Anti-aging use of RINL gene and antagonist thereof

Abstract

An anti-aging use of an RINL gene and an antagonist thereof. Provided is a use of an RINL gene or a protein encoded by the RINL gene. The RINL gene or the protein encoded by the RINL gene is: (1) used as a marker for diagnosis, detection, or prognosis of aging; or (2) used for preparing a reagent or kit for diagnosis or detection of an aging state; or (3) used for preparing a drug for delaying aging. Experimental results show that RINL-KO can reduce cellular senescence and improve the motor capability and metabolic capacity of aged mice.

Description

Anti-aging applications of the RINL gene and its antagonists

[0001] Cross-references to related applications

[0002] This disclosure claims priority to Chinese Patent Application No. 2024110400856, filed on July 31, 2024, the entire contents of which are incorporated herein by reference. Technical Field

[0003] This disclosure belongs to the field of biomedical technology, specifically relating to the anti-aging use of an RINL gene and its antagonist. Background Technology

[0004] Aging is a spontaneous and inevitable process that occurs naturally over time, primarily manifested as a decline in adaptability and resistance, impaired physiological functions, degenerative changes in tissues and organs, and ultimately, the inevitable process of death. Accompanying aging, the incidence of many chronic diseases, such as heart disease, cancer, diabetes, and Alzheimer's disease, increases dramatically. In recent years, with the increase in average life expectancy and the decline in birth rates, the global population age structure has been constantly changing, mainly characterized by a continuous decrease in the number of people aged 0-15 years, while the number of people aged 65 and above has been continuously increasing. Currently, the number of people aged 65 and above is significantly higher than the number of people under 5 years old, and this trend will continue, meaning that the global aging population is intensifying. This demographic change will have a significant impact on the operation of national healthcare systems and place a heavy burden on economic development. Therefore, delaying aging and preventing, delaying, mitigating, or even reversing the progression of age-related diseases have become key to solving the aging problem for countries worldwide, and the search for anti-aging drugs is urgent.

[0005] However, there is currently a lack of satisfactory anti-aging drugs. Therefore, there is an urgent need in the field to develop new, safe and effective anti-aging methods and pharmaceutical compositions. Summary of the Invention

[0006] In order to address the problems existing in the prior art, the purpose of this disclosure is to provide anti-aging uses of the RINL gene and its antagonists.

[0007] To achieve the above objectives, this disclosure adopts the following specific solutions:

[0008] In one aspect, this disclosure provides the use of the RINL gene or its encoded protein, wherein the RINL gene or its encoded protein is used as a biomarker for diagnosing, detecting or prognosing aging; or (2) for preparing reagents or kits for diagnosing or detecting aging states; or (3) for preparing drugs for delaying aging.

[0009] On the other hand, this disclosure provides the use of RINL antagonists in the preparation of medicaments for the prevention and / or relief of aging.

[0010] On the other hand, this disclosure provides a method for screening drugs to prevent and / or alleviate aging, which includes the following steps: detecting changes in the expression level of the RINL gene in the body before and after drug administration.

[0011] On the other hand, this disclosure provides an RINL antagonist for preventing and / or alleviating aging, said RINL antagonist specifically targeting the nucleotide sequence shown in SEQ ID NO.1 or specifically binding to the amino acid sequence shown in SEQ ID NO.2, and having any of the following functions:

[0012] (1) Inhibit RINL gene expression;

[0013] (2) Cause the RINL gene to be deleted or silenced;

[0014] (3) Knock out the RINL gene;

[0015] (4) Antagonize RINL protein.

[0016] On the other hand, this disclosure provides a pharmaceutical composition for preventing and / or alleviating aging, comprising an RINL antagonist that inhibits RINL expression, and optionally a pharmaceutically acceptable carrier or excipient.

[0017] The beneficial effects of this disclosure are at least as follows:

[0018] The applicant unexpectedly discovered that the expression level of the RINL gene may be related to the degree of individual aging. Experiments showed that RINL-KO can improve cellular and mouse aging, prolong mouse lifespan, and improve the movement and metabolic capacity of aged mice. Specifically, RINL-KO also reduced the expression level of β-galactosidase, an aging marker, in cells and mouse liver. Therefore, this invention discloses for the first time that RINL can be used as a novel anti-aging target, which is of great significance for screening new drugs and provides a new approach to anti-aging treatment. Attached Figure Description

[0019] Figure 1 shows the trend of RINL protein expression levels in various organs of male mice with increasing age.

[0020] Figure 2 shows the effect of knocking down RINL expression in HK2 cells with siRNA on cell viability and β-gal expression.

[0021] Figure 3 shows the effect of RINL-KO on lifespan extension in male and female mice.

[0022] Figure 4 shows the effects of RINL-KO on the metabolism of male and female aged (24 months) mice.

[0023] Figure 5 shows the effect of RINL-KO on the motor function of male and female aged (24 months) mice.

[0024] Figure 6 shows the effect of RINL-KO on alleviating Klotho expression levels in a D-gal-induced rapid aging model mouse.

[0025] Figure 7 shows the effects of RINL-KO on the metabolism of male and female rapidly aging mice.

[0026] Figure 8 shows the effects of RINL-KO on the motor function of male and female rapidly aging mice.

[0027] Figure 9 shows the effect of RINL-KO on the body weight ratio of various important organs in a D-gal-induced rapid aging model mouse.

[0028] Figure 10 shows the effect of RINL-KO on alleviating the aging of brown adipose tissue, liver, lungs, and kidneys in a D-gal-induced rapid aging model mouse.

[0029] Figure 11 shows the effect of RINL-KO on the protein expression of aging markers p16 and p21 in the heart, liver, lung, kidney, and brown adipose tissue of a D-gal-induced rapid aging model mouse.

[0030] Figure 12 shows the effect of RINL-KO on the expression of key proteins (p-AMPK, AMPK, p-ACC, ACC, LC3B-II / I, p62) in the liver and related cell lines of a D-gal-induced rapid aging model mouse. Detailed Implementation

[0031] I. Terminology

[0032] To facilitate understanding of this disclosure, certain technical and scientific terms are specifically defined below. Unless otherwise expressly defined elsewhere in this document, all other technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this disclosure pertains.

[0033] The articles “a” and “a kind” used in this article refer to one or more (i.e., at least one) grammatical objects. For example, “a kind of element” means one element or more elements.

[0034] As used herein, the term "RINL" stands for Ras and Rab Interactor Like, referring to Ras and Rab interaction factor-like proteins that participate in various biological processes within cells, particularly in vesicle-mediated transport and the regulation of Rab proteins. RINL-expressed proteins can activate Rab5 subfamily small GTPases, thereby playing a role in endocytosis. RINL is expressed in various tissues, with particularly high levels in the thymus and spleen.

[0035] As used in this article, the term "Klotho" refers to a gene located on human chromosome 13 (13q12), which is 50 kb in length and consists of 5 exons. Through alternative splicing, Klotho can produce two proteins: a membrane protein (primarily expressed in the kidneys, placenta, small intestine, and prostate) and a circulating protein (primarily expressed in tissues such as the brain, hippocampus, placenta, kidneys, prostate, and small intestine). The membrane protein form can also be hydrolyzed to produce the circulating protein form. Klotho has been shown to have anti-aging effects, and serum Klotho levels can reflect the degree of aging in mice. Higher Klotho levels are associated with lower levels of aging in mice. Klotho protein exists in two forms: αKlotho and βKlotho, which are major components of the endocrine fibroblast growth factor (FGF) receptor complex and are essential for the high-affinity binding of FGF19, FGF21, and FGF23 to their corresponding FGF receptors.

[0036] As used in this article, the term “Nutlin-3a (Rebemadlin)” is a small molecule MDM2 antagonist that blocks the interaction between MDM2 and p53, stabilizes and activates the p53 protein, and is often used to study p53-mediated cell cycle arrest, apoptosis and aging models.

[0037] The terms “cell,” “cell line,” and “cell culture” used herein are used interchangeably, and all such names include their progeny. Therefore, the words “transformation” and “transformed cell” include primary test cells and cultures derived from them, regardless of the number of transfections. It should also be understood that, due to intentional or unintentional mutations, all progeny cannot be exactly identical in terms of DNA content. This includes mutant progeny with the same function or biological activity as those screened from the original transformed cells. Where different names are used, the context will be clear.

[0038] "Optional" or "optionally" means that the event or circumstances described below may, but do not have to, occur, including the circumstances in which the event or circumstances may or may not occur.

[0039] When applied to animals, humans, experimental subjects, cells, tissues, organs, or biological fluids, "application," "giving," and "treatment" refer to the contact of an exogenous drug, therapeutic agent, diagnostic agent, or composition with the animal, human, subject, cell, tissue, organ, or biological fluid. "Application," "giving," and "treatment" can refer to, for example, therapeutic, pharmacokinetic, diagnostic, research, and experimental methods. Cellular treatment includes contact between a reagent and cells, as well as contact between a reagent and a fluid, wherein the fluid is in contact with the cells. "Application," "giving," and "treatment" also mean the treatment of, for example, cells, by means of a reagent, diagnostic agent, conjugate composition, or by means of another cell in vitro and ex vivo. When "treatment" is applied to humans, veterinary, or research subjects, it refers to therapeutic treatment, preventative or prophylactic measures, research, and diagnostic applications.

[0040] "Treatment" means administering an oral or topical therapeutic agent to a patient who has symptoms of one or more diseases, and the therapeutic agent is known to have a therapeutic effect on these symptoms. Typically, a therapeutic agent is administered in a treated patient or population in an amount that effectively relieves one or more symptoms of the disease, whether by inducing the regression of these symptoms or inhibiting their progression to any clinically unmeasurable degree. The amount of therapeutic agent that effectively relieves any specific disease symptom (also called the "therapeutic effective dose") can vary depending on a variety of factors, such as the patient's disease state, age, and weight, as well as the drug's ability to produce the desired therapeutic effect in the patient. Whether the disease symptoms have been relieved can be evaluated using any clinical test that a physician or other healthcare professional typically uses to assess the severity or progression of the symptoms. Although the embodiments of this disclosure (e.g., treatment methods or products) may be ineffective in alleviating the symptoms of the target disease present in every patient, they should reduce the symptoms of the target disease in a statistically significant number of patients, as determined by any statistical test known in the art, such as the Student t-test, chi-square test, U-test according to Mann and Whitney, Kruskal-Wallis test (H-test), Jonckheere-Terpstra test, and Wilcoxon test. The term "treatment" for an individual suffering from a disease or disease condition indicates that the individual's symptoms are partially or completely relieved, or remain unchanged after treatment. Therefore, treatment includes prevention, treatment, and / or cure. Prevention refers to preventing underlying disease and / or preventing the worsening of symptoms or the development of disease. Treatment also includes any chimeric antigen receptors provided and any pharmaceutical use of the compositions provided herein.

[0041] As used herein, the terms “effective amount” or “therapeutic effective amount” are used interchangeably and refer to the amount of a compound, formulation, substance or composition that effectively achieves a particular biological outcome or provides a therapeutic or preventive benefit as described herein.

[0042] The term “consistently of” or variations thereof, used throughout the specification and claims, means that all said elements or groups of elements are included, and optionally include other elements that are similar to or different in nature from said elements, which do not significantly alter or introduce new properties to the specified dosing regimen, method or composition.

[0043] The term "antagonist" refers to a substance that can act at the nucleic acid or protein level to reduce or inhibit the expression or activity of RINL. These include, but are not limited to, shRNA, siRNA, miRNA, dsRNA, small molecule compounds, stRNA, aptamers, or antibodies or their antigen-binding fragments.

[0044] A "vector" is a composition of substances containing isolated nucleic acids and capable of delivering those isolated nucleic acids into cells. Many vectors are known in the art, including, but not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses. Therefore, the term "vector" includes autonomously replicating plasmids or viruses. The term should also be interpreted to include non-plasmid and non-viral compounds that facilitate the transfer of nucleic acids into cells, such as polylysine compounds, liposomes, etc. Examples of viral vectors include, but are not limited to, adenovirus vectors, adeno-associated virus vectors, retroviral vectors, etc.

[0045] The terms “patient,” “subject,” “individual,” etc., are used interchangeably in this document and are intended to include any living organism (e.g., a mammal) that can elicit an immune response. Examples of subjects include humans, dogs, cats, mice, rats, and their transgenic species.

[0046] The term "kit" is any article (e.g., a package or container) containing at least one reagent (e.g., a therapeutic agent, probe, small molecule, etc.) for the specific detection and / or therapeutic effect on the expression of the biomarkers described in this invention. The kit may be marketed, distributed, or sold as a whole for performing the methods described in this invention. The kit may contain one or more reagents essential for the expression of the composition used in the methods described in this invention. In some embodiments, the kit may further contain reference standards, such as nucleic acids encoding proteins that do not affect or regulate signaling pathways controlling immune responses, cell growth, division, migration, survival, or apoptosis. Many such control proteins are conceivable to those skilled in the art, including but not limited to: common molecular tags (e.g., green fluorescent protein and β-galactosidase); proteins not included in any classification including cell growth, division, migration, survival, or apoptosis by GeneOntology reference; or ubiquitous housekeeping proteins. The reagents in the kit may be provided in a single container or in a mixture of two or more reagents in a single container. Additionally, explanatory material describing the use of the compositions in the kit may be included.

[0047] Table 1. Abbreviations

[0048] II. Detailed Implementation Plan

[0049] In one aspect, this disclosure provides for the use of the RINL gene or its encoded protein, wherein the RINL gene or its encoded protein is used as a biomarker for diagnosing, detecting or prognosing aging; (2) for preparing reagents or kits for diagnosing or detecting aging states; or (3) for preparing drugs for delaying aging.

[0050] On the other hand, this disclosure provides the use of RINL antagonists in the preparation of medicaments for the prevention and / or relief of aging.

[0051] In some implementations, the drug can also improve the subject's exercise and metabolic capabilities.

[0052] In some embodiments, the RINL antagonist specifically targets the nucleotide sequence shown in SEQ ID NO.1 or specifically binds to the amino acid sequence shown in SEQ ID NO.2, and has any of the following functions:

[0053] (1) Inhibit RINL gene expression;

[0054] (2) Cause the RINL gene to be deleted or silenced;

[0055] (3) Knock out the RINL gene;

[0056] (4) Antagonize RINL protein.

[0057] In some embodiments, the RINL antagonist is selected from shRNA, siRNA, miRNA, dsRNA, stRNA, small molecule compounds, aptamers, antibodies, or antigen-binding fragments thereof.

[0058] In some embodiments, the antibody or its antigen-binding fragment is selected from whole antibodies, F(ab)-fragments, F(ab)2-fragments, single-chain antibodies, chimeric antibodies, bivalent antibody-constructions, synthetic antibodies, bispecific single-chain antibodies, or cross-clonal antibodies.

[0059] In some implementations, the RINL antagonist is an anti-RINL antibody.

[0060] In some embodiments, the RINL antagonist is siRNA, which comprises a sense strand and an antisense strand, each 15-30 base pairs in length, wherein the antisense strand contains a complementary region that is complementary to the mRNA encoding RINL.

[0061] In some embodiments, the nucleotide sequence of the siRNA is selected from any combination of the following sense and antisense strands:

[0062] (1) A positive chain having the sequence shown in SEQ ID NO.3, and an antisense chain having the sequence shown in SEQ ID NO.4; or

[0063] (2) A positive chain having the sequence shown in SEQ ID NO.5, and an antisense chain having the sequence shown in SEQ ID NO.6; or

[0064] (3) A sense chain having the sequence shown in SEQ ID NO.7 and an antisense chain having the sequence shown in SEQ ID NO.8.

[0065] In some embodiments, the drug is able to reduce the expression of β-galactosidase in cells and individuals.

[0066] In some implementations, the drug is able to increase the expression of Klotho in cells and individuals.

[0067] In some implementations, the drug improves the degree of aging in brown adipose tissue, liver, lung, and kidney tissue in an individual.

[0068] In some embodiments, the drug includes an active ingredient RINL antagonist and its pharmaceutically acceptable carrier or excipient.

[0069] In some embodiments, the RINL antagonist is present in the drug at a concentration of 1% to 99%.

[0070] On the other hand, this disclosure provides a method for screening drugs to prevent and / or alleviate aging, which includes the following steps: detecting changes in the expression level of the RINL gene in the body before and after drug administration. In some embodiments, the method uses the expression level of the RINL gene as a screening indicator.

[0071] On the other hand, this disclosure provides an RINL antagonist for preventing and / or alleviating aging, wherein the RINL antagonist specifically targets the nucleotide sequence shown in SEQ ID NO.1 or specifically binds to the amino acid sequence shown in SEQ ID NO.2, and has any of the following functions:

[0072] (1) Inhibit RINL gene expression;

[0073] (2) Cause the RINL gene to be deleted or silenced;

[0074] (3) Knock out the RINL gene;

[0075] (4) Antagonize RINL protein.

[0076] In some embodiments, the RINL antagonist is shRNA, siRNA, miRNA, dsRNA, stRNA, small molecule compound, aptamer or antibody or its antigen-binding fragment.

[0077] In some embodiments, the RINL antagonist is siRNA, which comprises a sense strand and an antisense strand, each 15-30 base pairs in length, wherein the antisense strand contains a complementary region that is complementary to the mRNA encoding RINL.

[0078] In some embodiments, the nucleotide sequence of the siRNA is selected from any combination of the following sense and antisense strands:

[0079] (1) A positive chain having the sequence shown in SEQ ID NO.3, and an antisense chain having the sequence shown in SEQ ID NO.4; or

[0080] (2) A positive chain having the sequence shown in SEQ ID NO.5, and an antisense chain having the sequence shown in SEQ ID NO.6; or

[0081] (3) A sense chain having the sequence shown in SEQ ID NO.7 and an antisense chain having the sequence shown in SEQ ID NO.8.

[0082] On the other hand, this disclosure provides a pharmaceutical composition for preventing and / or alleviating aging, comprising the aforementioned RINL antagonist, and optionally a pharmaceutically acceptable carrier or excipient.

[0083] In some embodiments, the pharmaceutical composition further comprises one or more additional therapeutic components.

[0084] On the other hand, this disclosure provides nucleic acids encoding the aforementioned RINL antagonist.

[0085] On the other hand, this disclosure provides an expression vector containing the aforementioned nucleic acid.

[0086] On the other hand, this disclosure provides a cell comprising the aforementioned nucleic acid and expression vector.

[0087] On the other hand, this disclosure provides a composition comprising the aforementioned RINL antagonist, and optionally a pharmaceutically acceptable carrier or excipient.

[0088] On the other hand, this disclosure provides the use of the aforementioned method, the aforementioned RINL antagonist, and / or the aforementioned pharmaceutical composition in the preparation of a medicament for treating and / or preventing individual aging, wherein the medicament is administered to an individual in an effective amount for therapeutic purposes.

[0089] On the other hand, this disclosure provides an anti-aging treatment method by administering a therapeutically effective amount of the aforementioned RINL antagonist and / or the aforementioned composition to a subject.

[0090] Example

[0091] A further understanding of this disclosure can be obtained by referring to the specific embodiments given herein, which are for illustrative purposes only and are not intended to limit the scope of this disclosure in any way. It is obvious that various modifications and variations can be made to this disclosure without departing from its spirit, and therefore such modifications and variations are also within the scope of protection claimed in this application. All clinical trial data used in this disclosure have been obtained with the informed consent of the subjects.

[0092] Example 1: RINL protein expression levels increase with age.

[0093] Tissue samples were taken from male C57BL / 6 mice at four time points (2M, 8M, 13M, and 24M) from birth to 24 months of age, and the relative expression levels of RINL protein in four major organs (heart, liver, kidney, and lung) were determined by Western blot.

[0094] The results are shown in Figure 1. As can be seen from Figure 1, the expression of RINL protein in each organ showed significant age-dependent differences. The overall trend showed that the expression level of RINL protein increased with age, suggesting that RINL protein may be an indicator protein of aging.

[0095] Example 2: Knocking down RINL expression in HK2 cells using siRNA can alleviate cellular senescence.

[0096] Based on the above findings, it is hypothesized that the expression level of the RINL gene may be related to the degree of individual aging. HK2 cells, human renal proximal tubular cells, are a common model cell used in research related to kidney diseases. Considering the strong correlation between kidney diseases and the degree of individual aging, siRNA knockdown of RINL expression in HK2 cells was used to study the relationship between RINL and aging. Three groups of siRNAs targeting RINL were designed, and their sequence information is shown in Table 2 below. RINL was knocked down using siRNA via liposome transfection. HK2 cells were lysed and proteins were collected. Intracellular RINL expression levels were analyzed by immunoblotting. The results, as shown in 2A, indicate that siRNA significantly reduced RINL expression in HK2 cells.

[0097] In addition, after knocking down RINL in HK2 cells via liposome transfection using siRINL, the cells were subjected to a rapid senescence model (treated with 200 μmol / L hydrogen peroxide for 24 hours). Cell viability was then detected by methylthiazolyl diphenyl-tetrazolium bromide (MTT) staining (Beijing Cooler Master Technology Co., Ltd.; CM7461), and the expression of β-galactosidase (β-gal, a marker of cell senescence) was observed by staining (Beyotime; C0602).

[0098] As shown in Figures 2B-2C, hydrogen peroxide treatment significantly reduced cell viability and increased the expression of β-gal, a marker of cellular senescence. Knocking down RINL expression could alleviate the decrease in cell viability caused by hydrogen peroxide treatment and the abnormal increase in the intracellular marker of cellular senescence (β-gal), showing a tendency to restore cells to normal levels. In other words, reducing the expression of RINL can help HK2 cells resist the rapid cellular senescence caused by hydrogen peroxide.

[0099] Table 2. Sequence information of siRINL

[0100] Example 3: RINL-KO extends the lifespan of mice

[0101] To investigate whether RINL knockout can prolong the lifespan of mice, we performed systemic knockout of the RINL gene in both female and male mice (knockout at birth) and set up a blank control group. All mice in each group were naturally fed with normal diet and all feeding conditions were kept consistent. We conducted continuous observation and checked for any new mouse deaths daily (to ensure the accuracy of the death date statistics). There were two groups of mice in each group, one for RINL systemic knockout mice and one for control mice, with ten mice in each group, to observe the effect of the RINL gene on the lifespan of mice.

[0102] The survival curve statistics are shown in Figure 3. It can be found that RINL gene knockout has the effect of prolonging the lifespan of both female and male mice.

[0103] Example 4: RINL-KO improves metabolic and motor function in aged mice

[0104] The metabolic state and motor function of aged mice are important indicators for assessing their degree of aging. Higher metabolic activity and stronger motor function generally indicate better health in aged mice and may predict a longer life expectancy. Therefore, testing the motor and metabolic functions of aged mice has become a routine method in aging-related research.

[0105] To investigate whether RINL knockout can improve aging in mice, 24-month-old mice (including females and males) were used in this study. A blank control group (WT) and an RINL gene knockout group (RINL-KO) were established. A 16-channel metabolic monitoring system (Promethion Core) was used to monitor the mice's metabolism, and metabolic values ​​over 24 hours were recorded. The results are shown in Figure 4. Regardless of day or night, the 24-hour energy expenditure (EE) of both female and male RINL-KO mice was significantly higher than that of the control group. This indicates that RINL gene knockout enhances basal metabolism and improves oxygen consumption levels in mice, which is beneficial for improving the health of aged mice.

[0106] Motor and coordination abilities are also important indicators for assessing aging. In this example, the grip strength (Ugo47200) and running ability (Ugo Basile 47300) of aged mice in the above groups were further tested.

[0107] Figures 5A and 5B show that RINL-KO significantly improved the motor function of aged mice, which further confirms the positive effect of RINL-KO on improving the health of aged mice.

[0108] Example 5: Construction of rapid aging RINL-KO and WT mouse models

[0109] Because organ and serum sampling is impossible after the natural aging of mice, and further molecular marker detection is also limited, we constructed a rapid aging mouse model using D-galactose (D-gal) (Merck; G0750). D-gal increases the production of reactive oxygen species (ROS), leading to oxidative stress. Prolonged oxidative stress damages cell membranes, proteins, and DNA, accelerating cellular aging. We used RINL systemic knockout and wild-type C57BL / 6 mice, both males and females, aged eight weeks, in groups of eight. The mice were induced into a rapid aging model by intraperitoneal injection of D-gal at 120-200 mg / kg / day for 12 consecutive weeks (120 mg / kg for females, 200 mg / kg for males). The groups were: WT+NaCl treatment group, WT+D-gal treatment group, RINL-KO+NaCl treatment group, and RINL-KO+D-gal treatment group.

[0110] To verify the success of the modeling and the effect of RINL knockout, blood was collected from the submandibular region of mice, and serum was obtained by centrifugation. The Klotho expression level was detected using a kit (D721177-0048). The Klotho level in serum can also reflect the degree of aging in mice.

[0111] The results are shown in Figure 6. In both female and male mice, the serum Klotho level in the rapid aging model mice was significantly decreased. The decreasing trend of Klotho in the serum of RINL-KO aging mice was significantly alleviated, and the expression level of Klotho in their serum tended to be normal.

[0112] Example 6: RINL-KO improves metabolism and motor function in a rapidly aging mouse model

[0113] Assessing the physical and metabolic capabilities of aged mice has become an indispensable standard practice in aging research. To further explore the potential ameliorative effect of RINL gene knockout on the rapid aging process in mice, we examined four groups of mice with a rapid aging model: WT+Nacl treatment group, WT+D-gal treatment group, RINL-KO+Nacl treatment group, and RINL-KO+D-gal treatment group. The metabolic activity of the mice was tracked using a 16-channel metabolic monitoring device (Promethion Core), and their metabolic data were recorded over 24 hours.

[0114] The relevant results are shown in Figure 7. Regardless of whether it was day or night, the 24-hour energy expenditure (EE) of both female and male mice in the RINL-KO group was significantly higher than that of the control group. This indicates that knocking out the RINL gene increases the basal metabolic rate and optimizes oxygen consumption in mice, which is beneficial to improving the health of rapidly aging mice.

[0115] Further testing was conducted on the grip strength (Ugo47200) and running ability (Ugo Basile 47300) of the rapidly aging mice in the above groups. The test results in Figures 8A and 8B show that RINL-KO significantly enhanced the motor performance of rapidly aging mice, further validating the positive effect of RINL gene knockout on improving the health of rapidly aging mice. Furthermore, RINL-KO had virtually no effect on normal mice (comparative to the NaCl treatment group). These results indicate that RINL knockout has an anti-aging effect and does not affect other physiological functions of mice under normal conditions.

[0116] Example 7: RINL-KO improves the aging degree of brown adipose tissue, liver, lung, and kidney tissues in rapidly aging mice.

[0117] After the in vivo mouse experiment, the mice in each of the above groups were sacrificed and samples were collected. Molecular indicators were detected and possible molecular mechanisms were explored. Specifically, the female and male mice in the WT+NaCl treatment group, WT+D-gal treatment group, RINL-KO+NaCl treatment group, and RINL-KO+D-gal treatment group were observed and photographed. They were then sacrificed, and their important organs were taken and weighed. The organ weight ratio of each group was analyzed and compared.

[0118] The results are shown in Figure 9. Figure 9A shows the body weight ratio of each organ in male mice, and Figure 9B shows the body weight ratio of each organ in female mice. As shown in Figure 8, under the induction of rapid aging drugs, the liver of mice in the WT+D-gal treatment group showed abnormal enlargement, while RINL-KO could alleviate the abnormal liver enlargement induced by D-gal treatment. In addition, brown adipose tissue (BAT) showed significant atrophy and weight reduction after D-gal induction, while RINL-KO alleviated this phenomenon induced by D-gal treatment. Abnormal increase in liver weight in mice may indicate the occurrence of various conditions, including obesity, metabolic disorders, inflammation, and fibrosis, while the atrophy and weight reduction of brown adipose tissue may indicate physiological aging, mitochondrial dysfunction, and metabolic degeneration.

[0119] Experimental results showed that RINL-KO could improve abnormal changes in the liver and brown adipose tissue induced by D-gal in rapidly aging female and male mice, which helps maintain the health of the mice.

[0120] Frozen sections of brown adipose tissue, liver, lung, and kidney tissues from female and male mice in the WT+NaCl treatment group, WT+D-gal treatment group, RINL-KO+NaCl treatment group, and RINL-KO+D-gal treatment group were prepared. The expression levels of aging markers in the brown adipose tissue, liver, lung, and kidney tissues of each group of mice were observed using a β-galactosidase (β-gal) staining kit (Beyotime; C0602).

[0121] The experimental results are shown in Figure 10. Figure 10A shows the staining results of brown adipose tissue, liver, lung, and kidney tissues of male mice, and Figure 10B shows the staining results of brown adipose tissue, liver, lung, and kidney tissues of female mice. The results show that D-gal drug induction significantly increased the expression of aging markers in brown adipose tissue, liver, lung, and kidney tissues of mice, while RINL-KO could alleviate the degree of aging induced by D-gal, indicating that RINL knockout can alleviate the degree of aging in brown adipose tissue, liver, lung, and kidney.

[0122] Example 8: RINL deficiency alleviates D-galactose-induced aging in mice at multiple organ levels by downregulating p16 / p21 signaling.

[0123] After the mouse experiment, the heart, liver, lung, kidney and brown adipose tissue of four groups of female and male mice (WT+NaCl, WT+D-gal, RINL-KO+NaCl, and RINL-KO+D-gal) were collected. After extracting total protein, the expression levels of aging marker proteins p16 and p21 were detected by Western blot.

[0124] The results are shown in Figure 11. Figure 11A shows the Western blot (WB) results of the heart, liver, lung, kidney, and brown adipose tissue of male mice, and Figure 11B shows the WB results of the corresponding tissues of female mice. The data show that D-gal treatment significantly upregulated the protein expression of p16 and p21 in the above organs, while RINL knockout significantly inhibited the D-gal-induced increase in p16 and p21, suggesting that RINL deficiency can alleviate D-gal-induced cellular senescence at multiple organ levels, including the heart, liver, lung, kidney, and brown adipose tissue.

[0125] Example 9: RINL deficiency delays aging by activating the AMPK-autophagy axis

[0126] Further collection of livers from four groups of female mice and Western blot analysis of key proteins of the AMPK-autophagy axis revealed (Figure 12A) that D-gal significantly downregulated the p-AMPK / AMPK, p-ACC / ACC, and LC3B-II / I ratios, while increasing p62, suggesting impaired energy sensing and inhibition of autophagic flux. RINL knockout reversed these changes, restoring p-AMPK, p-ACC, and LC3-II / I and decreasing p62.

[0127] This model was repeatedly validated at the cellular level (Figure 12B). HepG2 is a human liver cancer cell line that retains the metabolic characteristics of normal hepatocytes and is widely used in energy sensing and drug research. MEF is a mouse embryonic fibroblast that proliferates rapidly and has a clear genetic background, making it a classic primary model for aging and autophagy research.

[0128] Studies on aging and longevity suggest that the AMPK-ACC pathway activates autophagy upon sensing energy deficiency, clearing damaged organelles and abnormal proteins to maintain mitochondrial quality and metabolic homeostasis. Elevated LC3B-II / I and decreased p62 levels, autophagy markers, represent enhanced autophagic flux and are significantly associated with increased lifespan. Therefore, RINL deficiency, by antagonizing D-gal-induced aging through the AMPK-ACC-autophagy axis, may be one of the molecular mechanisms by which it promotes longevity.

[0129] In summary, the applicant discovered that the expression level of the RINL gene may be related to the degree of individual aging. Experiments showed that RINL-KO can improve cellular and mouse aging, prolong mouse lifespan, and improve the movement and metabolic capacity of aged mice. Specifically, RINL-KO also reduced the expression levels of aging markers β-galactosidase, p16, and p21 in cells and in mouse brown adipose tissue, liver, lungs, and kidneys. Therefore, this invention discloses for the first time that RINL can be used as a novel anti-aging target, which is of great significance for screening new drugs and provides a new approach to anti-aging treatment.

[0130] The above description is merely an embodiment of the present invention and is not intended to limit the present invention in any form or substance. It should be noted that those skilled in the art can make various improvements and additions without departing from the method of the present invention, and these improvements and additions should also be considered within the scope of protection of the present invention. Any modifications, alterations, and equivalent changes made by those skilled in the art based on the above-disclosed technical content without departing from the spirit and scope of the present invention are equivalent embodiments of the present invention. Furthermore, any modifications, alterations, and evolutions made to the above embodiments based on the essential technology of the present invention still fall within the scope of the technical solution of the present invention.

[0131] Table 3. Sequence Information

Claims

1. Use of RINL gene or its encoded protein, which: (1) is used as a marker for diagnosing, detecting or prognosing aging; (2) is used for preparing a reagent or kit for diagnosing or detecting aging state; or (3) is used for preparing a drug for delaying aging.

2. Use of RINL antagonist in preparing a drug for preventing and / or alleviating aging.

3. Use according to claim 2, wherein, The RINL antagonist specifically targets the nucleotide sequence shown in SEQ ID NO. 1 or specifically binds to the amino acid sequence shown in SEQ ID NO. 2, and has any one of the following functions: (1) inhibits RINL gene expression; (2) causes RINL gene deletion or silencing; (3) knocks out RINL gene; (4) antagonizes RINL protein.

4. Use according to claim 2 or 3, wherein, The RINL antagonist is selected from shRNA, siRNA, miRNA, dsRNA, stRNA, small molecule compound, aptamer or antibody or antigen-binding fragment thereof.

5. Use according to claim 4, wherein, The antibody or antigen-binding fragment thereof is selected from whole antibody, F(ab)-fragment, F(ab)2-fragment, single-chain antibody, chimeric antibody, divalent antibody-construct, synthetic antibody, bispecific single-chain antibody or cross-clonal antibody.

6. Use according to any one of claims 2-5, wherein, The drug comprises active ingredient RINL antagonist and pharmaceutically acceptable carrier or excipient.

7. Use according to any one of claims 2 to 6, wherein, The content of the RINL antagonist in the drug is 1% to 99%.

8. A method for screening a drug for preventing and / or alleviating aging, comprising the step of detecting the change in the expression amount of RINL gene in the body before and after administration.

9. A RINL antagonist for use in preventing and / or alleviating senescence, wherein, The RINL antagonist specifically targets the nucleotide sequence shown in SEQ ID NO. 1 or specifically binds to the amino acid sequence shown in SEQ ID NO. 2, and has any one of the following functions: (1) inhibits RINL gene expression; (2) causes RINL gene deletion or silencing; (3) knocks out RINL gene; (4) antagonizes RINL protein.

10. A pharmaceutical composition for preventing and / or alleviating aging, comprising the RINL antagonist of claim 9, and optionally pharmaceutically acceptable carrier or excipient; Preferably, it further comprises one or more additional therapeutic components.

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