Application of Angulin-1 gene and / or Angulin-1 protein in preparation of products for resisting aging and treating aging-related diseases

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to application of an Angulin-1 gene and / or Angulin-1 protein in preparation of products for resisting aging and treating aging-related diseases. A new target spot for preventing and / or delaying senescence and treating senescence-related diseases is found in the invention, and experiments prove that the occurrence of senescence is delayed or inhibited from a plurality of aspects such as cellular level, tissues and organs and the like by overexpressing the Angulin-1 gene of a body or improving the expression quantity of Angulin-1 protein, and the healthy life of cells and tissues is prolonged.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of Angulin-1 gene and / or Angulin-1 protein in the preparation of anti-aging and age-related disease treatment products. Background Technology

[0002] Aging is the gradual decline in an organism's structure and physiological functions over time, leading to a decrease in adaptability and resistance. With the increasing global trend of aging, the incidence of aging itself and related diseases (such as heart disease, cancer, diabetes, and osteoporosis) has increased significantly. This not only severely reduces patients' quality of life but also places a heavy burden on healthcare systems and the socio-economic system.

[0003] The mechanisms of aging involve multiple factors such as genomic instability, telomere loss, and epigenetic changes. Furthermore, the process of aging is complex and involves changes at the cellular level and in multiple tissues.

[0004] At the cellular level, aging is characterized by the upregulation of p16 and p21 expression levels, cell cycle arrest, increased activity of aging-associated β-galactosidase (SA-β-Gal), and the release of aging-associated secretory phenotypes (SASPs). SASPs drive tissue microenvironment inflammation through paracrine secretion of inflammatory factors such as IL-6 and TNF-α, as well as MMPs and proteases, thus exacerbating the progression of age-related diseases such as Alzheimer's disease and atherosclerosis. At the tissue level, aging is generally accompanied by the decline of multiple system functions. For example, in the nervous system, it manifests as cognitive impairment caused by Aβ deposition and abnormal phosphorylation of Tau protein; in the cardiovascular system, endothelial dysfunction and mtDNA leakage activate the cGAS-STING pathway, accelerating plaque formation and myocardial damage; and in the metabolic system, insulin resistance and mitochondrial dysfunction induce type 2 diabetes and non-alcoholic fatty liver disease.

[0005] Currently, no drugs have been approved as "anti-aging" drugs, and most are still in the preclinical research stage.

[0006] Currently, the development of anti-aging drugs focuses on signaling pathway regulation, clearance of senescent cells, and epigenetic remodeling. However, existing therapies have shown significant limitations in clinical translation. For example, among small molecule compounds, rapamycin can prolong the lifespan of model organisms by inhibiting the mTOR pathway, but it also causes metabolic disorders and immunosuppression, increasing the risk of infection with long-term use. Although dasatinib combined with quercetin (D / Q) can selectively clear senescent cells, it may also damage senescent cells required for normal tissue repair, leading to a decline in muscle regeneration capacity. Among epigenetic intervention drugs, histone deacetylase inhibitors (HDACi) such as vorinostat can remodel chromatin structure, but they can cause side effects such as thrombocytopenia and gastrointestinal reactions.

[0007] Therefore, developing anti-aging drugs or compositions that combine innovative mechanisms with safety has become an urgent need to break through current treatment bottlenecks.

[0008] Angulin-1, a transmembrane protein widely distributed in various tissues and organs of the body, plays a central role in the regulation of lipid and energy metabolism. Studies have shown that Angulin-1 can effectively maintain lipid and energy homeostasis by promoting the clearance of cholesterol and triglycerides from the liver; it can also maintain normal brain function by regulating cholesterol transport in the nervous system, preventing cholesterol overload in neurons, and reducing susceptibility to amyloid protein; in bronchial epithelial cells, Angulin-1 not only regulates epithelial barrier function but also participates in regulating mitochondrial respiration and inhibiting inflammatory responses. Furthermore, in multiple organs such as the digestive and urinary systems, Angulin-1 can alleviate inflammation and protect tissues from damage by reducing the release of inflammatory factors such as TNF-α and IL-6.

[0009] However, to date, there have been no reports of applying the Angulin-1 gene and / or Angulin-1 protein to the field of anti-aging. Summary of the Invention

[0010] To address the aforementioned technical problems, this invention provides the application of the Angulin-1 gene and / or Angulin-1 protein in the preparation of anti-aging and age-related disease treatment products.

[0011] This invention aims to provide a new target for preventing and / or delaying aging and treating aging-related diseases. During the research process, the inventors discovered that by overexpressing the Angulin-1 gene and increasing its gene and / or protein expression levels, it is possible to significantly inhibit aging biomarkers at the cellular level and in various tissues and organs of the body, thereby providing a new strategy and direction for the regulation of aging and the prevention of aging-related diseases.

[0012] The most direct objective of this invention is to provide the application of the Angulin-1 gene and / or Angulin-1 protein in the preparation of anti-aging and age-related disease treatment products.

[0013] Preferably, in the above-mentioned applications provided by the present invention, the aging can be either physiological aging or pathological aging. Physiological aging is aging caused by physiological degeneration that occurs after maturity, while pathological aging is aging caused by external factors, including diseases.

[0014] Preferably, the aging includes the aging of cells and tissues, and the tissues include any one of blood vessels, heart, liver, lungs, kidneys, brain, bones, and skin.

[0015] Preferably, the product uses the Angulin-1 gene as its target or the Angulin-1 protein as its functional component, and the content of the functional component in the product is 1%-99% (by mass fraction).

[0016] Preferably, when the product targets the Angulin-1 gene, it achieves anti-aging or treatment of aging-related diseases by overexpressing the Angulin-1 gene and increasing the content of Angulin-1 protein in cells.

[0017] Preferably, the product includes any one of the following: medicine, food, health products, and cosmetics.

[0018] Preferably, the Angulin-1 gene and / or Angulin-1 protein function through any one or more of the following combinations: (1) Biomarkers that inhibit aging, including: aging-related secretory phenotype, expression level of aging-related cycle protein P16 / P21, and SA-β-galactosidase activity; (2) Inhibit the expression of GDF15 gene and / or GDF15 protein; (3) Downregulate the expression of γH2AX to improve DNA damage; (4) Relieves skin laxity and reduces skin damage; (5) Relieves osteoporosis; (6) Slows down the aging process of the heart; (7) Slows down the aging process of the liver; (8) Slows down the aging process of the lungs; (9) Slows down the aging process of the kidneys; (10) Slows down the brain aging process.

[0019] The present invention also provides the application of an agent capable of promoting the expression of the Angulin-1 gene and / or Angulin-1 protein in the preparation of drugs for anti-aging and treatment of aging-related diseases.

[0020] Preferably, the formulation comprises at least one of the following: a recombinant expression vector containing the Angulin-1 gene or a gene fragment having more than 90% homology with the Angulin-1 gene; exosomes containing the Angulin-1 gene and / or Angulin-1 protein and / or its bioactive fragments; polypeptides, proteins, nucleic acids and nucleic acid aptamers, natural active substances, and biological agents capable of upregulating the expression of the Angulin-1 gene and / or Angulin-1 protein.

[0021] The recombinant expression vectors include any one of the following: vector-based eukaryotic expression plasmids specifically targeting the Angulin-1 gene and / or Angulin-1 protein; adenovirus; adeno-associated virus; lentivirus; retrovirus; LNP liposomes; microinjection technology; gene editing system elements; and homologous recombinant vectors.

[0022] The recombinant expression vector is constructed by inserting the Angulin-1 gene into a backbone vector to construct an expression vector that overexpresses the Angulin-1 gene, and then packaging the expression vector that overexpresses the Angulin-1 gene with a common medical vector to obtain a recombinant expression vector that overexpresses the Angulin-1 gene.

[0023] As a preferred embodiment, the specific method of application is to directly inject the preparation that can promote the expression of Angulin-1 gene and / or Angulin-1 protein into a human or animal.

[0024] As a preferred embodiment, the functional component of the drug is a formulation that promotes the expression of the Angulin-1 gene and / or Angulin-1 protein, wherein the content of the functional component in the drug is 1%-99% (by mass fraction).

[0025] The present invention also provides a drug for anti-aging, the drug comprising a formulation that promotes the expression of Angulin-1 gene and / or Angulin-1 protein and pharmaceutical excipients, wherein the pharmaceutical excipients include at least one of diluents, excipients, fillers, binders, humectants, disintegrants, absorption promoters, surfactants, adsorbents, and lubricants, and the drug is any dosage form selected from tablets, capsules, powders, pills, granules, solutions, suspensions, syrups, injections, suppositories, inhalers, and sprays.

[0026] Furthermore, the inventors discovered during their research that when the expression levels of Angulin-1 gene mRNA and / or Angulin-1 protein in the body are upregulated after drug administration, the drug can be confirmed to have a good anti-aging or therapeutic effect on age-related diseases. Conversely, the effect is poor or there is no response. Therefore, the anti-aging / therapeutic effect of the drug on age-related diseases can be confirmed or screened based on the expression levels of Angulin-1 gene mRNA and / or Angulin-1 protein.

[0027] Therefore, the application of products used to detect the mRNA expression level and / or Angulin-1 protein expression level in the preparation of drugs for screening anti-aging and age-related diseases is also a key technical content protected by this invention.

[0028] The beneficial effects of this invention are as follows: A novel target, the Angulin-1 gene, has been developed for the prevention and / or delay of aging and the treatment of aging-related diseases. By overexpressing the Angulin-1 gene or increasing the content of Angulin-1 protein in the body, the occurrence of aging can be delayed or inhibited at multiple levels, including the cellular and tissue levels. Furthermore, since the Angulin-1 gene is an endogenous protein in the body, it is safer to use as a drug. Attached Figure Description

[0029] Figure 1 This is a comparison chart of Angulin-1 expression and quantification in serum of people of different genders and ages in Example 1 of the present invention. Among them, a is a chart of Angulin-1 expression in serum of women of different ages, b is a chart of Angulin-1 quantification in serum of women of different ages, c is a chart of Angulin-1 expression in serum of men of different ages, and d is a chart of Angulin-1 quantification in serum of men of different ages. Figure 2 This diagram illustrates the expression levels of cell cycle proteins and aging-related factors in normal hepatocytes and human lung microvascular endothelial cells before and after aging induction in Example 2 of the present invention. Specifically, a represents the relative expression level of hepatocyte mRNA before and after aging induction; b represents the protein expression of aging-related factors in hepatocytes before and after aging induction; c represents the relative expression level of aging mRNA in human lung microvascular endothelial cells before and after aging induction; and d represents the protein expression of aging-related factors in human lung microvascular endothelial cells before and after aging induction. Figure 3This diagram illustrates the effects of knocking down or overexpressing the Angulin-1 gene on the expression of cyclins and aging-related factors in Example 3 of this invention. Specifically, a represents the quantitative expression of Angulin-1 mRNA in the knockdown group, b represents the quantitative expression of P16 and P21 proteins in the knockdown group, c represents the quantitative expression of genes related to aging-related secretory phenotypes in the knockdown group, d represents the quantitative expression of Angulin-1 mRNA in the overexpression group, e represents the quantitative expression of p16 and p21 genes in the overexpression group, f represents the quantitative expression of genes related to aging-related secretory phenotypes in the overexpression group, g represents the RNA-seq sequencing analysis results, h represents the quantitative expression of GDF15 in the knockdown group, i represents the promoter region luciferase detection results, j represents the 3'-UTR region luciferase detection results, k represents the control group for related protein expression in the knockdown group, and l represents the control group for related protein expression in the overexpression group. Figure 4 This is an example of the effect of Angulin-1 overexpression on mouse survival rate and liver aging in Example 4 of the present invention; wherein, a is a comparison of mouse mortality rate before and after overexpression, b is a comparison of serum albumin content before and after overexpression, c is a comparison of serum AST content before and after overexpression, and d is a comparison of serum ALT content before and after overexpression. Figure 5 This is the effect of Angulin-1 overexpression on mouse skin damage and aging in Example 5 of the present invention; wherein, a is a gross photograph of mouse skin tissue, b is a quantitative expression map of p16 and p21 genes after Angulin-1 overexpression, c is an immunofluorescence map of mouse skin tissue before and after overexpression, and d is a HE staining result map. Figure 6 This diagram illustrates the effect of Angulin-1 overexpression on liver tissue aging in mice in Example 6 of this invention; where a is a quantitative expression map of p16 and p21 genes in liver tissue, b is an immunofluorescence map, and c is a HE staining result map. Figure 7 This is an example of the effect of Angulin-1 overexpression on the aging of mouse lung tissue in Example 7 of the present invention. Among them, a is a quantitative expression map of p16 and p21 genes in lung tissue, b is an immunofluorescence detection map, c is a comparison map of SA-β-galactosidase activity, and d is a detection result map of HE staining. Figure 8 This diagram illustrates the effect of Angulin-1 overexpression on kidney tissue aging in Example 8 of the present invention. In the diagram, a is a quantitative expression map of p16 and p21 genes in kidney tissue, b is a detection result map of immunofluorescence method, c is a comparison map of SA-β-galactosidase activity detection, and d is a detection result map of HE staining. Figure 9This is the effect of Angulin-1 overexpression on mouse heart tissue aging in Example 9 of the present invention. In this example, a is a quantitative graph of p16 and p21 gene expression in heart tissue, b is a graph of detection results by immunofluorescence method, and c is a graph of detection results by HE staining. Figure 10 This is an example of the effect of Angulin-1 overexpression on aging of mouse brain tissue in Example 10 of this invention, where a is a quantitative graph of p16 and p21 gene expression in brain tissue, and b is a graph of the detection results by immunofluorescence method. Figure 11 This is a diagram illustrating the effect of Angulin-1 overexpression on the morphology of mouse bone tissue in Example 11 of this invention. Detailed Implementation

[0030] To enable those skilled in the art to better understand the present invention, the present invention will now be further described in conjunction with specific embodiments.

[0031] Example 1: Screening and discovery process of Angulin-1 gene and / or Angulin-1 protein, key genes for anti-aging. Serum samples were collected from young adults (24 years old, n=14) and older adults (67 years old, n=14) (including 7 young women, 7 young men, 7 older women, and 7 older men). The specific steps are as follows: Blood samples were collected using pyrogen-free and endotoxin-free centrifuge tubes. After standing for 1 hour, the samples were centrifuged at 1000×g for 20 minutes at 2-8℃. Serum was collected for later use. 2 μL of serum sample was taken from each serum sample and added to 18 μL of sterile water, followed by 5 μL of protein loading buffer. The mixture was boiled at 100℃ for 10 minutes. Western blotting (WB) was used to detect the Angulin-1 protein content in the serum samples. The results are shown below. Figure 1 As shown.

[0032] Figure 1 In the figure, a and c represent the expression of Angulin-1 protein in the serum of female and male groups, respectively; b and d represent the quantitative analysis of Angulin-1 protein expression in the serum of female and male groups, respectively.

[0033] Figure 1 The results showed that, regardless of gender, the expression level of Angulin-1 protein in the serum of the elderly was significantly lower than that in younger people.

[0034] Example 2: Screening and validation process of Angulin-1 gene and / or Angulin-1 protein, key genes for anti-aging. Human pulmonary microvascular endothelial cells and normal human liver cells were seeded normally in 6-well plates and allowed to grow to approximately 85% confluence. Cells were collected according to the passage procedure and counted using a hemocytometer. The cells were then seeded into 96-well plates at a density of 5 × 10⁶ cells per well. 3 Each sample was incubated at 37°C and 5% CO2 for 24 h, then the culture medium was discarded. The samples were then induced with 100 μM doxorubicin for 3 days. The gene expression levels of Angulin-1 protein, as well as cyclins p16 and p21, were detected by RT-PCR. The expression levels of cyclins p16 and p21, the DNA damage molecule γH2AX, and Angulin-1 protein were also detected. The results are as follows: Figure 2 As shown.

[0035] In this diagram, a represents the relative expression level of hepatocyte mRNA before and after senescence induction, b represents the protein expression of senescence-related factors in hepatocytes before and after senescence induction, c represents the relative expression level of senescence-related mRNA in human pulmonary microvascular endothelial cells before and after senescence induction, and d represents the protein expression of senescence-related factors in human pulmonary microvascular endothelial cells before and after senescence induction. Figure 2 The results showed that in senescent cells induced by doxorubicin, the expression levels of p16 and p21 genes, as well as the protein expression levels of cell cycle proteins P16 and P21 and DNA damage molecule γH2AX, were significantly higher than those in normal cells (i.e., the control group), while the expression levels of Angulin-1 gene and Angulin-1 protein in senescent cells were significantly lower than those in normal cells.

[0036] It is evident that the expression level of Angulin-1 gene / protein is closely related to the occurrence of aging. The expression level of Angulin-1 gene / protein is significantly downregulated in senescent cells. Therefore, the expression level of Angulin-1 gene / protein plays an important role in the aging process and can serve as a target for delaying aging.

[0037] Example 3: Construction and identification of lentivirus overexpressing Angulin-1 gene In this embodiment, human lung microvascular endothelial cells (HPMEC) were used as the cell line overexpressing Angulin-1 for research, and human embryonic kidney cells 293T were used as tool cells for lentivirus packaging.

[0038] The Angulin-1 gene sequence was obtained from NCBI, with the sequence number NM_205834.4. The Angulin-1 gene sequence was synthesized by Sangon Biotech Co., Ltd., and then constructed into the mammalian cell overexpression vector plasmid PQCXIP.

[0039] 3.1 Preparation of experimental group cells (1) Construction of HPMEC overexpression with Angulin-1 293T cells were seeded into 15 cm culture dishes and cultured overnight until the cell density reached about 70%-80% the next day. Before transfection, the culture medium in the culture dish was replaced with 20 mL of fresh culture medium, and the overexpression lentivirus packaging system was prepared in sequence. The following transfection reagent needs to be prepared for a 15 cm culture dish to be transfected: Solution A: 500 μL DMEM medium, 10 μg PQCXIP-Angulin-1 overexpression plasmid / control plasmid, 10 μg packaging plasmid pMDlg / pRRE; 10 μg packaging plasmid pRSV-Rev and 10 μg envelope plasmid pMD2.G; Solution B: 500 μL DMEM medium, 120 μL polyethyleneimine (PEI) transfection reagent; After incubating solutions A and B at room temperature for 5 min, mix solutions B and A, incubate at room temperature for 20 min, and then evenly add the mixture to the corresponding culture dishes. Incubate at 37℃ and 5% CO2 for 12 h, then replace with fresh culture medium and continue incubation for 48 h. Collect the culture supernatant and centrifuge to concentrate the virus, which is the Angulin-1 overexpression lentivirus.

[0040] Lentiviral infection of HPMEC cells: Seed an appropriate amount of HPMEC cells into 6-well plates and culture overnight until the cell density reaches about 70%-80% the next day. Discard the old culture medium, add 2 mL of fresh culture medium and 0.1 mL of the Angulin-1 overexpressing lentivirus packaged in the previous step, mix gently, and incubate at 37℃, 5% CO2 for 12 h. Discard the old culture medium, add 2 mL of fresh culture medium, and continue culturing for 36 h. Add 1 μg / mL puromycin to screen for positive cells. After 2-3 days, all uninfected cells will be killed, leaving positive cells, which are the Angulin-1 overexpressing cells.

[0041] (2) Preparation of control group cells Following the viral packaging procedure described above, the empty vector PQCXIP plasmid was transferred into the tool cells, which were then infected and screened. The remaining positive cells were used as the control group cells.

[0042] (3) Angulin-1 gene knockout shRNA design and vector construction: The shRNA targeting Angulin-1 was synthesized by Sangon Biotech Co., Ltd., wherein the nucleotide sequence of the sense strand of the shRNA is SEQ ID NO.1: TTTGAAGGAACACTGATGA; and the nucleotide sequence of the antisense strand of the shRNA is SEQ ID NO.2: TCATCAGTGTTCCTTCAAA.

[0043] Take 5 μg of pSIREN-RetroQ vector, add 1 μL each of restriction endonucleases BamH1 and EcoR1, and incubate at 37℃ for 3 h. Mix the digested pSIREN-RetroQ vector with 6x DNA loading buffer and add to a 1% agarose gel. Electrophoresis at 180 V for 25 min. After gel excision, recover approximately 6.4 Kb of the excised vector. Mix the corresponding shRNA positive and negative strands according to the following system: Set the annealing program in the PCR instrument to 37℃ for 30 min, 95℃ for 5 min, 90℃ for 1 min, and 85℃ for 1 min for annealing and recombination. The positive oligonucleotide chain (F, 100 μM) is 2 μL, the negative oligonucleotide chain (R, 100 μM) is 2 μL, and ddH2O is 16 μL, for a total volume of 20 μL. The above gel-excised fragment and the annealed nucleotide recombinant fragment are then processed using T4. Ligase was used for ligation, and the ligated shRNA was added to competent cells. The cells were incubated on ice for 30 min, heat-shocked at 42°C for 90 s, and then 500 μL of LB medium was added. The cells were cultured on a shaker at 37°C for 1 h. The culture was then spread onto ampicillin-resistant LB plates and incubated overnight at 37°C. Single clones were picked and added to 500 μL of ampicillin-resistant LB liquid medium and cultured on a shaker at 37°C for 12 h. A portion of the bacterial culture was taken for biological sequencing. The sequencing results were compared to identify the correct clone. The vector of the correct clone was extracted using the Tiangen Mini-Protein Plasmid Extraction Kit, which is the Angulin-1 gene knockout plasmid, for subsequent lentivirus packaging.

[0044] In addition, Angulin-1 knockdown was used to construct HPMEC cells and package the virus, followed by lentivirus infection of HPMEC cells, using the same steps as above.

[0045] 3.2 The expression of Angulin-1 mRNA in the Angulin-1 knockdown group, Angulin-1 overexpression group, and control group cells prepared in 3.1 was detected. Then, the cells in each group were treated with doxorubicin to detect the effect of Angulin-1 expression on the expression of P16, P21, and senescence-associated secretory phenotype (SASP) related genes.

[0046] The specific steps are as follows: Remove control group cells, Angulin-1 knockdown group cells, and Angulin-1 overexpression group cells from the incubator. After removing the culture medium, digest the cells, resuspend them in culture medium, count them using a counting chamber, and set them aside. Dilute the different groups of cells to 2×10⁻⁶. 5 Add 0.5 mL of cell suspension per well to a 24-well plate, gently shake the plate to mix, and once the cells have adhered, remove the complete culture medium from the 24-well plate. Add 0.5 mL of complete culture medium containing 100 μM doxorubicin to a 25-well plate, mix well, and incubate at 37°C for 3 days. Then collect the total RNA from the cells.

[0047] The expression of Angulin-1 mRNA and aging biomarkers in cells of each group are shown in the figure. Figure 3 In the figure, a, b, and c represent the expression levels of Angulin-1 mRNA, P16 and P21, and aging-related molecules in the knockdown HPMEC cells, respectively, while d, e, and f represent the expression levels of Angulin-1 mRNA, P16 and P21, and aging-related molecules in the overexpression group HPMEC cells, respectively.

[0048] Figure 3 The results from the study showed that knocking down Angulin-1 upregulated the expression of genes related to the aging-associated secretory phenotype (SASP), P16, and P21, while overexpression of Angulin-1 significantly reversed the above effects.

[0049] 3.3 RNA-seq sequencing was performed on Angulin-1 knockdown cells and control cells to detect the effect of Angulin-1 on gene expression. The control group cells and Angulin-1 knockdown group cells obtained above were seeded in 6-well plates. RNA was then extracted from the cells using the Trizol assay and sent to Novogene Bioinformatics Technology Co., Ltd. for RNA-seq sequencing. The results are shown in the figure. Figure 3 As shown in Figure g, compared with the control group, the genes upregulated in the Angulin-1 knockdown group are related to cell senescence, cell cycle, and P53 signaling pathway. This result can also preliminarily prove that the reduction of Angulin-1 expression can accelerate the occurrence of senescence.

[0050] 3.4 Verification of the interaction between Angulin-1 and GDF15 According to relevant literature, GDF15 plays an important regulatory role in the aging process, and is usually upregulated in aging cells, tissues and organs.

[0051] In this invention, further analysis of RNA-seq results revealed that, compared to the control group, the GDF15 gene was significantly upregulated in the Angulin-1 knockdown group. It is speculated that the reduced expression of the Angulin-1 gene / protein accelerates aging by increasing GDF15 expression. Experimental results are shown below. Figure 3 As shown in h.

[0052] Furthermore, to further verify the interaction between Angulin-1 and GDF15, this invention employs a dual-luciferase reporter gene assay. The experimental method is as follows: The human GDF15 gene promoter (the first 2000 kb of the GDF15 open reading frame start codon, synthesized by Shanghai Sangon Biotech) was cloned into the pGL3-Basic luciferase reporter vector (Promega) via NheⅠ and HindⅢ restriction sites. The pGL3 reporter vector, pGL4.74 René luciferase control vector (Promega), and pCMV6-Lsr vector were co-transfected into HEK293 cells in 96-well culture plates using Lipofectamine 3000. The full-length 3'-UTR of the human GDF15 gene (synthesized by Shanghai Sangon Biotech) was inserted downstream of the Renilla luciferase gene in the psiCHECK-2 vector (Clontech) via the PmeⅠ / NotⅠ restriction site. Using Lipofectamine 3000, the psiCHECK-2-GDF15:3'-UTR was co-transfected with the pCMV6-Angulin-1 vector or its control vector into HEK293 cells in 96-well plates. Twenty-four hours after transfection, the activities of firefly luciferase and Renilla luciferase were measured using a dual-luciferase reporter gene assay kit (Promega). The results are as follows: Figure 3 As shown in the diagram, i and j.

[0053] As shown in the figure, knocking down Angulin-1 upregulates the activity of the GDF15 promoter. Figure 3 (i) rather than 3'UTR region ( Figure 3 (j) to regulate GDF15 to exert its anti-aging effect.

[0054] 3.5 Effects of Angulin-1 on DNA stability in senescent cells.

[0055] Since the mechanisms of aging involve multiple factors such as genomic instability and telomere loss—specifically, the gradual shortening of telomeres at the ends of chromosomes restricts cell proliferation, thereby triggering DNA damage and leading to cell cycle arrest—this invention investigates whether Angulin-1 affects DNA stability in senescent cells. Because γH2AX is a specific biomarker for DNA damage, the expression of γH2AX is detected in this invention, with the specific steps as follows: Cell selection and culture: HPMEC cells were routinely cultured in DMEM cell culture medium containing 10% fetal bovine serum, 1% penicillin, and streptomycin, and incubated at 37°C with 5% CO2. The medium was changed every 2 days, and the cells were passaged at a ratio of 1:3. HPMEC cells, HPMEC knockdown cells, and HPMEC overexpression cells were removed from the incubator. After removing the culture medium, the cells were digested and resuspended in culture medium. Cells were counted using a counting chamber and then cultured at a concentration of 1×10⁻⁶ cells / cells. 6 The cells were seeded at a rate of 1 cell per well in 6-well plates (n=3). After the cells adhered, the complete culture medium in the 6-well plates was removed, and 2 mL of complete culture medium containing 100 μM doxorubicin was added to the 6-well plates. After mixing, the plates were incubated at 37°C for 3 days, and the total protein from the cells was collected.

[0056] The expression of γH2AX protein in each group was detected by Western blotting, and the results are as follows: Figure 3 As shown in the figure, k represents the expression comparison of the Angulin-1 knockdown group and l represents the expression comparison of the Angulin-1 overexpression group. It can be seen from the figure that Angulin-1 knockdown can upregulate the expression level of γH2AX, while Angulin-1 overexpression significantly reverses the above effect.

[0057] It is evident that the Angulin-1 gene and / or protein delay aging by alleviating cellular DNA damage.

[0058] Example 4: Validation of Angulin-1's ability to alleviate aging by extending lifespan and improving liver function in mice. Animal grouping and administration: 20 male C57BL / 6 mice, aged 24 months, were randomly divided into two groups: control group and Angulin-1 overexpression group, with 10 mice in each group.

[0059] Among them, the Angulin-1 overexpression group: administered 1×10 via intraperitoneal injection. 9 MOI Angulin-1 overexpresses the viral particle AAV-Angulin-1, which was synthesized by Hanheng Company. The preparation method is as follows: plasmid construction (1) Full-length amplification of the Angulin-1 target gene The full-length coding sequence of the mouse Angulin-1 gene was obtained from the NCBI database, with the gene number NM_001164184.1. Primer sequences for the target gene were designed, including: AAV-m- Angulin -F:ttgacctccatagaagacaccgggatccAAGCTTGCCCACCATGGCGCC; AAV-m- Angulin -R: tgtagtcgttaattaaggtacCGAATTCGACGACTAAACTTTCCCGACT.

[0060] The mouse Angulin-1 gene fragment was recovered by PCR electrophoresis using the primers described above and then excised from the gel. (2) Construction and extraction of recombinant plasmids The vector pHBAAV-CMV-MCS-3flag-T2A-ZsGreen and the recovered mouse Angulin-1 gene fragment were double-digested using restriction endonucleases BamHI and KpnI, followed by ligation, plating, single colony picking, and AAV packaging, concentration, and purification according to standard procedures to obtain the Angulin-1 overexpressing viral particle AAV-Angulin-1.

[0061] Control group: administered via intraperitoneal injection of 1×10 9 The MOI control virus particle AAV-CTL differs from the overexpression virus particle AAV-Angulin-1 in that the plasmid used for the control virus is an unmodified vector plasmid: pHBAAV-CMV-MCS-3flag-T2A-ZsGreen.

[0062] After viral injection, the survival of the two groups of mice was observed. Sixteen weeks later, the mice in each group were anesthetized with isoflurane and their eyeballs were enucleated to collect blood. The blood was centrifuged at 4500 g / min for 15 min, and serum was collected. Serum albumin and liver damage markers (AST and ALT) were detected using ELISA. At the same time, the experimental data were statistically analyzed using Prism 8 software. Unpaired t-tests were used to compare two groups, and one-way ANOVA was used to compare multiple groups. Data are expressed as mean ± standard error. P < 0.05 was interpreted as statistically significant.

[0063] Experimental results are as follows Figure 4 As shown, Figure 4In the figure, a is the survival curve of the two groups of mice, and b, c, and d are the results of serum albumin, AST, and ALT content of the two groups of mice, respectively.

[0064] Figure 4 The results showed that, compared with the control group, the mortality rate of mice injected with Angulin-1 overexpression group was significantly reduced and the survival rate was significantly increased (the survival rate of Angulin-1 overexpression group was 80%, while the survival rate of the control group was 27%), and the death date of mice in the Angulin-1 overexpression group was significantly delayed.

[0065] Furthermore, compared with the control group, the serum albumin level of mice in the Angulin-1 overexpression group was significantly increased, while the expression levels of liver injury markers AST and ALT did not change significantly. This shows that Angulin-1 can alleviate aging by prolonging life and promoting liver function, without causing any damage to the liver.

[0066] Example 5: Verification of the effect of Angulin-1 on improving the degree of skin aging in aging mice The grouping and administration methods for experimental animals are the same as in Example 4.

[0067] Unlike Example 4, in this example, after euthanizing each group of mice by dislocation, the skin tissue from the back of the neck was immediately collected and fixed with a 4% (w / v) paraformaldehyde solution at 4°C for 24 h. The tissue was then dehydrated with a gradient of alcohol. The mouse skin tissue was then embedded in paraffin, and after being sectioned into 5 μm sections, immunofluorescence staining was performed. The changes in aging-related proteins P16 and P21 in the skin tissue were observed under a laser confocal microscope.

[0068] Experimental results are as follows Figure 5 As shown, a) is a diagram of the changes in the appearance of the skin of mice in each group, b) is a diagram of the relative expression of aging-related genes in the skin tissue of mice in each group, c) is a diagram of the immunofluorescence staining results of the skin tissue of mice in each group, and d) is a diagram of the staining results of the skin tissue of mice in each group using the hematoxylin-eosin (HE) staining method.

[0069] Figure 5 As observed in the results, the skin of mice in the control group was loose and wrinkled with multiple skin lesions, while the skin of mice in the Angulin-1 overexpression group was relatively firm with no obvious skin lesions. Figure 5 (a). Furthermore, compared to the control group mice, the relative expression levels of p16 and p21 genes and proteins in the skin tissue of the Angulin-1 overexpression group mice were significantly reduced ( Figure 5 In the middle bc), collagen fibers were significantly increased in the skin tissue of mice, and the skin was thicker ( Figure 5 (d). It can be seen that overexpression of Angulin-1 can slow down the progression of skin aging in mice.

[0070] Example 6: Verification of the degree of improvement in liver aging by Angulin-1 in aging mice The grouping and administration methods for experimental animals are the same as in Example 4.

[0071] Unlike Example 4, in this example, after euthanizing each group of mice by cervical dislocation, the abdominal cavity was immediately opened and exposed. Liver tissue was located and fixed in 4% (w / v) paraformaldehyde solution at 4°C for 24 h. The liver tissue was then dehydrated using an alcohol gradient. The liver tissue was then embedded in paraffin, and after being sectioned into 5 μm sections, immunofluorescence staining was performed. The changes in aging-related proteins P16 and P21 in the liver tissue were observed under a laser confocal microscope.

[0072] The experimental results are shown in Figure 6 As shown in the figure, a is a graph showing the relative expression of aging-related genes in the liver tissue of mice in each group, b is a graph showing the immunofluorescence staining results of the liver tissue of mice in each group, and c is a graph showing the HE staining results of the liver tissue of mice in each group.

[0073] Figure 6 The results showed that, compared with the control group, the relative expression levels of aging-related genes and proteins in the liver tissue of mice overexpressing Angulin-1 were significantly reduced. Figure 6 (as shown in ab).

[0074] Compared with the control group mice, the hepatocytes in the liver tissue of the Angulin-1 overexpression group mice were relatively well-organized, with no significant enlargement or lipid deposition in the liver. Figure 6 As shown in Figure c), overexpression of Angulin-1 can slow down the progression of liver tissue aging in mice.

[0075] Example 7: Validation of the effect of Angulin-1 on improving lung aging in aging mice The grouping and administration methods for the experimental animals are the same as in Example 4; Unlike Example 4, in this example, after euthanizing each group of mice by cervical dislocation, the abdominal cavity was immediately opened to expose the thoracic cavity, and lung tissue was located. The lung tissue was fixed in 4% (w / v) paraformaldehyde solution at 4°C for 24 h, dehydrated with an alcohol gradient, and then embedded in paraffin. After 5 μm of continuous sectioning, immunofluorescence staining was performed, and the changes of aging-related proteins P16 and P21 in the lung tissue were observed under a laser confocal microscope. The lung tissue of another group of mice was embedded in OCT and frozen sectioned for β-galactosidase staining.

[0076] The experimental results are shown in Figure 7As shown in the figure, a is a graph showing the relative expression of aging-related genes in the lung tissue of mice in each group, and b, c, and d are graphs showing the results of immunofluorescence staining, β-galactosidase staining, and HE staining of lung tissue of mice in each group, respectively.

[0077] As shown in the figure, compared with the control group mice, the relative expression levels of aging-related genes and proteins in the lung tissue of the Angulin-1 overexpression group mice were significantly reduced. Figure 7 (as shown in ab).

[0078] Furthermore, the β-galactosidase staining results of lung tissue from each group of mice showed that, compared with the control group, the number of senescent cells in the lung tissue of mice in the Angulin-1 overexpression group was significantly reduced, and Figure 7 The staining results of d showed that the alveoli in the mouse lung tissue were relatively neatly arranged and the number of inflammatory cells was significantly reduced.

[0079] The above results demonstrate that overexpression of Angulin-1 can slow down the progression of lung tissue aging in mice.

[0080] Example 8: Angulin-1 improves the degree of renal aging in aging mice. The grouping and administration methods for experimental animals are the same as in Example 4.

[0081] Unlike Example 4, in this example, after euthanizing each group of mice by cervical dislocation, the abdominal cavity was immediately opened and exposed. Kidney tissue was located and fixed in 4% (w / v) paraformaldehyde solution at 4°C for 24 h. After dehydration with alcohol gradient, the mouse kidney tissue was embedded in paraffin, and after 5 μm of continuous sectioning, immunofluorescence staining was performed. The changes of aging-related proteins P16 and P21 in the kidney tissue were observed under a laser confocal microscope. The kidney tissue of another group of mice was embedded in OCT and frozen sectioned for β-galactosidase staining.

[0082] The experimental results are shown in Figure 8 As shown in the figure, a is the detection result of the relative expression of aging-related genes in the kidney tissue of mice in each group, and b, c, and d are the results of immunofluorescence staining, β-galactosidase staining, and HE staining of kidney tissue of mice in each group, respectively.

[0083] Figure 8 The results showed that, compared with the control group mice, the relative expression levels of p16 and p21 genes, as well as P16 and P21 proteins, were significantly reduced in the kidney tissue of the Angulin-1 overexpression group mice.

[0084] In addition, the staining results showed that, compared with the control group mice, the number of senescent cells in the kidney tissue of the Angulin-1 overexpression group mice was significantly reduced, and the degree of glomerular sclerosis and renal tubular damage in the kidney tissue was significantly reduced.

[0085] The results show that overexpression of Angulin-1 can slow down the progression of kidney tissue aging in mice.

[0086] Example 9: Validation of the effect of Angulin-1 on improving cardiac aging in aging mice The grouping and administration methods for experimental animals are the same as in Example 4.

[0087] Unlike Example 4, in this example, after euthanizing each group of mice by dislocation, the abdominal cavity was immediately opened to expose the thoracic cavity, and the heart tissue was located. The heart tissue was fixed in a 4% (w / v) paraformaldehyde solution at 4°C for 24 h, dehydrated with an alcohol gradient, and then embedded in paraffin. After 5 μm of continuous sectioning, immunofluorescence staining was performed, and the changes of aging-related proteins P16 and P21 in the heart tissue were observed under a laser confocal microscope.

[0088] The experimental results are shown in Figure 9 As shown in the figure, a is the detection result of the relative expression of aging-related genes p16 and p21 in the heart tissue of mice in each group, and b and c are the results of immunofluorescence staining and HE staining of heart tissue of mice in each group, respectively.

[0089] Figure 9 The results showed that, compared with the control group, the relative expression levels of p16 and p21 genes and p16 and p21 proteins in the heart tissue of mice overexpressing Angulin-1 were significantly reduced. Furthermore, the degree of myocardial thickening in the heart tissue of mice overexpressing Angulin-1 was significantly reduced.

[0090] Example 10: Validation of the effect of Angulin-1 on improving brain aging in aging mice The grouping and administration methods for experimental animals are the same as in Example 4.

[0091] Unlike Example 4, in this example, after euthanizing mice by cervical dislocation, the skulls were immediately dissected to expose brain tissue, which was then fixed in a 4% (w / v) paraformaldehyde solution at 4°C for 24 h. The tissue was then dehydrated using an alcohol gradient, and subsequently embedded in paraffin. After 5 μm serial sections, immunofluorescence staining was performed, and changes in aging-related proteins P16 and P21 in the brain tissue were observed under a laser confocal microscope. Experimental results are shown below. Figure 10 As shown in the figure, a is the detection result of the relative expression of aging-related genes p16 and p21 in the brain tissue of mice in each group, and b is the immunofluorescence staining result of brain tissue of mice in each group.

[0092] Figure 10 The results showed that, compared with the control group, the relative expression levels of aging-related genes and proteins in the brain tissue of mice overexpressing Angulin-1 were significantly reduced, indicating that overexpression of Angulin-1 can slow down the progression of brain aging in mice.

[0093] Example 11: Validation of the effect of Angulin-1 on improving bone aging in aging mice The grouping and administration methods for experimental animals are the same as in Example 4.

[0094] Unlike Example 4, in this example, after euthanizing the mice by cervical dislocation, the hind leg bone tissue was immediately collected and fixed in a 4% (w / v) paraformaldehyde solution at 4°C for 24 h. The tissue was then dehydrated using an alcohol gradient, decalcified, embedded in paraffin, and sectioned at 5 μm for HE staining. Pathological changes in the bone tissue were observed under an optical microscope. The experimental results are as follows: Figure 11 As shown.

[0095] Staining can be used to observe structural changes in bone trabeculae and medullary cavities. The results in the figure show that, compared with the control group mice, the Angulin-1 overexpression group mice had a significantly increased number of bone trabeculae, significantly narrower spacing, increased thickness, and a relatively smaller medullary cavity.

[0096] In summary, this invention has discovered and verified that the expression level of Angulin-1 gene and / or protein is related to the degree of cellular or organismal aging. Experiments show that Angulin-1 overexpression can improve the aging status of cells and various tissues and organs in mice, prolong the lifespan of mice, and improve the metabolic capacity of aged mice. Specifically, Angulin-1 overexpression reduces the expression of aging-related genes and β-galactosidase in cells and various tissues and organs of mice by reducing GDF15 expression and improving DNA damage in senescent cells. It also significantly improves osteoporosis and skin damage in mice. Therefore, this invention is the first to disclose that Angulin-1 can be used as a novel anti-aging target, which is of great significance for screening new drugs and provides a new approach to anti-aging treatment.

Claims

1. Application of Angulin-1 gene and / or Angulin-1 protein in the preparation of anti-aging and age-related disease treatment products.

2. The application as described in claim 1, characterized in that, The aging mentioned includes either physiological aging or pathological aging; physiological aging is aging caused by physiological degeneration that occurs after maturity, and pathological aging is aging caused by external factors, including diseases.

3. The application as described in any one of claims 1-2, characterized in that, The aging process mentioned includes the aging of cells and tissues.

4. The application as described in claim 3, characterized in that, The tissues mentioned include any one of the following: blood vessels, heart, liver, lungs, kidneys, brain, bones, and skin.

5. The application as described in claim 1, characterized in that, The product targets the Angulin-1 gene or uses the Angulin-1 protein as a functional component, with the functional component comprising 1%-99% of the product by mass.

6. The application as described in claim 1, characterized in that, The products mentioned include any one of the following: pharmaceuticals, food, health products, and cosmetics.

7. Application of formulations that promote the expression of Angulin-1 gene and / or Angulin-1 protein in the preparation of drugs for anti-aging and treatment of aging-related diseases.

8. The application as described in claim 7, characterized in that, The formulation comprises at least one of the following: a recombinant expression vector containing the Angulin-1 gene or a gene fragment having more than 90% homology with the Angulin-1 gene; exosomes containing the Angulin-1 gene and / or Angulin-1 protein and / or its bioactive fragments; polypeptides, proteins, nucleic acids and nucleic acid aptamers, natural active substances, and biological agents capable of upregulating the expression of the Angulin-1 gene and / or Angulin-1 protein.

9. The application as described in claim 8, characterized in that, The recombinant expression vectors include any one of the following: vector-based eukaryotic expression plasmids specifically targeting the Angulin-1 gene and / or Angulin-1 protein; adenovirus; adeno-associated virus; lentivirus; retrovirus; LNP liposomes; microinjection technology; gene editing system elements; and homologous recombinant vectors.

10. The application as described in claim 7, characterized in that, The formulation that promotes the expression of the Angulin-1 gene and / or Angulin-1 protein is used as the functional component of the drug, and the mass fraction of the functional component in the drug is 1%-99%.

11. A drug for anti-aging, characterized in that, The drug comprises a formulation that promotes the expression of the Angulin-1 gene and / or Angulin-1 protein, as well as pharmaceutical excipients. The pharmaceutical excipients include at least one of diluents, excipients, fillers, binders, humectants, disintegrants, absorption enhancers, surfactants, adsorbents, and lubricants. The drug is any dosage form selected from tablets, capsules, powders, pills, granules, solutions, suspensions, syrups, injections, suppositories, inhalers, and sprays.

12. The application of products used to detect the mRNA expression level and / or Angulin-1 protein expression level in the preparation of drugs for screening anti-aging and treating age-related diseases, characterized in that, When the expression levels of Angulin-1 gene mRNA and / or Angulin-1 protein are upregulated in the body after drug administration, the drug can be confirmed to have a good anti-aging or therapeutic effect on age-related diseases; if the expression levels are downregulated, there is no effect.

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