of specifically amplifying the Zip11 gene. Nucleic acids The nucleic acid sequence is determined using a substance selected from the group consisting of nucleic acid probes capable of specifically recognizing the

[0008] In some embodiments, determining the effect of the candidate substance on Zip11 activity and / or expression comprises determining the effect of said candidate substance on the expression level and / or activity of said Zip11 protein.

[0009] In some embodiments, the expression level and / or activity of the Zip11 protein is determined using a substance selected from the group consisting of a reagent that specifically recognizes Zip11 protein and a substance that can measure the activity of Zip11 protein.

[0010] In some embodiments, candidate substances that result in an increase in Zip11 activity and / or expression are identified as substances that can delay or ameliorate aging.

[0011] In some embodiments, candidate substances that cause a decrease in Zip11 activity and / or expression are identified as substances that promote or aggravate senescence.

[0012] In some embodiments, the method includes administering the candidate substance to a non-human animal, organ, tissue and / or cell, determining the effect of the candidate substance on the activity and / or expression of Zip11 in the non-human animal, organ, tissue and / or cell, and identifying a candidate substance that causes a change in the activity and / or expression of Zip11 as a substance that affects aging.

[0013] In some embodiments, the method is an in vitro or ex vivo method.

[0014] In some embodiments, the aging status is a body shape, a hair status, an immune level status, a tumor status, a liver status, a spleen status, a fertility status, an athletic performance status, lifespan The condition is determined by assessing one or more of the following: cognitive status; and cognitive performance status.

[0015] In some embodiments, the body morphology is assessed by detecting spinal morphology.

[0016] In some embodiments, the hair condition is assessed by detecting hair loss.

[0017] In some embodiments, immune status is assessed by detecting cytokine levels.

[0018] In some embodiments, the tumor status is determined by tumor volume, tumor size, Growth status and / or by detecting the presence of tumor cells.

[0019] In some embodiments, the liver condition is: Alanine aminotransferase activity, aspartate aminotransferase activity and / or by detecting the liver / body weight ratio.

[0020] In some embodiments, the spleen status is measured by spleen to body weight. ratio of It is evaluated by measuring

[0021] In some embodiments, the fertility The condition is assessed by examining the percentage of fertile eggs.

[0022] In some embodiments, the exercise ability The state of Rotarod fatigue experiment In some embodiments, the results are evaluated by: lifespan The status is assessed by detecting survival time.

[0023] In some embodiments, cognitive status is assessed by an open field test.

[0024] On the other hand, the present application provides the use of a substance capable of increasing the activity and / or expression of Zip11 for the preparation of a medicament, wherein the medicament is used to delay or ameliorate aging.

[0025] In some embodiments, the slowing or amelioration of aging comprises preventing or treating spinal curvature, preventing or treating hair loss, increasing cytokine levels, improving immunity, preventing or treating tumors, extending lifespan, preventing or treating liver damage, preventing or treating spleen damage, preventing or treating decline in fertility, improving motor performance, and / or improving behavioral and cognitive performance.

[0026] In some embodiments, the increase refers to at least a 15-fold increase in Zip11 activity and / or expression following administration of the agent compared to when the agent is not administered.

[0027] In some embodiments, said activity and / or expression of Zip 11 comprises activity and / or expression at the level of Zip 11 nucleic acid and / or at the level of Zip 11 protein.

[0028] On the other hand, the present application provides a system for identifying substances that can affect aging, the system including a substance that can determine the effect of a candidate substance on the activity and / or expression of Zip11.

[0029] In some embodiments, the system comprises an agent capable of determining the level and / or activity of Zip11 nucleic acid expression.

[0030] In some embodiments, the substance capable of determining the level and / or activity of Zip11 nucleic acid expression is Zip11. Nucleic acids Primers capable of specifically amplifying Zip11 and / or Zip11 Nucleic acids It is composed of a probe that can specifically recognize

[0031] In some embodiments, the system comprises an agent capable of determining the expression level and / or activity of the Zip11 protein.

[0032] In some embodiments, the substance capable of determining the protein expression level and / or activity of Zip11 consists of a reagent capable of specifically recognizing Zip11 protein and / or a reagent capable of determining the activity of Zip11 protein.

[0033] Meanwhile, the present application also provides use of a nucleic acid molecule encoding Zip11 or its expression product for the preparation of a medicament, wherein the medicament is used for delaying or ameliorating aging.

[0034] In some embodiments, the slowing or amelioration of aging comprises preventing or treating spinal curvature, preventing or treating hair loss, increasing cytokine levels, improving immunity, preventing or treating tumors, extending lifespan, preventing or treating liver damage, preventing or treating spleen damage, preventing or treating decline in fertility, improving motor performance, and / or improving behavioral and cognitive performance.

[0035] In some embodiments, the nucleic acid molecule comprises the nucleotide sequence set forth in any one of SEQ ID No. 1.

[0036] On the other hand, the present application provides an effective amount of a nucleic acid molecule encoding Zip11 or its expression product to a subject in need thereof. subject Also provided is a method for slowing or reversing aging, comprising administering to

[0037] In some embodiments, the delay or amelioration of aging is prevention or treatment of curvature of the spine, prevention or treatment of hair loss, increasing cytokine levels, improving immunity, preventing or treating tumors, extending lifespan, preventing or treating liver damage, preventing or treating spleen damage, preventing or treating decline in fertility, improving athletic performance, delaying decline in athletic performance, and / or improving behavioral and cognitive performance. It consists of .

[0038] In some embodiments, the nucleic acid molecule consists of the nucleotide sequence set forth in SEQ ID No.1.

[0039] Meanwhile, the present application also provides a method for determining the aging status of a subject, the method comprising measuring Zip11 activity and / or expression in the subject.

[0040] In some embodiments, said Zip11 activity and / or expression comprises Zip11 nucleic acid expression levels and / or activity, and / or Zip11 protein expression levels and / or activity.

[0041] In some embodiments, the subject person The level and / or activity of Zip11 nucleic acid expression in Nucleic acids Primers capable of specifically amplifying Zip11 Nucleic acids The nucleic acid sequence is determined using a substance selected from the group consisting of nucleic acid probes capable of specifically recognizing the

[0042] In some embodiments, the subject person The protein expression level and / or activity of Zip11 in the subject is determined using a substance selected from the group consisting of a reagent that specifically recognizes Zip11 protein and a substance that can measure Zip11 protein activity.

[0043] In some embodiments, the aging condition is one or more of the following groups: body shape condition, hair condition, immune level condition, tumor of condition, liver condition, spleen condition, fertility condition, athletic ability condition, Lifespan This is determined by assessing the patient's condition and cognitive performance status.

[0044] In some embodiments, the body morphology is assessed by detecting spinal morphology.

[0045] In some embodiments, the hair condition is assessed by detecting hair loss.

[0046] In some embodiments, immune status is assessed by detecting cytokine levels.

[0047] In some embodiments, the tumor status is determined by tumor volume, tumor size, Growth status and / or by detecting the presence of tumor cells.

[0048] In some embodiments, the liver condition is: Alanine aminotransferase activity, aspartate aminotransferase activity and / or by detecting the liver / body weight ratio.

[0049] In some embodiments, the spleen status is assessed by determining the spleen to body weight ratio.

[0050] In some embodiments, the fertility The condition is assessed by examining the percentage of fertile eggs.

[0051] In some embodiments, the exercise ability The condition is Rotarod fatigue experiment It is evaluated by the results.

[0052] In some embodiments, lifespan The status is assessed by detecting survival time.

[0053] In some embodiments, the cognitive status is assessed by an open field experiment.

[0054] On the other hand, the present application also provides use of the substance for measuring Zip11 activity and / or expression in the preparation of a reagent, which is used to identify the aging state of a subject.

[0055] In some embodiments, said Zip11 activity and / or expression comprises Zip11 nucleic acid expression levels and / or activity, and / or Zip11 protein expression levels and / or activity.

[0056] In some embodiments, the substance is Zip11 Nucleic acids Primers that can amplify the of nucleic acids The Zip11 protein is composed of a nucleic acid probe capable of specifically recognizing the Zip11 protein, a reagent capable of specifically recognizing the Zip11 protein, and / or a substance capable of measuring the activity of the Zip11 protein.

[0057] In some embodiments, the aging condition is body form , hair condition, immune level, tumor of The condition is determined by assessing one or more of the following group: a state of the liver, a state of the spleen, a state of fertility, a state of motor function, a state of longevity, and a state of cognitive function.

[0058] In some embodiments, the body morphology is assessed by detecting spine morphology. In some embodiments, the hair condition is assessed by detecting hair loss.

[0059] In some embodiments, immune status is assessed by detecting cytokine levels.

[0060] In some embodiments, the tumor status is determined by tumor volume, tumor size, Growth status and / or by detecting the presence of tumor cells.

[0061] In some embodiments, the liver condition is: Alanine aminotransferase activity, aspartate aminotransferase activity and / or by detecting the liver / body weight ratio.

[0062] In some embodiments, the spleen status is measured by spleen to body weight. ratio of It is evaluated by measuring

[0063] In some embodiments, the Fertility The condition is assessed by examining the percentage of fertile eggs.

[0064] In some embodiments, exercise ability The state of Rotarod fatigue experiment The evaluation is based on the results.

[0065] In some embodiments, lifespan The status is assessed by detecting survival time.

[0066] In some embodiments, cognitive status is assessed by an open field test.

[0067] On the other hand, the present application provides a system for determining the aging state of a subject, which comprises a substance capable of measuring the expression and / or activity of Zip11.

[0068] In some embodiments, the system comprises an agent capable of determining the level and / or activity of Zip11 nucleic acid expression.

[0069] In some embodiments, the substance capable of determining the level and / or activity of Zip11 nucleic acid expression comprises: primers capable of amplifying Zip11 and / or a nucleic acid probe capable of specifically identifying Zip11.

[0070] In some embodiments, the system comprises an agent capable of determining the expression level and / or activity of the Zip11 protein.

[0071] In some embodiments, the substance capable of determining the expression level and / or activity of the Zip11 protein consists of a substance capable of specifically recognizing the Zip11 protein and / or a substance capable of determining the activity of the Zip11 protein.

[0072] In some embodiments, the aging condition is body form , hair condition, immune level, tumor of The condition is determined by assessing one or more of the following group: a state of the liver, a state of the spleen, a state of fertility, a state of motor function, a state of longevity, and a state of cognitive function.

[0073] In some embodiments, the body morphology is assessed by detecting spinal morphology.

[0074] In some embodiments, the hair condition is assessed by detecting hair loss.

[0075] In some embodiments, immune status is assessed by detecting cytokine levels.

[0076] In some embodiments, the tumor status is determined by tumor volume. , tumor growth status and / or the presence of tumor cells It is evaluated by detecting

[0077] In some embodiments, the liver condition is: Alanine aminotransferase activity, aspartate aminotransferase activity and / or by detecting the liver / body weight ratio.

[0078] In some embodiments, the spleen status is measured by spleen to body weight. ratio of It is evaluated by measuring

[0079] In some embodiments, the fertility status is assessed by examining the rate of fertile eggs.

[0080] In some embodiments, the exercise ability The state of Rotarod fatigue experiment The evaluation is based on the results.

[0081] In some embodiments, lifespan The status is assessed by detecting survival time.

[0082] In some embodiments, the cognitive status is assessed by detecting an absentee field test.

[0083] On the other hand, the present application aging A method for assessing the effect of a candidate substance on a condition is provided, said method comprising: 1) The subject Record administering a supplement; 2) comparing the change in Zip11 expression and / or activity in the subject before and after the administration; and 3) evaluating the effect of the candidate substance on the aging state of the subject based on the change in Zip11 expression and / or activity. Pu.

[0084] In some embodiments, said activity and / or expression of Zip11 comprises activity and / or expression at the level of Zip11 nucleic acid and / or at the level of Zip11 protein.

[0085] In some embodiments, the candidate substance is subject When the expression and / or activity of Zip11 is upregulated in subject It slows or improves the aging state of the body.

[0086] In some embodiments, the delay or amelioration of aging is prevention or treatment of curvature of the spine, prevention or treatment of hair loss, increasing cytokine levels, improving immunity, preventing or treating tumors, extending lifespan, preventing or treating liver damage, preventing or treating spleen damage, preventing or treating decline in fertility, improving athletic performance, delaying decline in athletic performance, and / or improving behavioral and cognitive performance. It consists of .

[0087] In some embodiments, the candidate substance is subject When Zip11 expression and / or activity is decreased in subject accelerates or worsens the aging process.

[0088] In some embodiments, the accelerated or worsened aging consists of worsening spinal curvature, worsening hair loss, decreased cytokine levels, decreased immunity, worsening or recurrence of tumors, shortened lifespan, worsening liver damage, worsening spleen damage, worsening decreased fertility, decreased motor performance, and / or decreased behavioral and cognitive performance.

[0089] In another aspect, the present application also provides a non-human animal or part thereof that overexpresses Zip11.

[0090] In some embodiments, the non-human animal or portion thereof comprises additional copies of the Zip11 gene compared to a wild-type counterpart.

[0091] In some embodiments, the Zip11 gene is regulatably expressed by a tissue-specific promoter.

[0092] In some embodiments, the additional copy number of the Zip11 gene is specifically expressed in the liver.

[0093] In some embodiments, the tissue-specific promoter comprises the nucleotide sequence set forth in SEQ ID NO.1.

[0094] In some embodiments, the non-human animal or portion thereof exhibits a phenotype that delays or ameliorates an aging condition.

[0095] In some embodiments, the delay or amelioration of aging is prevention or treatment of curvature of the spine, prevention or treatment of hair loss, increasing cytokine levels, improving immunity, preventing or treating tumors, extending lifespan, preventing or treating liver damage, preventing or treating spleen damage, preventing or treating decline in fertility, improving athletic performance, delaying decline in athletic performance, and / or improving behavioral and cognitive performance. It consists of .

[0096] In another aspect, the present application also provides a non-human animal or part thereof in which the expression and / or function of the Zip11 gene is disrupted.

[0097] In some embodiments, at least a portion of the Zip11 gene is knocked out.

[0098] In some embodiments, at least a portion of exon 5 of the Zip11 gene is knocked out.

[0099] In some embodiments, the non-human animal or portion thereof exhibits a phenotype of accelerated or exacerbated aging.

[0100] In some embodiments, the accelerated or worsened aging may include increased spinal curvature, increased hair loss, decreased cytokine levels, weakened immunity, tumor progression or recurrence, shortened lifespan, liver damage, or other conditions. hindrance Increased spleen hindrance This consists of increased risk of stroke, decreased reproductive ability, decreased motor skills, and / or decreased behavioral cognition.

[0101] Other aspects and advantages of the present application will become readily apparent to those skilled in the art from the following detailed description. The following detailed description shows and describes only exemplary embodiments of the present application. Those skilled in the art will recognize that the present application will enable those skilled in the art to make modifications to the particular embodiments disclosed without departing from the spirit and scope of the invention associated with the present application. Accordingly, the drawings and specification of the present application are illustrative only and not restrictive. [Brief explanation of the drawings]

[0102] The specific features relevant to this application are set forth in the appended claims. The features and advantages of the invention to which this application is directed can be better understood by reference to the following embodiments and drawings, a brief description of which follows:

[0103] [Figure 1A] FIG. 1 shows the results of a comparison of the sequences of human Zip 11 and zebrafish Zip 11. [Figure 1B] FIG. 1 shows the steps for constructing Zip11 knockout zebrafish according to the present invention. [Figure 2] FIG. 1 shows the morphological characteristics of Zip11 − / − zebrafish described in the present invention. [Figure 3] 1 shows the survival curve of Zip11 − / − zebrafish according to the present invention. [Figure 4] FIG. 1 shows the reproductive rate of the F2 generation of Zip11 − / − zebrafish described in the present invention. [Figure 5]FIG. 1 shows the construction process of the Zip11 − / − mouse model described in the present invention, where A: knockout schematic diagram, B: vector structure, and C: Zip11 mRNA level. [Figure 6-7] FIG. 1 shows the state of hair loss in Zip11 − / − mice described in the present invention. [Figure 8] FIG. 1 shows the state of hair loss and spleen in Zip11 − / − mice described in the present invention. [Figure 9] FIG. 1 shows the state of spleen sections from Zip11 − / − mice described in the present invention. [Figure 10] FIG. 1 shows the state of liver sections from Zip11 − / − mice described in the present invention. [Figure 11] FIG. 1 shows electropherograms of the appearance and genotype of Zip11 − / − mice described in the present invention. [Figure 12] FIG. 1 shows the growth status of tumors in Zip11 − / − mice according to the present invention. [Figure 13] FIG. 1 shows the secretion levels of inflammatory factors in Zip11 − / − mice according to the present invention. [Figure 14] FIG. 1 shows the motility of Zip11 − / − mice according to the present invention. [Figure 15] FIG. 1 shows the construction process of the ZIP11 KI mouse model described in the present invention. [Figure 16] The expression level of ZIP11 mRNA in wild-type mice during knife beetle protein A-induced inflammation is shown. [Figure 17] Comparison of liver (A), liver / body weight ratio (B), ALT (C), and AST (D) values ​​between wild-type and ZIP11 KI mice. [Figure 18] The morphology of hepatocytes in ZIP11 KI mice is shown using different staining methods. [Figure 19] This indicates that vitamins increase the expression level of the ZIP11 gene in human-derived cells. [Figure 20] Shown is the weight gain of Zip11 − / − mice fed a high-fat diet. [Figure 21] This shows the impaired learning and cognitive abilities of Zip11- / - male mice. [Figure 22] 1 is a graph showing the trajectory of a mouse in an open field experiment. [Figure 23] Figure 1 shows increased p53 protein levels in Zip11 − / − mice. [Figure 24] This shows a decrease in M1 macrophages (A) and an increase in M2 macrophages (B) in Zip11 − / − mice after LPS stimulation. [Figure 25] This shows the reduction of bilirubin in ZIP11-overexpressing mice. [Figure 26] This shows elevated bilirubin in Zip11- / - mice. DETAILED DESCRIPTION OF THE INVENTION

[0104] The following specific embodiments illustrate how the present application may be practiced, and other advantages and benefits of the present application may be readily apparent to those skilled in the art as disclosed herein.

[0105] Zip11 nucleic acid, Zip11 protein

[0106] As used herein, the terms "Zip11," "Zip11," "ZIP11," and "SLC39A11" are used interchangeably and generally refer to members of the Solute Carrier Protein Family 39 (SLC39). They may also refer to the gene encoding the Zip11 protein. Zip11 as used herein may include human Zip11, zebrafish Zip11 / or mouse Zip11, or functional fragments or variants thereof, or homologs in other species.

[0107] For example, human ZIP11 may be comprised of a 342 amino acid fragment, the amino acid sequence of which can be found in NCBI accession number NP_001153242.1, encoded by NCBI database Gene ID: 201266; zebrafish Zip11 protein may comprise a 123 amino acid fragment, the amino acid sequence of which can be found in NCBI accession number XP_021335877.1, and the zebrafish Zip11 gene sequence in NCBI accession number NC_007123.7; mouse Zip11 may comprise a 342 amino acid fragment, the amino acid sequence of which can be found in NCBI accession number NP_001159975.1; the mouse Zip11 gene is listed in MGI database ID: 1917056. In this application, Zip11 knockout may be referred to as Zip11 KO, Zip11 KO, or Zip11- / -. Knock-in or overexpression of Zip11 can be designated as ZIP11 KI or ZIP11 KI.

[0108] The inventors of this application used genome-wide association studies to compare the genomes of 2,178 centenarians (564 men, 1,614 women) and 2,299 middle-aged controls (773 men, 1,526 women). They found multiple sequence variations, including single nucleotide polymorphisms (SNPs), that were significantly associated with longevity (p<105) in both men and women. In men, multiple SNPs, including ZIP11, were found to be significantly associated with aging status (Zeng Y, et al. Sex Differences in Genetic Associations With Longevity, JAMA Netw Open. 2018 Aug;l(4).pii: el81670.).

[0109] aging phenomenon

[0110] As used herein, "aging" generally refers to the accumulation of changes that occur in an organism over time. These changes range from those that affect the function of genes and cells to those that affect the function of organs, organ systems, and the entire organism. In particular, aging refers to changes that occur after an organism has reached biological maturity and may progress until the organism dies. aging The status can be determined by assessing a group of statuses such as body shape, hair status, immune level status, tumor status, liver status, spleen status, fertility status, motor performance status, lifespan status, and cognitive performance status.

[0111] The present inventors have found that aging is delayed or ameliorated in individuals with increased Zip11 activity and / or expression. 「"Delaying or ameliorating" generally refers to preventing or treating by reducing, minimizing, or eliminating the progression of a condition, disease, disorder, or phenotype (including abnormalities or symptoms). The term "delaying or ameliorating aging" includes preventing or treating spinal curvature, preventing or treating hair loss, increasing cytokine levels, improving immunity, preventing or treating tumors, extending lifespan, preventing or treating liver damage, preventing or treating splenic damage, preventing or treating decline in fertility, enhancing athletic performance and / or delaying the time point at which athletic performance declines, and / or enhancing behavioral and cognitive performance. The prevention or treatment of spinal curvature may include reducing the degree of spinal curvature or straightening the spine. The prevention or treatment of hair loss may consist of reducing the degree of hair loss or eliminating hair loss altogether. The cytokines may be IL-6, IL-2, IL-1-β, IL-7, IL-10, IL-12, TNF-α, IFN-α, IFN-β, IFN-γ, GM-CSF, or G-CSF. The enhancement of immunity may include increasing cytokine levels. The enhancement of immunity may include increasing the proportion of M1 macrophages and decreasing the proportion of M2 macrophages. The prevention or treatment of tumors may include reducing tumor size or preventing tumor development. The extension of lifespan may include reducing the activity / expression level of senescence-associated genes. The extension of lifespan may include extending survival time or increasing population survival. The prevention or treatment of liver damage may include improving susceptibility to hepatitis, for example, by: Alanine aminotransferase and aspartate aminotransferase levels The evaluation can be based on the liver cell morphology as well as the liver function. fertility Prevention or treatment of the decline may include increasing the rate of fertile embryos. The improvement of motor ability may include extending the time for which motor ability can be maintained, delaying the time point at which motor ability declines, etc. The improvement of cognitive ability may include increasing the desire to explore unknown spaces (e.g., in an open field experiment).

[0112] Furthermore, the present inventors have found that aging is accelerated or exacerbated in individuals with reduced Zip11 activity and / or expression. "Promote "Accelerating or worsening" generally means the increase, enhancement, or maintenance of a condition, disease, disorder, or phenotype (including abnormalities or symptoms). Accelerated or worsening aging includes accelerated or worsening spinal curvature, accelerated or worsening hair loss, decreased cytokines, weakened immunity, accelerated or worsening tumors, shortened lifespan, accelerated or worsening liver damage, accelerated or worsening spleen damage, decreased reproductive function, decreased athletic performance, and / or decreased cognitive performance. promotion The accelerated or worsened spinal curvature may include an increase in the degree of spinal curvature. The accelerated or worsened hair loss may include abnormal or complete hair loss of scalp or body hair. Examples of the cytokines include IL-6, IL-2, IL-1-β, IL-7, IL-10, IL-12, TNF-α, IFN-α, IFN-β, IFN-Γ, GM-CSF, G-CSF, etc. cytokines The weakening of the immune system may include reducing cytokine levels. The weakening of the immune system may include reducing the proportion of M1 macrophages and increasing the proportion of M2 macrophages. promotion The deterioration may include an increase in tumor size. The shortening of life span may include a shortening of survival time or a shortening of silent population survival rate. promotion or worsening may constitute an enlarged liver. R The above promotion or worsened spleen hindrance may include an enlarged spleen. Reproductive function Decline of promotion Or, the deterioration may include a decrease in the rate of fertilizable eggs. The decline in motor ability may include a shortened time to maintain motor ability or an earlier onset of motor ability decline. The decline in cognitive ability may include a decreased desire to explore unknown spaces (e.g., in an open field experiment).

[0113] The changes in Zip11 activity and / or expression can be measured using methods well known to those skilled in the art, such as immunohistochemistry, PCR, RT-PCR, in situ hybridization, Southern blot, Western blot, Northern blot, spectrophotometry, gene chips, flow cytometry (FACS), protein microarrays, DNA sequencing, and ELISA. In some embodiments, the methods involve measuring Zip11 activity and / or expression. Nucleic acids and a primer capable of specifically amplifying Zip11. Nucleic acids The method may include a nucleic acid probe capable of specifically recognizing Zip11 protein. The probe may specifically bind to a specific site on the Zip11 gene and not to genes other than Zip11, thereby "specifically identifying" the Zip11 gene, and the probe provides a detectable signal. The primers amplify the Zip11 gene but not other genes, i.e., "specific amplification." In some embodiments, the candidate consists of an oligonucleotide fragment. Such a fragment must be sufficiently long to hybridize with the RNA or DNA of the sample. It may be 10-40 nucleotides or more, e.g., 45, 50, 100, 500, or even the full length. Optionally, the method may include a reagent that specifically recognizes Zip11 protein and a substance capable of measuring the activity of Zip11 protein. The substance may be a protein-binding molecule, such as an antibody or ligand, that specifically binds to Zip11 or its protein fragment.

[0114] Uses, methods, and systems

[0115] The present application provides methods for identifying substances that can affect aging, the methods including determining the effect of a candidate substance on activity and / or expression, and identifying a candidate substance that alters the activity and / or expression as a substance that can affect aging. In some embodiments, the methods can be used for drug screening. For example, a substance that increases Zip11 activity and / or expression can be identified as a substance that can affect aging, and the substance can be used to prepare a drug that can delay or ameliorate aging. For example, if a substance that decreases Zip11 activity and / or expression is identified as a substance that can increase Zip11 activity and / or expression and that can affect aging, the substance may have toxic side effects.

[0116] The present application provides a system for identifying substances capable of affecting aging, which may include a substance capable of determining the effect of a candidate substance on Zip11 activity and / or expression. The system may also include a kit. The kit may include a substance capable of affecting aging described herein in suitable packaging and may include written documentation, such as instructions for use, a discussion of clinical trials, and a list of side effects. Such kits may also include information demonstrating or confirming the activity and / or benefits of the composition, such as scientific literature, package inserts, clinical trial results, and / or summaries thereof, and / or references describing administration, dosage, side effects, drug interactions, or other information useful to healthcare professionals. The kit may also include an additional agent. In some cases, the substance capable of affecting aging and the agent are provided as separate compositions in separate containers within the kit. In some cases, the substance capable of affecting aging and the agent are provided as a single composition within the kit container. Suitable packaging and additional items (e.g., measuring cups for liquid formulations, foil packaging to minimize exposure to air, etc.) are known in the art and may be included in the kit. In some cases, the system may be offered, sold, and / or marketed to interested parties, including healthcare providers, doctors, nurses, pharmacists, prescribers, drug developers, drug manufacturers, etc. In other cases, the system may be sold directly to consumers.

[0117] The present application provides the use of a nucleic acid molecule encoding Zip11 or its expression product for the preparation of a drug, the drug being used to delay or ameliorate aging. In some embodiments, the nucleic acid molecule encoding Zip11 or its expression product can be used to prepare a drug for gene therapy. The drug may include a vector capable of expressing the nucleic acid molecule. The vector may be a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. Vectors that function as self-replicating nucleic acid structures and vectors that are integrated into the genome of a host cell into which they are introduced are also included. Certain vectors can induce the expression of a nucleic acid operably linked thereto. Examples of such drugs include substances that act on the promoter or enhancer region of Zip11 to promote expression, substances that promote transcription of Zip11 gene mRNA, or substances that exert similar effects with the aid of intracellular transcription factors. The drug may also be contained in a container that can be placed together with packaging materials that explain how to use the drug. Generally, these instructions include definitive statements describing the concentration of the drug and, in some embodiments, may include the relative amounts of excipient components or diluents (e.g., water, saline, or PBS) needed to reconstitute the pharmaceutical composition.

[0118] The present application also provides an effective amount of a nucleic acid molecule encoding Zip11 or its expression product, in a subject in need thereof. subject The present invention provides a method for slowing or reversing aging, comprising administering to a subject a composition comprising: a) a compound selected from the group consisting of ...

[0119] For purposes of this application, the term "effective amount" generally refers to a non-toxic but sufficient amount of an agent to produce a desired biological result, such as an amount sufficient to reduce and / or ameliorate the severity and / or duration of a condition (such as a symptom of aging or a condition associated with aging) or symptom, prevent the worsening of the condition, cause a reduction in the condition, prevent the recurrence, onset, or onset of one or more symptoms associated with the condition, or enhance or improve the prophylactic or therapeutic effects of another treatment (e.g., a prophylactic or therapeutic agent). For example, an effective amount may be an amount that slows or ameliorates aging.

[0120] In the context of this application, the term "administration" generally refers to the amount of a substance or pharmaceutical composition administered. subject (e.g., a patient with symptoms of aging) Administration can be by any suitable means, including, but not limited to, injection (subcutaneous, intravenous, parenteral, intraperitoneal, intrathecal, etc.), oral, inhalation, rectal, and intradermal.

[0121] For example, the substances described herein that increase Zip11 activity and / or expression, the nucleic acid molecule encoding Zip11 or its expression product, and the vector can be prepared, separately or together, in a form suitable for oral administration. Examples include tablets or capsules prepared by conventional means with the addition of pharmaceutically acceptable excipients such as binding agents, fillers, glidants, disintegrants, or wetting agents. Methods for encapsulating tablets are known to those skilled in the art. For oral administration, liquid preparations can take the form of, for example, solutions, syrups, or suspensions, or they can be provided in the form of dry products that are reconstituted with water or other suitable excipient carriers before use. Various delivery systems are known and can be used to administer the compounds of the invention, including encapsulation in liposomes, particles, or microcapsules, recombinant cells capable of expressing the compound, receptor-mediated cell division, and construction of nucleic acids as part of retroviruses or other vectors.

[0122] In some embodiments, nucleic acid molecules comprising a nucleic acid encoding Zip11 or its expression product may be administered by gene therapy to treat, inhibit, or prevent a disease or disorder associated with aberrant Zip11 expression and / or activity. Gene therapy refers to treatment performed by administering an expressed or expressible nucleic acid to a subject. The nucleic acid molecules described herein produce proteins encoded thereby that mediate a therapeutic effect. For example, Zip11 activity and / or expression of the encoding nucleic acid molecule or its expression product may be associated with a condition requiring it. subject can be increased after administration to

[0123] The present application provides a method for measuring Zip11 activity and / or expression of a subject using a substance capable of measuring Zip11 activity and / or expression or a reagent prepared from said substance, and comparing said Zip11 activity and / or expression with a standard activity and / or expression, thereby determining an increase or decrease in the activity or expression of the Zip11 gene. aging In some embodiments, the methods may be used in diagnostic / medical testing, for example by a medical practitioner: subject Based on Zip11 activity and / or expression in subject In some cases, the aging status of a subject can be determined in a study, e.g. aging The described methods can be used to evaluate the effect of candidate substances on a condition. In some cases, the described methods can also be used in pharmaceutical processes, such as identifying substances to be screened for their effect on an aging condition. In some cases, the methods can be used in diagnostic / medical processes, e.g., in the evaluation of a subject's aging It can also be used to identify a condition.

[0124] This application is aging The present invention provides the use of substances for determining Zip11 activity and / or expression for the preparation of reagents for use in identifying conditions. In some cases, reagents capable of detecting Zip11 expression / activity can be prepared and used in diagnostic and / or medical testing.

[0125] The present application provides a method for measuring Zip11 expression and / or activity in a subject, comprising administering to the subject a substance capable of measuring Zip11 expression and / or activity. aging In some embodiments, the system may be offered, sold, and / or marketed to interested parties, including healthcare providers, physicians, nurses, pharmacists, prescribers, drug developers, drug manufacturers, etc. The system may also be sold directly to consumers.

[0126] In addition, the present application provides a method for determining whether a candidate substance is a aging In some cases, the method can be used for drug screening. For example, Record A supplementary substance is administered to the subject, and the expression and / or activity of Zip11 in the subject before and after the administration is compared. If the expression and / or activity of Zip11 is increased, the subject is treated with the supplementary substance. Record Supplements are substances that can delay or improve aging and are used as anti-aging agents. aging The method described can be used to evaluate the safety of candidate substances. Record A supplementary substance is administered to the subject, and changes in Zip11 expression and / or activity are compared before and after the administration. If Zip11 expression and / or activity is decreased, the subject is considered to have a decreased Zip11 expression and / or activity compared to the pre-administration. Record The auxiliary substance aging It can be determined to be a promoter.

[0127] Non-human animals or parts thereof

[0128] The present application provides a non-human animal or portion thereof that overexpresses Zip11. As used herein, the term "overexpression" generally refers to a gene that is transcribed or translated at a detectably higher level in corresponding cells compared to normal cells of the same tissue type.

[0129] Thus, overexpression can refer to overexpression of proteins or RNAs (due to transcription, post-transcriptional processing, translation, post-translational processing, altered stability, or altered proteolysis), localized overexpression (increased nuclear localization), or enhanced functional activity due to altered protein trafficking patterns. For example, overexpression can refer to an increase in expression of 100%, 150%, 200%, 250%, 300%, 400%, 500%, or more compared to normal or control cells. Overexpression can be achieved by methods known in the art, such as introducing a foreign gene. For example, the Zip11 gene can be overexpressed by introducing a promoter-containing vector into the genome of a non-human animal. The promoter may be a strong CAG promoter. The vector can insert a target gene into the target gene region of a host by homologous recombination and can be any vector available for genetic engineering, such as a plasmid vector, a viral vector, a mucosal vector, a bacterial artificial chromosome (BAC), or a yeast artificial chromosome (YAC), as well as a vector other than a plasmid. In some embodiments, sgRNAs targeting the mouse H11 locus can be constructed using Cas9 / RNA system gene targeting technology. In this application, the term "H11" generally refers to a motif that allows the insertion of a strong exogenous promoter therein to drive higher expression. In other cases, a vector carrying the CAG-Loxp-Stop-Loxp-hZ / P7J-P2A-tdTomato fragment can be simultaneously constructed and co-injected with Cas9. After the DNA strand is cleaved by Cas9, the CAG-Loxp-Stop-Loxp-hZIP11-P2A-tdTomato fragment can be integrated into the target site by homologous recombination. The host cells into which the knock-in vector is introduced are cells of the non-human animal or cells (including cell clusters) capable of differentiating into the non-human animal cells. Various types of host cells can be used depending on the purpose, including pluripotent stem cells such as ES cells and iPS cells, germline stem cells such as spermatogonial stem cells capable of differentiating into germ cells, and fertilized eggs.The knock-in vector can be introduced into the host cell by a known method such as electroporation. The pluripotent stem cells can be injected into an early embryo by a known method such as microinjection, and then transplanted into a surrogate mother for development to obtain a chimeric animal. Alternatively, by mating the chimeric animals, individuals with purified knock-in arrays can be obtained from their offspring.

[0130] Non-human animals or parts (e.g., ZIP 11 KI mice) that overexpress the ZIP11 gene described herein exhibit increased ZIP11 activity and / or expression, increased sensitivity to immune stimulation (e.g., in knife bean protein-induced liver injury, ZIP 11 KI mice exhibit at least four-fold, e.g., four-fold, five-fold, six-fold, or more, increased levels of glutaminase and glutathione compared to wild-type mice), and / or exhibit greater resistance to aging-related diseases. Non-human animals or parts that overexpress the ZIP 11 gene as described can shrink or delay tumor growth. For example, female Zip11- / - mice bearing tumors also exhibit larger tumor volumes than wild-type mice.

[0131] The present application also provides a non-human animal or a part thereof, which may be a rodent or a fish, such as a mouse. The expression and / or function of the Zip11 gene of the non-human animal or part thereof may be disrupted. As used herein, the term "disrupted" generally means that the gene is not expressed and / or the expression product loses its function. It is conceivable that the gene may be structurally disrupted (e.g., by insertion of an Flp / FRT element or a Cre / Loxp element) so that the gene contains at least one mutation or structural change, such that the disrupted gene is not expressed or cannot direct efficient expression of an expression product. The disruption may be achieved by knocking out at least a portion of the Zip11 gene. The knockout may be constructed by any method known in the art.

[0132] In some cases, this can be achieved with the aid of constructs from the Crisper / Cas9 system. Specifically, by crossing a Zip11 allele mouse having a Flox locus with a tool mouse specifically expressing Cre, it is possible to obtain a mouse model in which the expression and / or function of the Zip11 gene is disrupted. It is also possible to knock out a target gene using the principle of homologous recombination. For example, the Slc39all gene can be Flox-modified using embryonic stem cell targeting. In some cases, at least a portion of exon 5 of the Zip11 gene is knocked out. For example, the non-human animal or a portion thereof may be a mouse model in which the Zip11 gene has been knocked out. Here, "disrupted" means that the activity and / or expression of Zip11 is significantly reduced compared to a wild-type mouse in which the Zip11 gene is not knocked out.

[0133] Non-human animals or tissues in which Zip11 gene expression and / or function are disrupted as described herein exhibit reduced Zip11 activity and / or expression, and accelerated or worsened aging phenotypes. For example, in a Zip11 KO zebrafish model, the reproductive rate of 6-12 month old female fertilized eggs is reported to be reduced by about 10% to about 100% (e.g., about 20% to about 90%, about 30% to about 80%, about 40% to about 70%, or about 50% to about 60%), while the reproductive rate of 0-3 month old male fertilized eggs is reported to be reduced by about 10% to about 100% (e.g., about 20% to about 80%, about 30% to about 70%, about 30% to about 60%, or about 40% to about 50%), and the reproductive rate of 6-12 month old males is reported to be reduced to less than 1%. For example, Zip11 KO zebrafish have a female survival rate of 85% and a male survival rate of only 25% at 10 months of age compared to wild-type mice.For example, Zip11 knockout mice can exhibit at least a 30% reduction in motor ability (e.g., at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or more).

[0134] In some embodiments, the non-human animal or part thereof may be used in research, such as evaluating the effect of a candidate substance on Zip11 activity and / or expression levels. In some embodiments, the non-human animal or part may be used in a pharmaceutical process, such as screening to identify substances that affect Zip11 activity and / or expression. In some embodiments, the non-human animal or part may be used in a diagnostic and / or medical testing process, such as identifying the aging status of a subject. In some cases, the non-human animal or part thereof may be provided, sold, and / or marketed to interested parties, such as researchers, sales representatives, pharmaceutical representatives, etc.

[0135] In this application, the term "comprising" generally refers to the inclusion of explicitly specified features but not the exclusion of other elements.

[0136] As used herein, "about" generally means within 0.5% to 10% of the specified value, e.g., 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, 10% above or below.

[0137] The following examples are not intended to be limited by theory, and are used only to illustrate the fusion proteins, preparation methods, uses, etc. of the present application, and are not intended to limit the scope of the present application.

[0138] Example

[0139] Example 1: Construction of a zebrafish Zip11 knockout (KO) model

[0140] For the zebrafish gene (sequence available in the NCBI database, Gene ID: 101882215), an sgRNA was designed, and its sequence is shown in SEQ ID NO: 4. The pT7-gRNA plasmid was used as a PCR template. The gRNA template was purified by gum cut and then in vitro transcribed using a T7 kit. 1% agarose gel electrophoresis confirmed the transcription of the sgRNA, and a gRNA stock solution suitable for microinjection was obtained by phenol-chloroform-isoamyl alcohol extraction and purification. The pXT7-Cas9 plasmid was linearized with the restriction enzyme Xbal. After confirming completion of linearization by agarose gel electrophoresis, column purification was performed using a T7 kit. Cas9 mRNA was used as an in vitro transcription template, and the gRNA was purified by agarose gel electrophoresis to prepare the Cas9 mRNA stock solution for microinjection. The appropriate concentrations of sgRNA and Cas9-mRNA were mixed and microinjected into fertilized zebrafish eggs. Uninjected wild-type zebrafish embryos served as controls. Following incubation, the F0 generation of Zip knockout zebrafish was obtained. Figure 1A shows the amino acid sequence comparison between human and zebrafish Zip11, and Figure 1B shows the location of the Cas / gRNA target site in the knockout zebrafish Zip11 gene.

[0141] Example 2: Zip11 KO zebrafish model aging phenotype

[0142] The F0 generation was crossed with male and female zebrafish to produce the F1 generation of pure zebrafish (Zip11- / -), and the F1 generation was crossed to produce the F2 generation of Zip11- / - mutants. These were cultured and their phenotypes were observed and recorded. The results are shown in Figure 2. Continuous comparative observation of Zip11 knockout and wild-type zebrafish revealed that 1.5-year-old F1 generation females had a curved spine. F2 generation male and female zebrafish had a curved spine and thin, degenerated fins, whereas wild-type fins were intact.

[0143] The survival rates of the F2 and wild-type zebrafish were recorded from 3 to 24 months of age, and survival curves were plotted. As shown in Figure 3, the survival rate of the Zip11- / - F2 generation from birth to 6 months of age was comparable to that of wild-type zebrafish. From 6 months of age, male F2 fish showed high mortality, and all died by 16 months of age. Female F2 fish died at 8 months of age, and the survival rate after 20 months of age was less than 60%. The survival rate of female Zip11- / - fish was higher than that of males.

[0144] Eggs or sperm from the F2 generation of male and female zebrafish of different ages (0-3 months old, 6-12 months old) were fertilized in vitro with sperm or eggs from wild-type zebrafish and cultured, and the percentage of fertilized eggs that developed into complete individuals was counted. The results are shown in Figure 4. Figure 4 shows that the percentage of fertilized eggs decreases with increasing age, and that F2 generation male zebrafish have a lower percentage of fertilized eggs than females.

[0145] Figures 2–4 show that Zip11-deficient zebrafish exhibit aging phenotypes, including spinal curvature, cachexia, impaired regenerative capacity, premature infertility, and shortened lifespan, and aging is more severe in males.

[0146] Example 3 Construction of Zip11 knockout (KO) mice

[0147] Using the principle of homologous recombination, we constructed a Zip11 knockout model mouse targeting gene (MGI No. 1917056 or Ensembl No. ENSMUSG00000041654) with its transcript version (ENSMUST00000071539). The embryonic stem cell targeting vector was constructed by Infusion Inc. and contained a 3.4 kb 5′ homology arm (5′ homology arm forward primer SEQ ID NO: 5, 5′ homology arm reverse primer SEQ ID NO: 9, 3′ homology arm forward primer SEQ ID NO: 9, 3′ homology arm reverse primer SEQ ID NO: 10), a 2.3 kb Flox region (SEQ ID NO: 13), PGK-Neo-polyA (SEQ ID NO: 14), a 3.3 kb 3′ homology arm, and an MCl-TK-polyA (SEQ ID NO: 15) negative screening marker, where Flox targets exon 5. Figure 5B shows the plasmid map of the targeting vector. This vector was linearized and introduced into Jm8a3 embryonic stem cells by electroporation. Resistant clones were obtained by screening with G418 and Ganc. Positive clones were screened for homologous recombination by long fragment PCR.

[0148] Positive embryonic stem cell clones were amplified and injected into blastocysts of C57BL / 6 mice. The injected blastocysts were then transplanted into the oviducts of pseudopregnant female mice (C57BL / 6J) according to standard procedures. After birth, the coat color was used to determine whether the mice were chimeric and the degree of chimerism, and chimeric mice were obtained.

[0149] These chimeric male mice were mated with Flp mice to generate F1 generation mice. Neo-containing heterozygote-positive mice were identified by PCR and confirmed by sequencing. Subsequently, positive F1 heterozygote mice were mated with Flp mice to generate F2 generation mice. De-neo-heterozygote-positive mice were identified by PCR and confirmed by sequencing. Flp-mediated homologous recombination was used to generate targeted mice with Flox sites at both ends of the Zip11 CKO (Conditional Knockout) gene, i.e., Zip11 allele mice with Flox sites. After mating Zip11 allele mice with the Flox locus with tool mice expressing Cre recombinase specifically in that tissue, knockout of the Flox locus in Flox-purified Cre-positive mice resulted in functional loss of the target gene Zip11 in specific tissues and cell types. This construct is shown in Figure 5A. Figure 5C shows that the KO mice had reduced Zip11 expression and almost no mRNA transcription, indicating successful knockdown.

[0150] Example 4 Phenotyping of the aging state of Zip11 KO mice

[0151] 4.1 Male and female Zip11 KO mice showing signs of hair loss

[0152] Mice were raised in an SPF animal house. 22 days after weaning, they were separated into male and female cages and allowed to eat and drink ad libitum. The animal house was maintained at a temperature of 22±3°C, humidity of 35-5%, and a 12-hour day / night cycle. The mice were regularly observed for changes in phenology.

[0153] Figure 6 shows that Zip11 male mice show signs of hair loss at 6 months of age, and Figure 7 shows that Zip11 KO female mice show signs of hair loss on both the back and head at 8 months of age.

[0154] 4.2 Zip11 mice showing signs of splenomegaly

[0155] Eight-month-old Zip11 knockout and wild-type mice were anesthetized and sacrificed, and their spleens and livers were quickly removed, fixed in 4% paraformaldehyde for 48 hours, embedded in paraffin, and then sectioned at 5 p.m., routinely stained with hematoxylin and eosin (H&E), sealed with neutral rubber, and observed for histological changes under a microscope.

[0156] Figure 8 shows that 8-month-old Zip11 KO female mice showed signs of splenomegaly and enlarged spleens compared with control wild-type mice; and spleen sections showed enlarged spleen tissue in Zip11 knockout mice (Figure 9).

[0157] 4.3 Zip11 KO mice showed signs of liver hypertrophy

[0158] When sections of the mouse liver were observed in the same manner as above, it was confirmed that the liver tissue of Zip11KO mice was enlarged (Fig. 10).

[0159] 4.4 Spontaneous tumor formation in Zip11 KO mice

[0160] Figure 11 shows a photograph of the experimental mouse on the left and an electropherogram showing the genotype of the female experimental mouse, indicating that this mouse was a Zip11 knockout mouse. At 5 months of age, a mass was observed in the left forelimb of this Zip11 knockout mouse, as well as a mammary tumor. No spontaneous tumor growth was observed in wild-type control mice in the same cage (Figure 12). As can be seen in Figure 12, the Zip11 KO mouse exhibited significant tumor growth.

[0161] 4.5 Zip11 KO mice have higher levels of inflammatory factors

[0162] RT-PCR analysis of liver tissue from Zip11- / - mice compared with that from wild-type mice (Fig. 13) revealed that the mRNA expression levels of inflammatory factors such as IL-6 and TNF-α were elevated in Zip11-deficient mice, suggesting a stronger inflammatory response.

[0163] 4.6 Decreased locomotion in Zip11 KO mice

[0164] The effects of Zip11 on motor function were assessed using a linear accelerometer (Panlab / Harvard Apparatus, Holliston, USA). Zip11 KO and wild-type mice were fitted with a linear accelerometer. The rotational speed was increased from 4 rpm to 40 rpm over a 300-second cycle. The acceleration protocol was adopted to eliminate adaptation to the accelerometer and minimize discrepancies between individual mice due to imbalances in performance. Each trial ended when the mouse fell from the baton swivel. Each mouse underwent three baton swivel acceleration runs per experimental day, spanning four experimental days. Animals were allowed to recover for 5 min between trials to prevent fatigue-induced declines in performance. The latency to fall from the baton swivel was compared between Zip11 KO and wild-type mice.

[0165] The results are shown in FIG. 14, and it was found that Zip11 KO mice had a shorter time to maintain rotation and weaker motor ability than wild-type mice.

[0166] Example 5 Zip11 KO mice induce obesity in male mice

[0167] A high-fat diet (HFD) was administered to Zip11 KO male mice and wild-type mice to establish a hyperlipidemia model. Three wild-type mice, four Zip11 + / - mice, and four Zip11 - / - mice were fed the high-fat diet and their body weights were measured weekly. The results are shown in Figure 20. Zip11 male mice were slightly heavier than wild-type mice, indicating that knockout of Zip11 in male mice leads to the development of obesity in male mice.

[0168] Example 6 Cognitive and learning impairments in Zip11 KO mice

[0169] After 7 weeks of high-fat diet, Zip11 knockout mice (4.5 months old) were subjected to an open-field experiment to probe a range of behavioral abilities. This experiment exploited mouse avoidance, i.e., mice tend to move to the periphery of the open field due to fear of open, unfamiliar environments, whereas mice have a natural tendency to explore novel environments, exploring in the center. Longer movement times and distances in the center indicate higher exploratory desire and cognitive ability, whereas shorter movement times and distances in the center indicate lower exploratory desire and cognitive ability.

[0170] Knockout male mice (four Zip11+ / - mice, four Zip11- / - mice) and wild-type male mice (three) were placed in the center of the open field apparatus and allowed to move freely within the apparatus for 15 minutes. The distance traveled in the open field, the distance traveled in the center, and the total distance and time traveled in the open field were recorded. After each mouse finished, the apparatus was wiped with 75% alcohol to remove feces and urine, so that the odor left by the previous mouse would not affect the behavioral performance of the next mouse.

[0171] The statistical results of the open field experiment are shown in Figure 21, and the movement trajectories of the mice are shown in Figure 22. Zip11 knockout mice showed reduced movement distance and time in the central area, suggesting that their exploratory motivation and cognitive ability were reduced, affecting their learning and cognitive abilities.

[0172] Example 7 Increased p53 protein levels in Zip11 KO mice

[0173] Eight-month-old male and female Zip11 knockout mice and their wild-type counterparts were anesthetized, and their tails were rapidly amputated. Protein was extracted from mouse tissue cells, and impurities such as cell debris were removed. The p53 protein expression level was detected by Western blot analysis. The p53 level was significantly increased in the knockout mice (Figure 23), suggesting that Zip11 knockout is associated with the promotion of p53 aging.

[0174] Example 8 Zip11 KO mice exhibit reduced macrophage immune function under LPS stimulation

[0175] Wild-type (WT) mice (n = 6) and Zip11 KO mice (n = 4) were intraperitoneally injected with LPS (2.5 mg / kg) and D-galactosamine (0.25 g / kg) to induce acute inflammation. Six hours later, the mice were anesthetized and dissected. Blood and spleens were collected, and labeled anti-CD86 and anti-CD206 antibodies were added, respectively. The proportions of keto-CD86-positive (M1-type macrophages) and CD206-positive (M2-type macrophages) cells were measured by flow cytometry.

[0176] M1 macrophages exhibit strong pro-inflammatory and antigen-presenting abilities and function as host immune clearance agents against pathogens and tumor cells. On the other hand, M2 macrophages have anti-inflammatory, wound healing, fibrosis, tissue repair, and tumor growth and invasion promotion functions. As a result, when mice were stimulated with LPS, M1 macrophages significantly decreased (Figure 24A), while M2 macrophages significantly increased, indicating a decline in the immune function of macrophages (Figure 24B).

[0177] Example 9 Construction of a ZIP11 knock-in mouse model

[0178] We constructed an sgRNA targeting the mouse H11 locus (the sequence of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125,

[0179] The vector was then microinjected into C57BL / 6J embryonic stem cells. The resulting blastocysts were then implanted into pseudopregnant C57BL / 6J female mice to generate chimeric mice. The microinjected mice were identified by PCR, sequencing, and Southern blot analysis to obtain positive F0 mice. These F0 mice were individually crossed with C57BL / 6J mice to obtain positive F1 mice by PCR, sequencing, and Southern blot analysis. Next, the heterozygous mice were crossed with tool mice specifically expressing Cre recombinase to obtain H11-ZIP11 knockin (KI) mice, thereby obtaining pure knockin mice. After anesthetization and sacrifice, the livers of ZIP11 KI mice were rapidly harvested and ZIP mRNA expression levels were measured. Figure 15B shows that the hepatic interstitial cells of Z7P77KI mice specifically overexpressed ZIP11 compared to wild-type mice, indicating successful knockin.

[0180] Example 10: Decrease in Zip11 expression level by concanavalin A

[0181] Wild-type C57BL / 6 mice were divided into two groups. The experimental group received 10 mg / kg concanavalin A (ConA) via tail vein injection. 18 hours later, liver tissue was excised and ZIP 11 expression levels were measured using RT-PCR with β-actin as a reference. The results are shown in Figure 16. ConA reduced ZIP 11 levels.

[0182] Example 11 ZIP11 KI mice are more susceptible to concanavalin A-induced hepatitis responses.

[0183] Eight-week-old large ZIP11 KI mice and wild-type control mice housed in the same cage were administered 10 mg / kg of squid protein (ConA) via the tail vein. 18 hours later, the mice were anesthetized and sacrificed via cardiac puncture to extract serum. Tissues were fixed in 4% paraformaldehyde, embedded in paraffin, stained with H&E and TUNEL, and mounted to observe histological changes.

[0184] in serum Aspartate aminotransferase (ALT) and Alanine aminotransferase The AST concentration was measured, and the results are shown in Figures 17 and 18.

[0185] Figure 17A shows a comparison of liver size and color appearance between wild-type and ZIP 11 KI mice in a ConA-induced hepatitis injury model. Figure 17B shows the liver weight-to-body weight ratio, and Figures 17C and 17D show elevated serum ALT and AST levels in ZIP 11 KI mice. Figure 18 shows hepatocyte morphology using different staining methods. These results indicate that ZIP 11 KI mice are more susceptible to ConA-induced liver injury.

[0186] Example 12 Reduction of bilirubin levels in ZIP11 KI male mice

[0187] Mice overexpressing ZIP11 in the liver, wild-type 8-week-old male mice, and ZIP11 knockout mice were fed a Methionine and Choline Deficient L-Amino Acid Diet (MCD), a diet lacking methionine and choline sources, and the mice were fed an ad libitum diet. After 4 weeks, the mice were anesthetized, their livers dissected, weighed, and subjected to a series of physiological tests.

[0188] Figure 25A compares the livers of wild-type and male overexpressing mice. Serum bilirubin levels were measured at the Zhejiang University Drug Safety Evaluation Research Center. The serum bilirubin levels of male mice overexpressing ZIP11 were significantly reduced compared with those of wild-type mice (Figure 25B). The serum bilirubin levels of male ZIP11 knockout mice were significantly increased compared with those of wild-type mice (Figures 26A-26C). These findings suggest that ZIP11 plays a protective role in mice fed MCD for NASH formation.

[0189] Example 13 Vitamins increase Zip11 expression

[0190] Human HEK293 and HT1080 cells were treated with different concentrations of vitamin B3 (10 μM, 50 μM, 100 μM), vitamin B12 (1 μM, 10 μM, 50 μM), and vitamin C (10 μM, 100 μM, 500 μM) for 24 hours, and the relative expression of Zip11 mRNA was detected. The results are shown in Figure 19. The results showed that vitamins increased the expression level.

[0191] The foregoing detailed description is provided by way of illustration and example and is not intended to limit the scope of the appended claims. Various modifications of the presently recited embodiments will be apparent to those skilled in the art and will remain within the scope of the appended claims and their equivalents.

Claims

1. 1. A method for identifying a substance capable of affecting aging, said method comprising: determining the effect of the candidate substance on Zip11 activity and / or expression, and identifying the candidate substance that alters Zip11 activity and / or expression as a substance that affects aging; wherein a candidate substance that increases Zip11 activity and / or expression is identified as a substance that can delay or ameliorate aging; Candidate substances that decrease Zip11 activity and / or expression are identified as substances that may promote or exacerbate aging.

2. determining the effect of the candidate substance on the activity and / or expression of Zip11 comprises determining the effect of the candidate substance on the expression level and / or activity of the Zip11 nucleic acid; determining the level and / or activity of the Zip11 nucleic acid expression using a substance selected from the group consisting of primers capable of specifically amplifying the Zip11 nucleic acid and a nucleic acid probe capable of specifically recognizing the Zip11 nucleic acid; and / or determining the effect of the candidate substance on the activity and / or expression of Zip11 comprises determining the effect of the candidate substance on the protein expression level and / or activity of Zip11; The expression level and / or activity of the Zip11 protein is determined using a substance selected from the group consisting of a reagent that specifically recognizes the Zip11 protein and a substance that can measure the activity of the Zip11 protein. The method of claim 1.

3. the method comprising administering the candidate substance to a non-human animal, organ, tissue and / or cell, determining the effect of the candidate substance on the activity and / or expression of Zip11 in the non-human animal, organ, tissue and / or cell, and identifying a candidate substance that alters the activity and / or expression of Zip11 as a substance that affects aging; and / or the method is an in vitro or ex vivo method, 3. The method according to claim 1 or 2.

4. the aging status is determined by assessing one or more of body morphology, hair status, immune level status, tumor status, liver status, spleen status, fertility status, motor status, lifespan status, and cognitive status; The body morphology status is evaluated by detecting a spinal morphology; the hair condition is assessed by detecting hair loss; the immune status is assessed by detecting cytokine levels; the state of the tumor is assessed by detecting tumor volume, tumor growth status, and / or the presence of tumor cells; the liver status is assessed by detecting alanine aminotransferase activity, aspartate aminotransferase activity, and / or liver / body weight ratio; the spleen status is assessed by measuring the spleen / body weight ratio; said fertility status being assessed by detecting a fertile egg rate; The state of exercise ability is evaluated by a rotarod fatigue test result; The lifespan status is assessed by detecting survival time; and / or The cognitive status is assessed by an open field test. The method according to any one of claims 1 to 3.

5. A pharmaceutical composition for use in delaying or ameliorating aging, comprising a nucleic acid molecule encoding Zip11 or an expression product thereof.

6. the delay or amelioration of aging consists of preventing or treating curvature of the spine, preventing or treating hair loss, increasing cytokine levels, improving immunity, preventing or treating tumors, extending lifespan, preventing or treating liver damage, preventing or treating spleen damage, preventing or treating decline in fertility, enhancing athletic performance, delaying decline in athletic performance and / or enhancing behavioral and cognitive performance; and / or The pharmaceutical composition of claim 5, wherein the nucleic acid molecule comprises the nucleotide sequence shown in SEQ ID No.

1.

7. 1. A method for evaluating the effect of a candidate substance on an aging state in a subject, the method comprising: 1) administering the candidate substance to the subject; 2) comparing the change in Zip11 expression and / or activity in the subject before and after the administration; and 3) evaluating the effect of the candidate substance on the aging status of the subject based on the change in Zip11 expression and / or activity; where: If the expression and / or activity of Zip11 in the subject increases after administration of the candidate substance, the aging state in the subject is evaluated as being delayed or improved, and if the expression and / or activity of Zip11 in the subject decreases after administration of the candidate substance, the aging state in the subject is evaluated as being accelerated or worsened; and The subject is a non-human animal.

8. the activity and / or expression of Zip11 comprises activity and / or expression at the nucleic acid level of Zip11 and / or at the protein level of Zip11; The delay or improvement of the aging condition includes preventing or treating curvature of the spine, preventing or treating hair loss, increasing cytokine levels, improving immunity, preventing or treating tumors, extending lifespan, preventing or treating liver damage, preventing or treating spleen damage, preventing or treating decline in fertility, enhancing athletic performance, enhancing behavioral and cognitive performance, and / or delaying the onset of decline in athletic performance; The accelerated or worsening state of aging is characterized by including worsening spinal curvature, worsening hair loss, decreased cytokine levels, weakened immunity, worsening or recurrence of tumors, shortened lifespan, worsening liver damage, worsening spleen damage, worsening decline in reproductive ability, decreased motor and / or behavioral cognitive abilities. The method of claim 7.

9. A non-human animal or part thereof overexpressing Zip11.

10. Compared with the corresponding wild type, there is an additional copy number of the Zip11 gene, the Zip11 gene is regulated by a tissue-specific promoter; the additional copy number of Zip11 gene is expressed specifically in the liver, the tissue-specific promoter consists of the nucleotide sequence shown in SEQ ID NO. 1, and / or exhibiting a phenotype in which aging is delayed or improved, The non-human animal or part thereof according to claim 9.

11. The non-human animal or part thereof of claim 10, characterized in that the delay or improvement of the aging condition includes prevention or treatment of curvature of the spine, prevention or treatment of hair loss, increase in cytokine levels, improvement of immunity, prevention or treatment of tumors, extension of lifespan, prevention or treatment of liver damage, prevention or treatment of spleen damage, prevention or treatment of decline in reproductive ability, improvement of motor ability, delay of the time point at which motor ability declines and / or improvement of behavioral and cognitive ability.

12. A non-human animal or part thereof in which the expression and / or function of the Zip11 gene is disrupted.

13. At least part of the Zip11 gene has been knocked out. At least a portion of exon 5 of the Zip11 gene is knocked out; exhibiting accelerated or exacerbated aging phenotypes, A non-human animal or part thereof according to claim 12.

14. The non-human animal or part thereof of claim 13, wherein the accelerated or worsening state of aging includes worsening spinal curvature, worsening hair loss, decreased cytokine levels, decreased immunity, worsening or recurrence of tumors, shortened lifespan, worsening liver damage, worsening spleen damage, worsening decline in reproductive ability, decreased motor ability, and decreased behavioral and cognitive ability.

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