WO2022121627A1 - 一种筛选具有抗衰老潜力天然产物的方法 - Google Patents

一种筛选具有抗衰老潜力天然产物的方法 Download PDF

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WO2022121627A1
WO2022121627A1 PCT/CN2021/130639 CN2021130639W WO2022121627A1 WO 2022121627 A1 WO2022121627 A1 WO 2022121627A1 CN 2021130639 W CN2021130639 W CN 2021130639W WO 2022121627 A1 WO2022121627 A1 WO 2022121627A1
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cells
senescent cells
treatment
contact treatment
gse
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French (fr)
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孙宇
许奇霞
张旭光
贺瑞坤
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汤臣倍健股份有限公司
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/105—Plant extracts, their artificial duplicates or their derivatives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/13—Amines
    • A61K31/135—Amines having aromatic rings, e.g. ketamine, nortriptyline
    • A61K31/136—Amines having aromatic rings, e.g. ketamine, nortriptyline having the amino group directly attached to the aromatic ring, e.g. benzeneamine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/87—Vitaceae or Ampelidaceae (Vine or Grape family), e.g. wine grapes, muscadine or peppervine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P39/00—General protective or antinoxious agents
    • A61P39/06—Free radical scavengers or antioxidants
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/10—Screening for compounds of potential therapeutic value involving cells

Definitions

  • Senescent cells are distinct from both resting and terminally differentiated cells, in which resting cells are able to re-enter the cell cycle. Senescent cells are characterized by morphological abnormalities, changes in metabolic activity, chromatin remodeling, altered gene expression, increased lipofuscin, prominent granularity, severe vacuolization, and the emergence of a senescence-associated secretory phenotype.
  • ROS reactive oxygen species
  • aging may have evolved as a mechanism to avoid malignant transformation of damaged cells
  • the occurrence of aging may lead to many age-related pathologies, including cancer, cardiovascular and cerebrovascular diseases, osteoporosis, arthritis, metabolic diseases, neurological Degenerative symptoms and a series of clinical problems that endanger human health and longevity.
  • Cell senescence is manifested by nuclear membrane infolding, chromatin pyknosis, cell volume increase, and activation of multiple downstream signals including p53, p16 INK4A /Rb, PI3K/Akt, FoxO transcription factors and mitochondrial SIRT3/4/5, etc. path.
  • senescent cells are often associated with a number of pathological features, including local inflammation. Cellular senescence occurs in damaged cells and prevents them from proliferating in an organism. Under the influence of various external stimuli and internal factors, cell damage to a certain extent can lead to obvious signs of cell aging. When the damage accumulates for a certain period of time, various tissue degenerative changes and physiological aging phenotypes can be seen in the tissue.
  • senescent cells have significantly elevated expression levels of inflammatory cytokines, a phenomenon known as the senescence-associated secretory phenotype (SASP).
  • SASP senescence-associated secretory phenotype
  • Senescent cells participate in various physiological and pathological processes of the body mainly through three pathways: (1) The gradual accumulation of gene expression and morphological changes in senescent cells can affect the function of corresponding tissues; (2) Senescent cells restrict the regeneration of stem cells and undifferentiated progenitor cells. (3) Senescent cells not only show growth cycle arrest, but also release a large number of cytokines, chemokines, growth factors and proteases through autocrine and paracrine pathways, affecting the regeneration of adjacent cells and tissues. The microenvironment causes and accelerates the aging of the body and related diseases. In recent years, a large number of studies have shown that SASP plays a core pathological role in this process.
  • SASP factors secreted by senescent cells can also affect surrounding normal cells, and inhibiting SASP can delay the aging of the body and delay various related diseases.
  • Typical SASP factors include tumor necrosis factor- ⁇ (TNF- ⁇ ), interleukin 6 (IL-6), interleukin 8 (IL-8), interleukin 1a (IL-1a), matrix metalloproteinases ( MMP), granulocyte-macrophage colony stimulating factor (GM-CSF), and plasminogen activator inhibitor-1 (PAI1), etc., which promote immune system activation, leading to abnormal factors such as senescent cells in the tissue microenvironment Cleared by the body, and then play a tumor suppressor function.
  • TNF- ⁇ tumor necrosis factor- ⁇
  • IL-6 interleukin 6
  • IL-8 interleukin 8
  • IL-1a interleukin 1a
  • MMP matrix metalloproteinases
  • GM-CSF granulocyte-macrophage colony stimulating factor
  • SASP can still promote tumors through specific secreted factors (eg, VEGF, ANGPTL4) that promote angiogenesis, extracellular matrix remodeling, or epithelial-to-mesenchymal transition (EMT) factors develop.
  • VEGF vascular endothelial growth factor
  • ANGPTL4 vascular endothelial growth factor 4
  • EMT epithelial-to-mesenchymal transition
  • aging-induced chronic inflammation can cause systemic immunosuppression, and this chronic inflammation can also promote the occurrence and development of aging-related tissue damage and degeneration, organ dysfunction, and cancer and other aging-related diseases.
  • Stimuli such as DNA damage, telomere dysfunction, oncogene activation, and oxidative stress can induce SASP in cells, and the mechanisms are closely related to transcriptional cascades, autocrine loops, and persistent DNA damage responses.
  • overexpression or inhibition of the canonical pathways of senescence p53 and p16 INK4A /Rb did not affect the expression of SASP, indicating that although cycle arrest in senescent cells and SASP often co-occur, the regulatory pathways of the two do not completely overlap.
  • the DNA damage response has been reported to increase the secretion of SASP factors IL-6 and IL-8 by activating the telangiectatic ataxia mutant gene, Nijmegen break syndrome protein 1, and checkpoint kinase 2.
  • DNA damage response is activated immediately after cell damage, and it takes about 1 week or even longer to develop mature SASP in senescent cells, and the transient DNA damage response cannot induce cellular senescence or SASP, indicating that in addition to In addition to the DNA damage response, there are other mechanisms co-inducing SASP.
  • NF- ⁇ B and c/EBP ⁇ transcription factors are increased in activity upon cellular senescence and are involved in the expression of cytokines that regulate cellular stress and inflammatory signaling.
  • cytokines that regulate cellular stress and inflammatory signaling.
  • the phosphorylated NF- ⁇ B/RelA subunit enters the nucleus, binds to the SASP promoter, and regulates the expression of SASP factors. Therefore, NF- ⁇ B is often called the master regulator of SASP.
  • GATA4 zinc finger transcription factor 4
  • GATA4 can affect SASP-related genes IL-6, IL-8, Expression of CXCL1.
  • p38MAPK is a member of the serine/threonine protein kinase family and is an important signal transduction molecule. Activating or blocking p38MAPK is sufficient to affect the formation of SASP in senescent cells.
  • p38 MAPK is activated a few days after the onset of the senescence program and indirectly activates NF- ⁇ B by activating mitogen and stress-activated protein kinases MSK1 and MSK2, resulting in the accumulation of p65 and p50 in the nucleus, which is consistent with the early development of SASP Consistent.
  • Senescent cells do not directly secrete the pro-inflammatory factor IL-1 ⁇ , but a large amount of IL-1 ⁇ is distributed on the cell membrane surface of senescent cells, which together with NF- ⁇ B forms a positive feedforward loop to promote the encoding and transcription of inflammatory factors, and establish and maintain SASP.
  • mTOR promotes the secretion of SASP factors by regulating the level of IL-1 ⁇ , while rapamycin and its analogs do not affect the mRNA level of IL-1 ⁇ , but significantly reduce the expression of IL-1 ⁇ protein on the surface of senescent cells.
  • mTOR can also regulate p38MAPK downstream signal MAPKAPK2 to affect SASP factor secretion.
  • MAPKAPK2 phosphorylates the RNA-binding protein ZFP36L1, thereby limiting its ability to degrade SASP factor transcripts.
  • Transcription factor c/EBP ⁇ is related to cell senescence induced by tumor gene activation.
  • HMGB2 targets c/EBP ⁇ to regulate SASP, and promotes the expression of SASP genes by inhibiting the spread of heterochromatin.
  • SAHF senescence-associated heterochromatin loci
  • SIRT1 is metabolism-related, NAD+-dependent sirtuins, and SIRT1 has been found to have lifespan-extending effects in various models.
  • SIRT1 inhibits the expression of SASP factors by deacetylating histones H3K9 and H4K16 in the promoter regions of IL-6 and IL-8.
  • SIRT1 is knocked out, the levels of acetylation in these regions during cell senescence are higher than those in control cells .
  • MicroRNAs are a class of highly conserved single-stranded non-coding RNAs, about 20-26 nucleotides in length, that regulate gene expression in eukaryotic cells.
  • the results showed that miR-146, miR-34, miR-21 and miR-183 could regulate the SASP of senescent cells and effectively inhibit the overproduction of inflammatory cytokines.
  • miR-146a/b can reduce the production of IL-1 receptor-related kinase in human umbilical vein endothelial cells; on the contrary, inhibiting miR-146a/b can increase the activity of IL-1 receptor-related kinase, activate the transcription factor NF- ⁇ B, induce IL-6 and IL-8 protein expression.
  • Drugs that delay senescence mainly selectively eliminate senescent cells by temporarily blocking survival pathways (senescent cell anti-apoptotic pathway SCAPs), which protect senescent cells from the regulation of apoptosis-inducing signals in the environment.
  • survival pathways senescent cell anti-apoptotic pathway SCAPs
  • a class of drugs, senolytics may be used in the future to delay, prevent or treat a variety of aging-related diseases.
  • SCAPs senescence-associated anti-apoptotic pathways
  • the SCAP required for senescent cell survival varies between cell types.
  • the SCAPs required for survival of senescent human primary adipose progenitor cells differ from those in senescent human embryonic venous endothelial cells (HUVECs). This difference means that drugs targeting a single SCAP may not be able to eliminate multiple senescent cell types.
  • UUVECs senescent human embryonic venous endothelial cells
  • navitoclax targets and kills senescent cells in the culture-adapted IMR-90 lung fibroblast-like cell line, but is less effective on senescent primary human lung fibroblasts. Therefore, extensive testing on a range of cell types is still required to determine the broad-spectrum effects of senolytics.
  • the frequency of senolytic drug use may depend on the rate of accumulation of senescent cells, which may vary depending on the environment in which cellular senescence occurs. Repeated exposure to DNA-damaging cancer therapies or a persistent high-fat diet, for example, may lead to the re-accumulation of senescent cells more rapidly than natural aging. Intermittent use of senolytics reduces the risk of adverse effects in patients and allows senolytics to be used during healthy periods. In addition, intermittent dosing can reduce side effects from senolytics and reduce the likelihood of patients developing drug resistance.
  • the inventor discovered a new method for screening drugs with anti-aging potential.
  • the method has undergone two rounds of screening, and in each round of screening, the density and culture time of senescent cells need to be controlled within an appropriate range; further, drugs with anti-aging potential can be easily, quickly and accurately screened.
  • the screening method successfully screened and obtained grape seed extract (GSE), a natural product with anti-aging potential.
  • GSE grape seed extract
  • the inventors also verified the anti-aging activity of grape seed extract through in vivo and in vitro experiments. Further, the inventors conducted more in-depth research on the anti-aging activity of the grape seed extract obtained by screening and found that a certain concentration of grape seed extract can effectively inhibit the expression of SASP marker factors or eliminate senescent cells under in vitro conditions. .
  • the inventors also found that the combination of grape seed extract and mitoxantrone (MIT) can effectively enhance the anti-tumor (such as prostate cancer) activity of mitoxantrone (MIT).
  • MIT mitoxantron
  • the present invention provides a method for screening a drug with anti-aging potential, comprising:
  • the senescent cells after the first contact treatment are subjected to apoptosis detection, the first contact treatment is carried out in a 96-well plate, and the density of the senescent cells in the first contact treatment is 4500-5500 / hole (such as 4500, 4600, 4700, 4800, 4900, 5000, 5100, 5200, 5300, 5400, 5500, 4900-5100, 4800-5200, 4700-5300 or 4600-5400 / hole; preferably 5000 / hole ), the first contact treatment time is 5-10 days (such as 5, 6, 7, 8, 9, 10, 6-8 or 5-9 days, preferably 7 days), and the senescent cells are treated by The control cells are obtained after senescence induction treatment;
  • the candidate drugs that meet the following conditions at the same time are the primary screening drugs:
  • the second contact treatment was carried out in a 96-well plate, and the density of the senescent cells in the second contact treatment was 15,000 -25000/hole (such as 15000, 16000, 17000, 18000, 19000, 20000, 21000, 22000, 23000, 24000, 25000, 19000-21000, 18000-22000, 17000-23000 or 16000-24000, preferably 2000/hole pcs/well), the second contact treatment time is 25-35 days (such as 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 29-31, 28-32, 27-33 or 26-34 days, preferably 30 days), the senescent
  • the primary screening drug that simultaneously meets the following conditions is the target drug:
  • the drug may be a natural product.
  • the "method for screening drugs with anti-aging potential” may be "a method for screening natural products with anti-aging potential”.
  • the senescent cells that need to be subjected to the first/second contact treatment are simultaneously cultured under normal conditions , except that no GSE was added, the rest of the cell culture conditions (such as initial cell density, culture time, medium type and its volume, medium replacement frequency, cell collection and reagent handling procedures, etc.) were contacted with the first/second The processing is exactly the same.
  • the control cells are proliferating cells with a similar number to senescent cells
  • the senescent cells are cells obtained by subjecting the control cells to senescence induction treatment.
  • the aging treatment is chemotherapeutic drug treatment or radiation treatment. In some preferred embodiments, the aging treatment is a chemotherapeutic drug treatment.
  • the chemotherapeutic drug is bleomycin (BLEO).
  • the bleomycin (BLEO) is treated at a concentration of 40-60 ⁇ g/mL (eg, 45 ⁇ g/mL, 50 ⁇ g/mL, 55 ⁇ g/mL, or 60 ⁇ g/mL, preferably 50 ⁇ g/mL).
  • the senescence treatment step is: adding 50 ⁇ g/mL bleomycin (BLEO) to the culture medium when the PSC27 cells grow to 80% (referred to as PSC27-CTRL). After 12 hours of BLEO treatment, cells were briefly washed 3 times with PBS and left in culture for 7-10 days.
  • BLEO bleomycin
  • control cell is a human prostate primary stromal cell line (eg, PSC27).
  • the medium for the first contact treatment and the second contact treatment is a basal medium routinely used by those skilled in the art.
  • the medium for the first contact treatment and the second contact treatment is DMEM (containing 10% FBS).
  • the concentration of the drug candidate is 1 ⁇ M-1 mM (preferably 1 ⁇ M-50 ⁇ M, such as 1 ⁇ M, 1.25 ⁇ M, 1.5 ⁇ M, 2 ⁇ M, 3 ⁇ M, 4 ⁇ M, 5 ⁇ M, 10 ⁇ M, 15 ⁇ M, 20 ⁇ M, 25 ⁇ M, 30 ⁇ M, 35 ⁇ M, 40 ⁇ M, 45 ⁇ M or 50 ⁇ M).
  • 1 ⁇ M-1 mM preferably 1 ⁇ M-50 ⁇ M, such as 1 ⁇ M, 1.25 ⁇ M, 1.5 ⁇ M, 2 ⁇ M, 3 ⁇ M, 4 ⁇ M, 5 ⁇ M, 10 ⁇ M, 15 ⁇ M, 20 ⁇ M, 25 ⁇ M, 30 ⁇ M, 35 ⁇ M, 40 ⁇ M, 45 ⁇ M or 50 ⁇ M).
  • the concentrations of the above-mentioned candidate drugs and preliminary screening drugs may be a single concentration or a gradient concentration.
  • the screening when performing the first or second round of screening, the screening can be carried out at a single concentration or at a gradient concentration.
  • each concentration must meet the predetermined screening conditions before the candidate drug can be determined as the primary screening drug, or the primary screening drug can be determined as the target drug.
  • the cell proliferation assay is performed using the CCK-8 Cell Counting Kit.
  • the detection steps are as follows: (1) Inoculate 100 ⁇ L of cell suspension in a 96-well plate and incubate in a cell incubator (37°C, 5% CO 2 ); (2) Take out the cells to be detected (ie, on the 7th day after drug treatment) or cells on the 30th day), add 0.1 volume of CCK-8 to the culture dish, mix well, ensure the uniformity of the color in the well, and avoid the appearance of air bubbles; (3) continue to culture in the incubator for 1-4 hours; (4) ) Before reading by the microplate reader, shake the 96-well plate on a shaker for 1 min to ensure that the color of the well plate is uniform; (5) Use the microplate reader to read the absorbance value at 450 nm to calculate the cell activity.
  • the apoptosis detection is performed using the Caspase 3/7 activity kit (Promega).
  • the assay steps are: (1) remove a 96-well plate containing cells (ie, cells on day 7 or 30 after drug treatment) from the incubator and allow the plate to equilibrate to room temperature; (2) place the 100 ⁇ L of reagents were added to each well of a white-walled 96-well plate containing 100 ⁇ L of blank, as well as negative control cells or cells treated in culture medium. Due to the sensitivity of this assay, avoid the tip of the pipette tip touching the well containing the sample to avoid cross-contamination.
  • the culture medium and the drug candidate are changed every two days.
  • the culture medium and the primary screening drug are changed every two days.
  • the present invention provides a composition comprising grape seed extract at a concentration of 1-1.5 [mu]M (preferably 1.25 [mu]M). In some embodiments, the balance is water.
  • the present invention provides the use of a composition in the preparation of a reagent for inhibiting the expression of a SASP marker factor, the composition comprising a grape seed extract whose The concentration is 1-1.5 [mu]M (preferably 1.25 [mu]M).
  • the balance is water.
  • the reagents can be used by scientific researchers or other researchers in need in in vitro detection and experiments.
  • the SASP factor is selected from the group consisting of tumor necrosis factor- ⁇ (TNF- ⁇ ), interleukin 6 (IL-6), interleukin 8 (IL-8), interleukin 1 ⁇ (IL- 1 ⁇ ), interleukin 1 ⁇ (IL-1 ⁇ ), matrix metalloproteinases (MMPs such as MMP3), granulocyte-macrophage colony stimulating factor (GM-CSF), plasminogen activator inhibitor-1 (PAI1) ), chemokine (CXC, such as CXCL3), bidirectional regulatory factor (AREG), serine peptidase inhibitor Kazal type 1 (SPINK1), or any combination thereof.
  • TNF- ⁇ tumor necrosis factor- ⁇
  • IL-6 interleukin 6
  • IL-8 interleukin 1 ⁇
  • IL-1 ⁇ interleukin 1 ⁇
  • IL-1 ⁇ matrix metalloproteinases
  • MMPs matrix metalloproteinases
  • GM-CSF granulocyte-macrophage colony
  • the SASP factor is interleukin 6 (IL-6), interleukin 8 (IL-8), interleukin 1 alpha (IL-1 alpha), interleukin 1 beta (IL-1 beta) , matrix metalloproteinase (MMP, such as MMP3), granulocyte-macrophage colony stimulating factor (GM-CSF), chemokine (CXC, such as CXCL3), bidirectional regulatory factor (AREG) and serine peptidase inhibitor Kazal type 1 (SPINK1) combination.
  • MMP matrix metalloproteinase
  • GM-CSF granulocyte-macrophage colony stimulating factor
  • CXC chemokine
  • SPINK1 serine peptidase inhibitor Kazal type 1
  • the present invention provides a composition comprising grape seed extract at a concentration of 5-50 ⁇ M (preferably 5-25 ⁇ M, more preferably 25 ⁇ M). In some embodiments, the balance is water.
  • the present invention provides the use of a composition for preparing a reagent for eliminating senescent cells, the composition comprising grape seed extract at a concentration of 5 -50 ⁇ M (preferably 5-25 ⁇ M, more preferably 25 ⁇ M).
  • the balance is water.
  • the reagents can be used by scientific researchers or other researchers in need in in vitro detection and experiments.
  • the agent clears senescent cells by inducing apoptosis of senescent cells.
  • the agent is used to induce apoptosis in senescent cells.
  • the present invention provides a composition comprising grape seed extract and mitoxantrone (MIT).
  • MIT mitoxantrone
  • the mass ratio of the grape seed extract and the mitoxantrone (MIT) is 10:0.1-10:0.3 (preferably 10:0.2).
  • the present invention provides the use of the aforementioned composition in the preparation of medicine, food or health care product, and the medicine is used for the treatment and/or prevention of cancer.
  • the present invention provides a method for treating and/or preventing cancer, which comprises administering to a subject in need thereof an effective amount of the aforementioned composition or a medicine, food or health product prepared from the aforementioned composition.
  • the present invention provides the aforementioned composition or a medicine, food or health product prepared from the aforementioned composition, which is used for the treatment and/or prevention of cancer.
  • the aforementioned cancer is a cancer associated with aging.
  • the aforementioned cancer is prostate cancer.
  • the grape seed extract (grape seed extract, GSE) of the present application is a mixture of polyphenols and flavonoids extracted from the seeds of grape cultivars.
  • GSE grape seed extract
  • Monomeric polyphenols in GSE mainly include gallic acid, catechin, epicatechin and epicatechin gallic acid.
  • Polymeric polyphenols are mainly procyanidins condensed from catechin or epicatechin as monomers, among which oligomers (dimers, trimers, tetramers) - also known as oligomers (oligomerie procyanidin, OPC) has the strongest biological activity.
  • Grape seed extract is commercially available.
  • the component content of GSE is shown in Table 3.
  • treating generally refers to obtaining a desired pharmacological and/or physiological effect.
  • the effect may be prophylactic in terms of complete or partial prevention of the disease or its symptoms; and/or therapeutic in terms of partial or complete stabilization or cure of the disease and/or side effects due to the disease.
  • Treatment encompasses any treatment of a disease in a patient, including: (a) prevention of disease or symptoms in a patient susceptible to a disease or condition but not yet diagnosed; (b) suppression of symptoms of disease, i.e. preventing its further development; or (c) alleviating the symptoms of the disease, i.e. leading to a reduction in the disease or regression of symptoms.
  • vertebrate refers to a mammal.
  • Mammals include, but are not limited to, livestock (such as cattle), pets (such as cats, dogs, and horses), primates, mice, and rats.
  • the mammal refers to a human.
  • an “effective amount” refers to an amount of a specific drug to be used at the necessary dose and time to achieve the desired therapeutic or prophylactic effect.
  • a “therapeutically effective amount” of a substance/molecule of the invention may vary depending on factors such as the disease state, age, sex and weight of the individual and the ability of the substance/molecule to elicit a desired response in the individual.
  • a therapeutically effective amount also encompasses an amount in which any toxic or detrimental consequences of the substance/molecule are outweighed by the therapeutically beneficial effects.
  • a “prophylactically effective amount” refers to an amount effective at the dose and time necessary to achieve the desired prophylactic effect.
  • a prophylactically effective amount will be less than a therapeutically effective amount because the prophylactic dose is administered to the subject prior to the onset of the disease or at an early stage of the disease.
  • a therapeutically effective amount of the drug reduces the number of cancer cells; shrinks the tumor size; inhibits (ie, slows to some extent, preferably stops) infiltration of cancer cells into surrounding organs; inhibits (ie, slows to some extent, preferably stops) ) tumor metastasis; some degree of inhibition of tumor growth; and/or some degree of alleviation of one or more symptoms associated with cancer.
  • the drug screening method of the present application can achieve the best effect of removing senescent cells in the shortest time and with the least amount of drugs, that is, the highest R&D efficiency. If these screening steps are abandoned or changed, the screening of anti-aging drugs will bring about a substantial increase in time and cost, which is not conducive to obtaining research data and key results with low input and high output. will be significantly reduced.
  • the cell density is the key. If it increases or decreases, it will affect the accuracy, stability and repeatability of the data. For example, in the first round of screening, using cell densities of 4000 or 6000 cells/well, inaccuracies or divergent results can occur in the SASP expression data (eg, see Figures 11H-11I).
  • time is the key. If the time of the first stage is selected for other time lengths than this application, it will increase the dosage of the drug or increase the cell death rate, or affect the accuracy of the method (for example, see FIGS. 11D-11E ). If a longer or shorter time is selected in the second stage, the reliability and clinical reference value of the data will be affected, and the safety and efficacy of the drug cannot be accurately judged (for example, see Figures 11F-11G ).
  • Figure 1 shows that proliferating human stromal cells PSC27 (early passage such as p10-20) were treated with the chemotherapeutic drug bleomycin (BLEO) at a concentration of 50 ⁇ g/ml on days 7-10 by SA- ⁇ -Gal staining in vitro results after.
  • Top panel representative image, bottom panel, statistical data.
  • CTRL control cells;
  • BLEO cells after bleomycin treatment. **, P ⁇ 0.01.
  • Figure 2 shows the results of BrdU staining of PSC27 cells treated with the chemotherapeutic drug bleomycin (BLEO). Top panel, representative image, bottom panel, statistical data. CTRL, control cells; BLEO, cells after bleomycin treatment. ***, P ⁇ 0.001.
  • Figure 3 shows the results of immunofluorescence staining using ⁇ H2AX after PSC27 cells were treated with the chemotherapeutic drug bleomycin (BLEO).
  • CTRL control cells
  • BLEO cells after bleomycin treatment.
  • *** P ⁇ 0.001. According to the number of fluorescent spots in the nucleus, they were divided into 4 categories, including 0 foci, 1-3 foci, 4-10 foci and single cells >10 foci.
  • Figure 4 shows the experimental flow chart of screening natural product drug library to obtain plant material with anti-aging activity.
  • Figure 5A shows the data obtained by the candidate drug A in the cell proliferation experiment.
  • the data shows that after a series of concentrations of A, compared with the results of the treatment of senescent cells and proliferating cells, A in the concentration range of 0-30 ⁇ M failed to Senescent cells were induced to die significantly (there was no significant difference between the two groups of cells with further increasing concentrations, results not shown).
  • candidate drug A is a culture of snow lotus.
  • the screening results of the remaining drug candidates were basically the same as those of candidate drug A, that is, the remaining drug candidates failed to induce significant death of senescent cells in the concentration range of 0-30 ⁇ M (there was no significant difference between the two groups of cells if the concentration continued to increase).
  • Figure 5B shows the results of changes in caspase-3/7 activity after candidate drug A treats cells.
  • A has the ability to induce apoptosis in senescent cells only at specific concentrations (for example, the 16th- 20 hours); outside this concentration range, no significant apoptosis of senescent cells was induced (in stark contrast to GSE).
  • candidate drug A is a culture of snow lotus.
  • the screening results of the remaining drug candidates are roughly the same as those of candidate drug A, that is, the remaining drug candidates can only induce apoptosis in senescent cells at a specific individual concentration. Significant apoptosis; even no significant apoptosis of senescent cells at all concentrations, in stark contrast to GSE.
  • Figure 6A shows that after software processing and bioinformatics analysis of RNA-seq data, it is found that GSE can significantly reduce the significantly up-regulated genes in senescent cells compared with proliferating cells. Compared with the BLEO group, 2644 genes were significantly down-regulated and 1472 genes were significantly up-regulated in the BLEO/GSE group (fold change>2, P ⁇ 0.01).
  • Figure 7 shows the results of GSEA analysis showing that the expression of SASP or NF- ⁇ B molecular marker-related factors was concentrated up-regulated in senescent cells caused by BLEO, but significantly decreased after GSE treatment of senescent cells.
  • SASP molecular marker Left, SASP molecular marker; right, NF- ⁇ B molecular marker.
  • Figure 8 shows the results of protein-protein interaction (PPI) bioinformatics analysis, showing that GSE significantly down-regulated senescent cell molecules form a relatively complex network, and there are multiple interactions between them.
  • PPI protein-protein interaction
  • Figure 9 shows a representative pathway of 100 molecules on biological process that GSE caused significant down-regulation in senescent cells by KEGG pathway analysis.
  • Left Y-axis percentage.
  • Right Y-axis log10(p-value).
  • Figure 10 shows a representative pathway of 100 molecules on the cellular component of KEGG pathway analysis GSE caused significant downregulation in senescent cells.
  • Left Y-axis percentage.
  • Right Y-axis log10(p-value).
  • Figure 11A shows the relative expression levels of a group of typical SASP molecules in BLEO-induced senescent cells treated with different concentrations of GSE after the first round of screening. All data are normalized results compared to the CTRL group. *,P ⁇ 0.05;**,P ⁇ 0.01;***,P ⁇ 0.001.
  • Figure 11B shows the relative expression levels of a group of typical SASP molecules in BLEO-induced senescent cells treated with different concentrations of GSE after the second round of screening. All data are normalized results compared to the CTRL group. *,P ⁇ 0.05;**,P ⁇ 0.01.
  • Figure 11C shows the relative expression levels of a group of typical SASP molecules in BLEO-induced senescent cells treated with different concentrations of GSE after the first round of screening for candidate drug A. All data are normalized results compared to the CTRL group. ⁇ , P>0.05; *, P ⁇ 0.05.
  • Figure 11D shows that in the first round of screening, the detection was carried out on the 15th day after drug treatment.
  • fluorescence quantitative PCR qRT-PCR
  • Figure 11E shows that in the first round of screening, detection was carried out on the 2nd day after drug treatment.
  • fluorescence quantitative PCR qRT-PCR
  • Figure 11F shows that in the second round of screening, the detection was performed on the 50th day after drug treatment.
  • fluorescence quantitative PCR qRT-PCR
  • Figure 11G shows that in the second round of screening, detection was carried out on the 15th day after drug treatment.
  • fluorescence quantitative PCR qRT-PCR
  • Figure 11H shows that in the first round of screening, the cell density was 4000 cells/well.
  • fluorescence quantitative PCR qRT-PCR
  • Figure 11I shows that in the first round of screening, the cell density was 6000 cells/well.
  • fluorescence quantitative PCR qRT-PCR
  • qRT-PCR fluorescence quantitative PCR
  • Figure 12A shows the determination of senescence of PSC27 by SA- ⁇ -Gal staining with increasing concentrations of GSE after the first round of screening.
  • the P values of GSE at concentrations of 1.25 ⁇ M, 2.5 ⁇ M, 5 ⁇ M, 12.5 ⁇ M, 25 ⁇ M and 50 ⁇ M were compared with the data at 0 ⁇ M. significant data.
  • Figure 12B shows that after the second round of screening, the senescence of PSC27 was determined by SA- ⁇ -Gal staining under the condition of increasing GSE concentration. ⁇ ,P>0.05;**,P ⁇ 0.01;****,P ⁇ 0.0001. Among them, the P values of GSE at the concentrations of 5 ⁇ M, 12.5 ⁇ M, 25 ⁇ M and 50 ⁇ M are statistically significant data obtained by comparing the positive proportion of cells in these experimental groups with the data at 0 ⁇ M.
  • Figure 13 shows representative pictures of PSC27 under various conditions after SA- ⁇ -Gal staining. 3 repetitions per set, up and down. Scale bar, 20 ⁇ m.
  • Figure 14 shows the survival rate of proliferating cells and senescent cells detected by CCK8 under increasing concentrations of GSE. P values at each GSE concentration are significant differences between the CTRL and BLEO groups after comparison. **,P ⁇ 0.01;***,P ⁇ 0.001;****,P ⁇ 0.0001.
  • Figure 15 represents a population doubling test for PSC27.
  • Cells at passage 10 (p10) were BLEO-injuriously treated, followed by the addition of GSE to the medium at day 8.
  • the effect of GSE on cell proliferation potential was determined by comparing the doubling value (PD) of CTRL group, BLEO group, GSE group and BLEO/GSE group. ⁇ ,P>0.05;***,P ⁇ 0.001.
  • Figure 16 shows the induction of caspase 3/7 activity during GSE treatment of senescent cells.
  • PSC27 cells gradually entered the senescence stage after being treated with BLEO under culture conditions for 12 h.
  • 5 ⁇ M GSE was added to the medium of senescent cells starting at day 7, NucLight Rapid Red reagent was used to label cells, and caspase 3/7 reagent (IncuCyte) was used for apoptosis detection.
  • FIG 17 shows that Pan-caspase inhibitor (20 ⁇ M QVD-OPH) reverses the senolytic activity of GSE (5 ⁇ M GSE was used for this experiment, while 1 ⁇ M ABT263 was used as a positive control; the latter is a senescent cell apoptosis inducer reported in recent years ). Statistical differences were obtained by two-way ANOVA (Tukey'test).
  • Figure 18 shows apoptosis of PSC27 under several conditions measured by flow cytometry. Q2, distribution area of early apoptotic cells; Q3, distribution area of late apoptotic cells.
  • Figure 19 shows a comparative analysis of the number of viable and apoptotic cells treated with BLEO and/or GSE. ***,P ⁇ 0.001;****,P ⁇ 0.0001.
  • Figure 20 shows a schematic diagram of the dosing regime for mice in the pre-clinical trial.
  • Human stromal cells PSC27 and cancer cells PC3 were mixed in vitro (1:4) and then transplanted into mice subcutaneously to form xenografts. After multiple treatment cycles under the condition of single-drug or combined administration, the mice were finally sacrificed, and the expression changes of related molecules in tumor tissue were analyzed pathologically.
  • Figure 21 shows that the CTRL group and the BLEO injury group of PSC27 cells were mixed with PC3 in vitro, or the PC3 cells were transplanted into the subcutaneous tissue of mice alone to form xenografts. Tumors were dissected and obtained at the end of the 8th week, and the volume of the tumors under the conditions of each group was measured and compared. **,P ⁇ 0.01;***,P ⁇ 0.001;****,P ⁇ 0.0001.
  • Fig. 22 is a schematic diagram showing the administration time and administration mode of preclinical mice. Every two weeks was a dosing cycle, and MIT (mitoxantrone, mitoxantrone) was intraperitoneally administered to the mice on the first day of the 3rd/5th/7th week respectively. Mice were dosed with intraperitoneal GSE starting on the first day of week 5, once a week. After the 8-week course of treatment, the mice were dissected for pathological identification and expression analysis.
  • MIT mitoxantrone, mitoxantrone
  • Figure 23 represents a statistical analysis of tumor terminal volume.
  • the chemotherapeutic drug MIT was administered to mice alone or together with the anti-aging drug GSE, and the tumor size of each group was compared and analyzed after the end of the 8th week.
  • Figure 24 shows the comparison of cellular senescence in lesions of PC3/PSC27 tumor-bearing animals in preclinical experiments. Representative pictures after SA- ⁇ -Gal staining. Scale bar, 100 ⁇ m.
  • Figure 25 shows a parallel analysis of the percentage of SA-beta-Gal staining positive cells in tumor tissue in mice. ⁇ , P>0.05; **, P ⁇ 0.01; ***, P ⁇ 0.001.
  • Figure 26 shows the expression of SASP typical factors in epithelial cancer cells and stromal cells in mouse lesions by real-time quantitative PCR (qRT-PCR).
  • the stromal cells and cancer cells were specifically isolated by LCM technology, and total RNA was prepared and used for the detection of SASP expression.
  • Figure 27 shows quantitative PCR (qRT-PCR) assay to analyze the expression status of SASP factor in stromal cells in mouse lesions after vehicle, MIT and MIT/GSE administration. *,P ⁇ 0.05;**,P ⁇ 0.01;***,P ⁇ 0.001.
  • Figure 28 shows the analysis of DNA damage and apoptosis ratios in each group of mice after the specific isolation of cancer cells in the lesions by LCM technology. ⁇ , P>0.05; *, P ⁇ 0.05; **, P ⁇ 0.01.
  • Figure 29 shows picture analysis after immunohistochemical staining.
  • Scale bar 200 ⁇ m.
  • Figure 30 shows the Kaplan Meier data comparison of disease-free survival in NOD/SCID mice after various administration treatments.
  • the tumor volume of the animals in Vehicle, MIT, GSE and MIT/GSE groups exceeds 2000mm 3 , it is considered that a serious disease has appeared, and the mice need to be killed in time and their tumor bearing status detected.
  • ⁇ P>0.05; **, P ⁇ 0.01.
  • Figure 31 shows the comparative analysis of mouse body weight data at the end of the course of treatment under various drug treatment conditions. ⁇ , P>0.05.
  • Figure 32 shows the comparative analysis of mouse serological data at the end of the course of treatment under the above different administration conditions. Creatinine, urine (renal index), ALP and ALT (liver index) data were compared in parallel. ⁇ , P>0.05.
  • Figure 33 shows the comparative analysis of body weight data of immune intact mice (C57BL/6J) at the end of the course of treatment under various administration conditions. ⁇ , P>0.05.
  • Figure 34 shows the comparative analysis of mouse blood cell counts at the end of the course of treatment under different administration conditions in the pre-clinical setting. WBC, lymphocyte and neutrophil unit volume numbers were compared in parallel. ⁇ , P>0.05.
  • Figure 35 shows in vivo bioluminescence (BLI) images showing the location and signal intensity of reporter cells.
  • PSC27 cells that continue to express luciferase and enter the senescence stage induced by BLEO were transplanted into mice in advance by intraperitoneal injection; 2 days after the last vehicle or GSE administration, they were obtained using Berthold LB983 (BERTHOLD Technologies) small animal in vivo molecular imaging system Luciferase signaling in mice. Ruler, 15mm.
  • Figure 36 shows in vivo bioluminescence (BLI) images showing the location and signal intensity of reporter cells.
  • PSC27 cells that continuously express luciferase were transplanted into mice in advance by intraperitoneal injection; 2 days after the last vehicle or GSE administration, Berthold LB983 (BERTHOLD Technologies) small animal in vivo molecular imaging system was used to obtain luciferase signals in mice. Ruler, 15mm.
  • Fig. 37 shows the test flow for testing the physical fitness of experimental mice in the pre-clinical stage. After 20-month-old mice were dosed with Vehicle or GSE every two weeks, relevant physical fitness tests were performed at the end of the 4th month.
  • Figure 38 shows a series of physical fitness measurements performed on experimental mice, including quantitative measurements of maximum walking speed, endurance, grip strength, treadmill endurance, daily activities, body weight, and food intake. ⁇ , P>0.05; *, P ⁇ 0.05.
  • Figure 39 shows a representative panel of relative mRNA expression levels of SASP factors for stromal cells in solid organ microenvironments. Mice were sacrificed at the age of 24 months and then dissected to obtain solid organs such as lung, prostate and colorectum, and extract total RNA in stromal tissue for quantitative analysis by qRT-PCR. 6-month-old (6M) mice served as controls, and the signals of the other two groups of 24-month-old (24M) mice were normalized and plotted. ⁇ , P>0.05; *, P ⁇ 0.05.
  • Figure 40 shows the mouse lifespan analysis experimental design. Mice aged 24 to 27 months were dosed biweekly with Vehicle or GSE, and their survival was continuously monitored and their maximal lifespan recorded.
  • Figure 43 shows that the male mice with the highest life span in each group of animals were selected to perform a comparative analysis of the highest walking speed, endurance and overall life span between groups.
  • N 5. ⁇ , P>0.05; **, P ⁇ 0.01.
  • FIG. 47 shows that GSE was identified by HPLC-ESI-QTOF-MS technique, and it was found that there were multiple natural compound components in GSE. The identification of each peak was based on their respective retention times and true standard and phytochemical broad-spectrum data.
  • the inventors used baseline body weights to assign mice to experimental groups (to achieve similar body weights between groups), so randomization was performed only within weight-matched groups.
  • the inventors determined the sample size based on past experiments, so statistical power analysis was not used. All replicates in this study were from different samples, each from a different experimental animal.
  • PSC27 a primary normal human prostate stromal cell line
  • non-fibroblast cell lines including endothelial cells and smooth muscle cells
  • PSC27 is a primary human stromal cell line in nature, and it is A typical SASP is formed after stress factors such as ionizing radiation.
  • the inventors treated these cells with a specific dose of bleomycin (BLEO), which had been optimized in the preliminary experiments, and observed a significant increase in the positive rate of senescence-associated ⁇ -galactosidase (SA- ⁇ -GAL) staining. high, the BrdU incorporation rate was greatly reduced, and the DNA damage repair foci (DDR foci) were significantly increased in the days following drug injury ( Figures 1-3).
  • the inventors performed a systematic screening to compare the effects of drug products on the expression profiles of senescent cells in parallel ( Figure 4).
  • the normal human prostate primary stromal cell line PSC27 obtained from Fred Hutchinson Cancer Research Center, USA was cultured in an incubator at 37°C and 5% CO 2 , and propagated and passaged in PSCC complete culture medium.
  • the cells in logarithmic growth phase were collected with 0.25% trypsin, centrifuged at 1000 rpm for 2 min, the supernatant was discarded, and the cells were resuspended in freshly prepared freezing medium. Aliquot cells into sterile cryovials as indicated. Then, it was cooled by gradient and finally transferred to liquid nitrogen for long-term storage.
  • PSC27-CTRL 50 ⁇ g/ml bleomycin
  • Nuclei were counterstained with 2 ⁇ g/ml of 4',6-diamidino-2-phenylindole (DAPI). Select the most representative image from the three observation fields for data analysis and result display.
  • a FV1000 laser scanning confocal microscope (Olympus) was used to acquire confocal fluorescence images of cells.
  • the inventors screened a natural product library (BY-HEALTH) containing at least 4 components, mostly medicinal plant extracts. According to the research purpose, the inventor established a standard screening process, with a total of two rounds of screening.
  • Various natural products to be screened were diluted to a 96-well plate according to a certain concentration gradient, and the density was 5000 cells per well.
  • the medium uses DMEM (10% FBS), and the working concentration of natural products (or compounds) is generally controlled at 1 ⁇ M-1 mM (the exemplary concentration used in this example is 10-50 ⁇ M).
  • cell proliferation was determined with CCK-8 Cell Counting Kit (based on WST-8 principle, Vazyme), and apoptosis activity was determined with Caspase 3/7 activity kit (Promega).
  • the medium and drug candidates were replaced every two days, and the proliferation and apoptosis activities were detected 7 days after the drug was used.
  • the drug candidates initially identified in the first round are further screened for 30 days (time extension), that is, validation and determination.
  • the drugs that entered the second round of candidates were diluted into 96-well plates, 20,000 cells per well, the medium was DMEM (10% FBS), and the working concentration of natural products (or compounds) was generally controlled at 1 ⁇ M-l mM (an exemplary concentration of 10-50 ⁇ M employed in this example).
  • cell proliferation was determined with CCK-8 Cell Counting Kit (based on WST-8 principle, Vazyme), and apoptosis activity was determined with Caspase 3/7 activity kit (Promega).
  • the medium and drug candidates were changed every other day (ie, every two days).
  • CCK-8 Cell Counting Kit was used to detect cell proliferation or survival 7 days or 30 days after drug treatment.
  • the specific detection steps are as follows: (1) Inoculate 100 ⁇ L of cell suspension in a 96-well plate and incubate in a cell incubator (37°C, 5% CO 2 ); (2) Take out the cells to be detected (ie, on the 7th day after drug treatment) or cells on the 30th day), add 0.1 volume of CCK-8 to the culture dish, mix well, ensure the uniformity of the color in the well, and avoid the appearance of air bubbles; (3) continue to culture in the incubator for 1-4 hours; (4) ) Before reading by the microplate reader, shake the 96-well plate on a shaker for 1 min to ensure that the color of the well plate is uniform; (5) Use the microplate reader to read the absorbance value at 450 nm to calculate the cell activity.
  • the apoptotic activity was determined with Caspase 3/7 activity kit (Promega) 7 days or 30 days after drug treatment.
  • Caspase 3/7 activity kit Promega
  • the procedure followed was: (1) 96 wells containing cells (ie, cells on day 7 or 30 after drug treatment) were removed from the incubator plate, and allow the plate temperature to equilibrate to room temperature; (2) put the 100 ⁇ L of reagents were added to each well of a white-walled 96-well plate containing 100 ⁇ L of blank, as well as negative control cells or cells treated in culture medium.
  • the above standard screening process is also suitable for the screening of other natural product libraries based on specific uses or biological activities (such as anti-aging potential).
  • the candidate drugs in the above-mentioned natural product library (BY-HEALTH) containing at least 4 components were phytochemical extraction products such as GSE (grape seed extract), snow lotus culture, resveratrol and curcumin.
  • the eligible drug candidate was identified as GSE (Grape Seed Extract).
  • Example 2 Combining the in vivo and in vitro verification analysis of Example 2 and Example 3, it can be determined that GSE indeed has anti-aging activity. Therefore, the screening method of the present application has high accuracy and is simple and easy to operate.
  • the inventor further verified the accuracy of the screening method of the present application by changing the individual processing conditions of the first round of screening and the second round of screening.
  • the specific experimental conditions and experimental results are shown in the following table:
  • the drug treatment time of the first round and the second round, and the cell density of the first round are very critical to the accuracy of the final test result, and the treatment time is too long or too short. , the cell density is too large or too small, the accuracy of the screening method is significantly reduced.
  • RNA samples were obtained from stromal cells. Its integrity was verified by Bioanalyzer 2100 (Agilent), RNA was sequenced by Illumina HiSeq X10, and gene expression levels were quantified by the software package rsem (https://deweylab.github.io/rsem/).
  • RNA samples were depleted of rRNA with the RiboMinus Eukaryote kit (Qiagen, Valencia, CA, USA); and with TruSeq Stranded Total RNA preparation kits (Illumina, San Diego, CA) prior to deep sequencing according to the manufacturer's instructions , USA) to construct strand-specific RNA-seq libraries.
  • RiboMinus Eukaryote kit Qiagen, Valencia, CA, USA
  • TruSeq Stranded Total RNA preparation kits Illumina, San Diego, CA
  • Paired-end transcriptomic reads were mapped to the reference genome (GRCh38/hg38) and reference annotated from Gencode v27 using the Bowtie tool. Use the picard tools (1.98) script to mark duplicates (https://github.com/broadinstitute/picard) to identify duplicate reads and keep only non-duplicate reads.
  • Reference splice junctions were provided by the reference transcriptome (Ensembl build 73).
  • FPKM values were calculated using Cufflinks, and differential gene expression was called using Cufflinks, the maximum likelihood estimation function. Genes with significantly altered expression were defined by false discovery rate (FDR)-corrected P value ⁇ 0.05, and downstream analysis was performed with only ensembl genes 73 with status "Known” and biotype "coding".
  • PPI Protein-protein interaction
  • GSEA Gene Set Enrichment Analysis
  • genes were ranked using "wald statistics" obtained from DESeq2, GSEA in MSigDB (http://software.broadinstitute.org/gsea) based on data obtained from preliminary RNA-seq analysis /msigdb) on these sorted lists of all planned gene sets available).
  • DESeq2independent filtering is based on the mean of normalized read counts to screen out genes with very low expression levels.
  • SASP and GSEA signatures are as described in the inventor's previous publications (Zhang et al., 2018a).
  • Trizol reagent Extract the total RNA of cells in growth phase or arrest phase with Trizol reagent, add 1ml Trizol to each T25 culture flask cell, scrape the cell layer with a cell scraper, transfer it to a centrifuge tube, and mix well until it is not viscous.
  • RNA status and quality After quantifying RNA by spectrophotometer, take a small amount of total RNA and perform 1% agarose electrophoresis to check RNA status and quality.
  • the reverse transcription reaction product cDNA was diluted 50-fold as a template.
  • reaction conditions are: pre-denaturation at 95°C for 15sec, then 95°C for 5sec, 60°C for 31sec, 40 cycles; melting curve conditions are 95°C for 15sec, 60°C for 30sec, and 95°C for 15sec.
  • the samples were reacted on an ABI ViiA7 (ABI) instrument.
  • the expression of ⁇ -actin was used as an internal reference.
  • the amplification of each gene was analyzed by software, the corresponding threshold cycle number was derived, and the 2- ⁇ Ct method was used to calculate the relative expression level of each gene. The peaks and waveforms of the melting curve were analyzed to determine whether the resulting amplification product was a specific single target fragment.
  • Senescence-associated beta-galactosidase (SA-beta-Gal) staining was performed using previously reported procedures (Debacq-Chainiaux et al., 2009). Briefly, cell culture dishes were washed with PBS and fixed at room temperature. Cells were fixed in 2% formaldehyde and 0.2 glutaraldehyde for 3 min. SA- ⁇ -Gal was then stained with freshly prepared staining solution overnight at 37°C. Images were taken the next day and the percentage of positive cells per unit area was calculated.
  • PSC27 cells were plated in 96-well dishes and cell senescence was induced under BLEO treatment at 50 ⁇ g/ml. GSE and ABT263 were added at concentrations of 5.0 ⁇ M and 1.0 ⁇ M, respectively. Cell culture medium was supplemented with Incucyte Nuclight Fast Red Reagent (Essen Bioscience) and Incucyte C-3/7 Apoptosis Reagent (Essen Bioscience). Select a representative field of view to take pictures.
  • the inventors performed RNA-seq sequencing on the cells after the second round of screening in Example 1.
  • GSE grape seed extract
  • 2644 genes were significantly down-regulated, while 1472 genes were up-regulated, and the fold change of each gene in the inventor's heatmap was 2.0 (P ⁇ 0.01) ( Figure 6A).
  • the expression of SASP factors was generally decreased in senescent cells after GSE treatment, and these SASP factors were generally significantly upregulated in senescent cells (Fig. 6B).
  • GSE a plant-based natural product, can be used to control the pro-inflammatory phenotype of senescent cells, namely SASP, especially at relatively low concentrations.
  • the inventors further performed the detection by changing the individual treatment conditions of the first and second rounds.
  • the test conditions and test results are shown in the table below.
  • GSE is a new type of senolytics when used in high concentrations
  • the inventors next investigated the potential of population doubling (PD) of stromal cells after genotoxic treatment.
  • the combined treatment group of BLEO and GSE exhibited significantly increased PD capacity compared to the BLEO group cells that rapidly entered a growth arrest state after the invasive treatment ( Figure 15).
  • GSE itself does not appear to affect the PD of proliferating cells, a data that further suggests the selectivity of GSE between senescent versus normal cells.
  • mice were fed a standard experimental diet followed by administration of the chemotherapeutic drugs mitoxantrone (MIT, 0.2 mg/kg dose) and/or grape seed extract (GSE) (500 ⁇ l after 2 weeks) , 10mg/kg dose) intraperitoneal administration.
  • the time points are: the former is on the first day of weeks 3, 5, and 7, and the latter is on the first day of weeks 5 and 7.
  • a total of 3 cycles of MIT were administered throughout the course of treatment, and each cycle was 2 weeks.
  • mouse tumors were collected for volume measurement and histological analysis. Each mouse received a cumulative total of 0.6 mg/kg of MIT and 30 mg/kg of GSE.
  • MIT was administered to mice by intravenous infusion according to the above steps and sequence, but the dose was reduced to 0.1 mg/kg body weight/each time (the cumulative dose of MIT received throughout the course of treatment was 0.3 mg/kg body weight) to reduce drug-related toxicity.
  • Chemotherapy experiments were carried out until the end of the eighth week, and the mice were dissected immediately after sacrifice, and their xenografts were collected and used for pathological system analysis.
  • the inventors obtained 16-month-old male C57BL/6 mice by continuous rearing on the SPF animal platform, with 4 to 5 animals per cage.
  • the inventors first sorted the mice by weight from low to high, and then selected mice of similar weight.
  • senescence (SEN) or control (CTRL) transplantation treatments using a random number generator, were assigned to mice at each interval, while mice in the middle were assigned to the other treatment modality, resulting in senescence and the body weight of control transplanted mice.
  • One month after cell transplantation when the mice were 18 months old, physical function tests were performed. After that, no further tests were performed on the mice, except to examine their cages. The earliest death occurred approximately 2 months after the last physical function test.
  • mice C57BL/6 mice aged 19 to 21 months were housed 3-5 per cage.
  • the mice were classified according to body weight and randomly assigned to each group to receive control (vehicle) or drug (GSE) treatment by humans unaware of the design of the preclinical trial. From 24-27 months of age, mice were treated with vehicle or GSE every 2 weeks by oral gavage for 3 consecutive days. During the course of the study, some mice were removed from their original cages to try to avoid the animal housing stress that comes with long-term housing in a single cage. RotaRod and hanging tests are performed monthly as these tests are sensitive and non-invasive.
  • mice At the end of the experiment, the inventors euthanized the mice; if they exhibited one of the following symptoms, the inventors considered them dead: (1) unable to drink or eat; (2) unwilling to move even when stimulated; (3) Rapid weight loss; (4) Severe balance disorder; or (5) Bleeding or ulceration of the body. During the trial, no mice were excluded due to fights, accidental death, or dermatitis. When performing biostatistics, the inventors used the Cox proportional hazard model for survival analysis.
  • Carcasses were opened (abdominal, thoracic and skull) within 24 hours of animal death and kept individually in 10% formalin for at least 7 days. Decomposed or destroyed bodies are excluded. Preserved bodies were transported to a dedicated Autopsy site for pathological examination. Tumor burden (sum of different tumor types per mouse), disease burden (sum of different histopathological changes in major organs of each mouse), severity of each lesion and inflammation (lymphocyte infiltration) were assessed.
  • mice were injected intraperitoneally with 3 mg of fluorescein (BioVision, Milpitas, CA), delivered in a volume of 200 ⁇ l of PBS. Mice were anesthetized with isoflurane and bioluminescent images were acquired using the Xenogen IVIS 200 System (Caliper Life Sciences, Hopkinton, MA).
  • Forelimb grip strength was determined using the Grip Strength Meter (Columbus Instruments, Columbus, OH) and results were averaged over 10 trials.
  • For the hanging endurance test mice were placed on a 2 mm thick metal wire 35 cm above the mat. Mice were only allowed to grasp the wire with their forelimbs, and hanging time was normalized to body weight and expressed as hanging duration (sec) ⁇ body weight (g). The results were averaged from 2 to 3 experiments per mouse. Daily activity and food intake were monitored for 24 hours (12 hours light and 12 hours dark) by Comprehensive Laboratory Animal Monitoring System (CLAMS). The CLAMS system was equipped with an Oxymax Open Circuit Calorimeter System (Columbus Instruments).
  • mice were acclimated to running on an electric treadmill (Columbus Instruments) at a 5° incline for 3 days for 5 min per day, starting at 5 m/min for 2 min and then accelerating to to 7 m/min for 2 minutes, then 9 m/min for 1 minute.
  • mice ran on a treadmill at an initial speed of 5 m/min for 2 minutes, and then increased the speed by 2 m/min every 2 minutes until the mice were exhausted.
  • Fatigue was defined as the inability of mice to return to the treadmill despite mild electrical and mechanical stimulation.
  • the distance was recorded, and the total work (KJ) was calculated by the following formula: mass (kg) ⁇ g (9.8m/s 2 ) ⁇ distance (m) ⁇ sin (5°).
  • Cancer is one of the major chronic diseases that seriously threaten human lifespan and endanger health.
  • cancer cell drug resistance limits the efficacy of most anticancer treatments in the clinic, and senescent cells often contribute to the development of therapeutic resistance by developing SASP in damaged tumor foci. Even so, the feasibility and safety of removing senescent cells from primary tumors to boost the cancer therapeutic index has so far been little explored by scientists.
  • the inventors constructed tissue recombinants by mixing PSC27 stromal cells with PC3 epithelial cells, which is a typical highly malignant prostate cancer cell line.
  • the ratio of stromal cells to epithelial cells was 1:4 before the recombinants were implanted subcutaneously in the posterior thigh of non-obese diabetic and severe combined immunodeficiency (NOD/SCID) mice.
  • Tumor size (volume) was measured in animals at the end of 8 weeks after recombinant implantation ( Figure 20).
  • the inventors next compared the survival of the animals in the different drug treatment groups, primarily in a time-extended manner to assess the consequences of tumor progression.
  • the inventors monitored animals for tumor growth, and once the tumor burden in the mouse was prominent (size ⁇ 2000 mm 3 ), severe disease was judged to have occurred, a method used in some Methods of disease progression in the case of tumors and other diseases.
  • Mice treated with the MIT/GSE combination exhibited the longest median survival with at least 48.1% longer survival compared to the group treated with MIT alone ( Figure 30, green vs. blue). However, treating tumor-bearing mice with GSE alone did not result in significant benefit, with only marginal survival extension.
  • GSE has the potency to clear senescent cells, reduce tumor resistance, and improve overall therapeutic efficacy in the tumor mouse microenvironment, is there some significant health-promoting or disease-delaying benefit in naturally aging animals as well?
  • the inventors first tested the potential of GSE to deplete senescent or control cells expressing luciferase (LUC) and injected intraperitoneally into wild-type (WT) mice. Compared with Vehicle treatment, the bioluminescence intensity of GSE-treated LUC senescent cell-transplanted mice was significantly reduced in the corresponding parts of the body (Fig. 35).
  • there was no significant reduction (( Figure 43, Figure 44).
  • the prevalence and tumor burden of several age-related diseases were not statistically different between the two groups at necropsy in mice ( Figure 44). 45, Figure 46).
  • GSE a biologically active anti-aging drug
  • Grape seed extract was dissolved in 1 ml DMSO and sonicated for 5 min. Vortex for 1 min, centrifuge for 5 min, 14,000 rpm, and filter through a 0.25 mm filter prior to HPLC analysis. GSEs were analyzed and characterized using an Agilent 1200 series fast-resolution LC system (Agilent Technologies, Palo Alto, CA, USA) equipped with a binary pump and autosampler. The HPLC system was coupled to a quadrupole time-of-flight (QTOF) mass spectrometer (Bruker Daltonics, Bremen, Germany) equipped with an electrospray ionization (ESI) interface (model G1607A from Agilent Technologies, Palo Alto, CA, USA).
  • QTOF quadrupole time-of-flight
  • ESI electrospray ionization
  • HPLC analysis was performed on a QTOF mass spectrometer equipped with an ESI interface. In negative ion mode, the capillary voltage works at 3.5KV. Other parameters of the source were set as: drying gas temperature 220°C; drying gas flow rate, 9 L/min; atomizing gas pressure, 2.5 bar. The detected mass range is 50 to 1200 m/z.
  • the elution column is to monitor the UV absorbance of each sample and individual polyphenols or compounds at the 270nm peak. By comparison with HPLC retention time, the real compounds at the peaks are collected and identified, physicochemical properties and UV, ir, MS, 1 H -NMR and 13 C-NMR spectral analysis.
  • the inventors analyzed 16 major compounds distributed in GSE: 1) phenolic acids; (2) polyphenolic flavonoids (including procyanidins and other flavonoids); (3) other compounds (such as quercetin) cortex and its derivatives).
  • the inventors' data shows the base peak chromatogram (BPC) of GSE ( Figure 47) and the major peaks observed are listed in the GSE composition summary table (Table 3 below).
  • BPC base peak chromatogram
  • Table 3 The specific information of each active ingredient in GSE obtained by HPLC-ESI-QTOF-MS component identification, including name, ratio (weight percentage) and molecular formula

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Abstract

本发明涉及一种筛选具有抗衰老潜力天然产物的方法，该方法经过两轮筛选，且每轮筛选中需要控制衰老细胞的密度和培养时间在适当的范围内，进而，可以简便、快速且准确地筛选获得具有抗衰老潜力的天然产物。

Description

一种筛选具有抗衰老潜力天然产物的方法 技术领域
本发明属于生物医药领域，具体涉及一种筛选具有抗衰老潜力天然产物的方法。
背景技术
细胞衰老是指真核细胞一种相对稳定且通常不可逆的细胞周期停滞的状态，在这种状态下增殖细胞会对促生长刺激产生耐受，通常由DNA损伤等胁迫性信号所引起。上个世纪60年代，美国科学家Leonard Hayflick首先描述了细胞衰老的存在和特征，他观察到人类胚胎成纤维细胞(WI38)最终会停止分裂，但在长时间培养后仍然具有活力和代谢活性。这一现象后来被称为复制性衰老，是指正常细胞在大约30-50次分裂(即“Hayflick极限”)后会停止连续分裂。复制性衰老本质上由端粒渐进缩短所诱导。在每一轮的DNA复制中，端粒都会逐渐缩短，最终达到一个临界长度，阻止进一步复制，从而停止细胞分裂。较短的无帽端粒会引起DNA损伤应答，通常直接触发衰老。
现在普遍认为，除了具有干细胞样特性的细胞类型外，只有转化的(包括恶性)细胞会无限复制，而非转化细胞则不会。衰老细胞与静息细胞和终末分化细胞均不同，其中静息细胞能够重新进入细胞周期。衰老细胞的特征是形态学异常、代谢活性变化、染色质重构、基因表达改变、脂褐素增加、颗粒性明显、空泡化严重以及出现一种称为衰老相关分泌表型(senescence-associated secretory phenotype,SASP)的促炎症表型。由于核纤层lamin B1表达丧失，可观察到核膜完整性破坏。同时，衰老细胞积累功能失调的线粒体，并表现出活性氧种属(ROS)水平升高。还可观察到溶酶体内含物增加和溶酶体活性改变，表现为pH为6.0时β-半乳糖苷酶染色阳性率上升，使其成为广被采用的细胞衰老标志物。衰老的生物学作用比较复杂，衰老细胞的保护作用和有害作用均已有描述，主要取决于病理生理学环境。例如，尽管衰老可能作为避免受损细胞恶性转化的机制进化而来，但衰老的发生可能会导致许多年龄相关病变，包括癌症、心脑血管疾病、骨质疏松、关节炎、代谢性疾病、神经退行性症状等一系列危害人类健康与寿命的临床问题。
细胞衰老表现为核膜内折，染色质固缩，细胞体积增大，激活下游包括p53、p16 INK4A/Rb、PI3K/Akt、FoxO转录因子和线粒体SIRT3/4/5等在内的多条信号通路。除了进入永久性增殖停滞，衰老细胞常关系到许多病理学特征，包括局部炎症。细胞衰老发生于受损细胞， 并防止其在生物体内增殖。在各种外界刺激和内部因素影响下，细胞损伤达到一定程度则可以导致明显的细胞衰老迹象。当损伤累积达到一定的时间，组织中呈现各种肉眼可辨的组织退行变化和生理上的衰老表型。
尤其值得注意的是，衰老细胞中炎症性细胞因子的表达水平显著升高，这一现象被称为衰老相关分泌表型(SASP)。SASP这一概念是由Coppe等人于2008年首次提出。他们发现衰老细胞能通过分泌胞外基质蛋白、炎症相关因子及癌细胞生长因子促进邻近癌前细胞发生癌变或恶性程度加剧，并称这些蛋白为SASP因子。
衰老细胞主要通过3个途径参与机体的各种生理和病理过程：(1)衰老细胞基因表达和形态改变逐步累积可影响相应组织的功能；(2)衰老细胞限制干细胞和未分化祖细胞的再生潜能，导致组织再生能力下降；(3)衰老细胞不仅表现为生长周期停滞，还通过自分泌和旁分泌途径释放大量的细胞因子、趋化因子、生长因子和蛋白酶等，影响邻近细胞和组织的微环境，导致和加速机体衰老及相关疾病，近年大量研究表明在这一过程中SASP起到核心的病理作用。此外，衰老细胞分泌的这些因子还会影响周围的正常细胞，而抑制SASP则能够延缓机体衰老、延缓各种相关疾病。典型的SASP因子包括肿瘤坏死因子-α(TNF-α)、白细胞介素6(IL-6)、白细胞介素8(IL-8)、白细胞介素1a(IL-1a)、基质金属蛋白酶(MMP)、粒细胞-巨噬细胞集落刺激因子(GM-CSF)和纤溶酶原激活物抑制因子-1(PAI1)等，这些因子促进免疫系统激活，导致组织微环境中衰老细胞等异常因素被机体清除，进而发挥肿瘤抑制功能。然而，十分矛盾的是，SASP尚可通过特定分泌因子(如VEGF,ANGPTL4)促进血管生成、细胞外基质重塑或上皮-间质转化(epithelial-to-mesenchymal transition,EMT)的因子来促进肿瘤发展。此外，衰老诱导的慢性炎症可引起系统性免疫抑制，这种慢性炎症还可促进衰老相关的组织损伤和变性、器官功能失调和癌症等多种衰老相关疾病的发生和发展。
DNA损伤、端粒功能障碍、癌基因激活、氧化应激等刺激均可诱导细胞出现SASP，其机制与转录级联、自分泌环路和持续DNA损伤反应密切相关。但是，过表达或者抑制衰老经典通路p53和p16 INK4A/Rb不能影响SASP的表达，表明尽管衰老细胞的周期停滞和SASP经常协同发生，两者的调控通路并不完全重叠。据报道，DNA损伤反应通过激活毛细血管扩张共济失调突变基因、奈梅亨断裂综合征蛋白1和检测点激酶2增加SASP因子IL-6和IL-8的分泌。DNA损伤反应(DDR)在细胞受损后立即被激活，衰老细胞出现成熟SASP则需要约1周甚至更长的时间，并且短暂的DNA损伤反应并不能诱导细胞衰老，也 不能诱导SASP，表明除了DNA损伤反应外还存在其它机制共同诱导SASP。
研究表明，DDR、p38MAPK和mTOR信号作为上游驱动因子，NF-κB和c/EBPβ作为下游转录因子，均被发现参与到衰老细胞SASP的调节过程中。NF-κB和c/EBPβ转录因子在细胞衰老时活性增加，参与调节细胞应激和炎症信号的细胞因子表达。细胞衰老时磷酸化的NF-κB/RelA亚基入核，与SASP启动基因结合，调控SASP因子表达，因此NF-κB通常被称为SASP的主调节器。小鼠肝脏、肾脏及老年人大脑组织的衰老细胞中锌指转录因子4(GATA4)水平较高，GATA4可以通过调节衰老细胞中NF-κB的活性影响SASP相关基因IL-6、IL-8、CXCL1的表达。p38MAPK是丝氨酸/苏氨酸蛋白激酶家族成员之一，是重要的信号转导分子，激活或者阻断p38MAPK足以影响衰老细胞SASP形成。p38MAPK在衰老程序开始几天后被激活，通过活化丝裂原和应激激活的蛋白激酶-MSK1和MSK2，间接激活NF-κB，使得p65和p50在核内聚集，这与SASP早期发展过程相一致。衰老细胞不直接分泌促炎因子IL-1α，但衰老细胞胞膜表面分布大量的IL-1α，与NF-κB共同形成正前馈环路促进炎性因子的编码转录，建立和维持SASP。mTOR通过调节IL-1α水平促进SASP因子分泌，而雷帕霉素(rapamycin)及其类似物不影响IL-1αmRNA水平，却明显降低衰老细胞表面IL-1α蛋白的表达。mTOR也能够调节p38MAPK下游信号MAPKAPK2影响SASP因子分泌，细胞衰老期间，MAPKAPK2磷酸化RNA结合蛋白ZFP36L1，从而限制其对SASP因子转录产物的降解能力。转录因子c/EBPβ与肿瘤基因激活诱导的细胞衰老有关，衰老时c/EBPβ募集到IL-6启动子上，直接促进SASP因子转录，c/EBPβ也是IL-6正前馈自分泌环路的重要组成部分，可以激活SASP的炎症网络，是SASP早期扩散的重要调节器。HMGB2靶向作用于c/EBPβ调控SASP，通过抑制异染色质的扩散来促进SASP基因的表达，细胞衰老期间大量HMGB2与染色质结合，消除了衰老相关异染色质位点(SAHF)对SASP基因的沉默作用，导致IL-6、IL-8等表达增加。
表观遗传学近年在SASP研究领域取得不少进展。Sirtuins是一种代谢相关、NAD+依赖的去乙酰化酶，在不同模型中已发现SIRT1有寿命延长的效应。衰老细胞中SIRT1通过脱乙酰化IL-6和IL-8启动子区组蛋白H3K9和H4K16实现对SASP因子的表达抑制，当敲除SIRT1后，细胞衰老期间这些区域乙酰化水平高于对照组细胞。microRNAs是一类高度保守的单链非编码RNA，长度大约为20～26个核苷酸，在真核细胞中调节基因的表达。研究结果表明，miR-146、miR-34、miR-21和miR-183等可以调节衰老细胞SASP，并能够有效地抑制炎性细胞因子的过度生成。miR-146a/b可以降低人脐静脉内皮细胞中IL-1受体 相关激酶的产生；相反抑制miR-146a/b可以提高IL-1受体相关激酶的活性，激活转录因子NF-κB，诱导IL-6和IL-8蛋白表达。
表观遗传改变通过影响DNA损伤修复、端粒长度和代谢途径或激活衰老相关基因和miRNAs的表达而影响衰老。多种证据表明染色质状态的改变与细胞衰老的控制密切相关。细胞可以感觉到不同的衰老刺激，这些刺激会激活信号通路，驱动染色质状态的改变。然而，衰老信号引起这种改变的途径仍然很大程度上是未知的。因此，从表观遗传角度揭示细胞衰老及其特定表型发生发展的调控机制，从进而揭示具有靶向价值的关键分子及其信号通路，是将来衰老生物学和老年医学的一个新兴方向，亟需深入开展相关探索，为临床医学提供重要科学依据和潜在的干预措施。
随着全球人口老龄化的日益加剧，人们对“主动健康、延缓衰老”的兴趣与日俱增，主要是基于一系列靶向衰老的基本机制可以延缓多种衰老相关慢性或非传染性疾病的发生或加剧的科学证据。因此，细胞衰老作为预防或治疗多种衰老相关疾病和提高健康寿命的潜在靶点已获得诸多关注。
延缓衰老的药物主要是通过短暂性阻断生存途径(衰老细胞抗凋亡途径SCAPs)选择性清除衰老细胞，该生存途径可保护衰老细胞免受环境中凋亡诱导信号的调控。临床前研究表明有一类药物，即senolytics有望将来应用于延缓、预防或治疗多种衰老相关的疾病。
尽管越来越多的实验支持靶向细胞衰老可以同时治疗多种衰老相关疾病，但还有待严谨的人体临床试验以帮助人们更好地评估延缓衰老药物的益处和风险。尽管国际已知的多种SASP抑制剂均可显著减弱SASP，但本质上不会杀死衰老细胞。为了在药理学上减轻衰老细胞的负担，科学家们正在开发“senolytics”(一种衰老细胞清除药物)这种性质的小分子、多肽和抗体来选择性地清除衰老细胞。研究者们自2015年发现senolytic药物以来，在鉴别其他小分子senolytic药物及其作用方面取得了相当大的进展。研究发现首个senolytic药物的文章是基于衰老细胞抵抗凋亡的假说，尽管衰老细胞会产生促凋亡SASP因子来触发自身死亡。
事实上，有研究表明在衰老细胞中促凋亡途径确实上调。因此，衰老细胞依赖于衰老相关的抗凋亡途径(SCAPs)来减轻SASP对自身的伤害，这一假说得到了验证。SCAPs是通过生物信息学方法(基于辐射诱导衰老的人前脂肪细胞的表达谱)来鉴定的。有研究通过体外RNA干扰实验发现衰老细胞对SCAPs具有依赖性，并将SCAPs确认为衰老细胞的致命弱点。这一研究发现最终促成了SCAP网络中潜在的senolytic靶点的发现以及第一 种senolytic药物的发现，其中senolytic药物包括达沙替尼(dasatinib)(一种FDA批准的酪氨酸激酶抑制剂)和槲皮素(quercetin)(一种存在于许多水果和蔬菜中的黄烷醇)的组合(D+Q)。此外，有研究将BCL-2家族中一种对抗凋亡的蛋白(BCL- XL)鉴定为SASP组分。在这一发现之后，第三种senolytic药物navitoclax(ABT263)也被鉴定出来，它是一种BCL-2家族抑制剂。研究人员目前已经鉴定出越来越多的senolytics，包括其它合成的小分子、从天然产物中提取的化合物以及靶向已知SCAPs肽的抑制剂。此外，SCAPs也作为潜在的senolytic靶点备受瞩目。
衰老细胞存活所需的SCAP在细胞类型之间有所不同。例如，衰老的人类原代脂肪祖细胞存活所需的SCAPs与衰老的人类胚胎静脉内皮细胞(HUVECs)中的SCAPs不同。这种差异意味着靶向单个SCAP的药物可能无法消除多种衰老细胞类型。而且大量研究表明大多数senolytics确实仅对有限的衰老细胞类型有效。例如，navitoclax能够靶向HUVECs，但对衰老的人类脂肪祖细胞无效。有证据表明，即使在一种特定类型的细胞内，senolytics的功效也可能不同。例如，在人肺成纤维细胞中，navitoclax能靶向并杀死适应培养的IMR-90肺成纤维细胞样细胞株中的衰老细胞，但对衰老的原代人肺成纤维细胞少有成效。因此，为确定senolytics的广谱作用，仍需要进行针对一系列细胞类型的广泛测试。
在特定条件下，senolytic药物的使用频率可能取决于衰老细胞的积累速度，而衰老细胞的积累速度可能会因细胞衰老发生的环境而异。例如，反复接触破坏DNA的癌症疗法或持续的高脂肪饮食，可能会比自然衰老更迅速地导致衰老细胞的重新累积。间歇性使用senolytics可以降低患者产生不良反应的风险，并允许在健康期间使用senolytics。此外，间歇给药还可以减少senolytics产生的副作用并降低患者产生耐药性的可能性。与抗癌药物或抗生素的情况不同，因为衰老细胞不发生分裂，因此机体无法依赖细胞增殖产生senolytics抗性，从而无法获得有利的突变，这位将来临床中广泛使用senolytics创造了良好的基础。
发明内容
发明人经过实验探索，发现了一种筛选具有抗衰老潜力的药物的新方法。该方法经过两轮筛选，且每轮筛选中需要控制衰老细胞的密度和培养时间在适当的范围内；进而，可以简便、快速且准确地筛选获得具有抗衰老潜力的药物，发明人通过本申请的筛选方法成功筛选并获得了具有抗衰老潜力的天然产物葡萄籽提取物(GSE)。同时，发明人还通过体内和体外实验验证了葡萄籽提取物的抗衰老活性。进一步地，发明人对筛选获得的葡萄籽 提取物的抗衰老活性进行了更为深入的研究后发现，一定浓度的葡萄籽提取物可以在体外条件下有效抑制SASP标志因子的表达或者消除衰老细胞。另外，发明人还发现，葡萄籽提取物与米托蒽醌(MIT)组合后，可以有效增强米托蒽醌(MIT)的抗肿瘤(如前列腺癌)活性。
为此，在本发明的第一方面，本发明提供了一种筛选具有抗衰老潜力药物的方法，其包括：
(1)将候选药物与衰老细胞进行第一接触处理，之后将未经过所述第一接触处理的衰老细胞和经过所述第一接触处理后的衰老细胞进行细胞增殖检测，同时将对照细胞和经过所述第一接触处理后的衰老细胞进行细胞凋亡检测，所述第一接触处理是在96孔板中进行的，所述第一接触处理中所述衰老细胞的密度为4500-5500个/孔(如4500、4600、4700、4800、4900、5000、5100、5200、5300、5400、5500、4900-5100、4800-5200、4700-5300或4600-5400个/孔；优选5000个/孔)，所述第一接触处理的时间为5-10天(如5、6、7、8、9、10、6-8或5-9天，优选7天)，所述衰老细胞是通过将所述对照细胞进行衰老诱导处理后获得的；
所述细胞增殖检测和细胞凋亡检测后，同时满足以下条件的候选药物为初筛药物：
A、所述细胞增殖检测后，经过所述第一接触处理后的衰老细胞与未经过所述第一接触处理的衰老细胞相比，检测结果具有显著性差异(P<0.05)；
B、所述细胞凋亡检测后，经过所述第一接触处理后的衰老细胞与对照细胞相比，检测结果具有显著性差异(P<0.05)；
(2)将所述初筛药物与衰老细胞进行第二接触处理，之后将未经过所述第二接触处理的衰老细胞和经过所述第二接触处理后的衰老细胞进行细胞增殖检测，同时将对照细胞和经过所述第二接触处理后的衰老细胞进行细胞凋亡检测，所述第二接触处理是在96孔板中进行的，所述第二接触处理中所述衰老细胞的密度为15000-25000个/孔(如15000、16000、17000、18000、19000、20000、21000、22000、23000、24000、25000、19000-21000、18000-22000、17000-23000或16000-24000个/孔，优选20000个/孔)，所述第二接触处理的时间为25-35天(如25、26、27、28、29、30、31、32、33、34、35、29-31、28-32、27-33或26-34天，优选30天)，所述衰老细胞是通过将所述对照细胞进行衰老诱导处理后获得的；
所述细胞增殖检测和细胞凋亡检测后，同时满足以下条件的初筛药物为目标药物：
A、所述细胞增殖检测后，经过所述第二接触处理后的衰老细胞与未经过所述第二接 触处理的衰老细胞相比，检测结果具有显著性差异(P<0.05)；
B、所述细胞凋亡检测后，经过所述第二接触处理后的衰老细胞与对照细胞相比，检测结果具有显著性差异(P<0.05)。
在一些实施方案中，所述药物可以为天然产物。进而可以理解，所述“筛选具有抗衰老潜力药物的方法”可以为“筛选具有抗衰老潜力天然产物的方法”。
需要说明的是，第一或第二接触处理后，细胞增殖检测或细胞凋亡检测时，未经过(第一/第二)接触处理的衰老细胞、对照细胞以及经过(第一/第二)接触处理的衰老细胞及其对照细胞的检测条件完全相同。另外，需要进行第一/第二接触处理的衰老细胞在进行第一/第二接触处理的过程中，对照细胞以及不进行第一/第二接触处理的衰老细胞同步进行常规条件下的细胞培养，除了不添加GSE之外，其余的细胞培养条件(如起始细胞密度、培养时间、培养基种类及其体积、培养基更换频率、细胞收集和试剂处理程序等)与第一/第二接触处理完全相同。其中，所述对照细胞即为与衰老细胞相近代数的增殖态细胞，所述衰老细胞是通过将所述对照细胞进行衰老诱导处理后获得的细胞。
在一些实施方案中，所述衰老处理为化疗药物处理或辐射处理。在一些优选的实施方案中，所述衰老处理为化疗药物处理。
在一些实施方案中，所述化疗药物为博莱霉素(BLEO)。
在一些实施方案中，所述博莱霉素(BLEO)的处理浓度为40-60μg/mL(如45μg/mL、50μg/mL、55μg/mL或60μg/mL，优选50μg/mL)。
在一些具体实施方案中，所述衰老处理的步骤为：PSC27细胞生长至80％(简称PSC27-CTRL)时培养液中加入50μg/mL博来霉素(bleomycin,BLEO)。BLEO处理12小时后，细胞被PBS简单洗过3次，留置于培养液中7-10天。
在一些实施方案中，所述对照细胞为人源前列腺原代基质细胞系(如PSC27)。
在一些实施方案中，所述第一接触处理和所述第二接触处理的培养基为本领域技术人员常规使用的基础培养基。在一些具体实施方案中，所述第一接触处理和所述第二接触处理的培养基为DMEM(含有10％FBS)。
在一些实施方案中，所述第一接触处理中，所述候选药物的浓度为1μM-l mM(优选1μM-50μM，如1μM、1.25μM、1.5μM、2μM、3μM、4μM、5μM、10μM、15μM、20μM、25μM、30μM、35μM、40μM、45μM或50μM)。
在一些实施方案中，所述第二接触处理中，所述初筛药物的浓度为1μM-l mM(优选1 μM-50μM，如1μM、1.25μM、1.5μM、2μM、3μM、4μM、5μM、10μM、15μM、20μM、25μM、30μM、35μM、40μM、45μM或50μM)。
需要说明的是，上述候选药物和初筛药物的浓度可以是单一浓度，也可以是梯度浓度。换句话说，进行第一轮或第二轮筛选时，可以是以单一浓度进行筛选，也可以是以梯度浓度进行筛选。以梯度浓度进行筛选时，每一个浓度均需符合预定筛选条件，才可以判定候选药物为初筛药物，或判定初筛药物为目标药物。
在一些实施方案中，所述细胞增殖检测是利用CCK-8 Cell Counting Kit试剂盒进行的。具体检测步骤可以参照检测说明进行。例如，检测步骤如下：(1)接种细胞悬液100μL于96孔板，细胞培养箱中(37℃,5％CO 2)孵育；(2)取出需要检测的细胞(即药物处理后第7天或第30天的细胞)，在培养皿中加入0.1体积的CCK-8，充分混合，保证孔中颜色的均一性，避免气泡出现；(3)培养箱中继续培养1-4小时；(4)酶标仪读数之前，将96孔板于摇床振荡1min，确保孔板颜色均匀；(5)使用酶标仪在450nm读取吸光值，计算细胞活性。当某种药物在特定浓度条件下造成药物处理后衰老细胞与未经药物处理衰老细胞相比，CCK-8指标之间达到显著性差异(P<0.05，每组至少3个重复样本)时，判定该药物在该浓度下具备靶向衰老细胞的有效性。
在一些实施方案中，所述细胞凋亡检测是利用Caspase 3/7 activity kit(Promega)进行的。具体检测步骤可以参照检测说明进行。例如，检测步骤为：(1)从孵化器中取出含有细胞(即药物处理后第7天或第30天的细胞)的96孔板，并允许该板温度平衡至室温；(2)将
Figure PCTCN2021130639-appb-000001
试剂的100μL添加到含有空白100μL的白壁96孔板的每孔中，以及阴性对照组细胞或培养基中处理的细胞。因这种测定的敏感性，避免枪头尖端触碰到含有样品的孔，以避免交叉污染。用板密封器盖住板或者盖子；(3)用平板摇床在300-500rpm转速下轻混孔内液体30秒，根据细胞培养系统，在室温下孵育持续30分钟至3小时。最佳孵育时间应根据预实验提前确定；(4)根据荧光光度计的使用指南，测定每个样品的生物荧光值。经过对比，在衰老细胞与相近代数的增殖态细胞之间出现显著性差异(P<0.05，每组至少3个重复样本)时，判定该药物在该浓度下具备特异性清除衰老细胞的效果或潜力。
在一些实施方案中，所述第一接触处理中，所述培养基和所述候选药物每两天更换一次。
在一些实施方案中，所述第二接触处理中，所述培养基和所述初筛药物每两天更换一次。
在本发明的第二方面，本发明提供了组合物，其包含葡萄籽提取物，所述葡萄籽提取物的浓度为1-1.5μM(优选1.25μM)。在一些实施方案中，余量为水。
在本发明的第三方面，本发明提供了组合物在制备试剂中的用途，所述试剂用于抑制SASP标志因子的表达，所述组合物包含葡萄籽提取物，所述葡萄籽提取物的浓度为1-1.5μM(优选1.25μM)。在一些实施方案中，余量为水。所述试剂可以供科研人员或其他有需要的研究人员在体外检测和实验中使用。
在一些实施方案中，所述SASP因子选自肿瘤坏死因子-α(TNF-α)、白细胞介素6(IL-6)、白细胞介素8(IL-8)、白细胞介素1α(IL-1α)、白细胞介素1β(IL-1β)、基质金属蛋白酶(MMP，如MMP3)、粒细胞-巨噬细胞集落刺激因子(GM-CSF)、纤溶酶原激活物抑制因子-1(PAI1)、趋化因子(CXC，如CXCL3)、双向调节因子(AREG)、丝氨酸肽酶抑制因子Kazal型1(SPINK1)，或其任何组合。
在一些实施方案中，所述SASP因子为白细胞介素6(IL-6)、白细胞介素8(IL-8)、白细胞介素1α(IL-1α)、白细胞介素1β(IL-1β)、基质金属蛋白酶(MMP，如MMP3)、粒细胞-巨噬细胞集落刺激因子(GM-CSF)、趋化因子(CXC，如CXCL3)、双向调节因子(AREG)和丝氨酸肽酶抑制因子Kazal型1(SPINK1)的组合。
在本发明的第四方面，本发明提供了组合物，其包含葡萄籽提取物，所述葡萄籽提取物的浓度为5-50μM(优选5-25μM，更优选25μM)。在一些实施方案中，余量为水。
在本发明的第五方面，本发明提供了组合物在制备试剂中的用途，所述试剂用于消除衰老细胞，所述组合物包含葡萄籽提取物，所述葡萄籽提取物的浓度为5-50μM(优选5-25μM，更优选25μM)。在一些实施方案中，余量为水。所述试剂可以供科研人员或其他有需要的研究人员在体外检测和实验中使用。
在一些实施方案中，所述试剂通过诱导衰老细胞凋亡来清除衰老细胞。
在一些实施方案中，所述试剂用于诱导衰老细胞凋亡。
在本发明的第六方面，本发明提供了组合物，其包含葡萄籽提取物和米托蒽醌(MIT)。
在一些实施方案中，所述葡萄籽提取物和所述米托蒽醌(MIT)的质量比为10:0.1-10:0.3(优选为10:0.2)。
在本发明的第七方面，本发明提供了前述的组合物在制备药物、食品或保健品中的用途，所述药物用于治疗和/或预防癌症。
在本发明的第八方面，本发明提供了治疗和/或预防癌症的方法，其包括给予有需要的 受试者有效量的前述组合物或者由前述组合物制备的药物、食品或保健品。
在本发明的第九方面，本发明提供了前述的组合物或者由前述的组合物制备的药物、食品或保健品，其用于治疗和/或预防癌症。
在一些实施方案中，上述癌症为与衰老相关的癌症。
在一些实施方案中，上述癌症为前列腺癌。
在本发明中，除非另有说明，否则本文中使用的科学和技术名词具有本领域技术人员所通常理解的含义。同时，为了更好地理解本发明，下面提供相关术语的定义和解释。
本申请的葡萄籽提取物(grape seed extract，GSE)是从葡萄栽培品种的种子中提取出来的一种多酚类和类黄酮混合物，1951年，Masquelier在法国获得了从松树皮提取OPC的专利技术，并于1970年从葡萄籽中提取成功，其包含单体多酚和聚合多酚。GSE中单体多酚主要包括没食子酸、儿茶素、表儿茶素和表儿茶素没食子酸。聚合多酚主要是以儿茶素或表儿茶素为单体缩合而成的原花青素，其中以低聚体(二聚、三聚、四聚体)-又称为寡聚体(oligomerie procyanidin，OPC)生物活性最强。葡萄籽提取物可以市购获得。GSE的成分含量详见表3。
本文使用的术语“治疗”一般是指获得需要的药理和/或生理效应。该效应根据完全或部分地预防疾病或其症状，可以是预防性的；和/或根据部分或完全稳定或治愈疾病和/或由于疾病产生的副作用，可以是治疗性的。本文使用的“治疗”涵盖了对患者疾病的任何治疗，包括：(a)预防易感染疾病或症状但还没诊断出患病的患者所发生的疾病或症状；(b)抑制疾病的症状，即阻止其进一步发展；或(c)缓解疾病的症状，即，导致疾病减轻或症状退化。
在本发明中，“受试者”指脊椎动物。在某些实施方案中，脊椎动物指哺乳动物。哺乳动物包括，但不限于，牲畜(诸如牛)、宠物(诸如猫、犬、和马)、灵长类动物、小鼠和大鼠。在某些实施方案中，哺乳动物指人。
在本发明中，“有效量”指在必需的剂量和时间上有效实现期望的治疗或预防效果的特定药物的使用量。本发明的物质/分子的“治疗有效量”可根据诸如个体的疾病状态、年龄、性别和体重及该物质/分子在个体中引发期望应答的能力等因素而变化。治疗有效量还涵盖该物质/分子的治疗有益效果胜过任何有毒或有害后果的量。“预防有效量”指在必需的剂量和时间上有效实现期望的预防效果的量。通常而非必然，由于预防剂量是在疾病发作之前或在疾病的早期用于受试者的，因此预防有效量会低于治疗有效量。在癌症的情况中，药物的治疗有效量可减少癌细胞数；缩小肿瘤体积；抑制(即一定程度的减缓，优选停止)癌细 胞浸润到周围器官中；抑制(即一定程度的减缓，优选停止)肿瘤转移；一定程度的抑制肿瘤生长；和/或一定程度的减轻与癌症有关的一种或多种症状。
有益效果：
1、本申请的药物筛选方法，可以在最短时间、以最少药物用量、获得达到清除衰老细胞的最佳效果，即最高的研发效率。如果舍弃或者改变这些筛选步骤，将对抗衰老药物的筛选带来时间和成本上的大幅增长，不利于低投入、高产出地获得研究数据和关键结果，最终使得筛选效率显著下降，且准确度将显著降低。
2、本申请的药物筛选方法中，细胞密度是关键，若增加或者减少，会影响数据的准确性、稳定性和可重复性。例如，第一轮筛选中，如使用4000或6000个细胞/孔的细胞密度，会在SASP表达数据上出现不准确或结果分歧(例如参见图11H-11I)。
3、本申请的药物筛选方法中，时间是关键。第一阶段时间若选择本申请以外其它时间长度，会增加药物用量或提高细胞死亡率，或者影响方法的准确度(例如参见图11D-11E)。第二阶段如选择更长或更短时间，将影响数据的可靠性和临床参考价值，不能准确判断药物的安全性和有效性(例如参见图11F-11G)。
4、本申请的药物筛选方法中，两轮筛选在技术上必要且关键。如果舍弃第二轮筛选，准确度将下降(例如参见本申请表1和表2)。另外，不需要进行第三轮或更多轮筛选，以免由于无限推演药物筛选时间和规模，造成不必要的时间成本和经济成本的上升。两轮筛选已经可以比较准确地反映将来临床条件下的微环境中衰老细胞的生存状态或靶向清除效果。
附图说明
图1表示增殖态人源基质细胞PSC27(早期代数如p10-20)在体外经过化疗药物博来霉素(BLEO)以50μg/ml浓度处理之后第7-10天，通过SA-β-Gal染色之后的结果。上图，代表性图片，下图，统计学数据。CTRL，对照细胞；BLEO，博来霉素处理后细胞。**，P<0.01。
图2表示PSC27细胞经过化疗药物博来霉素(BLEO)处理之后，经过BrdU染色之后的结果。上图，代表性图片，下图，统计学数据。CTRL，对照细胞；BLEO，博来霉素处理后的细胞。***，P<0.001。
图3表示PSC27细胞经过化疗药物博来霉素(BLEO)处理之后，使用γH2AX经过免疫荧光染色(immunofluorescence staining)之后的结果。CTRL，对照细胞；BLEO，博来霉素处理后的细胞。***，P<0.001。根据细胞核内荧光点的数量，将其分为4类，包括0foci,1～3foci,4～10foci和>10foci的单个细胞。
图4表示筛选天然产物药库以获得具有抗衰老活性植物原料的实验流程图。
图5A表示候选药物A在细胞增殖实验中所获得的数据，数据显示：经过一系列浓度条件下的A对于衰老细胞与增殖细胞处理的结果相比较，A在0-30μM浓度范围内均未能诱导衰老细胞显著死亡(继续提高浓度在两组细胞之间也未出现显著性差异，结果未显示)。其中，候选药物A为雪莲培养物。其余候选药物的筛选结果与候选药物A基本相同，即其余候选药物在0-30μM浓度范围内均未能诱导衰老细胞显著死亡(继续提高浓度在两组细胞之间也未出现显著性差异)。
图5B表示候选药物A处理细胞之后的caspase-3/7活性变化结果，结果显示：A仅在特定浓度下才有引起衰老细胞发生凋亡的能力(例如，从加药之后起的第16-20小时)；在此浓度范围之外，均不能造成衰老细胞的显著性凋亡(与GSE之间形成鲜明对比)。其中，候选药物A为雪莲培养物。其余候选药物的筛选结果与候选药物A大致相同，即其余候选药物最多仅能在特定的个别浓度下才有引起衰老细胞发生凋亡的能力，在此浓度范围之外，均不能造成衰老细胞的显著性凋亡；甚至是在所有浓度下均不能造成衰老细胞的显著性凋亡，这与GSE之间形成鲜明对比。
图6A表示RNA-seq数据经软件处理和生信分析之后发现GSE可以使得衰老细胞相比于增殖态细胞显著上调的基因出现明显回落。相比于BLEO组，BLEO/GSE组细胞有2644个基因显著下调，同时有1472个基因显著上调(fold change>2,P<0.01)。
图6B表示Heatmap显示BLEO损伤造成的衰老细胞中大量因子表达上调，但经过GSE处理之后有不少出现明显逆转。红星标识，典型SASP外泌因子。
图7表示GSEA分析结果显示SASP或NF-κB分子标记相关因子的表达在BLEO造成的衰老细胞中集中上调，但在GSE处理衰老细胞之后发生显著下降。左，SASP分子标记；右，NF-κB分子标记。
图8表示蛋白-蛋白相互作用(PPI)生信分析结果显示，GSE显著下调的衰老细胞分子形成一个相对复杂的network，彼此间存在着多种互作关系。
图9表示KEGG通路分析GSE在衰老细胞中造成显著下调的100个分子在biological process上的代表性通路。左侧Y轴，percentage。右侧Y轴，log10(p-value)。
图10表示KEGG通路分析GSE在衰老细胞中造成显著下调的100个分子在cellular component上的代表性通路。左侧Y轴，percentage。右侧Y轴，log10(p-value)。
图11A表示在第一轮筛选后，荧光定量PCR(qRT-PCR)检测分析一组典型SASP分子在BLEO诱导形成的衰老细胞、被不同浓度的GSE处理条件下的相对表达水平。所有数据均为相比于CTRL组后的规范化结果。*,P<0.05；**,P<0.01；***,P<0.001。
图11B表示在第二轮筛选后，荧光定量PCR(qRT-PCR)检测分析一组典型SASP分子在BLEO诱导形成的衰老细胞、被不同浓度的GSE处理条件下的相对表达水平。所有数据均为相比于CTRL组后的规范化结果。*,P<0.05；**,P<0.01。
图11C表示候选药物A在第一轮筛选后，荧光定量PCR(qRT-PCR)检测分析一组典型SASP分子在BLEO诱导形成的衰老细胞、被不同浓度的GSE处理条件下的相对表达水平。所有数据均为相比于CTRL组后的规范化结果。^,P>0.05；*,P<0.05。
图11D表示在第一轮筛选时，药物处理后第15天再进行检测，第一轮筛选后，荧光定量PCR(qRT-PCR)检测分析一组典型SASP分子在BLEO诱导形成的衰老细胞、被不同浓度的GSE处理条件下的相对表达水平。所有数据均为相比于CTRL组后的规范化结果。^,P>0.05；*,P<0.05；**,P<0.01。
图11E表示在第一轮筛选时，药物处理后第2天即进行检测，第一轮筛选后，荧光定量PCR(qRT-PCR)检测分析一组典型SASP分子在BLEO诱导形成的衰老细胞、被不同浓度的GSE处理条件下的相对表达水平。所有数据均为相比于CTRL组后的规范化结果。^,P>0.05；*,P<0.05。
图11F表示在第二轮筛选时，药物处理后第50天再进行检测，第二轮筛选后，荧光定量PCR(qRT-PCR)检测分析一组典型SASP分子在BLEO诱导形成的衰老细胞、被不同浓度的GSE处理条件下的相对表达水平。所有数据均为相比于CTRL组后的规范化结果。^,P>0.05；*,P<0.05。
图11G表示在第二轮筛选时，药物处理后第15天即进行检测，第二轮筛选后，荧光定量PCR(qRT-PCR)检测分析一组典型SASP分子在BLEO诱导形成的衰老细胞、被不同浓度的GSE处理条件下的相对表达水平。所有数据均为相比于CTRL组后的规范化结果。^,P>0.05；*,P<0.05。
图11H表示在第一轮筛选时，细胞密度为4000个/孔，第一轮筛选后，荧光定量PCR(qRT-PCR)检测分析一组典型SASP分子在BLEO诱导形成的衰老细胞、被不同浓度的GSE处理条件下的相对表达水平。所有数据均为相比于CTRL组后的规范化结果。^,P>0.05；*,P<0.05。
图11I表示在第一轮筛选时，细胞密度为6000个/孔，第一轮筛选后，荧光定量PCR(qRT-PCR)检测分析一组典型SASP分子在BLEO诱导形成的衰老细胞、被不同浓度的GSE处理条件下的相对表达水平。所有数据均为相比于CTRL组后的规范化结果。^,P>0.05；*,P<0.05。
图12A表示在第一轮筛选后，在GSE浓度递增的条件下，用SA-β-Gal染色确定PSC27的衰老与否。*,P<0.05；**,P<0.01；***,P<0.001；****,P<0.0001。其中，经过第一轮筛选，GSE在1.25μM，2.5μM，5μM，12.5μM，25μM和50μM浓度下的P值为这些实验组的细胞阳性比例同0μM时的数据相比后得出的统计学显著性数据。
图12B表示在第二轮筛选后，在GSE浓度递增的条件下，用SA-β-Gal染色确定PSC27的衰老与否。^,P>0.05；**,P<0.01；****,P<0.0001。其中，GSE在5μM，12.5μM，25μM和50μM浓度下的P值为这些实验组的细胞阳性比例同0μM时的数据相比得出的统计学显著性数据。
图13表示SA-β-Gal染色后PSC27在各种条件下的代表性图片。每组3个重复，上下排列。标尺，20μm。
图14表示CCK8检测增殖态细胞同衰老组细胞在GSE渐增浓度下的存活率。每一GSE浓度下的P值均为CTRL和BLEO组之间相比后的显著性差异。**,P<0.01；***,P<0.001；****,P<0.0001。
图15表示PSC27的群体倍增(population doubling)测试。细胞在第10代(p10)时，受到BLEO损伤性处理，随后GSE在第8天时加入培养基。通过比较分析CTRL组，BLEO组，GSE组和BLEO/GSE组的倍增值(PD)确定GSE对于细胞增殖潜力的影响。^,P>0.05；***,P<0.001。
图16表示GSE处理衰老细胞过程中诱导出现caspase 3/7活性。PSC27细胞经BLEO在培养条件下处理12h后逐渐进入衰老阶段。5μM的GSE在第7天开始加入衰老细胞的培养基，NucLight Rapid Red试剂用于标记细胞，而caspase 3/7试剂(IncuCyte)用于apoptosis检测。Caspase 3/7活性以每4小时的间隔检测一次(n＝3)。
图17表示Pan-caspase抑制剂(20μM QVD-OPH)逆转GSE的senolytic活性(5μM的GSE用于这一实验,而1μM的ABT263作为阳性对照；后者为近年被报道的衰老细胞凋亡诱导剂)。统计学差异通过two-way ANOVA(Tukey’test)获得。
图18表示流式细胞仪测定PSC27在几种条件下的细胞凋亡情况。Q2，早期凋亡细胞的分布区域；Q3，晚期凋亡细胞的分布区域。
图19表示对比分析细胞经过BLEO和/或GSE处理之后的存活和凋亡数量。***,P<0.001；****,P<0.0001。
图20表示预临床试验中小鼠的给药方式示意图。人源基质细胞PSC27同癌细胞PC3在体外混合(1:4)之后移植入小鼠皮下形成移植瘤。在单药或组合式给药条件下经过多个治疗周期的处理，最终小鼠处死、病理分析其肿瘤组织有关分子表达变化。
图21表示PSC27细胞的CTRL组和BLEO损伤组在体外同PC3混合之后，或者PC3细胞单独移植入小鼠皮下组织形成移植瘤。在第8周结束时解剖并获得肿瘤，检测、对比各组条件下肿瘤的体积。**,P<0.01；***,P<0.001；****,P<0.0001。
图22表示预临床试验小鼠的给药时间和给药方式示意图。每两周为一次给药周期，在第3/5/7周的第一天分别对小鼠腹腔给药MIT(mitoxantrone，米托蒽醌)。第5周第一天开始对小鼠进行腹腔GSE给药，每周一次。8周疗程结束后，解剖小鼠并进行病理鉴定与表达分析。
图23表示肿瘤终端体积统计分析。化疗药物MIT单独或与抗衰老药GSE一起用于对小鼠给药，第8周结束之后对比分析各组肿瘤大小。
图24表示临床前试验中PC3/PSC27荷瘤动物病灶中细胞衰老情况对比。SA-β-Gal染色之后代表性图片。标尺，100μm。
图25表示小鼠体内肿瘤组织中SA-β-Gal染色阳性细胞百分比平行分析。^,P>0.05；**,P<0.01；***,P<0.001。
图26表示荧光定量PCR(qRT-PCR)检测分析小鼠病灶中上皮癌细胞和基质细胞中SASP典型因子的表达情况。通过LCM技术将基质细胞和癌细胞分别进行特异分离、制备总RNA并用于SASP表达检测。^,P>0.05；*,P<0.05；**,P<0.01；***,P<0.001。
图27表示荧光定量PCR(qRT-PCR)检测分析vehicle、MIT和MIT/GSE给药之后的小鼠病灶中基质细胞SASP因子表达状态。*,P<0.05；**,P<0.01；***,P<0.001。
图28表示用LCM技术将病灶中癌细胞进行特异分离之后分析各组小鼠中DNA损伤和凋亡比例。^,P>0.05；*,P<0.05；**,P<0.01。
图29表示免疫组化染色(immunohistochemical staining)之后的图片分析。Caspase 3cleaved(CCL3)在各组小鼠病灶中的信号形成鲜明对比。标尺，200μm。
图30表示NOD/SCID小鼠在经过各种给药处理之后，无病生存期的Kaplan Meier数据对比。Vehicle,MIT,GSE和MIT/GSE组动物在体内肿瘤体积超过2000mm 3时，即被认为严重疾病已经出现，小鼠需要及时处死并检测其荷瘤情况。^,P>0.05；**,P<0.01。
图31表示各种不同给药处理条件下疗程结束时小鼠体重数据对比分析。^,P>0.05。
图32表示以上不同给药处理条件下疗程结束时小鼠血清学数据对比分析。Creatinine,urine(肾脏指标),ALP和ALT(肝脏指标)数据平行对比。^,P>0.05。
图33表示各种不同给药处理条件下疗程结束时免疫完整型小鼠(C57BL/6J)体重数据对比分析。^,P>0.05。
图34表示预临床中不同给药处理条件下疗程结束时小鼠血细胞计数对比分析。WBC,lymphocyte和neutrophil单位体积数量平行对比。^,P>0.05。
图35表示动物体内生物发光(BLI)图像显示报告细胞的位置与信号强度。持续表达luciferase、经BLEO诱导进入衰老阶段之后的PSC27细胞通过腹腔注射被提前移植于小鼠体内；最后一次Vehicle或GSE给药2天之后，使用Berthold LB983(BERTHOLD Technologies)小动物活体分子成像系统获取小鼠体内luciferase信号。标尺，15mm。
图36表示动物体内生物发光(BLI)图像显示报告细胞的位置与信号强度。持续表达luciferase的增殖态PSC27细胞通过腹腔注射被提前移植于小鼠体内；最后一次Vehicle或GSE给药2天之后，使用Berthold LB983(BERTHOLD Technologies)小动物活体分子成像系统获取小鼠体内luciferase信号。标尺，15mm。
图37表示预临床中对实验小鼠体能进行检测的试验流程。20月龄小鼠经Vehicle或GSE每两周一次给药之后，在第4个月末进行相关体能检测。
图38表示针对实验小鼠进行一系列体能测定，包括最大步行速度、持久力、握力、跑步机耐力、日常活动、体重与摄食量的定量检测。^，P>0.05；*，P<0.05。
图39表示实体器官微环境中基质细胞的一组代表性SASP因子相对mRNA表达水平。小鼠在24月龄时处死之后解剖并获得其肺脏、前列腺和结直肠等实体器官并提取其基质组 织中总RNA，进行qRT-PCR定量分析。6月龄(6M)小鼠作为对照，其它两组24月龄(24M)小鼠信号对其规范化之后作图。^，P>0.05；*，P<0.05。
图40表示小鼠寿命分析试验设计。24至27月龄小鼠经过Vehicle或GSE每两周一次给药，持续监测其生存情况并记录其最大寿命。
图41表示预临床阶段小鼠的疗后生存曲线。从24至27月龄时开始，C57BL/6小鼠每两周经受一次Vehicle或GSE腹腔给药(Vehicle组n＝80；GSE组n＝91)。每组动物的中位生存期(median survival)经过计算并予以标明。****，P<0.0001。
图42表示预临床阶段小鼠的总体(终生，或全长)生存曲线。从24至27月龄时开始，C57BL/6小鼠每两周经受一次Vehicle或GSE腹腔给药(Vehicle组n＝80；GSE组n＝91)。每组动物一生中的中位生存期(median survival)经过计算并予以标明。****，P<0.0001。
图43表示选取每组动物中寿命长度位于最高区间的雄性小鼠，进行组间最高步行速度、持久力和总体寿命的比较分析。N＝5。^，P>0.05；**，P<0.01。
图44表示选取每组动物中寿命长度位于最高区间的雌性小鼠，进行组间最高步行速度、持久力和总体寿命的比较分析。N＝5/组。^，P>0.05；***，P<0.001。
图45表示针对两组动物中每只小鼠在生命终端所罹患的疾病负担进行对比分析。N＝60/组。统计结果以box-and-whisker plots显示，每个box展示出median with interquartile range。^，P>0.05。
图46表示针对两组动物中每只小鼠在生命终端所罹患的肿瘤数量进行对比分析。N＝60/组。统计结果以box-and-whisker plots显示，每个box展示出median with interquartile range。^，P>0.05。
图47表示使用HPLC-ESI-QTOF-MS技术对GSE进行成分鉴定，发现GSE中存在多个天然化合物成分。每个峰值的鉴定，基于它们各自的滞留时间和真实的标准及植物化学的广谱数据。
具体实施方式
下面通过具体的实施例对本发明中的技术方案进行清楚、完整地描述。应当理解，此部分所描述的具体实施例仅可用于解释本发明，并不用于限定本发明。
需要说明的是，对于生物统计学方法，以下实施例中所有涉及细胞增殖率，存活率和SA-β-Gal染色等的体外实验和小鼠移植瘤及预临床药物处理的体内试验均重复3次以上，数 据以均值±标准误的形式呈现。统计学分析建立在原始数据的基础上，通过one-way analysis of variance(ANOVA)or a two-tailed Student's t-test进行计算，而P<0.05的结果认作具有显著性差异。
因素之间的相关性用Pearson’s correlation coefficients检验。当小鼠在几个队列中获得并分组在笼子中时，采用Cox proportional hazard model进行生存分析。该模型将治疗的性别和年龄作为固定效应，队列和初始笼分配作为随机效应。由于在研究中，一些小鼠被从最初的笼子中移动，以尽量减少来自单笼外壳的压力，发明人还进行了没有笼效应的分析。这两种分析的结果在方向性或统计意义上没有很大差异，增强了对发明人结果的自信度。生存分析使用statistical software R(version 3.4.1；library‘coxme’)。在大多数实验和结果评估中，研究者对分配采取盲选。发明人使用基线体重将小鼠分配至实验组(以实现组间相似的体重)，因此只在与体重匹配的组内进行随机化。发明人根据过往的实验确定样本量，因此没有使用statistical power analysis。本研究中的所有重复都来自不同的样本，每个样本来自不同的实验动物。
实施例1具有抗衰老潜力的药物的筛选
一、预备工作
为了筛选获得能有效调节衰老细胞表型的化合物，发明人利用一个包含至少4种植物衍生物组成的天然原料性药库开展了无偏倚性筛选。为了检测这些药物的药效和潜在的生物价值，发明人选择使用原发性正常人前列腺基质细胞系，即PSC27作为体外细胞模型。PSC27主要由成纤维细胞组成，而非成纤维细胞系(包括内皮细胞和平滑肌细胞)也存在，但比例较小，PSC27在性质上是人源原代基质细胞系，在暴露于基因毒性化疗或电离辐射等胁迫因素后形成典型的SASP。发明人用预实验中已经优化过的方式，即特定计量的博莱霉素(BLEO)处理这些细胞，并观察到衰老相关β-半乳糖苷酶(SA-β-GAL)染色阳性率明显升高，BrdU掺入率大幅降低，DNA损伤修复灶(DDR foci)在药物损伤后的数天内显著升高(图1-3)。发明人通过系统筛选的方式来平行比较药物产品对衰老细胞表达谱的影响(图4)。
1.细胞培养
(1)细胞系维持
正常人源前列腺原代基质细胞系PSC27(获自美国Fred Hutchinson Cancer研究Research Center)于37℃和5％CO 2条件的培养箱中培养，在PSCC完全培养液中增殖和 传代。
(2)细胞冻存与复苏
a.细胞冻存
以0.25％胰蛋白酶收集对数生长期细胞，1000rpm离心2min，弃去上清，重新悬浮细胞于新鲜配置的冻存液中。分装细胞于已标示的无菌冻存管中。然后经梯度降温，最后转入液氮中长期储存。
b.细胞复苏
取出液氮中冻存的细胞，立即放入37℃水浴，使其快速融化。直接加入2ml细胞培养液，使细胞均匀悬浮。待细胞贴壁后，更换新的培养液。
(3)体外实验处理
为造成细胞损伤，PSC27细胞生长至80％(简称PSC27-CTRL)时培养液中加入50μg/ml博来霉素(bleomycin,BLEO)。药物处理12小时后，细胞被PBS简单洗过3次，留置于培养液中7-10天，然后进行后续实验。
2.免疫印记和免疫荧光检测
用NuPAGE 4-12％Bis-Tris gel分离细胞裂解来源蛋白质，并转移到硝化纤维素膜(Life Technologies)上。用5％脱脂牛奶在室温下阻断印迹1h，在4℃下与所需的一抗在制造商协议的浓度下孵育一夜，然后在室温下与辣根过氧化物酶结合二抗(Santa Cruz)孵育1h，用增强化学发光(ECL)检测试剂(Millipore)按照制造商的协议开展膜印迹信号检测，并使用ImageQuant LAS 400 Phospho-Imager(GE Healthcare)。作为一种标准的蛋白质标记，发明人使用Thermo Fisher Scientific公司提供的PageRuler Plus Prestained Protein Ladder(no.26619)。
对于免疫荧光染色，目标细胞在培养皿中培养之后在coverslip上预种至少24h。在短暂洗涤后，用4％多聚甲醛在PBS中固定8min，用5％正常山羊血清(NGS,Thermo Fisher)阻断30min。小鼠单克隆抗体anti-phospho-Histone H2A.X(Ser139)(clone JBW301,Millipore)和小鼠单克隆抗体anti-BrdU(Cat#347580，BD Biosciences)，及二级抗体Alexa
Figure PCTCN2021130639-appb-000002
488(or 594)-conjugated F(ab')2按顺序加入到覆有固定细胞的载玻片上。细胞核用2μg/ml of 4',6-diamidino-2-phenylindole(DAPI)进行复染。从3个观察视野中选取最具代表性的一张图像进行数据分析和结果展示。FV1000激光扫描共聚焦显微镜(Olympus)用于获取细胞共聚焦荧光图像。
二、筛选方法
发明人以一个包含至少4种成分、多为药用植物提取物的天然产物库(BY-HEALTH)为例进行筛选。根据研究目的，发明人建立了一套标准的筛选流程，一共有两轮筛选。
第一轮：
各种待筛天然产物分别按照一定浓度梯度稀释至96孔板，密度为每孔5000个细胞。培养基使用DMEM(10％FBS)，天然产物(或化合物)的工作浓度一般控制在1μM-l mM(本实施例中采用的示例浓度为10-50μM)。药物处理后第7天，细胞增殖用CCK-8 Cell Counting Kit试剂盒(基于WST-8原理，Vazyme)测定，细胞凋亡活性以Caspase 3/7 activity kit(Promega)确定。
以上过程中培养基和候选药物每两天更换一次，增殖和凋亡活性在药物使用起7天后开始检测。
第二轮：
经第一轮初步确定的候选药物进一步经过筛选30天(时间延伸)，即验证确定。具体地，将进入第二轮候选范围的药物稀释到96孔板中，每孔20,000个细胞，培养基使用DMEM(10％FBS)，天然产物(或化合物)的工作浓度一般控制在1μM-l mM(本实施例中采用的示例浓度为10-50μM)。药物处理后第30天，细胞增殖用CCK-8 Cell Counting Kit试剂盒(基于WST-8原理，Vazyme)测定，细胞凋亡活性以Caspase 3/7 activity kit(Promega)确定。
以上培养过程中培养基和候选药物每隔一天更换一次(即每两天一次)。
以上两轮筛选中，药物处理细胞后7天或30天用CCK-8 Cell Counting Kit试剂盒检测细胞增殖或存活情况。具体的检测步骤如下：(1)接种细胞悬液100μL于96孔板，细胞培养箱中(37℃,5％CO 2)孵育；(2)取出需要检测的细胞(即药物处理后第7天或第30天的细胞)，在培养皿中加入0.1体积的CCK-8，充分混合，保证孔中颜色的均一性，避免气泡出现；(3)培养箱中继续培养1-4小时；(4)酶标仪读数之前，将96孔板于摇床振荡1min，确保孔板颜色均匀；(5)使用酶标仪在450nm读取吸光值，计算细胞活性。当某种药物在特定浓度条件下造成药物处理后衰老细胞与未经药物处理衰老细胞相比，CCK-8指标之间达到显著性差异(P<0.05，每组至少3个重复样本)时，判定该药物在该浓度下具备有效性。
以上两轮筛选中，药物处理细胞后7天或30天以Caspase 3/7 activity kit(Promega)确定细胞凋亡活性。在以Caspase 3/7 activity kit(Promega)测定细胞凋亡活性时，遵循的流程为：(1)从孵化器中取出含有细胞(即药物处理后第7天或第30天的细胞)的96孔板，并允许 该板温度平衡至室温；(2)将
Figure PCTCN2021130639-appb-000003
试剂的100μL添加到含有空白100μL的白壁96孔板的每孔中，以及阴性对照组细胞或培养基中处理的细胞。因这种测定的敏感性，避免枪头尖端触碰到含有样品的孔，以避免交叉污染。用板密封器盖住板或者盖子；(3)用平板摇床在300-500rpm转速下轻混孔内液体30秒，根据细胞培养系统，在室温下孵育持续30分钟至3小时。最佳孵育时间应根据预实验提前确定；(4)根据荧光光度计的使用指南，测定每个样品的生物荧光值。经过对比，在衰老细胞与相近代数的增殖态细胞之间出现显著性差异(P<0.05，每组至少3个重复样本)时，判定该药物在该浓度下具备特异性清除衰老细胞的效果或潜力。
以上标准筛选流程，同样适合于其它天然产物库根据特定用途或生物活性(如抗衰老潜力)的筛选。
另外，为进一步确定每种药物对细胞表型和存活率等的影响，发明人进行了进一步深入的验证性分析。
三、实验结果
筛选前，上述包含至少4种成分的天然产物库(BY-HEALTH)中的候选药物为GSE(葡萄籽提取物)、雪莲培养物、白藜芦醇和姜黄素等植物化学提取产物。
经过第一轮筛选，符合条件的候选药物只有GSE(葡萄籽提取物)。其余几种候选药物完全不符合筛选标准，即在第一轮筛选后即被淘汰。具体实验结果如图5A和图5B所示。
经过第二轮筛选，符合条件的候选药物确定为GSE(葡萄籽提取物)。
结合实施例2和实施例3的体内体外验证分析可以确定，GSE的确具有抗衰老活性。因此，本申请的筛选方法准确度高、且简便易操作。
另外，发明人基于上述实施例，通过变换第一轮筛选和第二轮筛选的个别处理条件，对本申请筛选方法的准确度进行了进一步验证。具体的实验条件和实验结果如下表所示：
Figure PCTCN2021130639-appb-000004
Figure PCTCN2021130639-appb-000005
由上表可以进一步确定，本申请的筛选方法中，第一轮和第二轮的药物处理时间，以及第一轮的细胞密度对于最终检测结果的准确性十分关键，处理时间过长或过短，细胞密度过大或过小，筛选方法的准确度均明显下降。
实施例2抗衰老药物的体外分析和验证
一、方法
1.全转录组测序分析(RNA-sequencing)
对不同处理条件下的人源前列腺原代基质细胞系PSC27进行全转录组测序。从基质细胞中获得总RNA样本。其完整性经Bioanalyzer 2100(Agilent)验证，RNA用Illumina HiSeq X10测序，基因表达水平由软件包rsem(https://deweylab.github.io/rsem/)进行量化。简而言之，以RiboMinus Eukaryote kit(Qiagen,Valencia,CA,USA)消除RNA样品中的rRNA；并根据制造商的指示，在深度测序前用TruSeq Stranded Total RNA preparation kits(Illumina,San Diego,CA,USA)构建链特异性RNA-seq文库。
Paired-end transcriptomic reads取被映射到参考基因组(GRCh38/hg38)，并使用Bowtie工具从Gencode v27进行参考注释。使用picard tools(1.98)脚本标记重复项(https://github.com/broadinstitute/picard)识别重复读取，只保留非重复读取。Reference splice junctions由参考转录组提供(Ensembl build 73)。用Cufflinks计算FPKM值，用Cufflinks,最大似然估计函数调用差异基因表达。表达显著变化的基因由false discovery rate(FDR)-corrected P value<0.05定义，仅用状态“Known”和生物型“coding”的ensembl genes 73 进行下游分析。
接下来使用Trim Galore(v0.3.0)(http://www.bioinformatics.babraham.ac.uk/projects/trim_galore/)修剪Reads，而质量评估使用FastQC(v0.10.0)(http://www.bioinformatics.bbsrc.ac.uk/projects/fastqc/)。随后，利用DAVID bioinformatics platform(https://david.ncifcrf.gov/),、Ingenuity Pathways Analysis(IPA)program(http://www.ingenuity.com/index.html)。在Majorbio I-Sanger Cloud Platform(www.i-sanger.com)免费在线平台上对原始数据进行了初步分析，并将原始数据存入NCBI Gene Expression Omnibus(GEO)database数据库，登录代码为GSE156301。
2.蛋白质-蛋白质相互作用网络分析
用STRING3.0进行蛋白质-蛋白质相互作用(PPI)分析。将符合标准的特定蛋白质导入在线分析软件(http://www.networkanalyst.ca)，选择一个最小交互网络进行进一步的集线器和模块分析。
3.基因集富集分析(GSEA)
基于RNA-seq初步分析所得数据，对于每个差异表达显著基因分析比较，基因是使用从DESeq2获得的“wald statistics”进行排序的，GSEA是在MSigDB(http://software.broadinstitute.org/gsea/msigdb)中可用的所有规划基因集的这些排序列表上进行的)。DESeq2independent filtering是基于归一化读取计数的平均值，筛选出表达水平很低的基因。SASP和GSEA signature如发明人过往发表文献所述(Zhang等人，2018a)。
4.定量PCR(RT-PCR)测定基因表达
(1)细胞总RNA的提取
以Trizol试剂抽提处于生长期或停滞期细胞的总RNA，每T25培养瓶细胞加入1ml Trizol，用细胞刮刀刮下细胞层后将其转移至离心管中，充分混匀至不粘稠。每1ml Trizol加0.2ml氯仿，剧烈震荡15sec，室温孵育5-10min；4℃，11,000g离心15min；将无色上清液移入一新的离心管中，按每1ml Trizol加0.5ml异丙醇，室温孵育10分钟，11,000g，4℃离心10min；倒掉上清，用75％乙醇(每1ml Trizol至少用1ml 75％乙醇)洗涤，4℃，7,500g离心5min；室温干燥RNA沉淀5-10分钟(RNA不能干燥)，用DEPC-H 2O溶解沉淀。
分光光度计定量RNA之后，取少量总RNA进行1％琼脂糖电泳，检查RNA状态和质量。
(2)逆转录反应
OligodT 23V N(50uM)      1ul
Total RNA              1-2ug
RNase Free ddH 2O       to 8ul
65℃加热5min，迅速置于冰上骤冷，并静置2min。
配置第一链cDNA合成液
2x RT Mix              10ul
HiScript II Enzyme Mix 2ul
按照以下条件进行第一链cDNA合成：
25℃              5min
50℃              45min
85℃              5min
(3)实时定量PCR反应
将逆转录反应产物cDNA稀释50倍作为模板。
Figure PCTCN2021130639-appb-000006
按照以上标准加样，反应条件为：95℃预变性15sec，然后95℃5sec，60℃31sec，40个循环；融解曲线条件为95℃15sec，60℃30sec，95℃15sec。样品于ABI ViiA7(ABI)仪上进行反应。以β-actin的表达作内参。反应完成后，经软件分析查看每个基因的扩增情况，导出相应的域值循环数，采用2-ΔΔCt方法，计算每个基因的相对表达量。对融解曲线(melting curve)的波峰和波形进行分析以确定得到的扩增产物是否为特异性单一目的片段。
5.SA-β-Gal染色
衰老相关β-半乳糖苷酶(SA-β-Gal)染色，操作执行以往报道程序(Debacq-Chainiaux等人，2009年)。简单地说，细胞培养皿中经PBS洗涤，在室温下固定。在2％甲醛和0.2戊二醛中作用3min，用以固定细胞。然后用新制备的染色液对SA-β-Gal进行染色，在37℃下过夜。第二天拍摄图像并计算单位面积内阳性细胞百分比。
6.克隆扩增实验
单细胞克隆扩增实验，按过往文献所述进行(Duan等人，2015年；Wu等人，2018年)。简单地说，细胞被铺板于明胶涂层的12孔板，密度为2000个细胞/孔。。结晶紫染色之后计算细胞克隆数。
7.药物诱导衰老细胞凋亡
将PSC27细胞铺板于96孔皿中，在50μg/ml的BLEO处理下诱导细胞衰老。分别以5.0μm和1.0μm的浓度加入GSE和ABT263。细胞培养基配以Incucyte Nuclight快速红色试剂(Essen Bioscience)和IncucyteC-3/7细胞凋亡试剂(Essen Bioscience)。选取代表性视野进行拍照。
二、结果
1.GSE在低浓度下使用时可以有效抑制SASP的表达
发明人对实施例1第二轮筛选后的细胞进行了RNA-seq测序。而随后获得的高通量数据表明，经过实施例1的筛选方法筛选获得的葡萄籽提取物(grape seed extract,GSE)，显著改变了衰老细胞的表达谱。其中2644个基因出现显著下调，同时1472个基因发生上调，发明人这里的heatmap中每个基因的倍数变化为2.0(P<0.01)(图6A)。重要的是，GSE处理之后的衰老细胞中SASP因子的表达普遍降低，而这些SASP因子一般会在衰老细胞中明显上调(图6B)。虽然一些SASP不相关基因的表达情况与那些典型的SASP因子表现出类似的趋势，但GSEA分析的数据进一步揭示了表征SASP表达或NF-KB激活的分子特征的显著抑制，后者是介导促炎SASP发展的主要转录性事件(图7)。基于蛋白质-蛋白质相互作用的生信分析结果显示了一个高度活跃的网络，其涉及多种因素在细胞衰老时显著上调，而一旦细胞处于GSE作用下，则反而呈现下调(图8)。进一步的GO生物信息学数据表明，这些分子在功能上参与了一组重要生物过程，包括信号转导、细胞间通讯、能量调节、细胞代谢和炎症反应(图9)。这些下调基因中的大多数，生化本质上属于表达后即释放至胞外空间的蛋白质，或位于内质网或高尔基体上，总体而言在特征上与这些分子的分泌性质相互一致(图10)。
为了进一步证实和研究GSE在体外条件下对SASP表达的影响，发明人在一系列体外梯度浓度下处理了PSC27细胞(PSC27细胞参见实施例1；另外，PSC27细胞的处理方法参见实施例1的两轮筛选)。两轮筛选的实验结果如图11A、图11B和表1所示。
表1：两轮筛选实验结果
Figure PCTCN2021130639-appb-000007
数据表明，工作浓度在1.25μM时的GSE以最大的效率抑制了SASP发生发展(图11B)。然而，较低或较高浓度的这种药物的疗效却不理想，尽管后者可能与这种药物的细胞毒性增加引起的细胞应激反应有关(图11B)。因此，GSE这一植物性天然产物，可用于控制衰老细胞的促炎表型，即SASP，尤其在相对低浓度下使用彰显最佳效果。
另外，发明人还基于上述体外检测对SASP表达的影响的方法，在仅将GSE替换为候选药物A(候选药物A参见实施例1)，其余条件不变的情况下进一步进行了检测。检测结果如图11C所示。由图11C可知，候选药物A在第一轮筛选后，绝大部分SASP标志因子的表达并未产生显著性差异，P>0.05，与GSE之间形成鲜明对比。其余候选药物与候选药物A的结果大致相同，即其余候选药物在第一轮筛选后，绝大部分甚至是全部SASP标志因子的表达均未产生显著性差异，P>0.05，与GSE之间形成鲜明对比。该结果验证了实施例1的筛选方法的准确性，除GSE以外的候选药物A和其余候选药物在第一轮筛选后即会被淘汰。
另外，发明人还基于上述体外检测对SASP表达的影响的方法，通过变换第一轮和第二轮的个别处理条件，进一步进行了检测。检测条件和检测结果如下表所示。
Figure PCTCN2021130639-appb-000008
Figure PCTCN2021130639-appb-000009
由上表可知，第一轮筛选中，若细胞密度过大或过小，或药物处理时间过长或过短，或者第二轮筛选中，药物处理时间过长或过短，会在SASP表达数据上出现不准确或结果分歧(例如，经过梯度浓度的GSE处理后的衰老细胞相比于未经GSE处理的衰老细胞，部分甚至大部分SASP标志因子的表达并未产生显著性差异，P>0.05，进而，根本无法筛选最优浓度)。以上结果进一步验证了实施例1中本申请筛选方法的准确性。
2.当在高浓度使用时GSE是一种新型的senolytics
鉴于GSE在控制SASP表达方面的显著疗效，发明人接下来进一步探究了这种天然产物在较高浓度下杀死衰老细胞的潜力。为此，发明人测量了随着GSE浓度的增加，体外条件下所处理(处理方法参见实施例1的两轮筛选)的衰老细胞(细胞参见实施例1)的生存百分比。两轮筛选的实验结果如图12A、图12B和表2所示。
表2：两轮筛选实验结果
Figure PCTCN2021130639-appb-000010
Figure PCTCN2021130639-appb-000011
SA-β-GAL染色数据表明，在GSE浓度达到5μM之前，衰老细胞不会被消除(图12B)。随着浓度的增加，GSE对衰老细胞(80％染色阳性)的杀伤效果进一步增强，而当GSE在25μM时达到阈值(衰老细胞此时剩余20％)；当其浓度升高到50μM时，GSE的杀伤效果没有进一步增强(图12B；图13)。
为了进一步剖析这些问题，发明人做了验证性实验。细胞活力测定(测定方法参见实施例1)表明，与其增殖态对照细胞相比较，GSE从5μM浓度开始诱导衰老细胞显著死亡(图14)。当GSE浓度增加到50μM时，存活衰老细胞的百分比下降到约10％。然而，即使在GSE的50μm时，增殖细胞也并未明显减少。这些结果，证实了GSE对衰老细胞高度的选择性和突出的特异性，而这种特征实际是senolytics作为一类独特的抗衰老药的基本要求。
发明人接下来研究了基质细胞经基因毒性处理后群体倍增(population doubling,PD)的潜力。与损伤性处理之后迅速进入生长停滞状态的BLEO组细胞相比，BLEO和GSE的联合治疗组表现出显著增高的PD能力(图15)。然而有趣的是，GSE本身似乎不影响增殖细胞的PD，这一数据进一步表明GSE在衰老细胞与正常细胞之间的选择性。
为了探究GSE是否通过诱导凋亡的方式造成衰老细胞丧失存活能力，发明人使用GSE在培养条件下分别处理增殖组细胞和衰老组细胞。随后观察到的caspase-3/7活性变化结果，表明GSE引起衰老细胞发生凋亡；从GSE加入之后的第16小时，衰老组开始与对照组之间出现统计学差异(图16)。此外，泛caspase抑制剂QVD可防止GSE对衰老细胞的杀伤，这一过程中的实际效果跟ABT263(一种目前已知的、十分有效的衰老细胞凋亡诱导剂)对衰老细胞的影响非常相似(图17)。上述一系列结果证实，GSE通过诱导凋亡的方式促使衰老细胞进入死亡程序，但增殖态细胞基本不被这一天然药物靶向。
鉴于GSE对衰老细胞产生的明显影响，发明人随后分析了GSE诱导细胞凋亡的潜力。流式细胞数据显示衰老PSC27细胞活力显著降低，而其凋亡比例显著升高，但增殖细胞的变化却并不明显(图18；图19)。因此，发明人的数据一致性支持GSE在体外条件下通过 诱导细胞凋亡的方式引起衰老细胞的消除，该天然产物在靶向衰老细胞方面具有突出的潜力。
实施例3抗衰老药物的体内分析和验证
一、方法
1.小鼠移植瘤接种和预临床治疗试验
所有实验小鼠实验均严格遵循中国科学院上海生命科学研究院实验动物看护和使用委员会(IACUC)的有关规章进行。年龄6-8周的免疫缺陷型小鼠(NOD-SCID mice,ICR)(体重约25g)用于本专利相关动物实验。基质细胞PSC27和上皮细胞PC3以1:4预先确定的比例混合，而每一移植体包含1.25×10 6细胞，用于组织重构。移植瘤通过皮下移植方式植入小鼠体内，移植手术结束之后8周末动物被执行安乐死。肿瘤体积按照如下公式计算：V＝(π/6)x((l+w)/2) 3(V,体积；l,长度；w,宽度)。
在预临床治疗试验中，经过皮下移植的小鼠被供给标准实验食谱，2周之后实施化疗药物米托蒽醌(MIT,0.2mg/kg剂量)和/或葡萄籽提取物(GSE)(500μl,10mg/kg剂量)腹腔给药。时间点为：前者在第3,5,7周的第一天，后者在第5,7周的第一天。整个疗程共进行3次MIT循环给药，每个循环为2周。疗程结束后，小鼠肿瘤被收集用于体积测量和组织学分析。每只小鼠累积性共接受MIT这一药物0.6mg/kg体重，GSE则为30mg/kg体重。为造成全身范围SASP因子在化疗诱导下表达，MIT按照以上步骤和顺序，经过静脉输注方式对小鼠给药，但剂量下降至0.1mg/kg体重/每次(整个疗程累计接受MIT剂量为0.3mg/kg体重)以减轻药物相关毒性。化疗试验进行到第8周末结束，小鼠处死之后立即解剖，其移植瘤被收集并用于病理系统分析。
2.小鼠寿命研究
在细胞移植研究中，发明人在SPF动物平台通过连续饲养获得了16个月大的雄性C57BL/6小鼠，每个笼子里有4到5只动物。发明人首先按体重从低到高对小鼠进行分类，然后选择了体重相似的小鼠。接下来，衰老(SEN)或对照(CTRL)移植治疗方式，则使用随机数产生器被分配给每间隔一次的小鼠，而中间的小鼠被分配到另一种治疗方式中，从而使衰老和对照移植小鼠的体重匹配。细胞移植1个月后，当小鼠年龄为18个月时，进行身体功能测试。在那之后，除了检查它们的笼子外，没有对这些老鼠进行进一步的测试。最早的死亡发生在上次身体功能测试后大约2个月。19至21个月大的C57BL/6小鼠，每个笼子里 安放有3-5只。与移植小鼠一样，小鼠根据体重进行分类，并随机分配给每一组，由不知道预临床试验设计的人进行对照组(vehicle)或药物组(GSE)组处理。从24-27个月龄开始，小鼠每2周用vehicle或GSE治疗一次，每次连续3天口服灌胃。在研究过程中，一些老鼠被从原来的笼子移走，以尽量避免在单一笼子中长期饲养产生的动物居住压力。RotaRod和hanging测试每月进行，因为这些测试是敏感和无创的。试验结束时，发明人对小鼠进行安乐死；如果它们表现出以下几种症状之一，发明人就认为它们已经死亡：(一)不能饮水或吃饭；(二)即使有刺激也不愿意移动；(三)快速减肥；(四)严重的平衡障碍；或(五)机体出血或出现溃疡肿瘤。试验过程中，没有老鼠因为打架、意外死亡或皮炎而被排除在外。进行生物统计时，发明人采用Cox proportional hazard model进行生存分析。
3.预临床动物死后病理检查
研究人员每天对笼子进行检查，并将死鼠从笼子中取出。在动物死亡24小时内，尸体被打开(腹腔、胸腔和颅骨)，并单独保存在10％福尔马林中至少7天。分解或破坏的身体被排除在外。保存的尸体被运到Autopsy专用地点进行病理检查。评估肿瘤负担(每个小鼠不同类型肿瘤的总和)，疾病负担(每个小鼠主要器官不同组织病理学变化的总和)，每个病变的严重程度和炎症(淋巴细胞浸润)。
4.生物发光成像
小鼠腹腔注射3mg荧光素(BioVision,Milpitas,CA)，以体积200μl的PBS递送。小鼠用异氟烷麻醉，使用Xenogen IVIS 200 System(Caliper Life Sciences,Hopkinton,MA)获取生物发光图像。
5.体能检测
所有检测均在最后一次安慰剂或药物处理后的第5天开始。最大步行速度采用加速RotaRod System(TSE System,Chesterfiled,MO)进行评估。在RotaRod上小鼠被训练3天，速度分别为4，6和8r.p.m，第1、2和3天历时200秒。在测试日，小鼠被放置在RotaRod上，在4r.p.m速度下开始。以5分钟为间隔，转速由4加速到40r.p.m。当老鼠从RotaRod上掉下来时，速度被记录下来。最终结果从3或4个试验中取平均值，并规范为基线速度。在前两个月内训练过的小鼠不再接受训练。
前肢握力(N)使用Grip Strength Meter(Columbus Instruments,Columbus,OH)测定，结果来自超过10个试验的平均值。对于悬挂耐力试验，小鼠被放置在一个2毫米厚的金属线上，后者位于垫子上方35厘米处。小鼠只被允许用前肢抓住电线，悬挂时间根据体重进行规范 化，表示为悬挂持续时间(sec)×体重(g)。结果取每只小鼠2到3次实验的平均值。通过Comprehensive Laboratory Animal Monitoring System(CLAMS)监测24小时(12小时光照和12小时黑暗)的日常活动和食物摄入量。CLAMS系统配备了Oxymax Open Circuit Calorimeter System(Columbus Instruments)。对于跑步机性能，小鼠在5°倾斜度下适应在电动跑补机(Columbus Instruments)上跑步，经过3天训练，每天持续5分钟，以5米/分钟的速度开始2分钟，继而加速至到7米/分钟2分钟，然后9米/分钟1分钟。在试验当日，小鼠在跑步机上以5米/分钟的初始速度跑步2分钟，然后每2分钟增加2米/分钟的速度，直到小鼠筋疲力尽。疲劳被定义为即便有轻微的电击刺激和机械刺激，小鼠仍无法回到跑步机上。试验结束后记录距离，用下列公式计算总功(KJ)：质量(kg)×g(9.8m/s 2)×距离(m)×sin(5°)。
二、结果
1.使用GSE治疗性靶向衰老细胞可促进肿瘤消退并能有效降低化疗耐药
鉴于GSE在体外较高浓度条件下清除衰老细胞中的突出选择性，发明人接下来考虑这种药物是否可以被利用来干预体内与增龄相关的疾病。癌症是严重威胁人类寿命和危害健康的主要慢性疾病之一。此外，临床中癌细胞耐药性限制了大多数抗癌治疗的效果，而衰老细胞往往通过在受损肿瘤灶中发展SASP来促进治疗性耐药的发生。即便如此，从原发肿瘤中清除衰老细胞以促进癌症治疗指数的可行性与安全性，至今几乎未被科学家们探索过。
首先，发明人通过将PSC27基质细胞与PC3上皮细胞混合构建成组织重组体，后者是一种典型的高度恶性前列腺癌细胞系。在非肥胖糖尿病和严重联合免疫缺陷(NOD/SCID)实验小鼠大腿后侧皮下植入重组体之前，基质细胞与上皮细胞的数量比例为1：4。动物在重组体植入体内之后8周结束时，测量肿瘤大小(体积)(图20)。同由PC3癌细胞和原代PSC27基质细胞组成的肿瘤相比，由PC3细胞和衰老PSC27细胞组成的异种移植物(xenograft)体积显著增加(P<0.001)，这一差异再次证实了衰老细胞在肿瘤进展中的关键促进作用(图21)。
为了更加接近临床条件，发明人特别设计了一种临床前方案，其中涉及基因毒化疗药物治疗和/或衰老药物干预(图22)。在皮下植入两周后，当观察到体内肿瘤已经稳定被摄取时，发明人在第3、第5和第7周的第一天分别向实验动物提供单次剂量的MIT(MITOXANTRONE，一种化疗剂)或安慰剂，直到8周方案全部结束。同安慰剂治疗组相比，MIT给药可显著延缓肿瘤生长，这证实了MIT作为化疗药物的疗效(肿瘤大小减少 44.0％，P<0.0001)(图23)。值得注意的是，虽然GSE本身并不会引起肿瘤收缩，但对治疗MIT后的小鼠，GSE给药却可显著减小肿瘤(与MIT相比，肿瘤体积减少55.2％，P<0.001；与安慰剂治疗相比，肿瘤体积减少74.9％，P<0.0001)(图23)。
接下来，发明人推断细胞衰老是否发生在这些动物的肿瘤灶中。检测结果证明，MIT给药过程诱导了肿瘤组织中大量衰老细胞的出现，尽管这毫不奇怪。然而，GSE给药则将这些化疗动物病灶内的大多数衰老细胞基本耗尽(图24；图25)。激光捕获显微解剖(LCM)和随后的定量PCR(条件参见实施例2)结果表明，SASP因子的表达显著升高，包括IL6、CXCL8、SPINK1、WNT16B、GM-CSF、MMP3、IL1Α，这一趋势伴随着化疗动物衰老标记p16 INK4A的上调(图26)。有趣的是，这些变化主要发生在基质细胞中，而不是它们邻近的癌细胞，这意味着残留癌细胞再增殖的可能性，而这些细胞在治疗损伤的TME中产生了获得性耐药。然而，在使用GSE给药时，这一变化在很大程度上被逆转，正如转录水平数据分析结果所展示的那样(图27)。
为了研究直接支持在MIT给药的小鼠中SASP的表达和逆转这种衰老相关模式的机制，发明人在第一次GSE给药7天后即解剖了这两种药物治疗的动物体内的荷瘤，选择给药7天后这一时间点主要是因为这时病灶中癌细胞耐药克隆尚未形成。与安慰剂相比，MIT给药导致DNA损伤和凋亡程度均显著增加。虽然GSE单独不能诱导DNA损伤或造成凋亡，但化疗药物MIT却可以高度上调这两个指标(图28)。然而，当MIT处理的动物与GSE一起使用时，DNA损伤或凋亡的指数明显增强，这意味着这些衰老药物处理条件下的动物体内肿瘤位点细胞毒性增强。作为支持性证据，当GSE在治疗上应用时，caspase 3 cleavage活性升高，这是细胞凋亡的一个典型标志(图29)。
接下来发明人比较了不同药物处理组动物的生存情况，主要以一种时间延长的方式来评估肿瘤进展的后果。在这一临床前队列中，发明人对动物进行了肿瘤生长监测，一旦小鼠内体肿瘤负担突出(大小≥2000mm 3)，就会判断为严重疾病已经发生，这是一种用于某些情况下肿瘤等疾病的病情进展的方法。接受MIT/GSE组合治疗的小鼠表现出最长的中位生存期，与仅接受MIT治疗的组相比，存活期延长了至少48.1％(图30，绿色与蓝色相比)。然而，仅用GSE治疗荷瘤小鼠并没有造成显著的好处，只有边际性生存延伸。
值得注意的是，在这些研究中进行的治疗似乎被实验小时很好地耐受。发明人没有观察到尿素、肌酐、肝酶或体重的显著波动(图31；图32)。更重要的是，在本研究设计的各药剂量下使用的化疗和抗衰老药物不会显著干扰免疫系统的完整性和关键器官的组织稳 态，即使在免疫完整型的野生小鼠中也是如此(图33；图34)。这些结果一致证实，抗衰老剂结合常规化疗药物有可能在普遍意义上增强肿瘤反应，而不引起严重的全身毒性。
2.GSE治疗造成的衰老细胞清除可以减轻机体功能障碍，延长老龄小鼠的晚年生存期，而不增加其在生命晚期阶段的发病率
既然GSE具有在肿瘤小鼠的微环境中清除衰老细胞、降低肿瘤耐药性和提高总体治疗效果的药效，那么对于自然衰老的动物是否也有某种促进健康或延缓疾病的显著益处？为回答这一问题，发明人首先测试了GSE将表达荧光素酶(luciferase,LUC)的、腹腔注射到野生型(WT)小鼠体内的衰老或对照细胞予以清除的潜力。与Vehicle处理相比，GSE处理过的LUC衰老细胞移植小鼠的体内相应部位生物发光强度明显降低(图35)。然而，体内有LUC对照细胞(增殖态，未衰老)移植的小鼠经过治疗后，Vehicle组和GSE组之间没有观察到显著差异(图36)。这些数据进一步支持GSE可以在组织微环境中选择性杀死衰老细胞。
为了确定衰老细胞在老年小鼠生理功能障碍中发挥的作用，发明人用GSE治疗20个月龄的非移植性WT小鼠，以安慰剂(Vehicle)为平行对照。治疗为间歇性的给药，历时4个月(图37)。结果表明，GSE减轻了小鼠的身体机能障碍，主要表现在显著提高的最大步行速度、悬挂耐力、握力、跑步机耐力和日常活动能力(图38)。相比于用Vehicle处理组，GSE给药组的食物摄入量也倾向于增高，尽管没有达到统计学显著程度(P＝0.1682，图38)。此外，GSE处理组的老龄小鼠内脏组织微环境中基质细胞几种重要SASP成分(qRT-PCR定量分析方法参见实施例2)的表达出现普遍降低(图39)，进一步说明组织水平的衰老细胞数量及其造成的影响均已被有效控制。
发明人进而考虑，是否可以使用一种具有潜在转化价值的方法来消除衰老细胞，即：从非常老龄的某一时间点开始进行间歇治疗，能否延长WT小鼠的剩余寿命(图40)？对此，一系列体内试验得以相应开展。值得发明人注意、也十分令人惊讶的是，在每两周服用一次药物的治疗方案下，从24-27个月年龄(相当于人类75-90岁的年龄)开始给药的GSE组，其治疗后中位生存期比Vehicle组延长了64.2％，同时具有较低的死亡危险(HR＝0.35，GSE组/Vehicle组；或HR＝2.857，Vehicle组/GSE组，P<0.0001)(图41，图42)。这一发现，表明GSE介导的衰老细胞清除可以降低老年小鼠的死亡风险，并有效延长其生存期。
为了进一步检验这种降低老年小鼠死亡率的治疗方案，是否以提高机体的晚期发病率为代价，发明人评估了这些小鼠的身体功能。尽管GSE组小鼠的剩余寿命较长，但经过GSE每两周一次给药处理的小鼠，在生命的最后2个月的身体功能跟Vehicle处理组的小鼠在雄、和雌两性之间分别比较时，并未出现显著降低((图43，图44)。此外在小鼠尸检中，几种年龄相关疾病的患病率和肿瘤负担，在两组之间也没有统计学差异(图45，图46)。因此，间歇性提供GSE这种具有生物活性的抗衰老药物，可以通过清除微环境中衰老细胞的方式，显著减少衰老机体的疾病负担，并可以增加治疗后阶段机体的寿命。这种治疗方式，并不会导致显著上升的机体发病率，可以在生命的晚期阶段安全使用。
实施例4基于ESI-QTOF-MS的天然药物实际组分鉴定
一、方法
1.样本制备
将葡萄籽提取物(GSE)溶解在1ml DMSO中，超声处理5min。涡旋1min，离心5min，每分钟14,000转，在HPLC分析前通过0.25mm过滤器过滤。用Agilent 1200系列快速分辨率LC系统(Agilent Technologies,Palo Alto,CA,USA)对GSE进行了分析鉴定，该系统配备了二元泵和自动采样器。将HPLC系统耦合到四极飞行时间质谱仪(QTOF)质谱仪(Bruker Daltonics,Bremen,Germany)，配备了电喷雾电离(ESI)界面(model G1607A from Agilent Technologies,Palo Alto,CA,USA)。荧光(ORAC)和吸光度(Folin-Ciocalteu,Vanillin assay,FRAP and TEAC)测量在协同Mx单色基多模微平板阅读器(Bio-Tek Instruments Inc.,Winooski,VT,USA)上进行，使用96孔聚苯乙烯microplates。
2.色谱条件
用Zorbax Eclipse Plus C18 column(1.8μm,150×4.6mm在室温下分离葡萄籽中的化合物。流动相为乙酸0.5％(溶剂a)和甲醇(溶剂b)。采用多步线性梯度：0min,0％B；5min,25％B；15min,35％B；20min,39％B；38min,60％B；40min,70％B；42min,80％B；44min,100％B；46min,0％B；48min,0％B。初始条件维持10min。注射体积为10μl。所用流量设定为0.3ml/min。
3.ESI-QTOF-MS检测
在配备ESI接口的QTOF质谱仪上进行了HPLC分析。在负离子模式下，毛细管电压工作在3.5KV条件下。源的其它参数设置为：干燥气体温度220℃；干燥气体流量，9L/min； 雾化气体压力，2.5bar。检测的质量范围为50～1200m/z。
利用测量的[M-H]-离子，提出了每种分析物的分子公式，并通过软件DataAnalysis 4.0(Bruker Daltonics)进行了处理，在5ppm时建立了确认元素组成的公认精度阈值。在HPLC方法的开发过程中，采用74900-00-05注射器泵(Vernon Hills,IL,USA)作为直接连接到界面的外部仪器校准，用乙酸钠团簇溶液。在每次运行开始时注入校准溶液，并在化合物鉴定之前对所有光谱进行校准。整个运行过程中的溶剂流量为0.6和1.5ml/min分析和半制备性HPLC。洗脱柱是在270nm波峰处监测每个样本和单个多酚或化合物的紫外吸光度通过与HPLC滞留时间的比较，收集和鉴定峰处所在的真实化合物，理化性质和紫外，i.r.，MS， 1H-NMR还有 13C-NMR谱分析。
二、结果
利用先进的化学分析技术对天然药物的实际成分(主要是各种植物来源的化合物)进行全面的分析和表征是至关重要的。这样，就需要建立合适的方法来表征GSE中的生物活性化合物。基于飞行时间质谱(QTOF)技术允许的精确质量和串联质谱对于元素组成的准确分析至关重要，发明人在这里需要获得有关包括小数在内的精确质量。
使用这一强大的技术，发明人分析了GSE中分布的16种主要化合物：1)酚酸；(2)多酚黄酮类化合物(包括原花青素和其它类黄酮)；(3)其它化合物(如槲皮素及其衍生物)。发明人的数据显示了GSE的基峰色谱图(BPC)(图47)，观察到的主要峰列于GSE成分总表中(下表3)。考虑到洗脱顺序，所有化合物的特征可用于解释它们由QTOF-MS获得的质谱数据。
表3：经过HPLC-ESI-QTOF-MS成分鉴定获得的GSE中各有效成分的具体信息，包括名称、比例(重量百分比)和分子公式
Figure PCTCN2021130639-appb-000012
Figure PCTCN2021130639-appb-000013
以上所述的具体实施例，对本发明的目的、技术方案和有益效果进行了进一步详细说明，所应理解的是，以上所述仅为本发明的具体实施例而已，并不用于限定本发明的保护范围，凡在本发明的精神和原则之内，所做的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。

Claims (10)

  1. 一种筛选具有抗衰老潜力天然产物的方法，其包括：
    (1)将候选天然产物与衰老细胞进行第一接触处理，之后将未经过所述第一接触处理的衰老细胞和经过所述第一接触处理后的衰老细胞进行细胞增殖检测，同时将对照细胞和经过所述第一接触处理后的衰老细胞进行细胞凋亡检测，所述第一接触处理是在96孔板中进行的，所述第一接触处理中所述衰老细胞的密度为4500-5500个/孔(优选5000个/孔)，所述第一接触处理的时间为5-10天(优选7天)，所述衰老细胞是通过将所述对照细胞进行衰老诱导处理后获得的；
    所述细胞增殖检测和细胞凋亡检测后，同时满足以下条件的候选天然产物为初筛天然产物：
    A、所述细胞增殖检测后，经过所述第一接触处理后的衰老细胞与未经过所述第一接触处理的衰老细胞相比，检测结果具有显著性差异(P<0.05)；
    B、所述细胞凋亡检测后，经过所述第一接触处理后的衰老细胞与对照细胞相比，检测结果具有显著性差异(P<0.05)；
    (2)将所述初筛天然产物与衰老细胞进行第二接触处理，之后将未经过所述第二接触处理的衰老细胞和经过所述第二接触处理后的衰老细胞进行细胞增殖检测，同时将对照细胞和经过所述第二接触处理后的衰老细胞进行细胞凋亡检测，所述第二接触处理是在96孔板中进行的，所述第二接触处理中所述衰老细胞的密度为15000-25000个/孔(优选20000个/孔)，所述第二接触处理的时间为25-35天(优选30天)，所述衰老细胞是通过将所述对照细胞进行衰老诱导处理后获得的；
    所述细胞增殖检测和细胞凋亡检测后，同时满足以下条件的初筛天然产物为目标天然产物：
    A、所述细胞增殖检测后，经过所述第二接触处理后的衰老细胞与未经过所述第二接触处理的衰老细胞相比，检测结果具有显著性差异(P<0.05)；
    B、所述细胞凋亡检测后，经过所述第二接触处理后的衰老细胞与对照细胞相比，检测结果具有显著性差异(P<0.05)。
  2. 权利要求1的方法，其中，所述衰老处理为化疗药物处理或辐射处理；
    优选地，所述化疗药物为博莱霉素(BLEO)；
    更优选地，所述博莱霉素(BLEO)的处理浓度为40-60μg/mL(优选50μg/mL)；
    或者，所述对照细胞为人源前列腺原代基质细胞系(如PSC27)；
    或者，所述第一接触处理和所述第二接触处理的培养基为DMEM(10％FBS)。
  3. 权利要求1的方法，其中，所述第一接触处理中，所述候选天然产物的浓度为1μM-l mM(优选1μM-50μM)；
    或者，所述第二接触处理中，所述初筛天然产物的浓度为1μM-l mM(优选1μM-50μM)。
  4. 权利要求1的方法，其中，所述细胞增殖检测是利用CCK-8 Cell Counting Kit试剂盒进行的；
    或者，所述细胞凋亡检测是利用Caspase 3/7 activity kit(Promega)进行的。
  5. 权利要求2的方法，其中，所述第一接触处理中，所述培养基和所述候选天然产物每两天更换一次；
    或者，所述第二接触处理中，所述培养基和所述初筛天然产物每两天更换一次。
  6. 组合物，其包含葡萄籽提取物，所述葡萄籽提取物的浓度为1-1.5μM(优选1.25μM)或者5-50μM(优选5-25μM，更优选25μM)。
  7. 组合物在制备试剂中的用途，所述试剂用于抑制SASP标志因子的表达，所述组合物包含葡萄籽提取物，所述葡萄籽提取物的浓度为1-1.5μM(优选1.25μM)；
    优选地，所述SASP因子选自肿瘤坏死因子-α(TNF-α)、白细胞介素6(IL-6)、白细胞介素8(IL-8)、白细胞介素1α(IL-1α)、白细胞介素1β(IL-1β)、基质金属蛋白酶(MMP，如MMP3)、粒细胞-巨噬细胞集落刺激因子(GM-CSF)、纤溶酶原激活物抑制因子-1(PAI1)、趋化因子(CXC，如CXCL3)、双向调节因子(AREG)、丝氨酸肽酶抑制因子Kazal型1(SPINK1)，或其任何组合。
  8. 组合物在制备试剂中的用途，所述试剂用于消除衰老细胞，所述组合物包含葡萄籽提取物，所述葡萄籽提取物的浓度为5-50μM(优选5-25μM，更优选25μM)；
    优选地，所述试剂通过诱导衰老细胞凋亡来清除衰老细胞；
    优选地，所述试剂用于诱导衰老细胞凋亡。
  9. 组合物，其包含葡萄籽提取物和米托蒽醌(MIT)；
    优选地，所述葡萄籽提取物和所述米托蒽醌(MIT)的质量比为10:0.1-10:0.3(优选为10:0.2)。
  10. 权利要求9的组合物在制备药物、食品或保健品中的用途，所述药物、食品或保健品用于治疗和/或预防癌症；
    优选地，所述癌症为与衰老相关的癌症；
    优选地，所述癌症为前列腺癌。
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