Use of senescence-related proteins for assessing senescence status

Abstract

The invention relates to the technical field of biology. In particular, the present invention relates to the use of senescence-related proteins for assessing the biological age of a subject, the degree of senescence of a subject, the effects of senescence intervention means and / or the risk of a subject in having a senescence-related disease. In addition, the invention further relates to application of the senescence-related protein to screening of drugs for delaying or reversing senescence and preventing and / or treating senescence-related diseases.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology. Specifically, the present invention relates to the use of aging-related proteins to assess the biological age of a subject, the degree of aging of a subject, the effect of aging intervention measures, and / or the risk of a subject suffering from aging-related diseases. In addition, the present invention also relates to the use of aging-related proteins to screen for drugs that delay or reverse aging, prevent and / or treat aging-related diseases. Background Art

[0002] "Population aging" has become an irresistible global trend. As the degree of population aging gradually deepens, it brings huge challenges to society, economy, culture, and medical care. Aging is a topic that has been around since ancient times. Some people in life always look younger than their peers, while others look older than their peers. We realize that calendar age is not equal to biological age. Whether we are aging prematurely or growing stronger in old age, we need to answer a question: How old are we? Is there a standard for measuring aging? With active health or drug intervention, can we become younger from a biological perspective?

[0003] At present, how to analyze aging and further intervene in aging has become an important direction to be tackled in the current field of aging research. Summary of the invention

[0004] The inventors of this application established a cohort of naturally aging people aged 20-65 in Quzhou, Zhejiang. After a series of questionnaires and health examinations, a total of 113 healthy volunteers with similar living backgrounds and unified dietary management were included. By collecting peripheral blood from volunteers for high-throughput proteomics analysis, a blood proteomics map of the aging population in China was established, and a series of age-related proteins were identified. According to the abundance of these proteins, the inventors used the elastic network machine learning model to predict the actual age of the subjects and established a protein age model for the aging of the Chinese population. The results showed that the model had good predictive performance, and after further screening, the protein molecules that were significantly upregulated with age were determined, namely APOC4, APOE and LILRA2, which can be used as biomarkers for population aging. By detecting the abundance of the protein in plasma, it can assist in measuring a person's biological age, aging status, risk of aging-related diseases, treatment effects of aging-related diseases, and / or the effects of aging interventions.

[0005] Therefore, in one aspect, the present application provides a reagent capable of detecting APOC4 levels, a reagent capable of detecting APOE levels, and / or a reagent capable of detecting LILRA2 levels for use in the preparation of a medicament, wherein the medicament is used for:

[0006] (i) assessing the biological age of the subject;

[0007] (ii) assessing the subject's aging status;

[0008] (iii) evaluate the effectiveness of aging interventions;

[0009] (iv) assessing a subject's risk of developing a disease associated with aging; and / or

[0010] (v) Evaluate the efficacy of treatments for age-related diseases.

[0011] In certain embodiments, the assessments described in (i) to (v) are independently auxiliary assessments.

[0012] In certain embodiments, the detection reagent measures the level of APOC4, APOE or LILRA2 by an in vitro detection method.

[0013] In certain embodiments, the in vitro detection method is selected from immunological detection, mass spectrometry, chromatography, or any combination thereof.

[0014] In certain embodiments, the immunological detection method is selected from enzyme-linked immunosorbent assay (ELISA), electrochemical immunoassay (ECIA), chemiluminescent immunoassay (CLIA), electrochemiluminescent immunoassay (ECLIA), fluorescent immunoassay (FIA) or radioimmunoassay (RIA).

[0015] In certain embodiments, the assessment comprises the steps of: (1) determining the level of APOC4, APOE and / or LILRA2 in a sample from the subject; (2) comparing the levels with their respective corresponding reference values; and (3) assessing the biological age of the subject, the degree of aging of the subject, the effect of aging intervention measures, the risk of the subject developing an aging-related disease and / or the effect of treatment of an aging-related disease.

[0016] In certain embodiments, the sample is selected from a blood sample, such as whole blood, serum, or plasma.

[0017] In certain embodiments, the reference value is the level (or average level) of APOC4, APOE or LILRA2 in the same type of sample from an individual (or population) who is in the same chronological age group as the subject and in a normal aging state; or, the subject is a subject who has received aging intervention or treatment for an aging-related disease, and the reference value is the level (or average level) of APOC4, APOE or LILRA2 in the same type of sample from an individual (or population) who is in the same chronological age group as the subject and has not received the aging intervention or treatment for the aging-related disease and is in a normal aging state; or, the subject is a subject who has received aging intervention or treatment for an aging-related disease, and the reference value is the level of APOC4, APOE or LILRA2 in the same type of sample from the subject before receiving the aging intervention or before receiving treatment for the aging-related disease.

[0018] In certain embodiments, the reference value of APOC4 level is calculated according to the following formula:

[0019] C 参照(APOC4) =0.2161×Y 时序 +50.49

[0020] Among them, "Y 时序 ” is the subject’s chronological age; “C 参照(APOC4) ” is the reference value, the unit is ng / mL.

[0021] In certain embodiments, the reference value of APOE level is calculated according to the following formula:

[0022] C 参照(APOE) =12.55×Y 时序 +687.3

[0023] Among them, "Y 时序 ” is the subject’s chronological age; “C 参照(APOE) " is a reference value, in μg / mL. In certain embodiments, the reference value of LILRA2 level is calculated according to the following formula:

[0024] C 参照(LILRA2) =0.0125×Y 时序 +1.044

[0025] Among them, "Y 时序 ” is the subject’s chronological age; “C 参照(LILRA2) ” is the reference value, the unit is ng / mL.

[0026] In certain embodiments, the extent of aging includes normal aging, accelerated aging, and / or delayed aging.

[0027] In certain embodiments, when the level of APOC4, APOE and / or LILRA2 in the sample from the subject is higher than their respective corresponding reference values, it indicates that the biological age of the subject is higher than its chronological age, the subject is in a state of accelerated aging, the effect of the aging intervention measure is not significant, the subject has a higher risk of developing an aging-related disease, and / or the treatment effect of the aging-related disease is not significant.

[0028] In certain embodiments, the subject is a mammal.

[0029] In certain embodiments, the subject is human, more preferably Asian.

[0030] In certain embodiments, the subject is Southeast Asian or East Asian (eg, Chinese, Japanese, Korean, Mongolian).

[0031] In certain embodiments, the aging is aging of the liver.

[0032] In another aspect, the present application provides a method for:

[0033] (i) assessing the biological age of the subject;

[0034] (ii) assessing the subject's aging status;

[0035] (iii) evaluate the effectiveness of aging interventions;

[0036] (iv) assessing the risk of a subject developing age-related diseases;

[0037] (v) Evaluate the efficacy of treatments for age-related diseases.

[0038] In certain embodiments, the assessments described in (i) to (v) are independently auxiliary assessments.

[0039] In certain embodiments, the method comprises the steps of: (1) determining the level of APOC4, APOE and / or LILRA2 in a sample from the subject; (2) comparing the levels with their respective corresponding reference values; and (3) assessing the biological age of the subject, the degree of aging of the subject, the effect of aging intervention measures, the risk of the subject developing an aging-related disease and / or the effect of treatment of an aging-related disease.

[0040] In certain embodiments, the reference value is the level (or average level) of APOC4, APOE or LILRA2 in the same type of sample from an individual (or population) who is in the same chronological age group as the subject and in a normal aging state; alternatively, the subject is a subject who has received aging intervention or treatment for an aging-related disease, and the reference value is the level (or average level) of APOC4, APOE or LILRA2 in the same type of sample from an individual (or population) who is in the same chronological age group as the subject and has not received the aging intervention or treatment for an aging-related disease and is in a normal aging state; alternatively, the subject is a subject who has received aging intervention or treatment for an aging-related disease, and the reference value is the level of APOC4, APOE or LILRA2 in the same type of sample from the subject before receiving the aging intervention or treatment for the aging-related disease.

[0041] In certain embodiments, when the level of APOC4, APOE and / or LILRA2 in the sample from the subject is higher than their respective corresponding reference values, it indicates that the biological age of the subject is higher than its chronological age, the subject is in a state of accelerated aging, the effect of the aging intervention measure is not significant, the subject has a higher risk of developing an aging-related disease, and / or the treatment effect of the aging-related disease is not significant.

[0042] In certain embodiments, the sample is selected from a blood sample, such as whole blood, serum, or plasma.

[0043] In certain embodiments, the extent of aging includes normal aging, accelerated aging, and / or delayed aging.

[0044] In certain embodiments, the aging is aging of the liver.

[0045] In another aspect, the present application provides a method for assessing the biological age of a subject, comprising: (1) determining APOC4, APOE and / or LILRA2 in a sample from the subject; and (2) assessing the biological age of the subject.

[0046] In certain embodiments, the assessment is a secondary assessment.

[0047] In certain embodiments, the method assesses the biological age of a subject based on the level of APOC4 in a plasma sample from the subject; wherein the assessment formula is:

[0048] Y 生物学 =(C APOC4 -50.49) / 0.2161

[0049] Among them, "Y 生物学" is the prediction of biological age; "C APOC4 ” is the concentration of APOC4 in plasma samples from subjects, in ng / mL.

[0050] In certain embodiments, the method assesses the biological age of a subject based on the level of APOE in a plasma sample from the subject; wherein the assessment formula is:

[0051] Y 生物学 =(C APOE –687.3) / 12.55

[0052] Among them, "Y 生物学 " is the prediction of biological age; "C APOE ” is the concentration of APOE in plasma samples from subjects, in μg / mL.

[0053] In certain embodiments, the method assesses the biological age of a subject based on the level of LILRA2 in a plasma sample from the subject; wherein the assessment formula is:

[0054] Y 生物学 =(C LILRA2 –1.044) / 0.0125

[0055] Among them, "Y 生物学 " is the prediction of biological age; "C LILRA2 ” is the concentration of LILRA2 in plasma samples from subjects, in ng / mL.

[0056] In certain embodiments, the method assesses the biological age of the subject based on any combination of the levels of APOC4, APOE, and LILRA2 in the subject's plasma sample. In certain embodiments, the method comprises: (a) obtaining the estimated biological age of the subject corresponding to APOC4, APOE, or LILRA2, respectively, based on the levels of APOC4, APOE, or LILRA2 in the subject's plasma sample according to the above method; (b) combining and evaluating the estimated biological ages in (a) to obtain the final estimated biological age of the subject.

[0057] In another aspect, the present application provides a drug screening method, comprising:

[0058] (1) Provide biological samples and divide them into test group and control group;

[0059] (2) exposing the test group biological samples to the candidate drug;

[0060] (3) analyzing the levels of APOC4, APOE, and / or LILRA2 in biological samples from the test group and comparing them with those of the control group;

[0061] wherein the control group is not exposed to the candidate drug (for example, the control group is not treated, or the control group is exposed to a control agent without an active ingredient);

[0062] If the level of APOC4, APOE and / or LILRA2 in the biological sample of the test group is lower than that of the control group, it indicates that the candidate drug has the potential to delay or reverse aging, and / or prevent and / or treat aging-related diseases.

[0063] In certain embodiments, the subject is a mammal.

[0064] In certain embodiments, the subject is a human.

[0065] In certain embodiments, the subject is Asian.

[0066] In certain embodiments, the subject is Southeast Asian or East Asian (eg, Chinese, Japanese, Korean, Mongolian).

[0067] In certain embodiments, the aging is aging of the liver.

[0068] In another aspect, the present application provides a reagent combination comprising any two or three of the following:

[0069] (i) a reagent capable of detecting APOC4 levels,

[0070] (ii) a reagent capable of detecting APOE levels,

[0071] (iii) A reagent capable of detecting the level of LILRA2.

[0072] In certain embodiments, the detection reagent measures the level of APOC4, APOE or LILRA2 by an in vitro detection method.

[0073] In certain embodiments, the in vitro detection method is selected from immunological detection, mass spectrometry, chromatography, or any combination thereof.

[0074] In certain embodiments, the immunological assay is selected from enzyme-linked immunosorbent assay (ELISA), electrochemical immunoassay (ECIA), chemiluminescent immunoassay (CLIA), electrochemiluminescent immunoassay (ECLIA), fluorescent immunoassay (FIA), or radioimmunoassay (RIA).

[0075] Definition of terms

[0076] In the present invention, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. In addition, the virology, biochemistry, and immunology laboratory operation steps used herein are conventional steps widely used in the corresponding fields. At the same time, in order to better understand the present invention, the definitions and explanations of the relevant terms are provided below.

[0077] When the terms "for example," "such as," "including," "comprising," or variations thereof are used herein, these terms will not be considered as limiting terms, but will be interpreted to mean "but not limited to" or "not limited to."

[0078] The terms "a" and "an" and "the" and similar referents in the context of describing the invention (especially in the context of the following claims) should be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context.

[0079] As used herein, the term "aging" has the meaning commonly understood by those skilled in the art, and is a common biological phenomenon in the growth and development of the body. Aging can be considered as a process of body function decline accompanied by aging, which is usually accompanied by a continuous decline in physical health and an increase in the risk of aging-related diseases, functional disorders and death.

[0080] As used herein, the term "aging-related diseases" refers to aging-related diseases whose risk of occurrence increases significantly with aging. In certain embodiments, the aging-related diseases include, but are not limited to, neurodegenerative diseases (such as Alzheimer's disease), diabetes, heart disease, cardiovascular and cerebrovascular diseases, hypertension, hyperlipidemia, osteoporosis, osteoarthritis, chronic kidney disease, cancer, and vision loss.

[0081] As used herein, the term "prevention" refers to a method implemented to prevent or delay the occurrence of a disease or condition or symptom (e.g., aging, aging-related diseases) in a subject. As used herein, the term "treatment" refers to a method implemented to obtain a beneficial or desired clinical result. For the purposes of the present invention, beneficial or desired clinical results include, but are not limited to, alleviating symptoms, reducing the scope of the disease, stabilizing (i.e., no longer worsening) the state of the disease, delaying or slowing the development of the disease, improving or alleviating the state of the disease, and alleviating symptoms (whether partial or complete), whether detectable or undetectable.

[0082] As used herein, the term "APOC4" refers to Apolipoprotein C4, which has a meaning generally understood by those skilled in the art. In certain embodiments, the APOC4 described herein comprises its wild type and naturally or artificially generated mutant types. In certain embodiments, the Uniprot number of the APOC4 described herein is P55056. In certain embodiments, the APOC4 described herein has: (i) an amino acid sequence corresponding to the Uniprot number, or (ii) an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity compared to (i).

[0083] As used herein, the term "APOE" refers to Apolipoprotein E, which has a meaning generally understood by those skilled in the art. In certain embodiments, the APOE described herein comprises its wild type and naturally or artificially produced mutant types. In certain embodiments, the Uniprot number of the APOE described herein is P02649. In certain embodiments, the APOE described herein has: (i) an amino acid sequence corresponding to the Uniprot number, or (ii) an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity compared to (i).

[0084] As used herein, the term "LILRA2" refers to Leukocyte Immunoglobulin Like Receptor A2, which has the meaning generally understood by those skilled in the art. In certain embodiments, the LILRA2 described herein comprises its wild type and naturally or artificially generated mutant types. In certain embodiments, the Uniprot number of the LILRA2 described herein is Q8N149. In certain embodiments, the LILRA2 described herein has: (i) an amino acid sequence corresponding to the Uniprot number, or (ii) an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity compared to (i).

[0085] As used herein, the term "identity" is used to refer to the matching of sequences between two polypeptides or between two nucleic acids. When a position in both sequences being compared is occupied by the same base or amino acid monomer subunit (e.g., a position in each of the two DNA molecules is occupied by adenine, or a position in each of the two polypeptides is occupied by lysine), then the molecules are identical at that position. The "percent identity" between two sequences is a function of the number of matching positions shared by the two sequences divided by the number of positions compared x 100. For example, if 6 out of 10 positions in two sequences match, then the two sequences have 60% identity. For example, the DNA sequences CTGACT and CAGGTT share 50% identity (3 out of a total of 6 positions match). Typically, the comparison is made when the two sequences are aligned to produce maximum identity. Such an alignment can be achieved by using, for example, the method of Needleman et al. (1970) J. Mol. Biol. 48: 443-453, which can be conveniently performed by a computer program such as the Align program (DNAstar, Inc.). The percent identity between two amino acid sequences can also be determined using the algorithm of E. Meyers and W. Miller (Comput. Appl Biosci., 4: 11-17 (1988)), which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. In addition, the percent identity between two amino acid sequences can be determined using the Needleman and Wunsch (J Mol. Biol. 48: 444-453 (1970)) algorithm, which has been incorporated into the GAP program in the GCG software package (available at www.gcg.com), using a Blossum 62 matrix or a PAM250 matrix and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6.

[0086] Advantageous Effects of the Invention

[0087] The protein markers (or protein marker combinations) provided in this application for evaluating the aging status of the body can more specifically and accurately evaluate the aging status of the Chinese population, and have the advantages of small sample usage, simple processing, short time course and easy-to-read results.

[0088] Embodiments of the present invention will be described in detail below in conjunction with the accompanying drawings and examples, but it will be appreciated by those skilled in the art that the following drawings and examples are only used to illustrate the present invention, rather than to limit the scope of the present invention. Various objects and advantages of the present invention will become apparent to those skilled in the art based on the following detailed description of the accompanying drawings and preferred embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0089] Figure 1 : Heatmaps showing age-related changes in plasma proteins (Pearson's correlation, corrected P value < 0.05, corrected for BMI).

[0090] Figure 2 : Individual differences in the abundance of plasma proteins based on age-related proteins (111 individuals). The color of the dots from light to dark indicates age from low to high.

[0091] Figure 3 : Dot plots show shared proteins between age-related proteins found in this study and proteins found in public datasets.

[0092] Figure 4 : Bar graphs showing the enrichment of proteins that increase with age (left) and decrease with age (right). Colors from light to dark represent -Log 10 P values ​​range from low to high.

[0093] Figure 5 :The dotted line graph shows the age-related changes in proteins. The upper row shows the data from the protein spectrum (the n values ​​of APOC4, APOE, and LILRA2 are 112, 112, 99, and 81, respectively), and the lower row shows the data from human plasma ELISA (the n values ​​of APOC4, APOE, and LILRA2 are 74, 71, 75, and 75, respectively). Confidence intervals are indicated by gray shading. Pearson correlation analysis was used to calculate correlation coefficients and P values.

[0094] Figure 6 : Annotation of the tissue origin of plasma proteins that accumulate (top) and decrease (bottom) with age. Left: The number and percentage of proteins from different tissues. Middle: Heatmap showing transcripts encoding age-associated proteins in different tissues (calculated per million reads (TPM)), data from the Human Protein Atlas.

[0095] Figure 7 :Western blot analysis of APOE protein levels in livers of young and old monkeys, GAPDH was used as an internal reference, data are presented as mean ± SEM. N = 8 monkeys per group, two-tailed t test. DETAILED DESCRIPTION

[0096] The embodiments of the present invention will be described in detail below in conjunction with the examples, but those skilled in the art will appreciate that the following examples are only used to illustrate the present invention and should not be considered to limit the scope of the present invention. If no specific conditions are specified in the examples, they are carried out according to normal conditions or the conditions recommended by the manufacturer. If the manufacturer is not specified for the reagents or instruments used, they are all conventional products that can be obtained commercially.

[0097] The technical route of the present invention mainly involves Queue creation , Plasma sample collection and processing , Plasma proteomics Sequence Analysis , Plasma ELISA test , Protein expression detection by WB Count 5 parts.

[0098] Example 1: Population cohort recruitment and establishment

[0099] The "Preliminary Screening Questionnaire" was published through the WeChat Mini Program to collect basic information of potential volunteers who are interested in joining this study based on the inclusion criteria, and to screen volunteers who meet the inclusion criteria; the inclusion criteria are as follows:

[0100] 1) Enrollment conditions

[0101] a) Aged 18 or above, with no major bad habits;

[0102] b) The applicant is in good health and has not undergone any major surgery within the past six months. Routine conditions related to the normal aging process are permitted, including:

[0103] i. Hypertension, well controlled (taking no more than 3 medications);

[0104] ii. Osteoporosis, osteopenia and / or osteoarthritis;

[0105] iii. Benign prostatic hypertrophy;

[0106] iv. Cataract, glaucoma, macular degeneration;

[0107] v.Dyslipidemia;

[0108] vi. Hypothyroidism;

[0109] vii. Prediabetes or abnormal fasting blood sugar (fasting blood sugar 100-126 mg / dL).

[0110] c) The subjects can communicate well with the researchers and cooperate with all tasks during the study;

[0111] i. Subjects must voluntarily sign a written informed consent.

[0112] 2) Exclusion criteria

[0113] a) Age does not meet the inclusion criteria;

[0114] b) Participants are currently undergoing other clinical trials or are receiving any research drugs or devices for treatment within 30 days prior to enrollment;

[0115] c) The investigator reports a history or current diagnosis of significant chronic medical conditions, including:

[0116] i. Any cancer (except polycythemia vera, basal cell or squamous cell skin cancer);

[0117] ii. Coronary artery disease / myocardial infarction / clinically significant congestive heart failure;

[0118] iii. Stroke / transient ischemic attack;

[0119] iv. Deep vein thrombosis / pulmonary embolism;

[0120] v. Serum creatinine>1.5mg / dl (male);

[0121] vi. poorly controlled hypertension (significant hypertension (systolic blood pressure > 160 mmHg, or diastolic blood pressure > 100 mmHg) despite treatment);

[0122] vii. History of active liver disease or metabolic acidosis;

[0123] viii. Chronic kidney disease / hemodialysis treatment, history of severe renal damage and / or eGFR≤45ml / min / 1.73m2;

[0124] ix. Severe autoimmune / inflammatory diseases, such as rheumatoid arthritis, lupus, Crohn's disease, etc.;

[0125] x. Neurological diseases such as dementia, such as Alzheimer's / Parkinson's, etc.;

[0126] xi. Diabetes (hemoglobin A1C>6.5% or fasting blood glucose>126 mg / dL or taking diabetes medication or insulin treatment);

[0127] xii. Recent (within 3 months) cardiovascular disease events (myocardial infarction, coronary artery intervention, coronary artery bypass grafting);

[0128] xiii. Infectious diseases such as HIV, hepatitis, tuberculosis, etc.

[0129] d) Currently taking the following medications regularly:

[0130] i. Chemotherapy drugs (such as tamoxifen, doxorubicin, mitoxantrone, bleomycin);

[0131] ii. Antiplatelet drugs (e.g. clopidogrel / Plavix, dipyridamole / Angeleno, ticlopidine / Ticlopidine, excluding aspirin);

[0132] iii. Cholinesterase inhibitors for Alzheimer's disease (donepezil / alicept).

[0133] e) Continuous alcohol or drug abuse;

[0134] f) Unable to provide informed consent;

[0135] g) The researcher believes that the physical factors of the participants may have an adverse effect on the research process or results.

[0136] 3) Termination criteria

[0137] a) Failure to follow the diet, work and rest schedule and physical examination requirements in accordance with the research content;

[0138] b) Experiencing abnormal physical conditions such as colds or fever during the study;

[0139] c) Accidental injury or physical discomfort during the research process;

[0140] d) Comply with the subject’s subjective wishes to terminate the program.

[0141] All volunteers who meet the inclusion criteria and are confirmed to be included in the group signed an informed consent form and joined the volunteer WeChat group for management and information communication. Within one month before the start of the project, volunteers need to maintain a normal schedule, eat a proper diet, avoid strenuous exercise, and refrain from excessive smoking or drinking. The day before the project, volunteers received a unified standardized diet (menu see Table 1) at the implementation hospital and were no longer allowed to eat other foods.

[0142] On the day of sample collection and physical examination, volunteers are required to wear clothes and shoes that are convenient for movement, are not allowed to wear makeup, and must bring identification documents (such as ID cards, etc.) to the hospital for physical examination and biological sample collection.

[0143] Table 1 Unified standardized diet menu

[0144]

[0145] Example 2: Plasma sample collection and processing

[0146] Prepare blood collection tubes (EDTA purple anticoagulant tubes), mark them, collect about 5 mL of peripheral blood from volunteers, let stand at room temperature for 10 minutes, then centrifuge (1800 rpm, 15 minutes, 4°C), the upper layer is plasma, and the lower layer is blood cells. Collect the upper plasma part and divide it into about 400 μL / tube, try to get plasma (but do not inhale the whole blood in the lower layer), divide it into 6 tubes / person, and transfer to -80°C refrigerator for freezing.

[0147] Example 3: Plasma proteomic sequencing analysis

[0148] Human plasma samples were taken out from the -80°C freezer, buried in dry ice and transported to the protein spectrum sequencing platform (Jingjie Bio). For protein extraction, Pierce TM The top 14 kit (ThermoFisher Scientific) was used to remove high-abundance proteins, and the protein was quantified using the BCA method after lysis. During digestion, 5mM dithiothreitol was first used for reduction at 56°C for 30 minutes, and 11mM iodoacetamide was added for treatment at room temperature in the dark for 15 minutes to produce an alkylation reaction, and then 8M urea was replaced three times, and then urea was replaced three times with buffer exchange. Finally, the digestion solution and trypsin (protease: protein = 1:50, m / m) were added for enzymatic digestion overnight. The samples were separated using an Agilent 300Extend C18 column (5μm particle diameter, 4.6mm ID, 250mm length) and high pH reversed-phase high performance liquid chromatography (HPLC), and the protein peptides were separated using an EASY-nLC 1200UPLC system (ThermoFisher Scientific). The separated peptides were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS / MS) in data independent acquisition (DIA) mode using an Orbitrap Exploris TM The separated peptides were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS / MS) on a 480 (ThermoFisher Scientific). The peptides were processed by capillary source and then analyzed by mass spectrometry on a timsTOF Pro (Bruker Daltonics) in parallel accumulation sequential fragmentation (PASEF) mode. The electrospray voltage was set to 1.60 kV. Precursors and fragments were analyzed on the TOF detector with an MS1 ​​scan range of 390 to 810 m / z, and 10 PASEF-MS / MS scans were acquired per cycle. The MS2 had an m / z range of 100 to 1700 with an isolation window of 25 m / z.

[0149] MS / MS data were processed using MaxQuant (version 1.6.15.0) and then searched against the human SwissProt database. The cleavage enzyme was set to trypsin / P, and a maximum of two missed cleavages were allowed. For precursor ions, the mass tolerance for the first search and the main search was 20 ppm and 5 ppm, respectively. For fragment ions, the mass tolerance was set to 0.02 Da. Fixed modifications were carbamidomethylation on cysteine, and variable modifications were acetylation on the protein N-terminus and oxidation on methionine. The FDR was adjusted to <1%. The raw intensity was divided by the median of each sample to obtain the normalized intensity for downstream analysis.

[0150] Example 4: Plasma ELISA detection

[0151] APOC4 (Human apolipoprotein C-IV (APOC4) ELISA kit, CUSABIO, Ca#.CSB-EL001934HU) The recommended method and steps for human apolipoprotein C-IV detection are as follows:

[0152] (1) All reagents were equilibrated at room temperature for 30 minutes before testing.

[0153] (2) Diluting the standard: Use sample diluent to dilute the standard. The final concentrations are 400 ng / mL, 200 ng / mL, 100 ng / mL, 50 ng / mL, 25 ng / mL, 12.5 ng / mL, 6.25 ng / mL and 0 ng / mL.

[0154] (3) Diluting plasma samples: According to the experimental design, 80 plasma samples were selected from four age groups: 20 samples in each age group: 20-29 years old, 30-39 years old, 40-49 years old, and >50 years old. The plasma was diluted at 1:1000 using sample diluent.

[0155] (4) Add samples. Add 50 μL of the sample to be tested and 50 μL of horseradish peroxidase labeling working solution to the standard well and sample well, respectively. Cover with a film and incubate at 37°C for 30 min. Do not add any liquid to the blank well.

[0156] (5) Discard the liquid, spin dry, and wash the plate 5 times, soaking for 2 min each time.

[0157] (6) Use a spray gun to add 90 μL of substrate solution in sequence and incubate at 37°C in the dark for about 20 min.

[0158] (7) Use a dispenser to add 50 μL of the stop solution in sequence, and use an ELISA reader to measure the absorbance (OD value) of each well at a wavelength of 450 nm.

[0159] (8) Data processing: Draw a curve by subtracting the blank well value from the standard and sample values. Calculate the regression equation of the standard curve with the concentration of the standard as the ordinate (logarithmic coordinate) and the OD value as the abscissa (logarithmic coordinate). Substitute the OD value to calculate the APOC4 concentration of each sample. Finally, draw a graph of the relationship between APOC4 and age with age as the abscissa and the APOC4 concentration calculated by ELISA as the ordinate.

[0160] (9) Correlation between APOC4 and age:

[0161] Y APOC4 =0.2161*X+50.49

[0162] Y (APOC4 concentration, ng / mL): predicts biological age

[0163] X(age): actual age

[0164] Explanation: According to the above formula, by inputting age, the biological age of the subject can be predicted.

[0165] *If the ELISA test result of APOC4 is higher than the Y value, it indicates that the biological age of the subject is greater than the actual age, that is, older than the actual age.

[0166] *If the ELISA test result of APOC4 is lower than the Y value, it indicates that the biological age of the subject is younger than the actual age, that is, younger than the actual age.

[0167] APOE (Apolipoprotein E Human ELISA Kit, Abcam, ab108813) The recommended method and steps for human apolipoprotein E detection are as follows:

[0168] (1) All reagents were equilibrated at room temperature for 20 minutes before testing.

[0169] (2) Dilution of standards: Use sample diluent to dilute the standards to final concentrations of 1 μg / mL, 0.5 μg / mL, 0.25 μg / mL, 0.125 μg / mL, 0.063 μg / mL, 0.031 μg / mL, 0.016 μg / mL, and 0 μg / mL, respectively.

[0170] (3) Diluting plasma samples: According to the experimental design, 80 plasma samples were selected from four age groups: 20 samples from each age group (20-29 years old, 30-39 years old, 40-49 years old, and >50 years old). The plasma was diluted at 1:400 using sample diluent.

[0171] (4) Sample addition: Add 50 μL of APOE standard or plasma sample to each well, apply the film, and incubate at room temperature for 2 h.

[0172] (5) Discard the liquid, spin dry, and wash the plate five times with 200 μL of 1× Wash Buffer, soaking for 2 min each time.

[0173] (6) Use a pipette to add 50 μL of 1× biotinylated APOE antibody to each well, apply the membrane, and incubate at room temperature for 1 h.

[0174] (7) Wash the plate according to the procedure in (5).

[0175] (8) Use a spray gun to add 50 μL of 1×SP Conjugate, apply the film, and incubate for 30 minutes.

[0176] (9) Wash the plate according to the procedure in (5).

[0177] (10) Use a spray gun to add 50 μL of color developing solution in sequence and develop the color for about 20 min in the dark.

[0178] (11) Use a dispenser to add 50 μL of the stop solution in sequence, and use an ELISA reader to measure the absorbance (OD value) of each well at 450 nm and 630 nm.

[0179] (12) Data processing: Draw a curve by subtracting the OD value at 630 nm from the OD value at 450 nm of the standard and sample. Calculate the regression equation of the standard curve with the concentration of the standard as the ordinate (logarithmic coordinate) and the OD value as the abscissa (logarithmic coordinate). Substitute the OD value to calculate the APOE concentration of each sample and multiply it by the dilution factor. Finally, draw a graph of the relationship between APOE and age with age as the abscissa and the APOE concentration calculated by ELISA as the ordinate.

[0180] (13) Correlation between APOE and age:

[0181] Y APOE =12.55*X+687.3

[0182] Y (APOE concentration, μg / mL): predicts biological age

[0183] X(age): actual age

[0184] Explanation: According to the above formula, by inputting age, the biological age of the subject can be predicted.

[0185] *If the APOE ELISA test result is higher than the Y value, it indicates that the subject's biological age is greater than the actual age, that is, older than the actual age.

[0186] *If the APOE ELISA test result is lower than the Y value, it indicates that the subject's biological age is younger than the actual age, that is, younger than the actual age.

[0187] LILRA2 (Human LILRA2 ELISA Kit, XY Biotechnology, XY9H2451) The recommended method and steps for human LILRA2 detection are as follows:

[0188] (1) All reagents were equilibrated at room temperature for 20 min before testing.

[0189] (2) Diluting the standard: Use sample diluent to dilute the standard. The final concentrations are 10 ng / mL, 5 ng / mL, 2.5 ng / mL, 1.25 ng / mL, 0.625 ng / mL, 0.3125 ng / mL, 0.156 ng / mL and 0 μg / mL.

[0190] (3) Diluting plasma samples: According to the experimental design, 80 plasma samples were selected from four age groups: 20 samples in each age group: 20-29 years old, 30-39 years old, 40-49 years old, and >50 years old. The plasma was diluted with sample diluent at a ratio of 1:3.

[0191] (4) Sample addition: Add 50 μL of LILRA2 standard or plasma sample to each well, and add only 50 μL of standard / sample diluent to the blank well. Cover with film and incubate at 37°C for 40 min.

[0192] (5) Discard the liquid, spin dry, and wash the plate five times with 300 μL of 1× Wash Buffer, soaking for 2 min each time.

[0193] (6) Use a dispenser to add 100 μL of 1× biotinylated LILRA2 antibody to each well in turn, and add only 100 μL of biotinylated antibody diluent to the blank well. Apply the film and incubate at 37°C for 30 min.

[0194] (7) Wash the plate according to the procedure in (5).

[0195] (8) Use a spray gun to add the SABC complex working solution in sequence, apply the film, and incubate at 37°C for 20 minutes. (9) Wash the plate according to the procedure in (5).

[0196] (10) Use a spray gun to add 100 μL of TMB colorimetric solution in sequence and allow to develop for about 10-15 min in the dark.

[0197] (11) Use a dispenser to add 100 μL of the stop solution in sequence, and use an ELISA reader to measure the absorbance (OD value) of each well at a wavelength of 450 nm.

[0198] (12) Data processing: The OD value of the blank well was subtracted from the OD value of the standard and sample to draw a curve. The regression equation of the standard curve was calculated with the concentration of the standard as the ordinate and the OD value as the abscissa. The OD value was substituted to calculate the LILRA2 concentration of each sample and multiplied by the dilution factor. Finally, a graph of the relationship between LILRA2 and age was drawn with age as the abscissa and the LILRA2 concentration calculated by ELISA as the ordinate.

[0199] (13) Correlation between LILRA2 and age:

[0200] Y LILRA2=0.01250*X+1.044

[0201] Y (LILRA2 concentration, ng / mL): predicts biological age

[0202] X(age): actual age

[0203] Explanation: According to the above formula, by inputting age, the biological age of the subject can be predicted.

[0204] *If the ELISA test result of LILRA2 is higher than the Y value, it indicates that the biological age of the subject is greater than the actual age, that is, older than the actual age.

[0205] *If the ELISA test result of LILRA2 is lower than the Y value, it indicates that the biological age of the subject is younger than the actual age, that is, younger than the actual age.

[0206] Example 5: WB detection of protein expression

[0207] (1) Protein lysis: Take out the tissue sample frozen in liquid nitrogen, grind it into fine powder in liquid nitrogen, add an appropriate amount of RIPA lysis buffer, lyse it in a shaker at 4°C for 60 min, boil it at 95°C for 10 min, and determine the protein concentration using the BCA protein quantification kit.

[0208] (2) Protein separation: Prepare 10% SDS-PAGE gel, add the quantified protein sample, separate by electrophoresis and transfer to a membrane.

[0209] (3) Protein quantitative analysis: For the separated protein samples, the target bands were cut out, diluted anti-APOE antibodies were added, and incubated overnight at 4°C on a shaker. After adding the secondary antibody and substrate, the target bands were collected using Bio-Rad Image Lab, and the grayscale values ​​of the bands were quantitatively analyzed using Image J software.

[0210] Proteins are effector molecules that directly regulate biological processes including aging. We analyzed plasma protein expression patterns according to chronological age and found that 119 proteins accumulated with age and 148 proteins decreased with age (Pearson correlation, BMI-corrected P value < 0.05) ( Figure 1 and Figure 2 In addition to circulating aging biomarkers previously identified in larger cohorts, such as GPNMB and CRP, we also identified several proteins that increased with age, including CCL16, SPP1, and CTSB, members of the senescence-associated secretory phenotype (SASP), whose increased secretion in part drives the age-related functional downregulation ( Figure 3Strikingly, proteins accumulated with age are clustered on plasma lipoproteins (e.g. APOF, APOC4, APOA4, APOB, APOC3), which are directly associated with elevated LDL and cholesterol concentrations and life processes related to chronic inflammation, such as the complement system, essential components of the innate immune system (e.g. C1R, C2, C4BPA, C5), and inflammatory and immune processes (e.g. LILRA2, LILRB1, CCL16, CCL18) ( Figure 4 and Figure 5 ). However, no studies have proven that the above molecules can indicate a person's biological age before this application. It is noteworthy that, through ELISA measurements of 80 volunteers' plasma, we confirmed that leukocyte immunoglobulin-like receptor subfamily A member 2 (LILRA2) accumulates most in the elderly and is closely related to chronic inflammation, increasing with age ( Figure 4 and Figure 5 ).

[0211] By analyzing plasma proteomics data from healthy people, we inferred that one-third of proteins accumulated with age come from the liver, while one-fifth of proteins decreased with aging are secreted by muscles ( Figure 6 Among them, liver-derived proteins that were positively correlated with aging included apolipoproteins (i.e., APOF, APOB, APOC3, APOC4), complement proteins, and inflammatory proteins, as well as well-known liver injury markers such as PCSK9 and ANGPTL3. Notably, we validated the age-dependent accumulation of APOC4, APOE, and LILRA2 using plasma ELISA ( Figure 5 ), and we also found APOE accumulation in liver tissue of aged primates ( Figure 7 ). Muscle-derived proteins negatively associated with aging include factors related to ATP metabolism and autophagy, and dysregulation of ATP metabolism and autophagy has been reported to induce sarcopenia.

[0212] The above analysis data suggest that tissue-specific proteins can serve as biomarkers for assessing tissue degeneration during aging. In addition, proteins released by other tissues such as the immune system and nervous system tissues have also been identified, which may help us narrow down the scope of more tissue-specific and systemic aging biomarkers. In summary, by analyzing the human plasma secretome, we revealed dysregulated lipid metabolism and increased systemic chronic inflammation in elderly plasma and identified clues to circulating biomarkers that help assess age-related organ-specific functional decline.

[0213] Although the specific embodiments of the present invention have been described in detail, it will be understood by those skilled in the art that various modifications and changes may be made to the details according to all the teachings that have been published, and these changes are within the scope of protection of the present invention. The entire invention is given by the attached claims and any equivalents thereof.

Claims

1. Use of a reagent capable of detecting APOC4 levels, a reagent capable of detecting APOE levels, and / or a reagent capable of detecting LILRA2 levels in the preparation of a medicament for: (i) assessing the biological age of the subject; (ii) assessing the subject's aging status; (iii) evaluate the effectiveness of aging interventions; (iv) assessing a subject's risk of developing a disease associated with aging; and / or (v) Evaluate the efficacy of treatments for age-related diseases.

2. The use of claim 1, wherein The assessments described in (i) to (v) are independently auxiliary assessments.

3. The use according to claim 1 or 2, wherein The detection reagent determines the level of APOC4, APOE or LILRA2 by an in vitro detection method; Preferably, the in vitro detection method is selected from immunological detection, mass spectrometry, chromatography or any combination thereof; Preferably, the immunological detection method is selected from enzyme-linked immunosorbent assay (ELISA), electrochemical immunoassay (ECIA), chemiluminescent immunoassay (CLIA), electrochemiluminescent immunoassay (ECLIA), fluorescent immunoassay (FIA) or radioimmunoassay (RIA).

4. The use according to any one of claims 1 to 3, wherein The assessment comprises the following steps: (1) determining the level of APOC4, APOE and / or LILRA2 in a sample from the subject; (2) comparing the level with its respective corresponding reference value; and (3) assessing the biological age of the subject, the degree of aging of the subject, the effect of aging interventions, the risk of the subject developing an aging-related disease, and / or the effect of treatment of an aging-related disease; Preferably, the sample is selected from a blood sample, such as whole blood, serum or plasma; Preferably, the reference value is the level (or average level) of APOC4, APOE or LILRA2 in the same type of sample from an individual (or group) who is in the same chronological age group as the subject and in a normal aging state; or, the subject is a subject who has received aging intervention or treatment for an aging-related disease, and the reference value is the level (or average level) of APOC4, APOE or LILRA2 in the same type of sample from an individual (or group) who is in the same chronological age group as the subject and has not received the aging intervention or treatment for the aging-related disease and is in a normal aging state; or, the subject is a subject who has received aging intervention or treatment for an aging-related disease, and the reference value is the level of APOC4, APOE or LILRA2 in the same type of sample from the subject before receiving the aging intervention or before receiving the aging-related disease treatment; Preferably, the degree of aging includes normal aging, accelerated aging, and / or delayed aging; Preferably, when the levels of APOC4, APOE and / or LILRA2 in the sample from the subject are higher than their respective corresponding reference values, it indicates that the biological age of the subject is higher than its chronological age, the subject is in a state of accelerated aging, the effect of the aging intervention measure is not significant, the subject has a higher risk of developing an aging-related disease, and / or the treatment effect of the aging-related disease is not significant.

5. A drug screening method comprising: (1) Provide biological samples and divide them into test group and control group; (2) exposing the test group biological samples to the candidate drug; (3) analyzing the levels of APOC4, APOE, and / or LILRA2 in biological samples from the test group and comparing them with those of the control group; wherein the control group is not exposed to the candidate drug (for example, the control group is not treated, or the control group is exposed to a control agent without an active ingredient); If the level of APOC4, APOE and / or LILRA2 in the biological sample of the test group is lower than that of the control group, it indicates that the candidate drug has the potential to delay or reverse aging, and / or prevent and / or treat aging-related diseases.

6. The use according to any one of claims 1 to 4 or the method according to claim 5, wherein The subject is a mammal; Preferably, the subject is a human, more preferably an Asian; Preferably, the subject is Southeast Asian or East Asian (eg, Chinese, Japanese, Korean, Mongolian).

7. A reagent combination comprising any two or three of the following: (i) a reagent capable of detecting the level of APOC4, (ii) a reagent capable of detecting APOE levels, (iii) A reagent capable of detecting the level of LILRA2.

8. The reagent combination of claim 7, wherein The detection reagent determines the level of APOC4, APOE or LILRA2 by an in vitro detection method; Preferably, the in vitro detection method is selected from immunological detection, mass spectrometry, chromatography or any combination thereof; Preferably, the immunological assay is selected from enzyme-linked immunosorbent assay (ELISA), electrochemical immunoassay (ECIA), chemiluminescent immunoassay (CLIA), electrochemiluminescent immunoassay (ECLIA), fluorescent immunoassay (FIA) or radioimmunoassay (RIA).

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