WO2026026919A1 - Rinl基因及其拮抗剂的抗衰老用途 - Google Patents

Rinl基因及其拮抗剂的抗衰老用途

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WO2026026919A1
WO2026026919A1 PCT/CN2025/111826 CN2025111826W WO2026026919A1 WO 2026026919 A1 WO2026026919 A1 WO 2026026919A1 CN 2025111826 W CN2025111826 W CN 2025111826W WO 2026026919 A1 WO2026026919 A1 WO 2026026919A1
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rinl
aging
gene
antagonist
mice
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French (fr)
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左正宏
何承勇
叶凌霄
韩长顺
杨春燕
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Xiamen University
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Xiamen University
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    • G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups

Definitions

  • This disclosure belongs to the field of biomedical technology, specifically relating to the anti-aging use of an RINL gene and its antagonist.
  • 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.
  • 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.
  • 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.
  • the purpose of this disclosure is to provide anti-aging uses of the RINL gene and its antagonists.
  • 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.
  • this disclosure provides the use of RINL antagonists in the preparation of medicaments for the prevention and/or relief of aging.
  • 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.
  • 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:
  • 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.
  • the expression level of the RINL gene may be related to the degree of individual aging.
  • RINL-KO can improve cellular and mouse aging, prolong mouse lifespan, and improve the movement and metabolic capacity of aged mice.
  • RINL-KO also reduced the expression level of ⁇ -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.
  • Figure 1 shows the trend of RINL protein expression levels in various organs of male mice with increasing age.
  • Figure 2 shows the effect of knocking down RINL expression in HK2 cells with siRNA on cell viability and ⁇ -gal expression.
  • Figure 3 shows the effect of RINL-KO on lifespan extension in male and female mice.
  • Figure 4 shows the effects of RINL-KO on the metabolism of male and female aged (24 months) mice.
  • Figure 5 shows the effect of RINL-KO on the motor function of male and female aged (24 months) mice.
  • Figure 6 shows the effect of RINL-KO on alleviating Klotho expression levels in a D-gal-induced rapid aging model mouse.
  • Figure 7 shows the effects of RINL-KO on the metabolism of male and female rapidly aging mice.
  • Figure 8 shows the effects of RINL-KO on the motor function of male and female rapidly aging mice.
  • 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.
  • 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.
  • 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.
  • 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.
  • key proteins p-AMPK, AMPK, p-ACC, ACC, LC3B-II/I, p62
  • RINL stands for Ras and Rab Interactor Like, a Ras and Rab interaction factor-like protein involved in various intracellular biological processes, particularly 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.
  • Klotho refers to a gene located on human chromosome 13 (13q12), which is 50 kb in length and consists of 5 exons.
  • a membrane protein primarily expressed in the kidneys, placenta, small intestine, and prostate
  • 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: ⁇ Klotho and ⁇ Klotho, 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.
  • FGF endocrine fibroblast growth factor
  • 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.
  • cell cell line
  • 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.
  • “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.
  • treatment When “treatment” is applied to humans, veterinary, or research subjects, it refers to therapeutic treatment, preventative or prophylactic measures, research, and diagnostic applications.
  • 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.
  • 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 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.
  • the embodiments of this disclosure e.g., treatment methods or products
  • 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.
  • 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.
  • 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.
  • 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.
  • patient refers to any living organism (e.g., a mammal) that can elicit an immune response.
  • subjects include humans, dogs, cats, mice, rats, and their transgenic species.
  • 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.
  • 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.
  • control proteins are conceivable to those skilled in the art, including but not limited to: common molecular tags (e.g., green fluorescent protein and ⁇ -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.
  • 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.
  • this disclosure provides the use of RINL antagonists in the preparation of medicaments for the prevention and/or relief of aging.
  • the drug can also improve the subject's exercise and metabolic capabilities.
  • 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:
  • the RINL antagonist is selected from shRNA, siRNA, miRNA, dsRNA, stRNA, small molecule compounds, aptamers, antibodies, or antigen-binding fragments thereof.
  • 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.
  • the RINL antagonist is an anti-RINL antibody.
  • 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.
  • the nucleotide sequence of the siRNA is selected from any combination of the following sense and antisense strands:
  • the drug is able to reduce the expression of ⁇ -galactosidase in cells and individuals.
  • the drug is able to increase the expression of Klotho in cells and individuals.
  • the drug improves the degree of aging in brown adipose tissue, liver, lung, and kidney tissue in an individual.
  • the drug includes an active ingredient RINL antagonist and its pharmaceutically acceptable carrier or excipient.
  • the RINL antagonist is present in the drug at a concentration of 1% to 99%.
  • 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.
  • the method uses the expression level of the RINL gene as a screening indicator.
  • 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:
  • the RINL antagonist is shRNA, siRNA, miRNA, dsRNA, stRNA, small molecule compound, aptamer or antibody or its antigen-binding fragment.
  • 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.
  • the nucleotide sequence of the siRNA is selected from any combination of the following sense and antisense strands:
  • 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.
  • the pharmaceutical composition further comprises one or more additional therapeutic components.
  • this disclosure provides nucleic acids encoding the aforementioned RINL antagonist.
  • this disclosure provides an expression vector containing the aforementioned nucleic acid.
  • this disclosure provides a cell comprising the aforementioned nucleic acid and expression vector.
  • this disclosure provides a composition comprising the aforementioned RINL antagonist, and optionally a pharmaceutically acceptable carrier or excipient.
  • 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.
  • 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.
  • Example 1 RINL protein expression levels increase with age.
  • 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 using Western blot.
  • Example 2 Knocking down RINL expression in HK2 cells using siRNA can alleviate cellular senescence.
  • the expression level of the RINL gene may be related to the degree of individual aging.
  • HK2 cells human renal proximal tubular cells
  • 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.
  • mice 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.
  • 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.
  • RINL knockout can improve aging in mice.
  • a blank control group (WT) and an RINL gene knockout group (RINL-KO) were established.
  • the metabolism of the mice was monitored using a 16-channel metabolic monitoring system (Promethion Core), and the metabolic values of the mice 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 the basal metabolism of mice and improves their oxygen consumption levels, which is beneficial for improving the health of aged mice.
  • 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.
  • D-gal D-galactose
  • ROS reactive oxygen species
  • Prolonged oxidative stress damages cell membranes, proteins, and DNA, accelerating cellular aging.
  • 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.
  • Example 6 RINL-KO improves metabolism and motor function in a rapidly aging mouse model
  • mice assessed the physical and metabolic capabilities of aged mice has become an indispensable standard practice in aging research.
  • WT+Nacl treatment group WT+D-gal treatment group
  • RINL-KO+Nacl treatment group WT+D-gal treatment group
  • RINL-KO+D-gal treatment group WT+D-gal treatment group
  • RINL-KO+D-gal treatment group WT+D-gal treatment group
  • RINL-KO+D-gal treatment group 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.
  • Example 7 RINL-KO improves the aging degree of brown adipose tissue, liver, lung, and kidney tissues in rapidly aging mice.
  • 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.
  • Figure 9A shows the body weight ratio of each organ in male mice
  • Figure 9B shows the body weight ratio of each organ in female mice.
  • the liver of mice in the WT+D-gal treatment group showed abnormal enlargement
  • RINL-KO could alleviate the abnormal liver enlargement induced by D-gal treatment.
  • brown adipose tissue BAT
  • BAT brown adipose tissue
  • 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.
  • 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.
  • Figure 10A shows the staining results of brown adipose tissue, liver, lung, and kidney tissues of male mice
  • 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.
  • Example 8 RINL deficiency alleviates D-galactose-induced aging in mice at multiple organ levels by downregulating p16/p21 signaling.
  • 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.
  • Figure 11A shows the Western blot (WB) results of the heart, liver, lung, kidney, and brown adipose tissue of male mice
  • 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.
  • Example 9 RINL deficiency delays aging by activating the AMPK-autophagy axis
  • 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.
  • the expression level of the RINL gene may be related to the degree of individual aging.
  • RINL-KO can improve cellular and mouse aging, prolong mouse lifespan, and improve the movement and metabolic capacity of aged mice.
  • RINL-KO also reduced the expression levels of aging markers ⁇ -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.

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Abstract

一种RINL基因及其拮抗剂的抗衰老用途。提供一种RINL基因或其编码蛋白的用途，所述RINL基因或其编码蛋白：(1)用作诊断、检测或预后衰老的标志物；或(2)用于制备诊断或检测衰老状态的试剂或试剂盒；或(3)用于制备延缓衰老的药物。实验结果表明，RINL-KO能够改善细胞的衰老情况，改善老龄小鼠的运动和代谢能力。

Description

RINL基因及其拮抗剂的抗衰老用途
相关申请的交叉引用
本公开要求于2024年7月31日提交的中国专利申请2024110400856号的优先权，上述中国专利申请的全文通过引用并入本文。
技术领域
本公开属于生物医药技术领域，具体涉及一种RINL基因及其拮抗剂的抗衰老用途。
背景技术
衰老是生命体随着时间推移自发的必然过程，主要表现为适应性和抵抗力减退、机体生理功能产生障碍、组织器官发生退行性变化、并最终不可避免走向死亡的过程。伴随着机体的衰老，许多慢性疾病例如心脏病、癌症、糖尿病、阿尔茨海默氏病等的发病率急剧升高。近年来，随着人类平均预期寿命的增加和出生率的下降，全球人口的年龄结构不断变化，主要表现为0–15岁之间的人口数目不断减少，同时伴随着65岁以上的人口数目不断增加。现如今65岁以上的老年人口数目已明显高于5岁以下的人口数目，而且这一趋势还将持续存在，这意味着全球老龄化程度加重。这种人口变化将对国家医疗体系运营产生重大的影响，给经济发展造成沉重的负担。因此延缓衰老，预防、延迟、减轻甚至逆转与年龄相关的疾病进程已成为世界各国解决老龄化问题的关键，寻找抗衰老药物刻不容缓。
然而，目前尚缺乏令人满意的抗衰老药物，因此，本领域迫切需要开发新的安全且有效的抗衰老的方法和药物组合物。
发明内容
为了解决现有技术中存在的问题，本公开的目的在于提供RINL基因及其拮抗剂的抗衰老用途。
为了实现上述目的，本公开采用以下具体方案：
在一方面，本公开提供了RINL基因或其编码蛋白的用途，所述RINL基因或其编码蛋白：(1)用作诊断、检测或预后衰老的标志物；或(2)用于制备诊断或检测衰老状态的试剂或试剂盒；或(3)用于制备延缓衰老的药物。
另一方面，本公开提供了RINL拮抗剂在制备预防和/或缓解衰老药物中的用途。
另一方面，本公开提供了一种筛选预防和/或缓解衰老药物的方法，其包括以下步骤，检测给药前后机体中RINL基因表达量的变化。
另一方面，本公开提供了一种预防和/或缓解衰老的RINL拮抗剂，所述RINL拮抗剂特异性靶向SEQ ID NO.1所示的核苷酸序列或特异性结合SEQ ID NO.2所示的氨基酸序列，并具有以下任一种功能：
(1)抑制RINL基因表达；
(2)使RINL基因缺失或沉默；
(3)敲除RINL基因；
(4)拮抗RINL蛋白。
另一方面，本公开提供了一种预防和/或缓解衰老的药物组合物，其包括抑制RINL表达的RINL拮抗剂，以及任选地药学上可接受的载体或辅料。
本公开的有益效果至少如下：
申请人意外地发现RINL基因的表达量可能与个体衰老程度相关，实验表明RINL-KO能够改善细胞和小鼠的衰老情况，延长小鼠寿命，改善老龄小鼠的运动和代谢能力。具体地，RINL-KO还降低了细胞内和小鼠肝脏中的衰老标志物β-半乳糖苷酶的表达量。因此，本发明首次公开可以将RINL作为抗衰的新靶点，对于筛选新药具有重要意义，也为抗衰的治疗提供了一种新思路。
附图说明
图1示出了雄性小鼠各器官中RINL蛋白表达量随年龄增加的变化趋势。
图2示出了使用siRNA敲低HK2细胞中RINL的表达后，对细胞活力和β-gal表达的影响。
图3示出了RINL-KO对雄性以及雌性小鼠寿命延长影响。
图4示出了RINL-KO对雄性以及雌性老龄小鼠(24月龄)代谢情况影响结果。
图5示出了RINL-KO对雄性以及雌性老龄小鼠(24月龄)的运动能力影响。
图6示出了RINL-KO缓解D-gal诱导快速衰老模型小鼠Klotho表达水平的影响。
图7示出了RINL-KO对雄性以及雌性快速衰老小鼠代谢情况影响结果。
图8示出了RINL-KO对雄性以及雌性快速衰老小鼠运动能力影响结果。
图9示出了RINL-KO对D-gal诱导快速衰老模型小鼠各重要器官体重比水平的影响。
图10示出了RINL-KO对D-gal诱导快速衰老模型小鼠的棕色脂肪组织、肝脏、肺、肾脏衰老程度的缓解作用。
图11示出了RINL-KO对D-gal诱导快速衰老模型小鼠的心脏、肝、肺、肾及棕色脂肪中衰老标志物p16与p21的蛋白表达的影响。
图12示出了RINL-KO对D-gal诱导快速衰老模型小鼠的肝脏以及相关细胞系中AMPK-自噬通路关键蛋白(p-AMPK、AMPK、p-ACC、ACC、LC3B-II/I、p62)表达的影响。
具体实施方式
I.术语
为了更容易理解本公开，以下具体定义了某些技术和科学术语。除显而易见在本文件中的它处另有明确定义，否则本文使用的所有其它技术和科学术语都具有本公开所属领域的一般技术人员通常理解的含义。
本文使用的冠词“一”和“一种”是指该冠词所指的一个或一个以上(即至少一个)的语法对象。举例来说，“一种元件”表示一个元件或一个以上的元件。
如本文所用，术语“RINL”全称为Ras And Rab Interactor Like，即Ras和Rab相互作用因子样蛋白，在细胞内参与多种生物学过程，特别是与囊泡介导的运输和Rab蛋白的调控有关。RINL表达的蛋白能够激活Rab5亚家族小GTP酶，从而在内吞作用中发挥作用。RINL在多种组织中表达，尤其在胸腺和脾脏中表达水平较高。
如本文所用，术语“Klotho”基因位于人类第13条染色体(13q12)上，全长50kb，由5个外显子组成。通过选择性剪接，Klotho可以产生两种蛋白，一种为膜蛋白(主要在肾脏、胎盘、小肠和前列腺中表达)，一种为循环蛋白(主要在大脑、海马、胎盘、肾脏、前列腺和小肠等组织中表达)。其中膜蛋白形式也可以通过水解作用产生循环形式的蛋白。Klotho已经被证明具有抗衰老的作用，而血清中Klotho水平也可以反映小鼠衰老程度。Klotho水平越高，小鼠衰老程度越低。Klotho蛋白有两种形式：αKlotho和βKlotho，它们作为内分泌成纤维细胞生长因子(FGF)受体复合物的主要部分，是FGF19、FGF21和FGF23与其相应的FGF受体高亲和力结合所必需的。
如本文所用，术语“Nutlin-3a(Rebemadlin)”是一种小分子MDM2拮抗剂，可阻断MDM2与p53的相互作用，稳定并激活p53蛋白，常用于研究p53介导的细胞周期阻滞、凋亡及衰老模型。
本文使用的表述“细胞”、“细胞系”和“细胞培养物”可互换使用，并且所有这类名称都包括其后代。因此，单词“转化体”和“转化细胞”包括原代受试细胞和由其衍生的培养物，而不考虑转移数目。还应当理解的是，由于故意或非有意的突变，所有后代在DNA含量方面不可能精确相同。包括具有与最初转化细胞中筛选的相同的功能或生物学活性的突变后代。在意指不同名称的情况下，其由上下文清楚可见。
“任选”或“任选地”意味着随后所描述地事件或环境可以但不必发生，该说明包括该事件或环境发生或不发生地场合。
“施用”、“给予”和“处理”当应用于动物、人、实验受试者、细胞、组织、器官或生物流体时，是指外源性药物、治疗剂、诊断剂或组合物与动物、人、受试者、细胞、组织、器官或生物流体的接触。“施用”、“给予”和“处理”可以指例如治疗、药物代谢动力学、诊断、研究和实验方法。细胞的处理包括试剂与细胞的接触，以及试剂与流体的接触，其中所述流体与细胞接触。“施用”、“给予”和“处理”还意指通过试剂、诊断、结合组合物或通过另一种细胞体外和离体处理例如细胞。当“处理”应用于人、兽医学或研究受试者时，是指治疗处理、预防或预防性措施，研究和诊断应用。
“治疗”意指给予患者内用或外用治疗剂，所述患者具有一种或多种疾病症状，而已知所述治疗剂对这些症状具有治疗作用。通常，在受治疗患者或群体中以有效缓解一种或多种疾病症状的量给予治疗剂，无论是通过诱导这类症状退化还是抑制这类症状发展到任何临床不可测量的程度。有效缓解任何具体疾病症状的治疗剂的量(也称作“治疗有效量”)可根据多种因素变化，例如患者的疾病状态、年龄和体重，以及药物在患者产生需要疗效的能力。通过医生或其它专业卫生保健人士通常用于评价该症状的严重性或进展状况的任何临床检测方法，可评价疾病症状是否已被减轻。尽管本公开的实施方案(例如治疗方法或制品)在缓解每个患者都有的目标疾病症状方面可能无效，但是根据本领域已知的任何统计学检验方法如Student t检验、卡方检验、依据Mann和Whitney的U检验、Kruskal-Wallis检验(H检验)、Jonckheere-Terpstra检验和Wilcoxon检验确定，其在统计学显著数目的患者中应当减轻目标疾病症状。术语“治疗”患有疾病或疾病状况的个体表示所述个体的症状部分或全部缓解，或者在治疗后保持不变。因此，治疗包括预防、治疗和/或治愈。预防指防止潜在疾病和/或防止症状恶化或疾病发展。治疗还包括所提供的任何嵌合抗原受体以及本文所提供的组合物的任何药学用途。
如本文所用，术语“有效量”或“治疗有效量”在本文中可互换使用，并指代如本文所述的有效实现特定生物学结果或提供治疗或预防益处的化合物、配制物、物质或组合物的量。
整个说明书和权利要求书中使用的术语“基本上由……组成”或其变形表示包括所有所述元件或元件组，并且任选包括与所述元件类似或不同性质的其它元件，所述其它元件非显著改变指定给药方案、方法或组合物的基本性质或新性质。
术语“拮抗剂”是指可以在核酸或蛋白质水平起作用的物质，以减少或抑制RINL的表达或活性。其包括但不限于shRNA、siRNA、miRNA、dsRNA、小分子化合物、stRNA、适配体、或抗体或其抗原结合片段。
“载体”是物质的组合物，其包含分离的核酸且可用于将所分离的核酸递送到细胞内。本领域中已知许多载体，包括但不限于线性多核苷酸、与离子或两亲性化合物有关的多核苷酸、质粒和病毒。因此，术语“载体”包括自主复制质粒或病毒。该术语也应解释为包括非质粒和非病毒化合物，它们促进核酸转移到细胞中，例如像，聚赖氨酸化合物、脂质体等。病毒载体的实例包括但不限于腺病毒载体、腺相关病毒载体、逆转录病毒载体等。
术语“患者”、“受试者”、“个体”等在本文中可互换使用且意图包括其中可引发免疫应答的活的生物体(例如哺乳动物)。受试者的实例包括人、狗、猫、小鼠、大鼠及其转基因物种。
术语“试剂盒”是包含至少一种试剂(例如治疗剂、探针、小分子等)的任意制品(例如包或容器)，其用于特异性检测和/或治疗性地影响本发明所述标志物的表达。所述试剂盒可以作为整体进行推广、分发或销售，用于执行本发明所述方法。所述试剂盒可包含一种或多种试剂，所述试剂对用于本发明所述方法的组合物的表达是必需的。在某些实施方案中，所述试剂盒可以进一步包含参考标准，例如，编码不影响或不调节信号通路的蛋白质的核酸，所述信号通路控制免疫应答、细胞生长、分裂、迁移、存活或凋亡。本领域技术人员可以设想许多这样的对照蛋白质，包括但不限于：常见的分子标签(例如，绿色荧光蛋白和β-半乳糖苷酶)；通过GeneOntology参考，未在包括细胞生长、分裂、迁移、存活或凋亡的任何通路的分类里的蛋白质；或者普遍存在的管家蛋白质。试剂盒中的试剂可以通过单个容器提供，或者通过单个容器中的两种或更多种试剂的混合物提供。另外，可以包括描述试剂盒中组合物的用途的说明材料。
表1.缩略词
II.具体实施方案详述
在一方面，本公开提供了RINL基因或其编码蛋白的用途，所述RINL基因或其编码蛋白：(1)用作诊断、检测或预后衰老的标志物；(2)用于制备诊断或检测衰老状态的试剂或试剂盒；或(3)用于制备延缓衰老的药物。
另一方面，本公开提供了RINL拮抗剂在制备预防和/或缓解衰老药物中的用途。
在一些实施方案中，所述药物还能改善受试者的运动和代谢能力。
在一些实施方案中，所述RINL拮抗剂特异性靶向SEQ ID NO.1所示的核苷酸序列或特异性结合SEQ ID NO.2所示的氨基酸序列，并具有以下任一种功能：
(1)抑制RINL基因表达；
(2)使RINL基因缺失或沉默；
(3)敲除RINL基因；
(4)拮抗RINL蛋白。
在一些实施方案中，所述RINL拮抗剂选自shRNA、siRNA、miRNA、dsRNA、stRNA、小分子化合物、适配体、抗体或其抗原结合片段。
在一些实施方案中，所述抗体或其抗原结合片段选自全抗体、F(ab)-片段、F(ab)2-片段、单链抗体、嵌合抗体、二价抗体-构建体、合成抗体、双特异性单链抗体或交叉克隆抗体。
在一些实施方案中，所述RINL拮抗剂为抗RINL抗体。
在一些实施方案中，所述RINL拮抗剂为siRNA，所述siRNA包含正义链和反义链，其长度为15-30个碱基对，所述反义链包含与编码RINL的mRNA互补的互补区域。
在一些实施方案中，所述siRNA的核苷酸序列选自下述正义链和反义链的组合中的任一种：
(1)具有如SEQ ID NO.3所示序列的正义链，和具有如SEQ ID NO.4所示序列的反义链；或
(2)具有如SEQ ID NO.5所示序列的正义链，和具有如SEQ ID NO.6所示序列的反义链；或
(3)具有如SEQ ID NO.7所示序列的正义链，和具有如SEQ ID NO.8所示序列的反义链。
在一些实施方案中，所述药物能够降低细胞和个体内β-半乳糖苷酶的表达。
在一些实施方案中，所述药物能够提高细胞和个体内Klotho的表达。
在一些实施方案中，所述药物改善个体中棕色脂肪组织、肝脏、肺、肾脏组织的衰老程度。
在一些实施方案中，所述药物包括活性成分RINL拮抗剂及其药学上可接受的载体或辅料。
在一些实施方案中，所述RINL拮抗剂在药物中的含量为1％～99％。
另一方面，本公开提供了一种筛选预防和/或缓解衰老药物的方法，其包括以下步骤，检测给药前后机体中RINL基因表达量的变化。在一些实施方案中，所述方法将RINL基因的表达水平作为筛查指标。
另一方面，本公开提供了一种预防和/或缓解衰老的RINL拮抗剂，其中，所述RINL拮抗剂特异性靶向SEQ ID NO.1所示的核苷酸序列或特异性结合SEQ ID NO.2所示的氨基酸序列，并具有以下任一种功能：
(1)抑制RINL基因表达；
(2)使RINL基因缺失或沉默；
(3)敲除RINL基因；
(4)拮抗RINL蛋白。
在一些实施方案中，所述RINL拮抗剂为shRNA、siRNA、miRNA、dsRNA、stRNA、小分子化合物、适配体或抗体或其抗原结合片段。
在一些实施方案中，所述RINL拮抗剂为siRNA，所述siRNA包含正义链和反义链，其长度为15-30个碱基对，所述反义链包含与编码RINL的mRNA互补的互补区域。
在一些实施方案中，所述siRNA的核苷酸序列选自下述正义链和反义链的组合中的任一种：
(1)具有如SEQ ID NO.3所示序列的正义链，和具有如SEQ ID NO.4所示序列的反义链；或
(2)具有如SEQ ID NO.5所示序列的正义链，和具有如SEQ ID NO.6所示序列的反义链；或
(3)具有如SEQ ID NO.7所示序列的正义链，和具有如SEQ ID NO.8所示序列的反义链。
另一方面，本公开提供了一种预防和/或缓解衰老的药物组合物，其包括前述的RINL拮抗剂，以及任选地药学上可接受的载体或辅料。
在一些实施方案中，所述药物组合物还包含一种或多种另外的治疗组分。
另一方面，本公开提供了编码前述RINL拮抗剂的核酸。
另一方面，本公开提供了一种包含前述核酸的表达载体。
另一方面，本公开提供了一种包含前述核酸和表达载体的细胞。
另一方面，本公开提供了一种组合物，其包含前述的RINL拮抗剂，以及任选地药学上可接受的载体或辅料。
另一方面，本公开提供了一种前述的方法、前述的RINL拮抗剂和/或前述的药物组合物在制备治疗和/或预防个体衰老的药物中的用途，其中，所述药物被治疗施用有效量到个体中。
另一方面，本公开提供了一种抗衰治疗方法，通过向受试者施用治疗有效量的前述RINL拮抗剂和/或前述组合物。
实施例
通过参考在此给出的一些具体实施例可获得对本公开的进一步的理解，这些实施例仅用于说明本公开，其无意于对本公开的范围做出任何限制。显然，可以对本公开做出多种改动和变化而不脱离本公开的实质，因此，这些改动和变化同样在本申请要求保护的范围内。本公开中所用的临床实验数据均已取得受试者的知情和同意。
实施例1：RINL蛋白表达水平随着年龄增长而增加
对C57BL/6雄性小鼠自出生后第1日至24月龄的4个时间点(2M、8M、13M、24M)组织取样，并采用蛋白免疫印迹(Western blot)对心、肝、肾、肺4个主要器官中RINL蛋白的相对表达量进行测定。
测定结果见图1，由图1可知，各器官中RINL蛋白表达均呈现显著的年龄依赖性差异。整体趋势表现为RINL蛋白表达水平随着年龄增加的增加，这提示RINL蛋白可能是一个衰老指针蛋白。
实施例2：使用siRNA敲低HK2细胞中RINL的表达可以缓解细胞衰老
基于上述发现，推测RINL基因的表达量可能与个体衰老程度相关。HK2细胞，人肾近曲小管细胞，是一种常见的用于肾脏疾病相关研究的模型细胞，考虑到肾脏疾病与个体衰老程度相关性较大，使用siRNA敲低HK2细胞中RINL表达来研究RINL与衰老程度的关系。设计三组针对RINL的siRNA，其序列信息见下表2，通过脂质体转染的方法，利用siRNA对RINL进行敲低。裂解HK2细胞收集蛋白，通过免疫蛋白印记法分析细胞内RINL表达水平，结果如2A显示，siRNA显著降低了HK2细胞内的RINL表达。
另外，在siRINL通过脂质体转染的方法敲低HK2细胞中的RINL后，对细胞进行快速衰老造模(使用200μmol/L浓度的过氧化氢处理细胞24小时)后，通过噻唑蓝染色法(Methylthiazolyldiphenyl-tetrazolium bromide，MTT)(北京酷来搏科技有限公司；CM7461)检测细胞活力，并对细胞进行β-半乳糖苷酶(β-galactosidase，β-gal，细胞衰老标志物)染色(碧云天；C0602)观察细胞内β-gal的表达情况。
由图2B-2C可知，过氧化氢处理造成细胞活力显著下降并增加细胞内细胞衰老标志物β-gal的表达，敲低RINL表达可以缓解双氧水处理带来的细胞活力下降并缓解细胞内细胞衰老标志物(β-gal)的异常升高，有使细胞恢复至正常水平的趋势，即RINL的表达量降低可以使HK2细胞抵抗双氧水导致的细胞快速衰老。
表2.siRINL的序列信息

实施例3：RINL-KO延长小鼠寿命
为了研究RINL敲除是否能延长小鼠寿命，我们在雌性和雄性小鼠中把RINL基因进行全身性敲除(出生即敲除)，并设置空白对照，各组小鼠自然喂养，保证其正常饮食，各项饲养条件均保持一致，并进行持续性观察，每日观察是否有新死亡小鼠(保证死亡日期统计的准确性)，雌雄各两组小鼠，分别为RINL全身性敲除小鼠与对照小鼠，每组十只，以观察RINL基因对小鼠寿命长度方面的影响。
生存曲线统计结果见图3，可以发现RINL基因敲除在雌性与雄性小鼠中，都有着延长小鼠寿命的作用。
实施例4：RINL-KO改善老龄小鼠的代谢能力与运动能力
老龄小鼠的代谢状态和运动能力是评估其衰老程度的重要指标。代谢活动越旺盛，运动能力越强，通常意味着老龄小鼠拥有更佳的健康状况，并可能预示着它们拥有更长的预期寿命。因此，对老龄小鼠的运动能力和代谢能力进行检测，已成为衰老相关研究中的常规手段。
为了研究RINL敲除是否能改善小鼠的衰老情况，本实施例中选用24月龄小鼠(包括雌性与雄性)，并设置空白对照组(WT)和RINL基因敲除组(RINL-KO)，使用16通道代谢监测系统(Promethion Core)监测小鼠代谢情况，并统计24小时内小鼠的代谢数值，结果见图4。不管白天还是黑夜，雌性和雄性RINL-KO组小鼠的24小时能量消耗(Energy Expenditure，EE)明显高于对照组。说明RINL基因敲除后增强了小鼠的基础代谢，改善了小鼠的氧耗水平，这有利于改善老龄小鼠的健康程度。
运动和协调能力也是评估衰老的重要指标之一，本实施例中继续检测了上述分组中老龄小鼠的抓握力(Ugo47200)以及跑动能力(Ugo Basile 47300)。
图5A、5B结果显示，RINL-KO显著提升了老龄小鼠的运动能力，这一结果进一步证实了RINL-KO对改善老龄小鼠健康程度的积极作用。
实施例5：构建快速衰老的RINL-KO以及WT小鼠模型
因为自然衰老小鼠死亡后无法进行器官与血清取样，也无法进行更多的分子指标检测，于是我们构建了快速衰老小鼠，使用D半乳糖(D-Galactose，D-gal)(Merck；G0750)药物进行诱导，D-gal能够增加活性氧(reactive oxygen species,ROS)的产生，导致氧化应激。长期的氧化应激会损伤细胞膜、蛋白质和DNA，加速细胞老化。选用RINL全身性敲除和野生型的C57BL/6小鼠，雌性雄性均为八周龄，每组8只，通过腹腔注射D-gal 120-200mg/kg/天的方式连续注射12周(雌性120mg/kg，雄性200mg/kg)，诱导快速衰老模型。分组情况为：WT+NaCl处理组、WT+D-gal处理组、RINL-KO+NaCl处理组、RINL-KO+D-gal处理组。
为了验证是否造模成功以及RINL敲除的作用，对小鼠进行颌下取血，离心得到血清，使用试剂盒(D721177-0048)检测其Klotho表达水平。血清中Klotho水平也可以反映小鼠衰老程度。
检测结果见图6，不论在雌鼠还是雄鼠中，快速衰老模型小鼠血清中Klotho水平显著下降，RINL-KO衰老小鼠血清中Klotho的下降趋势得到显著缓解，其血清中Klotho的表达量趋向于正常水平。
实施例6：RINL-KO改善快速衰老小鼠模型的代谢和运动能力
检测老年小鼠的体能和代谢能力已成为研究衰老不可或缺的标准做法。为了进一步探究RINL基因敲除对小鼠快速衰老进程的潜在改善效果，我们对快速衰老造模的小鼠的四组小鼠进行检测，包括：WT+Nacl处理组、WT+D-gal处理组、RINL-KO+Nacl处理组、RINL-KO+D-gal处理组。利用16通道的代谢监测设备(Promethion Core)对小鼠的代谢活动进行跟踪，并记录了它们在24小时内的代谢数据。
相关结果见图7，不论是在日间还是夜间，RINL-KO组的雌性和雄性小鼠所展示的24小时能量消耗(Energy Expenditure，EE)均显著超过对照组。这表明敲除RINL基因后，小鼠的基础代谢速率得到提升，氧耗水平得到优化，有益于提高快速衰老小鼠的健康水平。
进一步测试了上述分组中快速衰老小鼠的抓握力(Ugo47200)和跑步能力(Ugo Basile 47300)。图8A和8B的测试结果表明，RINL-KO显著增强了快速衰老小鼠的运动性能，这进一步验证了RINL基因敲除对提升快速衰老小鼠健康水平的正面影响。并且RINL-KO对正常小鼠(NaCl处理组内对比)基本无影响，以上结果说明敲除RINL有抵抗衰老的作用，且正常条件下不影响小鼠的其他生理功能。
实施例7：RINL-KO改善快速衰老小鼠的棕色脂肪组织、肝脏、肺、肾脏组织衰老程度
小鼠活体实验结束后，对前述各组小鼠处死收样，收集各器官，检测分子指标以及探究可能的分子机制，具体为：WT+NaCl处理组、WT+D-gal处理组、RINL-KO+NaCl处理组、RINL-KO+D-gal处理组的雌性和雄性小鼠，观察其个体体积并拍照记录，后处死，并取其重要器官并称重，分析对比各组的器官体重占比。
结果见图9，图9A为雄性小鼠各器官体重比，图9B为雌性小鼠各器官体重比，由图8可知，快速衰老药物诱导下，WT+D-gal处理组小鼠的肝脏出现异常肿大，而RINL-KO能够缓解D-gal处理诱导的小鼠肝脏异常肿大。此外，棕色脂肪组织(brown adipose tissue，BAT)经D-gal诱导后出现了显著萎缩以及重量减少，而RINL-KO缓解D-gal处理诱导下的这一现象。小鼠肝脏重量异常增加可能表明多种情况的发生，包括肥胖、代谢紊乱、炎症和纤维化等，而棕色脂肪的萎缩和重量减少可能表明出现生理老化、线粒体功能障碍以及代谢功能退化等。
实验结果表明，RINL-KO能够改善D-gal诱导快速衰老雌性以及雄性小鼠的肝脏以及棕色脂肪的异常变化，这有助于保持小鼠的健康状况。
对WT+NaCl处理组、WT+D-gal处理组、RINL-KO+NaCl处理组、RINL-KO+D-gal处理组的雌性和雄性小鼠的棕色脂肪组织、肝脏、肺、肾脏组织进行冰冻切片，并使用β-半乳糖苷酶(β-galactosidase，β-gal)染色试剂盒(碧云天；C0602)，观察各组小鼠棕色脂肪组织、肝脏、肺、肾脏组织中衰老标志物的表达量高低。
实验结果见图10，图10A为雄性小鼠棕色脂肪组织、肝脏、肺、肾脏组织染色结果图，图10B为雌性小鼠棕色脂肪组织、肝脏、肺、肾脏组织染色结果图，结果显示：D-gal药物诱导使小鼠棕色脂肪组织、肝脏、肝脏、肺、肾脏组织中衰老标志物的表达显著升高，而RINL-KO可以缓解D-gal诱导带来的衰老程度，说明RINL敲除可使棕色脂肪组织、肝脏、肺、肾脏的衰老程度得到缓解。
实施例8：RINL缺失通过下调p16/p21信号在多器官水平缓解D-半乳糖诱导的小鼠衰老
小鼠实验结束后，采集WT+NaCl、WT+D-gal、RINL-KO+NaCl、RINL-KO+D-gal四组雌、雄小鼠的心脏、肝脏、肺、肾脏及棕色脂肪组织，提取总蛋白后利用Western blot检测衰老标志物蛋白p16与p21的表达水平。
结果见图11，图11A为雄性小鼠心脏、肝脏、肺、肾脏、棕色脂肪组织的WB结果，图11B为雌性小鼠相应组织的WB结果。数据显示，D-gal处理显著上调上述器官中p16与p21的蛋白表达，而RINL敲除可明显抑制D-gal诱导的p16、p21升高，提示RINL缺失可在心脏、肝脏、肺、肾脏及棕色脂肪组织等多器官水平缓解D-gal诱导的细胞衰老。
实施例9：RINL缺失通过激活AMPK-自噬轴延缓衰老
进一步收集四组雌性小鼠肝脏，Western blot检测AMPK-自噬轴关键蛋白发现(图12A)D-gal显著下调p-AMPK/AMPK、p-ACC/ACC及LC3B-II/I比值，同时升高p62，提示能量感应受阻、自噬流抑制；RINL敲除则逆转上述变化，恢复p-AMPK、p-ACC及LC3-II/I并降低p62。
细胞水平(图12B)重复验证了这一模式，HepG2为人源肝癌细胞系，保留正常肝细胞代谢特征，广泛用于能量感应与药物研究；MEF为小鼠胚胎成纤维细胞，增殖快、遗传背景清晰，是衰老与自噬研究的经典原代模型。
衰老与长寿研究提示，AMPK-ACC通路感知能量不足后激活自噬，清除受损细胞器与异常蛋白，维持线粒体质量与代谢稳态；自噬标志物LC3B-II/I升高、p62降低代表自噬流增强，与寿命延长显著相关。因此，RINL缺失通过“AMPK-ACC-自噬”轴拮抗D-gal诱导的衰老，可能是其促长寿的分子机制之一。
综上，申请人发现RINL基因的表达量可能与个体衰老程度相关，实验表明RINL-KO能够改善细胞和小鼠的衰老情况，延长小鼠寿命，改善老龄小鼠的运动和代谢能力。具体地，RINL-KO还降低了细胞内和小鼠棕色脂肪组织、肝脏、肺、肾脏中的衰老标志物β-半乳糖苷酶、p16、p21的表达量。因此，本发明首次公开可以将RINL作为抗衰的新靶点，对于筛选新药具有重要意义，也为抗衰的治疗提供了一种新思路。
以上所述，仅为本发明的实施例，并非对本发明任何形式上和实质上的限制，应当指出，对于本技术领域的普通技术人员，在不脱离本发明方法的前提下，还将可以做出若干改进和补充，这些改进和补充也应视为本发明的保护范围。凡熟悉本专业的技术人员，在不脱离本发明的精神和范围的情况下，当可利用以上所揭示的技术内容而做出的些许更动、修饰与演变的等同变化，均为本发明的等效实施例；同时，凡依据本发明的实质技术对上述实施例所作的任何等同变化的更动、修饰与演变，均仍属于本发明的技术方案的范围内。
表3.序列信息

Claims (10)

  1. RINL基因或其编码蛋白的用途，所述RINL基因或其编码蛋白：(1)用作诊断、检测或预后衰老的标志物；(2)用于制备诊断或检测衰老状态的试剂或试剂盒；或(3)用于制备延缓衰老的药物。
  2. RINL拮抗剂在制备预防和/或缓解衰老药物中的用途。
  3. 根据权利要求2所述的用途，其中，所述RINL拮抗剂特异性靶向SEQ ID NO.1所示的核苷酸序列或特异性结合SEQ ID NO.2所示的氨基酸序列，并具有以下任一种功能：
    (1)抑制RINL基因表达；
    (2)使RINL基因缺失或沉默；
    (3)敲除RINL基因；
    (4)拮抗RINL蛋白。
  4. 根据权利要求2或3所述的用途，其中，所述RINL拮抗剂选自shRNA、siRNA、miRNA、dsRNA、stRNA、小分子化合物、适配体或抗体或其抗原结合片段。
  5. 根据权利要求4所述的用途，其中，所述抗体或其抗原结合片段选自全抗体、F(ab)-片段、F(ab)2-片段、单链抗体、嵌合抗体、二价抗体-构建体、合成抗体、双特异性单链抗体或交叉克隆抗体。
  6. 根据权利要求2-5任一项所述的用途，其中，所述药物包括活性成分RINL拮抗剂及药学上可接受的载体或辅料。
  7. 根据权利要求2-6任一项所述的用途，其中，所述RINL拮抗剂在药物中的含量为1％～99％。
  8. 一种筛选预防和/或缓解衰老药物的方法，其包括以下步骤，检测给药前后机体中RINL基因表达量的变化。
  9. 一种预防和/或缓解衰老的RINL拮抗剂，其中，所述RINL拮抗剂特异性靶向SEQ ID NO.1所示的核苷酸序列或特异性结合SEQ ID NO.2所示的氨基酸序列，并具有以下任一种功能：
    (1)抑制RINL基因表达；
    (2)使RINL基因缺失或沉默；
    (3)敲除RINL基因；
    (4)拮抗RINL蛋白。
  10. 预防和/或缓解衰老的药物组合物，其包括权利要求9所述的RINL拮抗剂，以及任选地药学上可接受的载体或辅料；
    优选地，其还包含一种或多种另外的治疗组分。
PCT/CN2025/111826 2024-07-31 2025-07-31 Rinl基因及其拮抗剂的抗衰老用途 Pending WO2026026919A1 (zh)

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