Marker for evaluating or assisting in evaluating adrenal gland tissue aging and application thereof

Abstract

The invention discloses a method for evaluating or assisting in evaluating adrenal gland tissue aging and a marker thereof. Experiments prove that any one of p21Cip1, Aggresome, A beta 1-40, GPNMB and LDLR can be used as the marker to evaluate the adrenal gland tissue aging degree of primates. The LDLR as a marker can distinguish senescent adrenal gland cells from young adrenal gland cells. The human adrenal gland secretion and endocrine regulation and control functions can be evaluated by utilizing the marker, the human adrenal gland aging degree can also be evaluated, important molecular indications are provided for research and intervention of adrenal gland aging related diseases, especially human adrenal gland function evaluation and related systemic disease diagnosis, and the marker has important application value.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and specifically relates to a marker for evaluating or assisting in evaluating adrenal tissue aging, thereby targeting and delaying adrenal aging-related diseases. Background Art

[0002] The adrenal glands are an important part of the human endocrine system. They are located at the top of the left and right kidneys. They mainly undertake important functions such as human metabolism, rhythm, immunity, blood pressure, and stress by synthesizing and secreting steroid hormones and catecholamines. The adrenal glands are structurally divided into the cortex and medulla. The cortex is divided into the zona glomerulosa, zona fasciculata, and zona reticularis from the outside to the inside. They synthesize and secrete specific hormones respectively: for example, the zona glomerulosa mainly secretes aldosterone to regulate water and salt balance, the zona fasciculata mainly secretes glucocorticoids that widely regulate metabolism and immunity, and the zona reticularis mainly produces the sex hormone precursor dehydroepiandrosterone. The loss of adrenal endocrine homeostasis plays a central role in regulating systemic aging. Studies have shown that changes in adrenal hormone levels are part of normal aging, and this change is associated with a wide range of aging-related pathophysiology, including sarcopenia, hypertension, osteopenia, diabetes, and memory, cognitive and sleep dysfunction. Adrenal dysfunction also increases the risk of common health problems, such as decreased immune response and subsequent diseases or infections, decreased ability of the elderly to recover from injuries and diseases, and general weakness and fatigue. In addition, during the aging process, changes in adrenal physiological function are thought to be related to changes in adrenal hormone output. In particular, the content of DHEA and DHEA-S (dehydroepiandrosterone and its sulfide) will drop to 80% of the peak value in youth in old age, and this feature has been considered a hallmark of aging.

[0003] Therefore, in-depth analysis of the cellular and molecular mechanisms of the adrenal aging process can provide important interventional target cells and key regulatory factors for delaying adrenal aging and endocrine disorders, and further provide an important theoretical basis for the systemic pathological mechanisms caused by adrenal aging, which has important scientific and clinical significance. Summary of the invention

[0004] The purpose of the present invention is to provide primate adrenal aging markers and further study diseases related to adrenal aging.

[0005] The present invention claims a marker for use in preparing a product for identifying or assisting in identifying the degree of aging of different adrenal cells or preparing a product for evaluating or predicting adrenal function damage or related diseases caused by aging, wherein the marker is p21 Cip1 , Aggresome, Aβ1-40, GPNMB, LDLR.

[0006] The present invention claims to protect the use of substances for testing the above-mentioned markers in the preparation of products for identifying or assisting in identifying the degree of aging of different adrenal cells or in the preparation of products for evaluating or predicting related diseases caused by adrenal function damage or aging.

[0007] Wherein, the substance used to test the marker is a substance for detecting the expression amount of the marker.

[0008] Further, 1) the substance for testing the marker is to detect p21 in the adrenal tissue to be tested. Cip1 The expression level of substances, p21 Cip1 The senescence degree of the adrenal tissue under test with high expression level is greater than or candidate greater than p21 Cip1 Adrenal tissue to be tested with low expression level;

[0009] 2) the material for testing the marker is to detect the expression of Aβ1-40 in the adrenal tissue to be tested, and the aging degree of the adrenal tissue to be tested with a low expression of Aβ1-40 is greater than or is a candidate greater than the adrenal tissue to be tested with a high expression of Aβ1-40;

[0010] 3) The substance for testing the marker is a substance for detecting the expression of GPNMB in the adrenal tissue to be tested, and the aging degree of the adrenal tissue to be tested with a high expression of GPNMB is greater than or candidate greater than the adrenal tissue to be tested with a low expression of GPNMB;

[0011] 4) the substance for testing the marker is a substance for detecting the content of Aggresome in the adrenal tissue to be tested, and the aging degree of the adrenal tissue to be tested with a high Aggresome content is greater than or candidate greater than that of the adrenal tissue to be tested with a low Aggresome content;

[0012] 5) The substance for testing the marker is a substance for detecting the expression level of LDLR in the adrenal tissue to be tested, and the aging degree of the adrenal tissue to be tested with a low LDLR expression level is greater than or is candidate greater than that of the adrenal tissue to be tested with a high LDLR expression level.

[0013] further,

[0014] The present invention claims a product for identifying or assisting in identifying the aging degree of different adrenal cells, wherein the product comprises a substance for detecting the marker.

[0015] Furthermore, the product is a kit.

[0016] The substance for testing the marker is any one of the following 1)-5),

[0017] 1) The substance for testing the marker is to detect p21 in the adrenal tissue to be tested. Cip1 The expression level of substances, p21Cip1 The senescence degree of the adrenal tissue under test with high expression level is greater than or candidate greater than p21 Cip1 Adrenal tissue to be tested with low expression level;

[0018] 2) the material for testing the marker is to detect the expression of Aβ1-40 in the adrenal tissue to be tested, and the aging degree of the adrenal tissue to be tested with a low expression of Aβ1-40 is greater than or is a candidate greater than the adrenal tissue to be tested with a high expression of Aβ1-40;

[0019] 3) The substance for testing the marker is a substance for detecting the expression of GPNMB in the adrenal tissue to be tested, and the aging degree of the adrenal tissue to be tested with a high expression of GPNMB is greater than or candidate greater than the adrenal tissue to be tested with a low expression of GPNMB;

[0020] 4) The substance for testing the marker is a substance for detecting the expression of Aggresome in the adrenal tissue to be tested, and the aging degree of the adrenal tissue to be tested with high Aggresome expression is greater than or candidate greater than the adrenal tissue to be tested with low Aggresome expression;

[0021] 5) The substance for testing the marker is a substance for detecting the expression level of LDLR in the adrenal tissue to be tested, and the aging degree of the adrenal tissue to be tested with a low LDLR expression level is greater than or is candidate greater than that of the adrenal tissue to be tested with a high LDLR expression level.

[0022] Wherein, the detection sample of the product may be adrenal cells.

[0023] Furthermore, the test sample of the product may be an adrenal tissue sample isolated from a mammal, a paraffin section thereof or a frozen section thereof.

[0024] The application provided by the present invention is any of the following applications:

[0025] The present invention first protects p21 Cip1 , Aggresome, Aβ1-40, GPNMB as a marker for detecting the degree of aging of adrenal tissue and / or evaluating or predicting related diseases caused by adrenal function damage or aging.

[0026] The present invention provides the use of at least one of the following 1)-4) in the following AD:

[0027] A) Detect the aging degree of adrenal tissue;

[0028] B) Assess or predict diseases related to adrenal function damage or aging;

[0029] C) preparing a product for detecting the degree of aging of adrenal tissue;

[0030] D) Preparation of products for assessing or predicting diseases related to adrenal dysfunction or aging;

[0031] 1) Detection of p21 Cip1 The substance expressing the amount;

[0032] 2) substances for detecting the expression of Aggresome;

[0033] 3) a substance for detecting the expression of β-Amyloid (abbreviated as Aβ) (1-40) protein;

[0034] 4) a substance for detecting the expression of GPNMB;

[0035] In the above application, the test sample of the product can be an adrenal tissue sample separated from a mammal, a paraffin section thereof or a frozen section thereof.

[0036] Use of a substance for knocking down LDLR expression in any of the following:

[0037] 1) Application in the preparation of products for regulating adrenal aging;

[0038] 2) Use in the preparation of products for reducing the content of DHEA-S in epithelial cells of the adrenal gland;

[0039] 3) Use in the preparation of products for reducing cholesterol content in adrenal glands;

[0040] 4) Application in the preparation of products for regulating HAECs cell senescence.

[0041] Application of DHEA-S in the preparation of products for delaying HAECs cell senescence.

[0042] In the above application, the test sample of the product can be adrenal cells. Adrenal cells refer to cells separated from adrenal tissue.

[0043] In the above application, the adrenal cell may be at least one of an adrenal cortical cell, an adrenal medullary cell, or an adrenal cell model derived from a human tumor cell line.

[0044] Another object of the present invention is to provide a method for identifying or assisting in identifying the degree of aging of adrenal tissue to be tested.

[0045] The method provided by the present invention is at least one of the following 1)-4):

[0046] 1) The method comprises the following steps: detecting p21 in the adrenal tissue to be tested Cip1 Expression level, p21 Cip1 The senescence degree of the adrenal tissue under test with high expression level is greater than or candidate greater than p21Cip1 Adrenal tissue to be tested with low expression level;

[0047] 2) The method shown comprises the following steps: detecting the expression level of Aggresome in the adrenal tissue to be tested, wherein the aging degree of the adrenal tissue to be tested with high Aggresome expression level is greater than or candidate greater than that of the adrenal tissue to be tested with low Aggresome expression level;

[0048] 3) The method shown comprises the following steps: detecting the expression level of β-Amyloid (1-40) in the adrenal tissue to be tested, wherein the aging degree of the adrenal tissue to be tested with a high expression level of β-Amyloid (1-40) is greater than or is a candidate greater than that of the adrenal tissue to be tested with a low expression level of β-Amyloid (1-40);

[0049] 4) The method shown comprises the following steps: detecting the expression level of GPNMB in the adrenal tissue to be tested, wherein the aging degree of the adrenal tissue to be tested with a high expression level of GPNMB is greater than or is greater than that of the adrenal tissue to be tested with a low expression level of GPNMB;

[0050] The present invention also protects the use of LDLR as a marker for distinguishing aging adrenal cells from young adrenal cells.

[0051] The present invention provides a method for identifying or assisting in identifying whether a tested adrenal cell is an aged adrenal cell or a young adrenal cell, which is (D1) or (D2).

[0052] (D1) Compare the expression levels of the target protein in the adrenal cells to be tested and in the young adrenal cells, where the target protein is LDLR protein; then make the following judgment: if the expression level of the target protein in the adrenal cells to be tested is lower than that in the young adrenal cells, the adrenal cells to be tested are or are suspected to be aged adrenal cells; otherwise, they are or are suspected to be young adrenal cells.

[0053] (D2) Compare the expression levels of the target gene in the adrenal cells to be tested and in the young adrenal cells, where the target gene is the LDLR gene; then make the following judgment: if the expression level of the target gene in the adrenal cells to be tested is lower than that in the young adrenal cells, the adrenal cells to be tested are or are suspected to be aging adrenal cells; otherwise, they are or are suspected to be young adrenal cells.

[0054] The present invention also protects the use of a system for detecting the expression level of LDLR in distinguishing aging adrenal cells from young adrenal cells.

[0055] In any of the above-mentioned applications, the system for detecting the expression of LDLR may include a reagent for detecting the expression of LDLR. The reagent for detecting the expression of LDLR may include "a substance that can specifically bind to the LDLR protein" and / or "a substance that can specifically bind to the LDLR gene transcript". The system for detecting the expression of LDLR may specifically consist of a reagent for detecting the expression of LDLR. The reagent for detecting the expression of LDLR may specifically consist of "a substance that can specifically bind to the LDLR protein" and / or "a substance that can specifically bind to the LDLR gene transcript".

[0056] In any of the above applications, the system for detecting the expression level of LDLR may be a system for detecting the expression level of LDLR gene by quantitative PCR.

[0057] The present invention also protects the use of a system for detecting the expression amount of LDLR in preparing a product for distinguishing between aged adrenal cells and young adrenal cells.

[0058] In the above application, the detection sample of the product may be adrenal cells.

[0059] In the above application, the test sample of the product can be an adrenal tissue sample separated from a mammal, a paraffin section thereof or a frozen section thereof.

[0060] Any of the above-mentioned aged adrenal cells refers to the adrenal cells of old mammals. Any of the above-mentioned young adrenal cells refers to the adrenal cells of young mammals.

[0061] The present invention further provides an application of DHEA-S to delay HAECs cell senescence, comprising the following steps:

[0062] 1, 5 or 25 μg / mL DHEA-S treatment for 72 hours can delay the cell senescence of replicatively aged HAECs (human arterial endothelial cells).

[0063] The above-mentioned delay of cell aging has the following manifestations:

[0064] E1) Increased cell migration ability.

[0065] E2) Decreased levels of senescence-associated β-galactosidase (SA-β-gal).

[0066] E3) The mRNA levels of aging inflammatory genes and aging marker genes were reduced.

[0067] In the above, the lower than is statistically lower than.

[0068] The mammal is a cynomolgus monkey or a human.

[0069] When the mammal is a cynomolgus monkey, old means 18-21 years old, and young means 4-5 years old. When the mammal is a human, old means more than 70 years old, and young means 18-20 years old.

[0070] In the above text, p21 Cip1 (Gene ID: 1026, updated on 19 February 2023): This gene encodes a potent cyclin-dependent kinase inhibitor. The protein encoded by this gene mediates p53-dependent cell cycle G1 arrest in response to various stress stimuli and may contribute to the execution of apoptosis after caspase activation.

[0071] Aggresome: is an accumulation of misfolded proteins, chaperones and proteasomes around the centrosome. Aggresomes can isolate harmful protein aggregates and promote their degradation by aggregating them with proteasome subunits or triggering them to enter autophagosomes and deliver them to lysosomes, thereby protecting cells. It is a prominent feature of many age-related organ degenerative diseases.

[0072] Aβ1-40: Also written as β-Amyloid (1-40), β-amyloid protein (1-40) is the main component of Alzheimer's disease plaque amyloid protein and is a physiological peptide present in the brain, cerebrospinal fluid and plasma.

[0073] GPNMB (Gene ID: 10457, updated on December 4, 2022): The protein encoded by this gene is a type I transmembrane glycoprotein. GPNMB is upregulated by lysosomal stress associated with cellular aging and is a key protective factor in maintaining lysosomal integrity.

[0074] LDLR (Gene ID: 3949, updated on 29-Jan-2023): The low-density lipoprotein receptor (LDLR) gene family consists of cell surface proteins involved in receptor-mediated endocytosis of specific ligands. The encoded protein is normally bound to the cell membrane, binds to low-density lipoprotein / cholesterol and is taken into the cell. Lysosomal release of cholesterol occurs, and at the same time, stimulated cholesterol ester synthesis occurs.

[0075] The inventors of the present invention constructed and integrated the analysis of the single nuclear transcriptome of the adrenal gland of cynomolgus monkeys and immunohistochemistry or fluorescence detection and found that p21 Cip1Any one of Aβ, Aggresome, Aβ1-40, GPNMB, and LDLR can be used as a marker to evaluate the degree of adrenal aging, and LDLR can be used as a marker to distinguish between aged adrenal cells and young adrenal cells. The marker of the present invention plays an important role in the screening and identification of adrenal cells, can evaluate the secretion and endocrine regulation function of human adrenal glands, and can also evaluate the degree of human adrenal aging, for the research and intervention of diseases related to adrenal aging, especially in the evaluation of human adrenal function and the diagnosis of related systemic diseases. It provides important molecular indications and has important application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0076] Figure 1 Schematic diagram of adrenal tissue structure and construction and analysis of cynomolgus monkey single-cell nuclear sequencing library (where a represents the structural division of the adrenal gland, and b represents the experimental design of construction and analysis of cynomolgus monkey single-cell nuclear sequencing library).

[0077] Figure 2 It is a bioinformatics analysis of adrenal cell subpopulations and the expression levels of adrenal cell markers in adrenal cell subpopulations (where a represents adrenal cell subpopulations, and b represents the expression levels of adrenal cell markers in adrenal cell subpopulations).

[0078] Figure 3 Bioinformatics analysis of adrenal cell aging-related transcriptional noise analysis, cell identity score, high-frequency differentially expressed genes, and pathway enrichment (where a represents the cell identity score associated with adrenal cell aging, b represents the transcriptional noise analysis associated with adrenal cell aging, c represents the high-frequency differentially expressed genes associated with adrenal cell aging, and d represents the pathway differentially expressed genes associated with adrenal cortex cell aging).

[0079] Figure 4 Immunohistochemistry or fluorescence staining was used to identify the expression and localization of adrenal cell senescence molecular markers (a represents p21 Cip1 a represents the distribution of positive signals in various zones of the adrenal gland, b represents the distribution of positive signals of Aggresome in various zones of the adrenal gland, c represents the distribution of positive signals of Aβ1-40 in various zones of the adrenal gland, and d represents the distribution of positive signals of GPNMB in various zones of the adrenal gland).

[0080] Figure 5 Real-time fluorescence quantitative PCR was used to detect the expression of LDLR gene in adrenal cells of young and old cynomolgus monkeys.

[0081] Figure 6 Western blot was used to detect the protein level of LDLR protein in the adrenal tissue of young and old cynomolgus monkeys.

[0082] Figure 7Schematic diagram of lentiviral transduction of non-targeting or LDLR-targeting sgRNA in NCI-H295R cell line for CRISPR / cas9-mediated knockout system and western blot analysis of LDLR protein levels.

[0083] Figure 8 Enzyme-linked immunosorbent assay (ELISA) was used to detect the DHEA-S content in the supernatant of NCI-H295R cells with non-targeting or LDLR-targeting sgRNA transduced by lentivirus in the CRISPR / cas9-mediated knockout system.

[0084] Fig. 9 Filipin III fluorescence staining was used to quantify free cholesterol levels in NCI-H295R cells transfected with lentiviruses containing non-targeting or LDLR-targeting sgRNA in the CRISPR / Cas9-mediated knockout system.

[0085] Fig.10 SA-β-gal analysis of HAECs after treatment with different concentrations of DHEA-S for 72 hours.

[0086] Fig.11 This is an analysis of cell migration ability after HAECs were treated with different concentrations of DHEA-S for 72 hours.

[0087] Fig.12 Real-time fluorescence quantitative PCR was used to detect the expression of mRNA levels of aging inflammatory genes and aging marker genes in HAECs treated with 1 μg / mL DHEA-S for 72 hours. DETAILED DESCRIPTION

[0088] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0089] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0090] In the quantitative tests in the following examples, the experimental data are expressed as mean ± standard deviation, and the experiments were repeated three times. The results were averaged and analyzed using GraphPad Prism 8.0 statistical software.

[0091] The sources of the biological materials in the following embodiments are as follows: Human embryonic kidney cell 293T cells were purchased from ATCC, with the catalog number CRL-3216. Human adrenocortical carcinoma cell NCI-H295R was purchased from iCell, with the catalog number iCell-h296. Human arterial endothelial cells were purchased from Lonza, with the catalog number CC-2535. Lentiviral packaging vector psPAX2 (referred to as plasmid psPAX2): Addgene product, #12260. Lentiviral packaging vector pMD2G (referred to as plasmid pMD2G): Addgene product, #12259. The above biological materials are only used for repeating the relevant experiments of the present invention and cannot be used for other purposes.

[0092] Rabbit anti-p21 (CDKN1A / CIP1) was purchased from Cell Signaling Technology, catalog number 2947S;

[0093] Rabbit anti-Aβ1-40 was purchased from Cell Signaling Technology, catalog number 12990S;

[0094] Rabbit anti-GPNMB was purchased from Abcam, catalog number ab175427;

[0095] Rabbit anti-LDLR was purchased from Bioworld, catalog number BS72690;

[0096] Mouse anti-GAPDH was purchased from Santa Cruz, catalog number sc-365062;

[0097] Filipin III dye was purchased from APExBIO, catalog number B6034;

[0098] MβCD (Methyl-beta-cyclodextrin) powder was purchased from Sigma, catalog number C4555; human DHEA-S enzyme-linked immunosorbent assay (ELISA) detection kit was purchased from DRG, catalog number EIA-1562;

[0099] The antifade mounting medium was purchased from Vector Laboratories, catalog number H-1000;

[0100] Protein loading buffer was purchased from Dingguo Changsheng, catalog number WB-0091;

[0101] RNase inhibitor, SuperScriptII reverse transcriptase, 5×SuperScriptII first-chain synthesis buffer, dithiothreitol (DTT),

[0102] Rabbit anti-protein and mouse anti-protein secondary antibodies were purchased from Zhongshan Jinqiao, with catalog numbers ZB-2301 and ZB-2307, respectively.

[0103] TritonX-100 was purchased from Sigma-Aldrich, catalog number T9284;

[0104] Accumax digestion solution was purchased from Millpore, catalog number SCR006;

[0105] Trypsin-EDTA (0.25%) / trypsin-EDTA cell digestion solution was purchased from Gibco, catalog number 25200072;

[0106] DPBS was purchased from Gibco with catalog number 21600069.

[0107] The adrenal tissues in the following examples were derived from young and old cynomolgus monkeys, respectively. The cynomolgus monkey experiments in the examples were conducted based on the Guidelines for the Ethical Treatment of Non-human Primates and have passed the ethical approval of the Institute of Zoology, Chinese Academy of Sciences.

[0108] In the following examples, the primer names and nucleotide sequences for constructing the CRISPR / cas9-mediated knockout system and real-time fluorescence quantitative PCR detection are shown in Table 1.

[0109] Table 1

[0110]

[0111] Example 1. Acquisition and detection of molecular markers of senescent adrenal cells

[0112] Schematic diagram of the process of single-cell nuclear transcriptome sequencing and RNA sequencing library construction and analysis of cynomolgus monkeys Figure 1 shown.

[0113] 1. Bioinformatic analysis of single-cell nuclear transcriptome data from cynomolgus monkey adrenal gland

[0114] 1. Based on the sequencing libraries of skeletal muscle cells of young and old cynomolgus monkeys in the database, 12 cell types including four subpopulations of adrenal cortical cells were obtained in combination with classical marker gene annotations, such as Figure 2 As shown, they are zona glomerulosa cells, transitional cells, zona fasciculata cells, zona reticularis cells, chromaffin cells, neurons, fibroblasts, smooth muscle cells, endothelial cells, adipocytes, T cells and macrophages.

[0115] 2. Through transcriptional noise, cell identity score, high-frequency differential genes, and pathway enrichment analysis, such as Figure 3As shown, it was found that the expression of LDLR in adrenal cortical cells, especially zona reticularis cells, was significantly downregulated with aging.

[0116] 2. Immunohistochemical staining

[0117] 1. Dewax and hydrate the paraffin sections of adrenal tissue with 100%, 100% and 100% (volume percentage) of xylene and 100%, 100%, 90%, 90%, 80%, 70% (volume percentage) of gradient ethanol in sequence;

[0118] 2. Place the sections in 1x citrate buffer, pH 6.0 (purchased from Zhongshan Jinqiao, catalog number ZLI-9065) and heat for 5 times, 5 minutes each time, for antigen retrieval;

[0119] 3. After cooling to room temperature, the slices were rinsed three times in 1xPBS solution (pH 7.3, PBS powder purchased from Zhongshan Jinqiao, item number ZLI-9063);

[0120] 4. Permeabilize with 0.4% (volume percentage, diluted in 1x PBS solution (pH 7.3, PBS powder purchased from Zhongshan Jinqiao, item number ZLI-9063)) Triton X-100 solution for 2 hours;

[0121] 5. Then rinse again in 1×PBS, pH 7.3 for 3 times, 5 minutes each time. Then incubate the sections with 3% (volume percentage) H2O2 for 15 minutes to inactivate endogenous peroxidase;

[0122] 6. Then rinse again in 1× PBS, pH 7.3 for 3 times, 5 minutes each time.

[0123] 7. Tissue sections were incubated with primary antibodies dissolved in 1% donkey serum at 4°C overnight. The primary antibodies were rabbit anti-p21 (CDKN1A / CIP1), rabbit anti-Aβ1-40 or rabbit anti-GPNMB (different markers correspond to different antibodies);

[0124] 8. The next day, the sections were incubated with the corresponding secondary antibody conjugated to HRP (donkey anti-rabbit IgG (H+L) secondary antibody) at room temperature for 1 hour;

[0125] 9. Use DAB kit (purchased from Zhongshan Jinqiao, catalog number ZLI-9018) for color development according to the manufacturer's instructions;

[0126] 10. Wash the slices several times in running water;

[0127] 11. Stain with hematoxylin staining solution (purchased from Zhongshan Jinqiao, product number ZLI-9620) for 1 minute, and wash off the hematoxylin staining solution with running water for 1-3 seconds;

[0128] 12.1% (volume percentage) hydrochloric acid ethanol for 1-3 seconds, wash with water for 10-30 seconds;

[0129] 13. Finally, the sections were dehydrated in a series of graded ethanol (50%, 70%, 80%, 90%, 100% and 100%) and xylene;

[0130] 14. Use neutral gum to seal the slides.

[0131] 15. Images were taken using a PerkinElmer Vectra Polaris system.

[0132] Following the above steps, immunohistochemical staining was performed on paraffin sections of the adrenal glands of seven young and eight old cynomolgus monkeys. The test results and quantitative results are shown in Figure 4 , Figure 4 a is the expression and localization of the molecular marker p21 (CDKN1A / CIP1) in senescent adrenal tissue identified by immunohistochemical staining. Figure 4 b is the expression and localization of Aβ1-40, a molecular marker of aging adrenal tissue, identified by immunohistochemical staining. Figure 4 c is the expression and localization of GPNMB, a molecular marker of aging adrenal tissue, identified by immunohistochemical staining; the left picture of each figure is the detection result, and the right picture is the quantitative result (in each group of bar graphs, green is young and orange is old), ZG is the adrenal zona glomerulosa area, ZF is the adrenal zona fasciculata area, ZR is the adrenal zona reticularis area, and M is the adrenal medulla area. The ordinates are the proportion of cells positively stained with p21 (CDKN1A / CIP1), Aβ1-40, GPNMB, and the proportion of positive staining areas. The results showed that compared with young adrenal tissue, p21 in old adrenal tissue was higher than that in young adrenal tissue. Cip1 The proportion of cells with positive staining increased significantly overall ( Figure 4 ), the Aβ1-40 and GPNMB positive staining areas were significantly reduced overall ( Figure 4 ).

[0133] 3. Aggresome Fluorescence Staining

[0134] 1. Dewax and hydrate the paraffin sections of adrenal tissue obtained in the above two with 100%, 100% and 100% (volume percentage) of xylene and 100%, 100%, 90%, 90%, 80%, 70% (volume percentage) of gradient ethanol in sequence;

[0135] 2. Permeabilize the slices with 0.4% (volume percentage) Triton X-100 on ice for 30 minutes;

[0136] 3. Incubate with Aggresome dye (Proteostat, ENZ-51035-K100 diluted 1:2000 in 1xPBS buffer pH 7.3) in the dark for 30 minutes;

[0137] 4. Rinse the sections in 1× PBS three times, 5 minutes each time;

[0138] 5. Counterstain with Hoechst33342 (Thermo Fisher Scientific).

[0139] 6. Images were taken using the PerkinElmer Vectra Polaris system.

[0140] Adrenal paraffin sections from 7 young and 8 old cynomolgus monkeys were stained for Aggresome fluorescence according to the above procedure. The detection and quantitative results are shown in Figure 4 d, the left picture is the detection result, the right picture is the quantitative result (in each group of bar graphs, green is young, orange is old), ZG is the adrenal zona glomerulosa area, ZF is the adrenal zona fasciculata area, ZR is the adrenal zona reticularis area, M is the adrenal medulla area, and the ordinate is the proportion of cells with positive Aggresome staining. The results show that compared with young adrenal tissue, the proportion of cells with positive Aggresome staining in old adrenal tissue increased significantly.

[0141] These results indicate that p21 Cip1 , Aggresome, Aβ1-40, and GPNMB can all be used as markers to detect the degree of adrenal aging, as follows:

[0142] Detection of p21 in adrenal tissue Cip1 Expression level, p21 Cip1 The senescence degree of the adrenal tissue under test with high expression (high proportion of cells with positive staining) is greater than or candidate greater than p21 Cip1 Adrenal tissue with low expression (low percentage of cells with positive staining) Cip1 The substances used for expression detection include: rabbit anti-p21 Cip1 .

[0143] The expression of Aβ1-40 in the adrenal tissue to be tested is detected, and the aging degree of the adrenal tissue to be tested with a low ratio of Aβ1-40 positive staining areas is greater or candidate greater than that of the adrenal tissue to be tested with a high ratio of Aβ1-40 positive staining areas. The substances used to detect the ratio of Aβ1-40 positive staining areas in the adrenal tissue to be tested include: rabbit anti-Aβ1-40 for immunofluorescence staining detection.

[0144] The proportion of β-Amyloid (1-40)-positive staining areas in the adrenal tissue to be tested is detected. The degree of aging of the adrenal tissue to be tested with a high proportion of cells positively stained with β-Amyloid (1-40) is greater than or candidate greater than that of the adrenal tissue to be tested with a low proportion of cells positively stained with β-Amyloid (1-40). The substances used to detect the proportion of cells positively stained with β-Amyloid (1-40) in the adrenal tissue to be tested include: rabbit anti-β-Amyloid (1-40) for immunofluorescence staining detection.

[0145] The Aggresome expression level in the adrenal tissue to be tested is detected (the expression level is measured by the proportion of cells with positive staining). The degree of aging of the adrenal tissue to be tested with high Aggresome expression level (high proportion of cells with positive staining) is greater than or potentially greater than that of the adrenal tissue to be tested with low Aggresome expression level (low proportion of cells with positive staining).

[0146] The material used for detecting the proportion of Aggresome-positive cells in the adrenal tissue to be tested includes: containing Aggresome staining-related reagents.

[0147] 4. Real-time fluorescence quantitative PCR detection

[0148] 1. Search the primer sequence of the target gene on PrimerBank and synthesize the primer. Dissolve the primer in ddH2O with a final concentration of 10nM. Take 2μg of the reverse transcribed cDNA template and then prepare the RT-qPCR system according to the instructions.

[0149] 2. Prepare the reaction system, mix thoroughly, place it in the fluorescence quantitative PCR instrument and react according to the procedure.

[0150] 3. The primer sequences of real-time fluorescence quantitative PCR are shown in Table 1.

[0151] Real-time fluorescence quantitative PCR was used to detect the mRNA expression of LDLR in the adrenal tissues of 7 young and 8 old cynomolgus monkeys. Figure 5 As shown, quantitative PCR confirmed that the expression of LDLR mRNA in the adrenal gland of cynomolgus monkeys decreased with aging ( Figure 5 ).

[0152] 5. Western blot detection

[0153] Western blot was used to detect the expression of LDLR protein in the adrenal tissues of 7 young and 8 old cynomolgus monkeys.

[0154] The adrenal tissues of the 15 cynomolgus monkeys were placed in a 1% SDS solution and the cell pellet was blown several times using a tissue grinder to fully lyse it, and then heated to 105°C for 10 minutes to fully denature the protein. Then, an equal amount of 20 μg of protein was taken for Western blot (with GAPDH expression as a reference).

[0155] The specific steps of Western blot are as follows:

[0156] 1. Add an appropriate amount of concentrated protein loading buffer (Dingguo) to an equal amount of protein sample. Load the prepared protein sample into the SDS-PAGE gel well and perform protein electrophoresis experiment.

[0157] 2. Stop electrophoresis when the bromophenol blue in the protein loading buffer reaches near the bottom of the gel.

[0158] 3. After completing step 2, use a membrane transfer apparatus to transfer the protein for 2.5 hours.

[0159] 4. After completing step 3, immediately use skimmed milk powder for blocking at room temperature for 1 hour.

[0160] 5. After completing step 4, use LDLR antibody and internal reference protein antibody GAPDH for primary antibody incubation at 4°C overnight.

[0161] 6. After completing step 5, incubate with a secondary antibody of the corresponding resistance at room temperature for 1 hour. After washing, store at 4°C for 30 minutes to reduce the background before developing.

[0162] Western blot results Figure 6 The results showed that the protein expression of LDLR in the adrenal tissue of aged cynomolgus monkeys was significantly reduced.

[0163] The above results confirm that LDLR is a new molecular marker of primate adrenal aging.

[0164] Example 2

[0165] 1. Plasmid construction

[0166] Lentivirus CRISPR / cas9-mediated knockout of LDLR was performed. Non-targeting control (NTC) sgRNA (sg-NTC) or LDLR targeting sgRNA (sg-LDLR) was cloned into lentiCRISPRv2 plasmid (#52961, Addgene).

[0167] 2. Lentivirus Packaging

[0168] 1. For lentiviral production, HEK293T cells were placed in 10 cm tissue culture dishes and transfected with 9 μg of lentiviral knockout plasmids with the specified sequences, 6 μg of psPAX2 plasmid (Addgene, #12260), and 3 μg of pMD2G plasmid (Addgene, #12259) using PEI transfection reagent (Polysciences, 24765). The medium was replaced after 8 hours, and viral particles were collected after 72 hours and purified by ultracentrifugation at 19400g for 2.5 hours at 4°C.

[0169] 2. Use Western blot method to detect the expression level of LDLR in cells.

[0170] Test results such as Figure 7 As shown, the expression of LDLR in cells of the sg-LDLR group was significantly lower than that of the sg-NTC group, and the results showed that the expression of the LDLR gene was successfully inhibited.

[0171] 3. Human DHEA-S enzyme-linked immunosorbent assay (ELISA) detection

[0172] The DHEA-S content in the supernatant of the culture medium of CRISPR / cas9-mediated LDLR knockout human adrenocortical carcinoma cells was identified by enzyme-linked immunosorbent assay (ELISA).

[0173] 1. Before the experiment, place all reagents at room temperature.

[0174] 2. Take out the 96-well plate and add the standard and sample to the test wells in sequence according to the concentration gradient. Add 25 μL of standard or sample to the well plate.

[0175] 3. Place 200 μL of enzyme conjugate into each well.

[0176] 4. Incubate at room temperature for 1 hour (without covering the plate) on a shaker.

[0177] 5. Shake out the water in the wells. Rinse the wells three times with diluted 1× washing solution (400 μL per well). Tap the wells vigorously on absorbent paper to remove any remaining droplets.

[0178] 6. Add 100 μL of substrate solution to each well and incubate at room temperature for 15 minutes.

[0179] 7. Add 50 μL of stop solution to each well to stop the enzymatic reaction.

[0180] 7. Use an enzyme-labeled instrument to detect the absorbance at 450nm and 690nm respectively. The actual absorbance is the value read at 450nm minus the value read at 690nm. Draw the corresponding standard curve based on the standard and calculate the concentration of the target protein in the sample.

[0181] The results are as follows Figure 8 As shown, the DHEA-S content in the culture supernatant of CRISPR / cas9-mediated LDLR knockout human adrenocortical carcinoma cells was reduced.

[0182] 8. Filipin III free cholesterol staining

[0183] The free cholesterol level of CRISPR / Cas9-mediated LDLR knockout human adrenocortical carcinoma cells was detected by labeling cellular free cholesterol with Filipin III dye.

[0184] Cultured cells were incubated in serum-free medium containing 10 mM βCD solution at 37 ° C for 30 minutes before sampling. The cells were then fixed with 4% PFA for 10 minutes and permeabilized with TritonX-100 (0.4% PBS) for 10 minutes. After multiple washes in PBS, the cells were fixed and incubated in the dark with 0.05 mg / mL Filipin III (APE×BIO) and 1 μM PI in PBS for 1 hour. Images were captured by confocal laser microscopy (Leica DM5000).

[0185] The results are as follows Fig. 9 As shown, CRISPR / Cas9-mediated LDLR knockout in human adrenocortical carcinoma cells reduced free cholesterol levels.

[0186] The above results indicate that LDLR can be used as a marker to detect the degree of adrenal aging, as follows:

[0187] The LDLR gene mRNA level and protein expression in the adrenal tissue to be tested are detected, and the aging degree of the adrenal tissue to be tested with low LDLR gene mRNA level and protein expression is greater than or candidate greater than the adrenal tissue to be tested with low LDLR gene mRNA level and protein expression. The substances used to detect the LDLR expression in the adrenal tissue to be tested include: rabbit anti-LDLR for protein blotting detection, and primers for real-time fluorescence quantitative PCR detection of LDLR.

[0188] The substances used to detect the LDLR function in the adrenal tissue to be tested include: CRISPR / cas9-mediated LDLR knockout lentiviral packaging system, containing Filipin III staining related reagents. DHEA-S enzyme-linked immunosorbent assay kit.

[0189] Example 3: Verification of DHEA-S in delaying aging of human vascular endothelial cells HAECs

[0190] HAECs cultured to passage 12 were treated with 0 (control group), 1, 5, and 25 μg / mL DHEA-S for 72 hours and then subjected to the following experiment to detect the cell senescence phenotype.

[0191] 1. SA-β-Gal staining analysis

[0192] SA-β-gal staining analysis was performed on human arterial endothelial cells. The specific steps are as follows:

[0193] Aspirate the medium and wash the cultured cells twice with 1×PBS. Fix in 2% formaldehyde and 0.2% glutaraldehyde at room temperature for 5 minutes, then incubate with 1mg / mL fresh X-gal staining solution at 37°C for 12 hours in the dark. The staining solution formula is as follows: 40mM citric acid / sodium phosphate buffer, 5mM K4[Fe(CN)6]·6H2O, 5mM K3[Fe(CN)6], 150mM NaCl, 2mM MgCl2, 1mg / ml X-gal.

[0194] SA-β-Gal staining was observed under a normal microscope. Using X-Gal as a substrate, a dark blue product was generated under the catalysis of senescence-specific β-galactosidase. Hoechst33342 (Thermo Fisher Scientific) was used to counterstain the cell nucleus, and the proportion of SA-β-gal staining positive cells was further quantitatively analyzed.

[0195] The results are as follows Fig.10 As shown, after HAECs were treated with different concentrations of DHEA-S for 72 hours, the proportion of SA-β-gal positive cells was significantly reduced compared with that of the control group.

[0196] 2. Cell Migration Assay

[0197] The cell migration ability of HAECs was measured by scratch healing assay. HAECs were plated and cultured in collagen (Advanced BioMatrix)-coated 24-well plates. When the cells reached 100% confluence, a linear scratch was created using a sterile 20 μL pipette tip, and the cells were then cultured and monitored using the IncuCyte S3 Live Cell Analysis System (Essen Bioscience). Cell migration ability was quantified using ImageJ.

[0198] The results are as follows Fig.11 As shown, after HAECs were treated with different concentrations of DHEA-S for 72 hours, the cell migration ability was significantly increased compared with the control group.

[0199] 3. Real-time fluorescence quantitative PCR detection

[0200] The method was the same as that of detecting the mRNA expression of LDLR in the adrenal tissue of cynomolgus monkeys. Fig.12 As shown, the expression of mRNA levels of aging inflammatory genes and aging marker genes was significantly lower than that of the control group.

[0201] The present invention has been described in detail above. It will be apparent to those skilled in the art that the present invention may be implemented in a wide range under equivalent parameters, concentrations and conditions without departing from the spirit and scope of the present invention and without the need for unnecessary experimentation. Although the present invention provides specific embodiments, it should be understood that further improvements may be made to the present invention. In short, according to the principles of the present invention, this application intends to include any changes, uses or improvements to the present invention, including changes made by conventional techniques known in the art that depart from the scope disclosed in this application. Applications of some of the basic features may be made within the scope of the following appended claims.

Claims

1. Use of a marker in the preparation of a product for identifying or assisting in identifying the degree of aging of different adrenal cells or in the preparation of a product for evaluating or predicting adrenal function damage or related diseases caused by aging, wherein the marker is p21 Cip1 , Aggresome, Aβ1-40, GPNMB, LDLR.

2. Use of a substance for testing the marker described in claim 1 in the preparation of a product for identifying or assisting in identifying the degree of aging of different adrenal cells or in the preparation of a product for evaluating or predicting related diseases caused by adrenal function damage or aging.

3. The use according to claim 2, wherein the substance used to test the marker according to claim 1 is a substance for detecting the expression amount of the marker.

4. A product for identifying or assisting in identifying the degree of aging of different adrenal cells, characterized in that: The product comprises the marker described in claim 1. The product according to claim 4 , which is a marker or a kit.

6. The product according to claim 5, wherein the product is a kit, and the substance for testing the marker is any one of the following 1)-5), 1) The substance for testing the marker is to detect p21 in the adrenal tissue to be tested. Cip1 The expression level of substances, p21 Cip1 The senescence degree of the adrenal tissue under test with high expression level is greater than or candidate greater than p21 Cip1 Adrenal tissue to be tested with low expression level; 2) the material for testing the marker is to detect the expression of Aβ1-40 in the adrenal tissue to be tested, and the aging degree of the adrenal tissue to be tested with a low expression of Aβ1-40 is greater than or is a candidate greater than the adrenal tissue to be tested with a high expression of Aβ1-40; 3) The substance for testing the marker is a substance for detecting the expression of GPNMB in the adrenal tissue to be tested, and the aging degree of the adrenal tissue to be tested with a high expression of GPNMB is greater than or candidate greater than the adrenal tissue to be tested with a low expression of GPNMB; 4) The substance for testing the marker is a substance for detecting the expression of Aggresome in the adrenal tissue to be tested, and the aging degree of the adrenal tissue to be tested with high Aggresome expression is greater than or candidate greater than the adrenal tissue to be tested with low Aggresome expression; 5) The substance for testing the marker is a substance for detecting the expression level of LDLR in the adrenal tissue to be tested, and the aging degree of the adrenal tissue to be tested with a low LDLR expression level is greater than or is candidate greater than that of the adrenal tissue to be tested with a high LDLR expression level.

7. The product according to any one of claims 4 to 6, characterized in that: The test sample of the product may be adrenal cells.

8. The product according to claim 7, characterized in that The test sample of the product can be an adrenal tissue sample isolated from a mammal, or a paraffin section or a frozen section thereof.

9. Use of a substance for knocking down LDLR expression in any of the following: 1) Application in the preparation of products for regulating adrenal aging; 2) Use in the preparation of products for reducing the content of DHEA-S in epithelial cells of the adrenal gland; 3) Use in the preparation of products for reducing cholesterol content in adrenal glands; 4) Application in the preparation of products for regulating HAECs cell senescence.

10. Application of DHEA-S in the preparation of products for delaying HAECs cell aging.

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